ES2702246T3 - Stable IgG4 binding agent formulations - Google Patents

Abstract

A formulation comprising: - from 5 mg / ml to 280 mg / ml of an IgG4 antibody, wherein the antibody binds to the chemokine receptor CXC type 5 (CXCR5) and comprises a variable region of heavy chain and a variable region of light chain, wherein the antibody is a humanized anti-CXCR5 IgG4 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26, - from 5 to 50 mM citrate as buffer agent; - from 0.001% to 0.1% w / v of polysorbate 20; the pH of the formulation being from pH 5 to pH 6; and wherein the monomeric antibody has a purity of 90% after storage for at least 6 months at + 5 ° C.

Description

DESCRIPTION

Stable IgG4 binding agent formulations

BACKGROUND OF THE INVENTION

The human LIGHT antigen is a potential target of cytokines that has been implicated in the processes of chronic inflammatory autoimmune diseases. As a member of the TNF superfamily (TNFSF) of ligands, LIGHT is also known as TNFSF14 or CD258. LIGHT is expressed on the surface of T cells after activation in a strictly regulated manner. However, LIGHT is also present in constitutively detectable levels on the surface of immature dendritic cells and on T cells and natural killer (NK) cells of the intestine. LIGHT mediates its biological effects by binding to three receptors of the TNF superfamily, including the lymphotoxin p receptor (LTpR), the herpes virus entry mediator (HVEM) and decoy receptor 3 (DcR3). Lymphocytes that express LIGHT can induce symptoms similar to IBD in humans, and increases in LIGHT expression have been observed in patients with active Crohn's disease and other inflammatory disorders such as graft-versus-host disease.

CXCR5, also known as Burkitt's lymphoma receptor (BLR1), CD185, MDR15 and MGC117347, is a G-protein coupled receptor that is a member of the CXC chemokine receptor family. The unprocessed CXCR5 precursor has a length of 372 amino acids with a molecular weight of 42 K ^d . CXCR5 plays a role in the migration and localization of B cells within particular anatomical compartments. The "inactivated" mice lack peripheral lymph nodes, have fewer Peyer's patches and have reduced B cell levels. CXCL13, also known as BLC, is a ligand for CXCR5. CXCL13 is a chemoattractant of B cells.

The anti-LIGHT binding agents and anti-CXCR5 binding agents are each therapeutically relevant, and there is a need to formulate each of these binding agents into pharmacological products that can be administered to subjects, particularly human subjects, for treatment of inflammatory diseases. In order to develop a pharmaceutical formulation containing an anti-LIGHT binding agent or an anti-CXCR5 binding agent suitable for intravenous or subcutaneous administration, the binding agent should be concentrated to about 20 mg / ml or more, generally at about 100-150 mg / ml, and even up to 250 mg / ml. Many complications can arise at such high concentrations, including an increase in viscosity, a shift in pH, a change in the color of the solution and the formation of visible and subvisible particles.

The formulation of these binding agents is further complicated by the fact that these agents are highly prone to aggregation at said high concentrations.

The formulation of IgG4 antibodies is further complicated by the fact that IgG4 antibodies tend to form half molecules at high concentrations in solution. However, IgG4 antibodies are of therapeutic interest because they have a reduced effector function.

WO2004 / 071439 (example 15) describes a formulation comprising 2-50 mM ^citrate buffer ^{, pH} 6 ^{+/- 0.5 and 0.001% to 2.0% surfactant. This document also discloses that a} lyophilized formulation comprising 20-200 mg / ml of an IgG4 antibody (natalizumab; anti-4a-integrin) can be obtained by using: i) a buffer agent selected from the group consisting of phosphate, histidine, citrate, acetate or succinate in the ^{range of 2-50 mM concentration to provide a pH preferably} 6 ^{+/- 0.5 and ii) a surfactant such as a polysorbate at a concentration of 0, 001% to 2, 0% to improve stability and reduce the time of} reconstitution.

SUMMARY OF THE INVENTION

To meet these and other needs, formulations of highly stable IgG4 binding agents are provided herein. It has surprisingly been discovered that highly stable IgG4 binding agent formulations in the form of lyophilized liquids and powders comprising an IgG4 binding agent and a ^{citrate buffer, wherein the pH of the formulation is equal to or less than about pH} 6 ^{and the} isoelectric ^point of the binding agent. These formulations improve with respect to conventional formulations, which often lead to the dimerization of the binding agent, such as an antibody, by increasing the concentration of the binding agent, such as an antibody, in the formulation. In particular, the formulations of the invention reduce the amount of unwanted by-products, including aggregates, half molecules, degradation products, low molecular weight proteins (LMWP), high molecular weight proteins (HMWP) and rearrangements of acid, basic isoforms. and neutral of the binding agent, such as an antibody, component of the formulation. The present invention is defined in the claims.

In certain aspects, the specification provides a stable formulation comprising: an agent of binding comprising at least a portion of an Fc region of an IgG4 antibody; and from about 5 to about 50 mM citrate as a buffering agent; the pH of the formulation being equal to or less than ^{about pH} 6 ^{and the pI of the binding agent. According to the invention, the binding agent is an antibody.}

In certain aspects, the binding agent or antibody binds to lymphotoxin-like substances, shows an inducible expression and competes with herpes virus glycoprotein D for the herpes virus entry mediator, a receptor expressed in lymphocytes (LIGHT ). In specific aspects, the binding agent or the anti-LIGHT antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions (CDRs) comprising the amino acid sequences of SEQ ID NO: 1, 2 and 3, and the light chain variable region ^{comprises CDRs comprising the amino acid sequences of SEQ ID NO: 4, 5 and} 6 ^{. In other aspects, the} antibody is a fully human anti-LIGHT IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the ^{amino acid} sequence of ^{SEQ ID NO:} 8 ^.

In certain aspects, the binding agent or antibody binds to the chemokine receptor C-X-C type 5 (CXCR5). In specific aspects, the binding agent or anti-CXCR5 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions (CDRs) comprising the sequences of amino acids of SEQ ID NOS: 15, 16 and 17, and the variable region of the light chain comprises CDRs comprising the amino acid sequences of SEQ ID NOS: 18, 19 and 20. In other specific embodiments of the invention, the antibody is a humanized anti-CXCR5 IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26.

According to the invention, the concentration of antibodies is from 5 to 280 mg / ml. In certain specific embodiments of the invention, the antibody concentration is about 150 mg / ml. In other specific embodiments of the invention, the antibody concentration is about 50 mg / ml. In ^{further specific embodiments of the invention, the antibody concentration is about} 20 mg / ml. In still other specific embodiments of the invention, the antibody concentration is ^about 100 ^{mg / ml.}

In certain embodiments of the invention, the citrate concentration is from about 5 to about 15 mM. In some embodiments of the invention, the citrate concentration is about 10 mM. In some embodiments of the invention, the citrate buffer is sodium citrate dihydrate.

^{In certain embodiments of the invention, the pH of the formulation is between pH 5 and pH} 6 ^{. In} specific embodiments of the invention, the pH of the formulation is selected from the group consisting of about pH 5.0, about pH 5.5 and about pH 6.0.

In a specific aspect, the pI of the binding agent or antibody is between about 6.8 ^{and about 7.2. In an alternative specific aspect, the pI of the binding agent or antibody} is between about 7.6 and about 8.4.

According to the invention, the formulation additionally comprises a surfactant. According to the invention, the concentration of surfactant is between 0.001% and 0.1% w / v. According to the invention, the polysorbate is polysorbate 20. In ^{some specific embodiments of the invention, the concentration of polysorbate} 20 ^is about 0.005% w / v. In alternative specific embodiments of the invention, the concentration of polysorbate 20 is about 0.01% w / v. In other forms of specific alternative embodiment of the ^{invention, the concentration of polysorbate} 20 ^{is about} 0 ^{02% w / v.}

In certain embodiments of the invention, the formulation additionally comprises a tonicity agent. In certain specific embodiments of the invention, the concentration of tonicity agent is between about 0.1% and about 10% w / v. In certain specific embodiments of the invention, the tonicity agent is a saccharide. In some specific embodiments of the invention, the saccharide is mannitol. In other specific embodiments of the invention, the concentration of mannitol is between about 1% and about 10% w / v. In other specific embodiments of the invention, the concentration of mannitol is about 4%. In alternative specific embodiments of the invention, the saccharide is sucrose. In some ^{specific embodiments of the invention, the sucrose concentration is between about} 1 ^{% and} about 10% w / v. In some specific embodiments of the invention, the sucrose concentration is about 5% w / v. In alternative specific embodiments of the invention, the ^{sucrose concentration is about} 6 ^{% w / v. In still other specific embodiments} of the invention, the sucrose concentration is about 4.5% w / v. In additional specific alternative embodiments of the invention, the tonicity agent is sodium chloride. In In some specific embodiments of the invention, the concentration of sodium chloride is between about 0.01% and about 1%. In some specific embodiments of the invention, the concentration of sodium chloride is about 0.2%. In other specific embodiments of the invention, the tonicity agent is a combination of sucrose and sodium chloride. In ^specific embodiments ^{of the invention, the sucrose concentration is between about} 1 ^{% and} about 10% w / v. In other specific embodiments of the invention, the sodium chloride concentration is between about 0.01% and about 1%. In ^{alternative specific} embodiments ^{of the invention, the concentration of sucrose is about} 6 ^{% w / v and the} concentration of sodium chloride is about 0.2%. In other specific embodiments further alternative of the invention, the sucrose concentration is about 4.5% w / v ^{concentration of sodium chloride is about 0, 2%.}

In certain embodiments of the invention, the formulation additionally comprises an amino acid. In certain specific embodiments of the invention, the concentration of amino acids is between about 0.1% and about 5% w / v. In certain specific embodiments of the invention, the amino acid is proline or arginine. In specific embodiments of the invention, the concentration of proline or arginine is between about 1% and about 2% w / v. In other specific embodiments of the invention, the proline concentration is about 1.5% w / v. In ^{alternative specific embodiments of the invention, the concentration of arginine is about} 1 ^% w / v.

In certain embodiments of the invention, the formulation is a liquid formulation. In other specific embodiments of the invention, the formulation is a lyophilized formulation.

^{In certain embodiments of the invention, the formulation is stable for at least} 6 ^{months at} 5 ° C. In alternative embodiments of the invention, the formulation is stable for at least 9 months at 5 ° C.

In certain aspects described herein, the formulation shows a reduced amount of at least one by-product selected from the group consisting of aggregates, half molecules, degradation products, low molecular weight proteins, high molecular weight proteins and rearrangements of isoforms. acidic / basic / neutral antibodies compared to a reference anti-LIGHT formulation comprising an anti-LIGHT antibody in phosphate buffered saline at pH 7.3 or a reference anti-CXCR5 formulation comprising an anti-LIGHT antibody in phosphate buffered saline at pH 7.3.

The specification discloses a stable liquid antibody formulation suitable for subcutaneous administration, the formulation comprising:

a) 150 mg / ml of an anti-LIGHT IgG4 antibody (lymphotoxin type, shows inducible expression and competes with the completely human HSV glycoprotein D by HVEM, a receptor expressed by T lymphocytes) comprising a heavy chain comprising the sequence of amino acids of SEQ ID NO: 7 and a ^light chain ^{comprising the amino acid sequence of SEQ ID NO:} 8 ^;

b) 10 mM citrate addition;

c) 0.005% polysorbate 20 and

d) 4% mannitol;

the pH of the formulation being pH 5.5.

The specification discloses a stable liquid antibody formulation suitable for intravenous administration, the formulation comprising:

a) 50 mg / ml of an anti-LIGHT IgG4 antibody (lymphotoxin type, shows inducible expression and competes with the completely human HSV glycoprotein D by HVEM, a receptor expressed by T lymphocytes) comprising a heavy chain comprising the sequence of amino acids of SEQ ID NO: 7 and a ^light chain ^{comprising the amino acid sequence of SEQ ID NO:} 8 ^;

b) 10 mM citrate buffer and

^c) 0 ^{01% polysorbate 20;}

the pH of the formulation being approximately pH 5.5.

The specification discloses a stable formulation of lyophilized antibodies suitable for administration intravenous, comprising the formulation:

a) 50 mg / ml of an anti-LIGHT IgG4 antibody (lymphotoxin type, shows inducible expression and competes with the completely human HSV glycoprotein D by HVEM, a receptor expressed by T lymphocytes) comprising a heavy chain comprising the sequence of amino acids of SEQ ID NO: 7 and a ^light chain ^{comprising the amino acid sequence of SEQ ID NO:} 8 ^;

b) 10 mM citrate addition;

^c) 0 ^{01% polysorbate 20;}

d) 5% sucrose and

e) 1.5% proline;

the pH of the formulation being pH 5.5.

In alternative specific embodiments of the invention, the invention provides a stable antibody formulation, comprising:

a) 20 mg / ml of a humanized IgG4 anti-CXCR5 antibody (chemokine receptor CXC type 5) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;

b) 10 mM citrate addition;

^c) 0 ^{02% polysorbate 20;}

^d) 6 ^{% sucrose and}

^{e) 0, 2% sodium chloride;}

the pH of the formulation being pH 6.0.

In further alternative specific embodiments of the invention, the invention provides a stable antibody formulation comprising:

a) 100 mg / ml of a humanized IgG4 anti-CXCR5 antibody (chemokine receptor CXC type 5) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;

b) 10 mM citrate addition;

^c) 0 ^{01% polysorbate 20;}

d) 4.5% sucrose;

^{e) 0, 2% sodium chloride and}

^f) 1 ^{% arginine;}

the pH of the formulation being pH 6.0.

In a certain aspect described herein, the specification provides a kit ^{comprising a container comprising:} 1 ^{) the formulation of any one of the preceding claims and} 2 ⁾ a label or instructions for the administration and use of the formulation . In certain aspects described herein, the label comprises one or more of the following: instructions for the administration of the formulation, instructions for the use of the formulation, instructions on the storage conditions of the formulation, information on the batch and the batch number of the formulation and / or kit, information on the composition of the formulation, safety information, information on possible adverse reactions, side effects and / or side effects related to the administration of the formulation, or information on possible indications and / or contraindications of the formulation.

In certain embodiments of the invention, the invention provides a pre-filled device or a pre-filled container, such as a syringe, a cartridge, a vial, an ampoule or an auto-injector comprising the formulation of the invention. In certain other aspects described herein, the invention provides a kit comprising said pre-filled syringe, cartridge, vial, ampoule or autoinjector.

The specification provides a method for treating an inflammatory bowel disease comprising administering to a subject in need thereof a formulation as described herein.

The specification provides a method for treating rheumatoid arthritis comprising administering to a subject in need thereof a formulation as described herein.

In certain aspects, the specification discloses a formulation for use in a diagnostic or treatment procedure of the human or animal body. In specific aspects, the formulation is used in the treatment of an inflammatory bowel disease. In alternative aspects, the formulation is used in the treatment of rheumatoid arthritis.

In certain aspects, the specification provides a method for preparing a formulation as described herein comprising mixing the components of the formulation and adjusting the pH, in which the preparation is carried out under sterile conditions or the formulation is sterilized after mixing the components and adjusting the pH or both.

In certain specific aspects, the specification provides a method for preparing a stable antibody formulation comprising: a) providing an anti-LIGHT binding agent, b) resuspending the anti-LIGHT binding agent in from about 5 to about 50 mM of citrate buffer and c) adjusting the pH of the formulation to from about pH 5.0 to about pH 6.0.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a gel image showing the results of the denatured isoelectric focusing experiments that were used to determine the isoelectric point (pI) of the fully human anti-LIGHT IgG4 antibody comprising a heavy chain comprising the amino acid sequence of ^{SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO:} 8 ^{formulated in} phosphate buffered saline at pH 7.3 at a concentration of 5.5 mg / ml (the "Formulation original "," PBS Formulation "or the" Reference Lot "). Lanes 1 and 5: high-range IEF calibration kit of pI 5-10.5; lanes 2 and 4: a first batch of reference lot; lanes 3 and 4: a second batch of reference lot. The pI values are indicated by numbers.

Figure 2 is an image of an SDS-PAGE gel comparing different batches of reference batches under reducing and nonreducing conditions. Lanes 1 and 10: Biorad Precision Plus Protein standard; lane 5: empty; lane 2: a first batch of reference lot under non-reduced conditions; lanes 3 and 4: a second batch of ^reference lot ^{under non-reduced conditions; lane} 6 ^{: a first batch of reference lot under reduced conditions; lanes 7 and} 8 ^{: a second batch of Reference lot in reduced conditions; and lane 9: control of the system. The} sizes are indicated by numbers within the rows.

Figure 3 shows an ELISA graph that was used to determine the antigen binding activity of the first and second batches of the Reference Lot.

Figure 4 shows a chromatography of size exclusion chromatography (SEC) of the first batch of the Reference Lot. As shown in Figure 4, the SEC detected high molecular weight proteins (HMWP), for example di- / oligomers (RRT0.8) or aggregates, and low molecular weight proteins (LMWP) or degradation products. The first lot batch of Reference lot had a purity of 97% monomer content.

Figure 5 shows a weak cation exchange chromatogram for the first lot of reference lot. As shown in Figure 5, rearrangements of acidic, neutral and basic isoforms were produced during the stability studies. The first batch of reference Lot had a distribution of acidic / neutral / basic isoforms of 42.3 / 55.6 / 1.9%.

Figure 7 shows a dynamic light scattering pattern of the first lot of reference Lot, which was unfiltered. DLS was used to determine the hydrodynamic diameter of the first batch of antibody monomer from the Reference batch and potential soluble aggregates.

^Figure 8 ^{shows a light scattering pattern of the dynamic first lot of Reference Lot, which was} filtered. DLS was used to determine the hydrodynamic diameter of the first batch of antibody monomer from the Reference batch and potential soluble aggregates.

Figure 9 is a flow diagram of the manufacturing process of the pharmacological product for the formulation of high concentration of antibodies.

Figure 10 shows a dynamic light scattering pattern of Formulation 14. DLS was used to determine the hydrodynamic diameter of the antibody monomer and the soluble aggregate potentials.

Figure 11 is an image of a gel showing the results of isoelectric focusing to determine the pI (isoelectric point) of the Initial CXCR5 Antibody. Lanes 1.6: IEF Calibration High Range pI kit; Lanes 2.4: Initial standard reference antibody LP08031; and lanes 3.5: Pharmacological substance of Initial Antibody, RSN0151.

Figure 12 is an image of an SDS-PAGE gel comparing different batches of pharmacological substances under reducing and non-reducing conditions. The gel was also used to determine the molecular weight of the Initial CXCR5 Antibody and the presence of any aggregate.

Figure 13 is an ELISA graph that was used to determine the antigen binding activity of the Initial CXCR5 Antibody to a 28-mer peptide of the CXCR5 antigen.

Figure 14 is a SEC chromatogram of the stressed CXCR5 Antibody. The SEC was able to detect high molecular weight proteins (HMWP), for example di- / oligomers or aggregates, and low molecular weight proteins (LMWP) or degradation products. The Initial CXCR5 Antibody had a purity of 99% monomer content. Figure 15 is a WCX chromatogram that was used to determine acidic, neutral and basic isoforms of the Initial CXCR5 Antibody. The Initial CXCR5 Antibody had a distribution of acidic / neutral / basic isoforms of 14/85/1%.

Figure 16 is a measurement by DLS that was used to determine the hydrodynamic diameter of the antibody monomer and the soluble aggregate potentials.

Figure 17 is an image of the Initial CXCR5 Antibody in an acetate buffer of pH 5.0 (left) and pH 5.5 (right); every v. WFI (water for injection) and after thermal stress. This figure shows that acetate is a suitable buffer system.

Figure 18 is an image of the Initial CXCR5 Antibody in histidine buffer pH 6.0 (left), pH 5.5 (center) and pH 5.0 (right); every v. WFI (water for injection) and after thermal stress. This figure shows that histidine is a suitable buffer.

Figure 19 is an image of the Initial CXCR5 Antibody in a TRIS buffer of pH 7.5 after UF / DF (left) and after filtration (right); every v. WFI (water for injection) and after thermal stress. This figure shows that TRIS is an incompatible buffer system.

Figure 20 is an image of the Initial CXCR5 Antibody in a citrate buffer of pH 6.0 after UF / DF and filtration.

Figure 21 is an image of the Initial CXCR5 Antibody in an acetate buffer of pH 5.5 after UF / DF and filtration.

Figure 22 is an image of the Initial CXCR5 Antibody in a succinate buffer of pH 5.0 after UF / DF and filtration.

Figure 23 is an image of the Initial CXCR5 Antibody in a histidine buffer of pH 5.0 after UF / DF and filtration.

Figure 24 is an image of the Initial CXCR5 Antibody in an arginine buffer pH 6.0 after UF / DF and filtration.

Figure 25 is an image of the appearance of the solutions of the Initial CXCR5 Antibody LA_09_016 with different ^{surfactants (without surfactant, polysorbate 20, polysorbate 80, Lutrol f} 68 ^{, Cremophor RH40, Solutol HS15 and SDS)} after mechanical stress (350 rpm, 2 , 5 h, TA).

Figure 26 is a graph showing an increase in dimers under accelerated conditions, analyzed by SEC. An ^{increase in the formation of dimers of up to} 10 ^{% can be observed after three months of} storage in the four histidine formulations. The acetate formulations showed an increase in the ^{content of dimers of up to} 6 ^{%. In the four citrate formulations, the dimer concentration was less} than 2%, even after three months at 40 ° C.

Figure 27 is a graph showing an increase of basic isoforms under accelerated conditions, analyzed by WCX. Histidine is more deficient for the stability of the Initial CXCR5 Antibody under accelerated conditions. A slight increase in basic isoforms can be observed for the four acetate formulations. It is interesting to note that it was not possible to discriminate between the four citrate formulations.

Figure 28 is a graph showing a decrease of neutral isoforms under accelerated conditions, analyzed by WCX. This figure shows a strong decrease of neutral isoforms for histidine formulations. A slight decrease in acetate was observed. Citrate was the least affected.

Figure 29 shows the delta pH of the four formulations (A-D) in citrate buffer under accelerated conditions. The formulations that most stabilize the pH are those buffered with citrate, and especially the formulations B and D.

Figure 30 shows the delta of the pH of the four formulations (A-D) in acetate buffer under accelerated conditions. In buffered solutions with acetate of the Initial CXCR5 Antibody, the pH moved to a higher value.

Figure 31 shows the delta of the pH of the four formulations (A-D) in histidine buffer under accelerated conditions. In the histidine buffered solutions of the Initial CXCR5 Antibody, the pH decreased slightly. Figure 32 is a graph showing the hydrodynamic diameter of CXCR5 LA_09_027 A-D after 3 months of storage at 40 ° C. The citrate-buffered formulations showed only small aggregates after three weeks in formulation C, and after six weeks of storage in formulation A. Some aggregates could also be detected after three months in formulation B. But, compared to the formulations buffered with acetate, the amount was very small.

Figure 33 is a graph showing the hydrodynamic diameter of CXCR5 LA_09_028 A-D after 3 months of storage at 40 ° C. Formulation C of the acetate buffer showed some aggregates <200 nm after three weeks. Formulation A showed some aggregates after three months.

Figure 34 is a graph showing the effect of increasing the concentration of the Initial CXCR5 Antibody on the average Z. The Initial CXCR5 Antibody showed a significant increase in the hydrodynamic diameter (average Z) as the concentration of the antibody increased.

Figure 35 is a graph showing the effect of different stabilizers (excipients) on the average Z at 100 mg / ml of Initial CXCR5 Antibody after thermal stress. The average Z was measured before and after thermal stress. The stabilizing effect was similar in all the excipients analyzed, but the increase in the Z-average was generally reduced by using amino acids as stabilizers (arginine, lysine or glycine). Lysine was excluded due to a higher content of aggregates after stress. Arginine showed a better effect than glycine.

Figure 36 is a graph showing the effect of different stabilizers on the average Z at 100 mg / ml of Initial CXCR5 Antibody after mechanical stress. The average Z was measured before and after mechanical stress. The same reduction in the average Z was observed in the presence of amino acids. Sucrose had a better protective effect than trehalose against mechanical stress. Arginine and glycine had a better performance in combination with NaCl.

Figure 37 is a set of graphs showing the distribution of particle size, measured by DLS, of the ^{Initial CXCR5 Antibody formulated in 10 mM citrate buffer at pH} 6 ^{before mechanical stress (A) and after} mechanical stress (B) . A higher molecular weight species was measured by DLS after the mechanical stress of DS.

Figure 38 is a set of graphs showing the distribution of the particle size, measured by DLS, of the prototype formulations of pharmacological products of the Initial CXCR5 Antibody (AD, Table 111) before (A) and after (B) mechanical stress .

DETAILED DESCRIPTION

A. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art.

It should be noted herein that, as used herein and in the appended claims, the singular forms "a,""an,""the" and "the" also include reference to the plural, unless the context clearly indicates otherwise.

The term "about" or the term "about" means within 10%, such as within 5% (or 1 ^{% or less) of a certain value or range.}

The terms "administer" or "administration" refer to the act of physically injecting or otherwise administering a substance as it exists outside the body (eg, a formulation of the invention) to a patient, such as by mucosal, intradermal administration , intravenous, subcutaneous, intramuscular and / or any other physical administration procedure described herein or known in the art. When treating a disease, or a symptom thereof, administration of the substance is typically performed after the onset of the disease or the symptoms thereof. When a disease or its symptoms are prevented, administration of the substance is usually done before the onset of the disease or its symptoms.

In the context of a polypeptide, the term "analog" refers to a polypeptide that has a function similar or identical to a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, an LIGHT epitope or CXCR5, or an anti-human antibody. -LIGHT or anti-CXCR5, but which does not necessarily comprise an amino acid sequence similar or identical to that of a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, an LIGHT or CXCR5 epitope, or an anti-LIGHT antibody or anti-CXCR5, or having a structure similar or identical to that of a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, an LIGHT epitope or CXCR5, or an anti-LIGHT or anti-CXCR5 antibody. A polypeptide having a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following points: (a) a polypeptide having an amino acid sequence that is at least 30%, at least 35%, at less 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least the 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of a LIGHT or CXCR5 polypeptide (e.g., SEQ ID NO: 9 or SEQ ID NO: 14, respectively), a fragment of a LIGHt or CXCR5 polypeptide, an LIGHT or CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody described herein; (b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding an LIGHT or CXCR5 polypeptide, a fragment of an LIGHT or CXCR5 polypeptide, an LIGHT or CXCR5 epitope, or an anti-cancer antibody. LIGHT or anti-CXCR5 (or the VH or VL region thereof) described herein of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, minus 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues (see, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY); and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least less 99% identical to the nucleotide sequence encoding a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, an LIGHT or CXCR5 epitope, or an anti-LIGHT or anti-CXCR5 antibody (or a VH or VL region) of the same) described in the present document. A polypeptide with a structure similar to a LIGHT or CXCR5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, an LIGHT epitope or CXCR5, or an anti-LIGHT or anti-CXCR5 antibody refers to a polypeptide having a secondary, tertiary structure or quaternary similar to that of a LIGHT or CxCr5 polypeptide, a fragment of a LIGHT or CXCR5 polypeptide, an LIGHT epitope or CXCR5, or an LIGHT or CXCR5 antibody. The structure of a polypeptide can be determined by methods known to those skilled in the art, including, but not limited to, X-ray crystallography, nuclear magnetic resonance and crystallographic electron microscopy.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are optimally aligned for comparison purposes (for example, gaps can be introduced into the sequence of a first amino acid or nucleic acid sequence). for an optimal alignment with a second sequence of amino acids or nucleic acids). The amino acid residues or nucleotides of the amino acid positions or the corresponding nucleotide positions are then compared. When a position of the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position of the second sequence, then the molecules are identical in that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e.% identity = number of identical overlapping positions / total number of X positions 100%). In one embodiment, the two sequences have the same length.

The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can also be performed using a mathematical algorithm. A non-limiting example of a mathematical algorithm that is used for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl Acad Sci. USA 87: 22642268, modified as in Karlin and Altschul, 1993, Proc. Natl Acad Sci. USA 90: 58735877. Said algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403. Nucleotide searches by BLAST can be performed with the set of parameters of the NBLAST nucleotide program, for example, for score = 100, word length = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of interest. Searches of proteins by BLAST can be performed with the set of parameters of the XBLAST program, for example, for a score of 50, word length = 3 to obtain amino acid sequences homologous to a protein molecule of interest. To obtain alignments with gaps for comparison purposes, Gapped BLAST can be used as described by Altschul et al., 1997, Nucleic Acids Res. 25: 33893402. Alternatively, PSI BLAST can be used to perform an iterative search that detects distant relationships between molecules (Id.). When using the BLAST, Gapped BLAST and PSI Blast programs, the default parameters of the respective programs (for example, of XBLAST and NBLAST) can be used (see, for example, National Center for Biotechnology Information (NCBI) on the Internet at ncbi.nlm .nih.gov). Another non-limiting example of a mathematical algorithm that is used for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17. This algorithm is incorporated in the ALIGN program (version 2.0), which is part of the package Computer sequence alignment GCG. When the ALIGN program is used to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowed gaps. In the calculation of the identity percentage, usually only exact matches are counted.

An "antagonist" or "inhibitor" refers to a molecule capable of inhibiting one or more biological activities of a target molecule. Antagonists can interfere with the binding of a receptor to a ligand and vice versa, incapacitating or destroying cells activated by a ligand and / or interfering with receptor or ligand activation (eg, tyrosine kinase activation) or signal transduction after binding of the ligand to a receptor. The antagonist can completely block receptor-ligand interactions or can substantially reduce such interactions. All said points of intervention of an antagonist will be considered equivalent for the purposes of the present invention.

For example, an "antagonist" or "inhibitor" of LIGHT refers to a molecule that is capable of inhibiting or otherwise reducing one or more of the biological activities of LIGHT, such as in a cell expressing LIGHT or in a cell which expresses a LIGHT ligand, such as a LIGHT receptor. For example, in certain embodiments, the antibodies described herein are antagonist antibodies that inhibit or ^otherwise reduce ^{the secretion of CCL20, IL-} 8 ^{and / or RANTES from a cell having a LIGHT receptor expressed} on the surface. cellular (e.g., HVEM, LTpR and / or DcR3) when said antibody is contacted with said cell. In some embodiments, a LIGHT antagonist can act, for example, by inhibiting or reducing the activation and / or cellular signaling pathways of the cell expressing a LIGHT receptor, thereby inhibiting a cell-mediated biological activity of LIGHT. with respect to biological activity mediated by LIGHT in the absence of an antagonist. In certain aspects described herein, anti-LIGHT antibodies are fully human anti-LIGHT antibodies, such as fully human monoclonal anti-LIGHT antibodies.

For example, an "antagonist" or "inhibitor" of CXCR5 refers to a molecule capable of inhibiting one or more biological activities, such as signaling, by CXCR5. Therefore, antagonists (e.g., neutralizing antibodies) that bind to CXCR5, CXCL13 or other CXCR5 ligands, or a complex of CXCR5 and a ligand thereof, such as CXCL13; sequence variants of amino acids or derivatives of CXCR5 or CXCL13 that antagonize the interaction between CXCR5 and a ligand, such as CXCL13; Soluble CXCR5, optionally fused to a heterologous molecule such as an immunoglobulin region (e.g. an immunoadhesin); a complex comprising CXCR5 in association with another receptor or biological molecule; peptides of native or synthetic sequence that bind to CXCR5; etc.

The terms "antibody", "immunoglobulin" or "Ig" can be used interchangeably herein. The term "antibody" includes, but is not limited to, synthetic antibodies, monoclonal antibodies, antibodies produced recombinantly, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single chain Fv (scFv) (e.g. monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F (ab ') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic antibodies (anti-Id) and epitope-binding fragments of any of the foregoing. In particular, the antibodies include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., antigen-binding domains or molecules that contain an antigen-binding site that specifically binds to an LIGHT antigen (e.g. more complementarity determining regions (CDRs) of an anti-LIGHT antibody) or a CXCR5 antigen (eg, one or more complementarity determining regions (CDRs) of an anti-CXCR5 antibody). The anti-LIGHT or anti-CXCR5 antibodies can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), any class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. In some aspects, anti-LIGHT antibodies are completely human, such as fully human anti-LIGHT monoclonal antibodies. In certain aspects, the anti-LIGHT antibodies are IgG antibodies, human IgG4 antibodies. As an alternative, in some embodiments, the Anti-CXCR5 antibodies are humanized, such as humanized monoclonal anti-CXCR5 antibodies. In certain embodiments, the anti-CXCR5 antibodies are IgG antibodies, humanized IgG4 antibodies.

As used herein, the term "anti-LIGHT antibody" means an antibody or polypeptide derived therefrom (a derivative) that specifically binds to human LIGHT as defined herein, including, but not limited to. , molecules that inhibit or substantially reduce the binding of LIGHT to its ligands or inhibit LIGHT activity.

As used herein, the term "anti-CXCR5 antibody" means an antibody or a polypeptide derived therefrom (a derivative) that specifically binds to human CXCR5 as defined herein, including, but not limitation, molecules that inhibit or substantially reduce the binding of CXCR5 to its ligands or inhibit the activity of CXCR5.

The term "B cell activity" means higher than normal levels of B cells, which may be local, or evidence of a biological manifestation or function of a B cell, such as the expression of antibodies, the presence or activity of B cells. Bruton tyrosine kinase, the expression or presence of CD19, the expression or the presence of B cell activation factor, etc.

The term "binding agent" means any molecule, such as an antibody, an siRNA, a nucleic acid, an aptamer, a protein or a small molecule organic compound, that binds or binds specifically to LIGHT or CXCR5, or a variant or a fragment thereof.

The term "by-product" includes undesired products, which reduce or decrease the proportion of therapeutic / prophylactic binding agent, such as an antibody, in a given formulation. For example, typical by-products include aggregates of the antibody, fragments of the antibody, produced, for example, by degradation of the antibody by deamidation or hydrolysis, or mixtures thereof. Typically, the aggregates are complexes that have a molecular weight superior to that of the monomeric antibody. Antibody degradation products may include, for example, fragments of the antibody, for example, produced by deamidation or hydrolysis. Typically, the degradation products are complexes that have a lower molecular weight than the monomeric antibody. In the case of an IgG antibody, said degradation products have a molecular weight less than about 150 kD.

The terms "composition" and "formulation" are intended to encompass a product that contains the specified ingredients (eg, an anti-LIGHT antibody or an anti-CXCR5 antibody) in, optionally, the specified amounts, as well as any product that is obtain, directly or indirectly, from the combination of the ingredients specified in, optionally, the specified quantities.

The terms "constant region" or "constant domain" refer to a carboxy-terminal portion of the light and heavy chain that is not directly involved in the binding of the antibody to the antigen, but which shows various effector functions, such as interaction with the Fc receptor. The terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence with respect to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain.

The term "CXCR5" refers to the known molecule of natural origin that is found in lymphocytes, particularly in B cells, and particularly in virgin B cells; to said molecule isolated from said cells; to said molecule manufactured recombinantly using known materials and means, and using a nucleic acid encoding a CXCR5; as well as parts of CXCR5, such as the extracellular domain (EC), which retain the characteristics and properties relevant to the implementation of the present invention, such as binding to CXCL13. A soluble CXCR5 molecule can consist essentially of the EC domain of CXCR5, which includes, in general, approximately the first sixty amino acids of the molecule, ie, the amino-terminal portion of CXCR5.

CXCR5 is a non-promiscuous receptor. CXCL13 is a ligand of CXCR5 and is constitutively expressed in stromal cells, such as follicular dendritic cells, and in lymphoid tissues. CXCL13 specifically attracts B cells and a small subset of T cells called follicular T cells auxiliary to B, TFH. This may not be unexpected given the many interactions between T cell and B cell populations in the immune system. In addition, activated T cells induce or upregulate the expression of CXCR5. It has been found that the infiltration of lymphocytes in tertiary ectopic germ centers (GC) correlates well with the increase in the severity of the disease and the degradation of tolerance in certain disorders that occur with these atypical structures similar to lymph nodes. Using in vivo murine models , such as CXCR5 - / - and CXCL13 - / - mice, the absence of the receptor or the ligand results in a fine architecture of altered GC due to localization, and possibly interaction, of altered cells T and B. These mice are also protected against the development of severe collagen-induced arthritis (CIA). As CXCR5 is selectively expressed in mature B cells, which are associated with AR pathogenesis, blocking this receptor will modulate the arthritogenic response in affected individuals. The treatment of rheumatoid arthritis with biological products (ie, anti-TNFa and anti-CD20 antibodies, rituximab) has been shown to be clinically effective; in particular, patients under treatment targeting B cells have shown lasting improvements in clinical signs and symptoms. Targeted selection of CXCR5, which is expressed only in mature B cells and B helper T cells, will not affect the development of B cells or immunocompromise the patient. Unlike rituximab, the instant anti-CXCR5 antibody is a neutralizing antibody that does not mediate cellular cytotoxicity.

A "CXCR5 disease" is a malaise, a disorder, a disease, a condition, an abnormality, etc., which is characterized, or is provoked, by an overexpression or an increase in the levels of CXCL13 or another CXCR5 ligand, increased levels of B cells, increased levels of B cell activity, increased levels of CXCR5, or inadequate metabolism and activity of CXCR5.

The term "epitope" refers to a region located on the surface of an antigen, such as a LIGHT or CXCR5 polypeptide, or a LIGHT or CXCR5 polypeptide fragment, which is capable of binding to one or more antigen-binding regions of an antigen. a binding agent, such as an antibody, and having antigenic or immunogenic activity in an animal, such as a mammal, such as in a human, that is capable of eliciting an immune response. An epitope that possesses immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope that possesses antigenic activity is a portion of a polypeptide to which an antibody specifically binds, determined by any method well known in the art, for example, such as an immunoassay. Antigenic epitopes do not necessarily need to be immunogenic. Epitopes usually consist of surface groupings of chemically active molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics, as well as specific loading characteristics. A region of a polypeptide that contributes to an epitope can be contiguous amino acids of the polypeptide, or the epitope can come from two or more non-contiguous regions of the polypeptide. The epitope may or may not be a three-dimensional surface characteristic of the antigen. In certain embodiments, an LIGHT or CXCR5 epitope is a three-dimensional surface feature of an LIGHT or CXCR5 polypeptide (e.g., in a trimeric form of an LIGHT polypeptide). In other embodiments, an LIGHT epitope is a linear characteristic of a LIGHt or CXCR5 polypeptide (e.g., in a trimeric form or a monomeric form of the LIGHT polypeptide). Anti-LIGHT or anti-CXCR5 antibodies can specifically bind to an epitope of the monomeric (denatured) form of LIGHT or CXCR5, an epitope of the trimeric (native) form of LigHt or CXCR5, or both to the monomeric form (denatured) as to the trimeric (native) form of LIGHT or CXCR5. In specific aspects described herein, anti-LIGHT antibodies specifically bind to an epitope of the trimeric form of LIGHT but do not specifically bind to the monomeric form of LIGHT.

The term "excipients" refers to inert substances that are commonly used as a diluent, carrier, preservative, binder, stabilizing agent, etc., for drugs and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (eg, aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (eg, alkyl sulfonates, caprylate, etc.), surfactants (eg, SDS, polysorbate, surfactant) nonionic, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.

In the context of a peptide or a polypeptide, the term "fragment" refers to a peptide or a polypeptide that comprises less than the full-length amino acid sequence. Said fragment may arise, for example, from a truncation at the amino-terminal end, a truncation at the carboxy-terminal end and / or an internal deletion of a residue (s) of the amino acid sequence. The fragments may be the result, for example, of alternative RNA splicing or protease activity in vivo. In certain aspects described herein, the hLIGHT or hCXCR5 fragments include polypeptides comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 residues of contiguous amino acids, contiguous amino acids, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues or at least 250 contiguous amino acid residues of the sequence of amino acids of a LIGHT or CXCR5 polypeptide or an antibody that specifically binds to a LIGHT or CXCR5 polypeptide. In a specific aspect, a fragment of a LIGHT or CXCR5 polypeptide or an antibody that specifically binds to an LIGHT or CXCR5 antigen retains at least 1, at least 2, or at least 3 functions of the polypeptide or antibody.

The terms "fully human antibody" or "human antibody" are used interchangeably herein and refer to an antibody comprising a human variable region and, possibly, a human constant region. In specific embodiments, the expressions refer to an antibody that it comprises a variable region and a constant region of human origin. "Completely human" anti-LIGHT antibodies, in certain aspects, may also encompass antibodies that bind to LIGHT polypeptides and are encoded by nucleic acid sequences that are naturally occurring somatic variants of a germline immunoglobulin nucleic acid sequence. human In a specific aspect, anti-LIGHT antibodies are fully human antibodies. The term "completely human antibody" includes antibodies that have variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (See Kabat et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH Publication 91-3242). Methods for producing fully human antibodies are known in the art.

The term "recombinant human antibody" includes human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected in a host cell, antibodies isolated from a human antibody library recombinant combinatorials, antibodies isolated from an animal (e.g., a mouse or a cow) that is transgenic and / or transchromosomal to human immunoglobulin genes (see, for example, Taylor, LD et al. (1992) Nucl. 20: 6287-6295) or antibodies prepared, expressed, created or isolated by any other means involving the splicing of human immunoglobulin gene sequences to other DNA sequences. Said recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, however, said recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, somatic mutagenesis in vivo) and, therefore, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences which, although derived from and related to human germline VH and VL sequences, can not exist naturally within the germline repertoire of the human antibody in vivo .

An "IgG4 binding agent" or a "binding agent comprising at least a portion of an Fc region of IgG4" both refer to the binding agents described herein that include at least one Fc fragment of IgG4. In certain aspects, the fragment comprises 10, 20, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 or 220 amino acids of the Fc region of IgG4. In other aspects, the fragment includes 10-50, 50-100, 100-150 or 150-200 amino acids of the Fc region of IgG4. In other aspects, the portion of the Fc region of IgG4 may have a certain homology with the Fc region of IgG4. For example, the IgG4 binding agent can include a portion of a protein with more than 50, 60, 70, 80, 90, 93, 95, 96, 97, 98, 99 or 100% homology with the Fc region of IgG4. The exemplary Fc regions of IgG4 are described throughout the specification.

The term "heavy chain", when used with reference to an antibody, refers to five different types, called alpha (a), delta (A), epsilon (£), gamma (y) and mu (| j), based on the amino acid sequence of the constant domain of the heavy chain. These various types of heavy chains are well known in the art and result in five classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely, IgG1, IgG1, IgG3 and IgG4. In some embodiments, the heavy chain is a human heavy chain.

The "humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as F ^v , F ^ab , F ^{ab '} , F ^{(ab') 2} or other sub-sequences of target binding antibodies) containing sequences derived from non-human immunoglobulin, as compared to a human antibody. In general, the humanized antibody will substantially comprise all of one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin template sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (F ^c ), typically that of the human immunoglobulin template chosen. In general, the goal is to possess an antibody molecule that is minimally immunogenic in a human. Therefore, it is possible that one or more amino acids of one or more CDRs can also be exchanged for one that is less immunogenic to a human host, without substantially minimizing the specific binding function of one or more CDRs with respect to CXCR5 or CXCL13. Alternatively, FR can be non-human, but more immunogenic amino acids are replaced by less immunogenic ones. However, CDR grafting, as discussed above, is not the only way to obtain a humanized antibody. For example, modification of only CDR regions may be insufficient, since it is not uncommon for structural frame residues to have a role in determining the three-dimensional structure of CDR loops and the general affinity of the antibody for its ligand. Therefore, any means can be implemented so that the non-human parent antibody molecule is modified to be one that is less immunogenic to a human being, and the overall sequence identity with a human antibody is not always a need. Therefore, humanization can also be achieved, for example, by the mere substitution of only a few residues, in particular those that are exposed in the antibody molecule, and are not buried within the molecule, and therefore, are not easily accessible to the host's immune system. Said method is taught herein with respect to the replacement of "mobile" or "flexible" residues of the antibody molecule, with the aim of reducing or buffering the immunogenicity of the resulting molecule without compromising the specificity of the antibody for its epitope. or ^{determinant. See, for example, Studnicka et al., Prot Eng 7 (} 6 ^{) 805-814, 1994; Mol Imm 44: 1986-1988, 2007;} Sims et al., J Immunol 151: 2296 (1993); Chothia et al., J Mol Biol 196: 901 (1987); Carter et al., Proc Natl Acad Sci USA 89: 4285 (1992); Presta et al., J Immunol 151: 2623 (1993), WO 2006/042333 and U.S. Patent No. 5,869,619.

An "isolated" or "purified" binding agent, such as an antibody, is substantially free of cellular material or other contaminating proteins from the cellular or tissue source from which the binding agent is derived, or substantially free of chemical precursors or other chemical products when chemically synthesized. For example, the expression "substantially free of cellular material" includes preparations of an antibody in which the antibody is separated from the cellular components of the cells from which it is isolated or produced recombinantly. Therefore, an antibody that is substantially free of cellular material includes antibody preparations having less than about 30%, 20%, 10% or 5% (by dry weight) of heterologous protein (also referred to herein). polluting protein "). When the antibody is produced recombinantly, it is also desirable that it be substantially free of culture medium, ie, that the culture medium represents less than about 20%, 10% or 5% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, in some embodiments it is substantially free of chemical precursors or other chemicals, ie, it is separated from the chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly, said antibody preparations have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In some aspects described herein, anti-LIGHT or anti-CXCR5 antibodies are isolated or purified.

The terms "human LIGHT", "hLIGHT" or "hLIGHT polypeptide" and similar terms and expressions refer to polypeptides ("polypeptides", "peptides" and "proteins" are used interchangeably herein) comprising the amino acid sequence of SEQ ID NO: 9 and related polypeptides, including SNP variants thereof. Related polypeptides include allelic variants (ie, SNP variants); cutting and splicing variants; fragments; derivatives; substitution, deletion and insertion variants; fusion polypeptides and homologs between species, in some aspects described herein, which retain the LIGHT activity and / or are sufficient to generate an anti-LIGHT immune response. Soluble forms of LIGHT are also described which are sufficient to generate an anti-LIGHT immune response. As will be appreciated by those skilled in the art, an anti-LIGHT binding agent, such as an antibody, can be attached to a LIGHT polypeptide, polypeptide fragment, antigen and / or epitope, since an epitope is part of the larger antigen, which it is part of the larger polypeptide fragment, which, in turn, is part of the larger polypeptide. The hLIGHT can exist in trimeric (native) or monomeric (denatured) form.

The terms "human CXCR5", "hCXCR5" or "hCXCR5 polypeptide" and similar terms and expressions refer to the polypeptides ("polypeptides", "peptides" and "proteins" are used interchangeably herein) comprising the amino acid sequence of SEQ ID NO: 14 and related polypeptides, including SNP variants thereof. Related polypeptides include allelic variants (ie, SNP variants); cutting and splicing variants; fragments; derivatives; substitution, deletion and insertion variants; fusion polypeptides; and homologues between species, in some aspects described herein, which retain the activity of CXCR5 and / or are sufficient to generate an anti-CXCR5 immune response. Soluble forms of CXCR5 are also included that are sufficient to generate an anti-CXCR5 immune response. As will be appreciated by those skilled in the art, an anti-CXCR5 binding agent, such as an antibody, can bind to a polypeptide, polypeptide fragment, antigen and / or CXCR5 epitope, since an epitope is part of the larger antigen, which it is part of the larger polypeptide fragment, which, in turn, is part of the larger polypeptide.

The term "Kabat numbering" and similar expressions are recognized in the art and refer to a numbering system of amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues of the heavy chain variable regions and light of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190: 382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). For the variable region of the heavy chain, the hypervariable region typically varies between amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2 and amino acid positions 95 to 102 for CDR3. For the variable region of the light chain, the hypervariable region typically varies from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

The term "light chain", when used with reference to an antibody, refers to two distinct types, called kappa (k) or lambda (A) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In some embodiments, the Light chain is a human light chain.

The terms "manage", "managing" and "management" refer to the beneficial effects that a subject obtains from a treatment (for example, a prophylactic or therapeutic agent), which do not result in the cure of the infection. In certain aspects, a subject is administered one or more treatments (ie, prophylactic or therapeutic agents, such as a formulation of the invention) to "manage" a disease mediated by LIGHT (i.e., a chronic intestinal disease, IBD, Crohn's disease, ulcerative colitis or GVHD) or a disease mediated by CXCR5 (ie, rheumatoid arthritis), one or more symptoms thereof, to prevent the progression or worsening of the disease.

The term "monoclonal antibody" refers to an antibody obtained from a population of homogeneous or substantially homogeneous antibodies, and each monoclonal antibody will typically recognize a single epitope of the antigen. In some embodiments, a "monoclonal antibody" is an antibody produced by a single hybridoma or another cell. The term "monoclonal" is not limited to any particular procedure for producing the antibody. For example, monoclonal antibodies can be produced by the hybridoma method as described by Kohler et al .; Nature, 256: 495 (1975) or can be isolated from phage libraries. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed by them are well known in the art (see, for example, Chapter 11 of: Short Protocols in Molecular Biology, (2002) 5th ed., Ausubel et al. ., eds., John Wiley and Sons, New York).

The term "pharmaceutically acceptable" means that it is approved by a regulatory agency of the federal government or a state government, or that it appears in the US Pharmacopoeia. UU., European Pharmacopoeia or other Pharmacopoeia generally recognized for use in animals, and more particularly in humans.

By "pharmaceutically acceptable excipient" is meant any inert substance that is combined with an active molecule, such as a monoclonal antibody, to prepare a convenient or convenient dosage form. The "pharmaceutically acceptable excipient" is an excipient that is not toxic to the receptors at the doses and concentrations employed, and is compatible with other ingredients of the formulation comprising the monoclonal antibody.

The terms "prevent", "preventing" and "prevention" refer to the total or partial inhibition of development, recurrence, onset or spread of a disease mediated by LIGHT or mediated by CXCR5 and / or symptoms related to the same, which is obtained by administering a treatment or combination of treatments provided herein (e.g., a combination of prophylactic or therapeutic agents, such as a formulation of the invention).

The term "prophylactic agent" refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a disease mediated by LIGHT or mediated by CXCR5 and / or related symptoms in a subject. In certain embodiments, the term "prophylactic agent" refers to a formulation of the invention. In other specific embodiments, the term "prophylactic agent" refers to an agent other than a formulation of the invention. In some embodiments, a prophylactic agent is an agent that is known to be used or is currently being used to prevent an illness mediated by LIGHT or mediated by CXCR5 and / or a symptom related thereto, or which prevents the occurrence , the development, progression and / or severity of a disease mediated by LIGHT or mediated by CXCR5 and / or a symptom related thereto. In specific embodiments, the prophylactic agent is a fully human anti-LIGHT antibody, such as a fully human anti-LIGHT monoclonal antibody, or a humanized anti-CXCR5 antibody, such as a humanized anti-CXCR5 monoclonal antibody.

The term "LIGHT antigen" refers to the portion of an LIGHT polypeptide to which a binding agent, such as an antibody, specifically binds. A LIGHT antigen also refers to an analog or a derivative of an LIGHT polypeptide or a fragment thereof to which a binding agent, such as an antibody, specifically binds. In some embodiments, a LIGHT antigen is a monomeric LIGHT antigen or a trimeric LIGHT antigen. A region of a LIGHT polypeptide that contributes to an epitope can be contiguous amino acids of the polypeptide, or the epitope can come from two or more non-contiguous regions of the polypeptide. The epitope may or may not be a three-dimensional surface characteristic of the antigen. A region located on the surface of an LIGHT antigen that is capable of eliciting an immune response is an LIGHT epitope. The epitope may or may not be a three-dimensional surface characteristic of the antigen.

The term "CXCR5 antigen" refers to the portion of a CXCR5 polypeptide to which a binding agent, such as an antibody, specifically binds. A CXCR5 antigen also refers to an analog or a derivative of a CXCR5 polypeptide or a fragment thereof to which a binding agent specifically binds, such as an antibody. A region of a CXCR5 polypeptide that contributes to an epitope can be contiguous amino acids of the polypeptide, or the epitope can come from two or more non-contiguous regions of the polypeptide. The epitope may or may not be a three-dimensional surface characteristic of the antigen. A region located on the surface of a CXCR5 antigen that is capable of eliciting an immune response is a CXCR5 epitope. The epitope may or may not be a three-dimensional surface characteristic of the antigen.

The terms "LIGHT-mediated disease" and "LIGHT-mediated disorder" are used interchangeably and refer to any disease that is wholly or partly caused by, or is the result of, LIGHT. In certain embodiments, the LIGHT is expressed aberrant (eg, highly) on the surface of a cell. In some embodiments, LIGHT may be aberrantly up-regulated in a particular cell type. In other embodiments, normal, aberrant or excessive cell signaling is caused by the binding of LIGHT to an LIGHT ligand. In certain embodiments, the LIGHT ligand is a LIGHT receptor (e.g., HVEM, LTpR or DCR3), for example, which is expressed on the surface of a cell, such as a colon epithelial cell. In certain embodiments, the disease mediated by LIGHT is a chronic intestinal disease, an inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC). In other embodiments, the disease mediated by LIGHT is a graft-versus-host disease (GVHD).

The terms "CXCR5 mediated disease" and "CXCR5 mediated disorder" are used interchangeably and refer to any disease that is wholly or partially caused by or resulting from CXCR5. In certain embodiments, CXCR5 is expressed aberrant (eg, highly) on the surface of a cell. In some embodiments, the CXCR5 may be upregulated in an aberrant manner in a particular cell type. In other embodiments, normal, aberrant or excessive cell signaling is caused by the binding of CXCR5 to a CXCR5 ligand. In certain embodiments, the ligand CXCR5 is CXCL13. In certain embodiments, the disease mediated by CXCR5 is rheumatoid arthritis (RA).

The term "saccharide" refers to a class of molecules that are derivatives of polyhydric alcohols. Saccharides are commonly referred to as carbohydrates and may contain different amounts of sugar units (saccharide), for example, monosaccharides, disaccharides and polysaccharides.

The terms "specifically binds" or "specifically binds" mean that it binds specifically to an antigen or a fragment thereof and does not bind specifically to other antigens. For example, an antibody that specifically binds to an antigen can bind to other peptides or polypeptides with lower affinity, as determined by, for example, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), BIACORE, or other assays known in the art. The technique. Antibodies or variants or fragments thereof that specifically bind to an antigen can have cross-reactivity with related antigens. In some embodiments, antibodies or variants or fragments thereof that specifically bind to an antigen do not cross-react with other antigens. An antibody or a variant or fragment thereof that specifically binds to an LIGHT or CXCR5 antigen can be identified, for example, by immunoassays, BIAcore or other techniques known to those skilled in the art. Typically, a ^{specific or selective} reaction ^{will be at least twice the signal or background noise, and more typically more than} 10 ^{times the} background. See, for example, Paul, ed., 1989, Fundamental Immunology, second edition, Raven Press, New York, at pages 332-336, for a discussion regarding antibody specificity.

A "stable" or "stabilized" formulation is one in which the binding agent, such as an antibody, essentially retains its physical stability, identity, integrity and / or chemical stability, identity, integrity and / or biological activity. during storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., 247-301, New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. The stability can be measured at a selected temperature and other storage conditions for a selected period of time. The stability can be determined by at least one of the methods selected from the group consisting of visual inspection, SDS-PAGE, IEF, HPSEC, RFFIT and kappa / lambda ELISA. For example, an antibody "retains its physical stability" in a pharmaceutical formulation, if it does not show signs of aggregation, precipitation and / or denaturation after a visual examination of color and / or transparency, or measured by UV light scattering, SDS PAGE or by size exclusion chromatography (high pressure) (HPSEC). In some embodiments, when the formulations of the invention are used, 5% or less, typically 4% or less, typically 3% or less, more typically 2% or less and particularly 1% or less of The antibodies form aggregates, measured by HPSEC or any other suitable procedure to measure aggregation formation. For example, an antibody is considered stable in a particular formulation if the antibody monomer has a purity of about 90%, typically about 95%, in particular about 98% after a predetermined period of time in certain storage conditions in a particular formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical alteration may involve size modification (eg, clipping), which can be evaluated using (HP) SEC, SDS-PAGE and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS), for example. Other types of chemical alteration include charge alteration (which occurs, for example, as a result of deamidation), which can be assessed by ion exchange chromatography, for example. An antibody "preserves its biological activity" in a pharmaceutical formulation at a given time, if the biological activity of the antibody at any given time is at less about 90% (within the assay errors) of the biological activity exhibited at the time the pharmaceutical formulation was prepared, determined in an antigen binding assay or a virus neutralization assay, for example.

The terms "subject" and "patient" are used interchangeably. As used herein, a subject is typically a mammal, such as a non-primate mammal (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g. human being), and in some embodiments a human being. In one embodiment, the subject is a mammal, such as a human being, suffering from a disease mediated by LIGHT or mediated by CXCR5. In another embodiment, the subject is a mammal, such as a human being, at risk of developing a disease mediated by LIGHT or mediated by CXCR5.

The term "therapeutically effective amount" refers to the amount of a treatment (e.g., a formulation of the invention) that is sufficient to reduce and / or improve the severity and / or duration of a given disease and / or a symptom. related to it. This expression also covers a quantity necessary for the reduction or improvement of the advance or progression of a given disease, the reduction or improvement of recurrence, the development or the appearance of a certain disease, and / or to improve or enhance the effect or the prophylactic or therapeutic effects of another treatment (eg, a treatment other than a formulation of the invention). In some embodiments, the therapeutically effective amount of an antibody of the invention is from about 0.1 mg / kg (mg of antibody per kg of subject weight) to about 100 mg / kg. In certain embodiments, a therapeutically effective amount of an antibody provided herein is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg , about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, approximately 60 mg / kg kg, approximately 70 mg / kg, approximately 80 mg / kg approximately 90 mg / kg or approximately 100 mg / kg (or a range between them). In some embodiments, "therapeutically effective amount," as used herein, also refers to the amount of an antibody described herein to achieve a specific result (e.g., inhibition of a biological activity. of LIGHT from a cell, such as ^{inhibiting the secretion of CCL20, IL-} 8 ^{or RANTES from the cell, or inhibiting the biological activity of CXCR5} from a cell, such as binding to CXCL13).

The term "therapeutic agent" refers to any agent that can be used in the treatment, management or amelioration of a disease mediated by LIGHT or mediated by CXCR5 and / or a symptom related thereto. In certain embodiments, the term "therapeutic agent" refers to a formulation of the invention. In certain other embodiments, the term "therapeutic agent" refers to an agent other than a formulation of the invention. In some embodiments, a therapeutic agent is an agent that is known to be used, or has been or is currently being used, for the treatment, management or amelioration of a disease mediated by LIGHT or mediated by CXCR5 or a or more symptoms related to it.

The term "therapy" refers to any protocol, procedure and / or agent that can be used in the prevention, management, treatment and / or improvement of a disease mediated by LIGHT (eg, IBD or GVHD) or disease mediated by CXCR5 (for example, rheumatoid arthritis). In certain embodiments, the terms "therapies" and "therapy" refer to biological therapy, supportive therapy and / or other therapies useful in the prevention, management, treatment and / or amelioration of a disease mediated by LIGHT or mediated by CXCR5 known to one skilled in the art, such as medical personnel.

The terms "treat", "treatment" and "treating" refer to the reduction or improvement of the progression, severity and / or duration of a disease mediated by LIGHT (eg, a chronic intestinal disease, IBD or GVHD). ) or a disease mediated by CXCR5 (eg, rheumatoid arthritis) resulting from the administration of one or more therapies (including, among others, the administration of one or more prophylactic or therapeutic agents, such as a formulation of the invention). In specific embodiments for LIGHT, said terms refer to the reduction or inhibition of the binding of LIGHT to HVEM, the reduction or inhibition of the binding of LIGHT to LTpR, the reduction or inhibition of LIGHT binding to DcR3, the reduction or inhibition of the production or secretion of CCL20 from a cell that expresses a LIGHT receptor of a subject, the reduction ^{or inhibition of the production or secretion of IL-} 8 ^{from a cell that expresses a LIGHT receptor of a} subject, reducing or inhibiting the production or secretion of RANTES from a cell expressing a LIGHT receptor of a subject and / or inhibiting or reducing one or more symptoms associated with a disease mediated by LIGHT, such as a chronic intestinal disease, IBD or GVHD. In specific embodiments for CXCR5, such terms refer to the reduction or inhibition of the binding of CXCR5 to CXCL13, and / or the inhibition or reduction of one or more symptoms associated with a disease mediated by CXCR5, such as arthritis. rheumatoid

The terms "variable region" or "variable domain" refer to a portion of the light and heavy chains, typically about the amino-terminal amino acids 120 to 130 of the heavy chain and ^{about amino acids} 100 ^to 110 ^{of the light chain, whose The sequence differs widely between} antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. The Sequence variability is concentrated in the regions termed complementarity determining regions (CDR), while the more highly conserved regions in the variable domain are called structural regions (FR). The CDRs of the light and heavy chains are mainly responsible for the interaction of the antibody with the antigen. The numbering of amino acid positions is according to the EU Index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (US Department of Health and Human Services, Washington, DC) 5th ed. ("Kabat et al! ') In some embodiments, the variable region is a human variable region.

B. Formulation formulations and components

As indicated above, the formulations of the invention have surprisingly been found in the form of lyophilized liquids and powders comprising an IgG4 binding agent and a citrate buffer, the pH of the ^{formulation being equal to or less than about pH} 6 ^{and to the extent isoelectric (pl) of the binding agent. The} formulations of the invention provide significant improvements over conventional IgG4 binding agent formulations (e.g., phosphate buffered saline (PBS) formulations), which form undesired byproducts by increasing the concentration of the binding agent in the formulation. In particular, the formulations of the invention have a reduced amount of aggregates, half molecules, degradation products, low molecular weight proteins (LMWP), high molecular weight proteins (HMWP) and rearrangements of acidic, basic and neutral isoforms of the agent of union in the formulations.

i. Anti-LIGHT binding agents, and variants and fragments thereof

In certain aspects described herein, the formulations include an anti-LIGHT binding agent. The binding agents can be any molecule, such as an antibody, a siRNA, a nucleic acid, an aptamer, a protein or a small molecule organic compound, which binds or binds specifically to LIGHT, or a variant or fragment of the same. In some aspects described herein, the binding agent is an anti-LIGHT antibody, or a variant thereof, or an antigen-binding fragment thereof. Anti-LIGHT antibodies specifically bind to a protein, polypeptide fragment or LIGHT epitope (lymphotoxin type, show inducible expression and compete with v Hs glycoprotein D for HVEM, a receptor expressed by T lymphocytes). The LIGHT molecule can be of any species. In some aspects, the LIGHT molecule is from a human being, known in the present document as "hLIGHT". The hLIGHT has the following amino acid sequence, which is identified as SEQ ID NO: 9:

1 MEESVVRPSV FVVDGQTDIP FTRLGRSHRR QSCSVARVGL GLLLLLMGAG

51 LAVQGWFLLQ LHWRLGEMVT RLPDGPAGSW EQLTQERRSH EVNPAAHLTG

101 ANSSLTGSGG PLLWETQLGL AFLRGLSYHD GALVVTKAGY YYIYSKVQLG

150 GVGCPLGLAS TITHGLYKRT PRYPEELELL VSQQSPCGRA TSSSRVWWDS

200 SFLGGVVHLE AGEEVVVRVL DERLVRLRDG TRSYFGAFMV ^{(SEQ ID NO: 9)}

In certain exemplary aspects described herein, the anti-LIGHT antibody is a humanized antibody, a fully human antibody, or a variant thereof or an antigen-binding fragment thereof. In some aspects described herein, anti-LIGHT antibodies prevent the binding of LIGHT to its receptors and inhibit the biological activity of LIGHT (e.g., production or ^{LIGHT-mediated secretion of CCL20, IL-} 8 ^{or RANTES to from cells expressing a LIGHT ligand, such} as a LIGHT receptor (e.g., HVEM, LT ^ R and / or DcR3).

In certain specific aspects described herein, the anti-LIGHT antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of one or more of the following complementarity determining regions (CDRs):

HCDR1 - GYNWH ( SEQ ID NO: 1 );

HCDR2 - EITHSGSTNYNPSLKS ( SEQ ID NO: 2 ) or

HCDR3 - EIAVAGTGYYGMDV ( SEQ ID NO: 3 ).

In other specific aspects described herein, the anti-LIGHT antibody comprises a light chain variable region (VL) comprising the amino acid sequence of one or more of the following complementarity determining regions (CDRs):

LCDR1 - RASQGINSAFA ( SEQ ID NO: 4 );

LCDR2 - DASSLES ( SEQ ID NO: 5 ) or

LCDR3 - QQFNSYPLT ( SEQ ID NO: 6 ).

In a specific aspect described herein, the anti-LIGHT antibody comprises a heavy chain variable region (VH) comprising the amino acid sequences of SEQ ID NO: 1, 2 and 3.

In another specific aspect, the anti-LIGHT antibody comprises a light chain variable region (VL) ^{comprising the amino acid sequences of SEQ ID NO: 4, 5 and} 6 ^.

In more specific aspects, the anti-LIGHT antibody comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NO: 1, 2 and 3; and a light chain variable region ^{comprising the amino acid sequences of SEQ ID NO: 4, 5 and} 6 ^.

In specific aspects, the anti-LIGHT antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7:

1 QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYNWHWIRQP PGKGLEWIGE

51 ITHSGSTNYN PSLKSRVTIS VDTSKNQFSL KLSSVTAADT AVYYCVREIA

101 VAGTGYYGMD VWGQGTTVTV SSASTKGPSV FPLAPCSRST SESTAALGCL

151 VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT

201 KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFEGGP SVFLFPPKPK

251 DTLMISRTPE VTCWVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS

301 TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL P S SIE K T ISK AKGQPREPQV

351 YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL

401 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLG ^{(SEQ ID NO: 7)}

Positions 1-122: variable region of the heavy chain (VH). The CDRs (regions determining complementarity, according to the Kabat definition) are underlined.

Positions 123-448: constant region of human IgG4 (SwissProt IGHG4_HUMAN with the two mutations S241P and L248E, according to the Kabat numbering).

In other specific aspects described herein, the anti-LIGHT antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 8:

1 AIQLTQSPSS LSASVGDRVT IT C RASQGIN SAFAWYQQKP GKAPKLLIYD

51 ASSLESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ FNSYPLTFGG

101 GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV

151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG

201 LSSPVTKSFN RGEC ^{(SEQ ID NO: 8)}

Positions 1-107: variable region of the light chain (VL). The CDRs (regions determining complementarity, according to the Kabat definition) are underlined.

Positions 108-214: human C ^k constant region.

In other aspects described herein, the anti-LIGHT antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of SEQ ID NO: 8 .

In certain aspects described herein, the anti-LIGHT antibody comprises a heavy chain derived from the amino acid sequence of SEQ ID NO: 10 , which may be encoded by the nucleic acid sequence of SEQ ID NO: 11 .

M K H L W F F L L L V A A P R W V L S Q V Q L Q Q W G -1 AIGAAGCACCIGIGGIICIIICIGCIGCIGGIGGCCGCICCIAGATGGGIGCTGICCCAGGIGCAGCIGCAGCAGIGGGG - A G L L K P S E T L S L T C A V Y G G S F S G Y N W H -81 CGCTGGCCTGCTGAAGCCTTCCGAGACACTGTCCCTGACCTGCGCCGTGTACGGCGGCTCCTTCTCCGGCTACAACl'GGC - W I R Q P P G K G L E W I G E I T H S G S T N Y N P 161 ACTGGATCAGGCAGCCTCCCGGCAAGGGCCTGGAATGGATCGGCGAGATCACCCACTCCGGCTCCACCAACTACAACCCT S L K S R V T I S V D T S K N Q F S L K L S S V T A A -241 AGCCTGAAGTCCAGAGTGACCATCTCCGTGGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCTGTGACCGCCGC • D T A V Y Y C V R E I A V A G G G Y Y G M D V W G Q G 321 TGACACCGCCGTGTACTACTGTGTGCGGGAGATCGCCGTGGCTGGCACCGGCTACTACGGCATGGATGTGTGGGGCCAGG - T T V T V S S A S T K G P S V F P L A P C S R S T S 401 GCACCACCGTGACCGTGTCCAGCGCTTCTACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCC E S T A A L G C L V K D Y F P E P V T V S W N S G A L

481 GAGTCCACCGCCGCTCTGGGCTGCCTGGTGAñGGñCTñCTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCT • T S G V H T F P A V L Q S S G L Y S L S S V V T V P S 561 GACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGPACTCCCTGTCCTCCGTGGTGACCGTGCCTT - S S L G T K T Y T C N V D H K P S N T K V D K R V E 641 CCTCCTCCCTGGGCACCAAGACCIACACCIGTAACGIGGACCACAAGCCTICCAACACCAAGGIGGACAAGCGGGIGGAG S K Y G P P C P P C P A P E F E G G P S V F L F P P K

721 TCCAAGTACGGCCCTCCTTGCCCTCCCTGCCCTGCCCCTGAGTTCGAGGGCGGACCTAGCGTGTTCCTGTTCCCTCCTAA • P K D T L M I S R T P E V T C V V V D V S Q E D P E V 801 GCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGG - Q F N W Y V D G V E V H N A K I K P R E E Q F N S T 881 T CCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTTCAAT TCCACC Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K

961 TACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGTAAGGTCTCCAACAA • G L P S S I E K I I S K A K G Q P R E P Q V Y I L P P 1041 GGGCCTGCCCTCCTCCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAGCCTCAGGTGTACACCCTGCCTC - S Q E E M T K N Q V S L T C L V K G F Y P S D I A V 1121 CTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTG E W E S N G Q P E N N Y K T T P P V L D S D G S F F L

1201 GAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCT - Y S R L T V D K S R W Q E G N V F S C S V M H E A L H 1281 GIACICCAGGCIGACCGIGGACAAGTCCCGGTGGCAGGAGGGCAACGICriIICCIGCICCGIGAIGCACGAGGCCCIGC - N H Y T Q K S L S L S L G ^ (SEQ ID NO: 10)

1361 ACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCTGGGCTGA (SEQ ID NO: 11) Amino Acids 1-19: Signal peptide

Amino Acids 20-141: variable region (VH) of 124F19k2

Amino Acid 142-end: constant region of hIgG4 PE

Nucleic acids 1-57: nucleic acids that encode the signal peptide

Nucleic acids 58-423: nucleic acids encoding the variable region (VH) of 124F19k2

Nucleic acids 424-fin: nucleic acids that encode the constant region of hIgG4 PE

In specific alternative aspects described herein, the anti-LIGHT antibody comprises a light chain derived from the amino acid sequence of SEQ ID NO: 12 , which may be encoded by the nucleic acid sequence of SEQ ID NO: 13 .

M D M R V P A Q L L G L L L L W L P G A R C A I Q L T -1 ATGGACATGAGAGTGCCTGCTCAGCTGCTGGGACTGCTGCTGCTGTGGCTGCCTGGCGCTAGATGCGCCATCCAGCTGAC

• Q S P S S L S S V G D R V T I I C R A S Q G I N Y A -81 CCAGTCCCCCTCCTCTCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGGGCATCAACTCCG

• F A W Y Q Q K P G K A P K L L I Y D A S S L E S G V

161 CCTTCGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACGACGCCTCCTCCCTGGAATCCGGCGTG

P S R S G S G S G I D F I L I I S S L Q P E D F A I -241 CCCTCCAGATTTTCCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCAC

• Y Y C Q Q F N S Y P L T F G G G I K V E I K R I V A A -321 CTACTACTGCCAGCAGTTCAACTCCTACCCTCTGACCTTCGGCGGAGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTG

• P S V F I F P P S D E Q L K S G T S S V V C L L N N

401 CACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC

F Y P R E A K V Q W K V D N A L Q S G N S Q E S V T E -481 TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA

• Q D S K D S I Y S L S S I L T L S K A D Y E K H K V Y -561 GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCT

• A C E V T H Q G L S S P V T K S F N R G E C * (SEQ ID NO: 12)

641 ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 13)

Amino acids 1-22: signal peptide

Amino acids 23-129: variable region (VL) of 124F19k2

Amino Acids 130-end: constant region of hKappa

^{Nucleic acids} 1-66 ^{: nucleic acids that encode the signal peptide}

Nucleic acids 67-387: nucleic acids encoding the variable region (VL) of 124F19k2

388-end nucleic acids: nucleic acids that encode the hKappa constant region

In one aspect, the anti-LIGHT antibody is a fully human antibody. Examples of completely human antibody isotypes include IgA, IgD, IgE, IgG and IgM. In some aspects, the anti-LIGHT antibody is an IgG antibody. There are four forms of IgG. In some aspects, the anti-LIGHT antibody is an IgG4 antibody. In some aspects described herein, the anti-LIGHT antibody is a fully human IgG4 antibody.

In some aspects, the anti-LIGHT antibody additionally comprises a constant region, for example a constant region of human IgG. In some aspects, the constant region is a constant region of human IgG4. In additional aspects, the constant region is a constant region of modified human IgG4. In other aspects, the constant region is a constant region of human Ck.

In some aspects described herein, the anti-LIGHT antibody is a fully human anti-LIGHT IgG4 antibody comprising a heavy chain comprising the amino acid sequence of ^{SEQ ID NO: 7 and a light chain comprising the sequence of amino acids of SEQ ID NO:} 8 ^(The "Initial LIGHT Antibody"). In alternative aspects described herein, the anti-LIGHT antibody is a fully human anti-LIGHT IgG4 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions (CDR) comprising the amino acid sequences of SEQ ID NO: 1, 2 and 3, and wherein the variable region of the light chain comprises 3 CDRs comprising the ^{amino acid} sequences of ^{SEQ ID NO: 4, 5 and} 6 ^{. The identification, isolation, preparation and} characterization of anti-LIGHT antibodies, including anti-LIGHT antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO: 7 and an amino acid sequence, ^{have been described in detail.} of light chain ^{comprising SEQ ID NO:} 8 ^{, in U.S. Patent No. 8,058,402, corresponding to} PCT ^publication WO 2008/027338.

Certain formulations described herein also include variants of anti-LIGHT binding agents, such as antibodies. Specifically, the formulations described herein may include variants of the anti-LIGHT antibody which is a fully human anti-LIGHT IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a ^light chain ^{which it comprises the amino acid sequence of SEQ ID NO:} 8 ^{. Anti-LIGHT antibody variants} may have similar physicochemical properties depending on their high similarity. Variants are defined as antibodies with an amino acid sequence that is at least 95%, at least 97%, for example at least 98% or 99% homologous to an anti-LIGHT antibody, and capable of competing for binding to a LIGHT polypeptide, a LIGHT polypeptide fragment, or an LIGHT epitope. In some aspects, the variants will enhance, neutralize or otherwise inhibit the biological activity of LIGHT (eg, the production or ^LIGHT- mediated secretion ^{of CCL20, IL-} 8 ^{or RANTES from cells expressing a LIGHT ligand, such as a LIGHT receptor} (e.g., HVEM, LTpR, and / or DcR3.) The determination of competition for binding to the target can be made by routine procedures known to those skilled in the art.In some aspects described herein , the variants are human antibodies and, in some aspects described herein, are IgG4 molecules.In some aspects described herein, a variant is at least 95%, 96%, 97%, 98% or 99 % identical in amino acid sequence with the Initial Antibody The term "variant" refers to an antibody comprising an amino acid sequence that is modified with one or more amino acids compared to those amino acid sequences of the anti-LIGHT antibody. The variant may have conservative sequence modifications, including substitutions, modifications, additions and deletions of amino acids.

Examples of modifications include, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known blocking / blocking groups, proteolytic cleavage and binding to a cellular ligand or other protein. Amino acid modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis, molecular cloning, oligonucleotide-directed mutagenesis and random PCR-mediated mutagenesis in the nucleic acid encoding the antibodies. Conservative amino acid substitutions include those in which the amino acid residue is replaced by an amino acid residue having similar chemical or structural properties. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acid side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine) and side chains aromatics (for example, tyrosine, phenylalanine, tryptophan). It will be apparent to the expert that classifications of amino acid residue families other than those previously used can also be employed. In addition, a variant may have non-conservative amino acid substitutions, for example, the replacement of an amino acid with an amino acid residue having different structural or chemical properties. Similar secondary variations may also include amino acid deletions or insertions, or both. Guidance for determining which amino acid residues can be substituted, modified, inserted or deleted without eliminating the immunological activity can be found using computer programs well known in the art. Computer algorithms, such as, among others, Gap or Bestfit, which are known to those skilled in the art, can be used to optimally align the amino acid sequences to be compared and to define similar or identical amino acid residues. The variants may have the same or different binding affinities, both higher and lower, compared to an anti-LIGHT antibody, but are still capable of specifically binding to LIGHT, and may have a biological activity equal to, greater or less than the anti-L antibody. -LIGHT.

Also disclosed herein are antigen binding fragments of anti-LIGHT binding agents, such as antibodies. The term "antigen-binding domain", "antigen-binding region", "antigen-binding fragment" and similar expressions refer to the portion of an antibody that comprises the amino acid residues that interact with an antigen and confer to the binding agent its specificity and its affinity for the antigen (for example, the regions determining complementarity (CDR)). The antigen-binding region can be derived from any animal species, such as rodents (e.g., rabbit, rat or hamster) and humans. In some aspects described herein, the antigen binding region will be of human origin. Non-limiting examples of antigen-binding fragments include: Fab fragments, F (ab ') 2 fragments, Fd fragments, Fv fragments, single chain Fv molecules (scFv), dAb fragments and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of the antibody.

In some aspects, the anti-LIGHT binding agents (or a variant thereof or an antigen-binding fragment thereof) will improve, neutralize or otherwise inhibit the biological activity of LIGHT in vivo ^{(eg, the production or the LIGHT-mediated secretion of CCL20, IL-} 8 ^{or RANTES from a cell} expressing a LIGHT receptor, eg, HVEM, LTpR, and / or DcR3).

In some aspects, the anti-LIGHT binding agents (or a variant thereof or an antigen-binding fragment thereof) are antagonistic binding agents that enhance, neutralize or otherwise inhibit the ^{biological activity of LIGHT in vivo. (e.g., production mediated by LIGHT or secretion of CCL20, IL-} 8 ^or RANTES from cells expressing a LIGHT ligand, such as a LIGHT receptor, (e.g., HVEM, LTPR, and / or DcR3).

According to the invention, the anti-LIGHT binding agent (or a variant thereof or an antigen-binding fragment thereof) is present in the formulations in an amount of 5 mg / ml to 280 mg / m, for example, 5 mg / ml to about 200 mg / ml, from about 50 mg / ml to about 250 mg / ml, from about 100 mg / ml to about 250 mg / ml, from about 50 mg / ml to about 200 mg / ml and from about 100 mg / ml to about 200 mg / ml. For example, the anti-LIGHT binding agent may be present in the formulation in an amount of about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, approximately 35 mg / ml, approximately 40 mg / ml, approximately 45 mg / ml, approximately 50 mg / ml, approximately 55 mg / ml, approximately 60 mg / ml, approximately 65 mg / ml, approximately 70 mg / ml ml, approximately 75 mg / ml, approximately 80 mg / ml, approximately 85 mg / ml, approximately 90 mg / ml, approximately 95 mg / ml, approximately 100 mg / ml, approximately 105 mg / ml, approximately 110 mg / ml ml, approximately 115 mg / ml, approximately 120 mg / ml, approximately 125 mg / ml, approximately 130 mg / ml, approximately 135 mg / ml, approximately 140 mg / ml, approximately 145 mg / ml, approximately 150 mg / ml, approximately 155 mg / ml, approximately 160 mg / ml, approximately 165 mg / ml, approximately 170 mg / ml, approximately 175 mg / ml, approximately 180 mg / ml, approximately 185 mg / ml, approximately 190 mg / ml, approximately 195 mg / ml ml, approximately 200 mg / ml, approximately 205 mg / ml, approximately 210 mg / ml, approximately 215 mg / ml, approximately 220 mg / ml, approximately 225 mg / ml, approximately 230 mg / ml, approximately 235 mg / ml, approximately 240 mg / ml, approximately 245 mg / ml, approximately 250 mg / ml, approximately 255 mg / ml, approximately 260 mg / ml, approximately 265 mg / ml, approximately 270 mg / ml, approximately 275 mg / ml, or 280 mg / ml.

In alternative aspects, the anti-LIGHT binding agent can be present in the formulation in an amount of from 5 to about 25 mg / ml, from about 26 to about 50 mg / ml, from about 51 to about 75 mg / ml, of about 76 to about 100 mg / ml, about 101 to about 125 mg / ml, about 126 to about 150 mg / ml, about 151 to about 175 mg / ml, about 176 to about 200 mg / ml, about 201 mg / ml to about 225 mg / ml, about 226 mg / ml to about 250 mg / ml, about 251 to 280 mg / ml, about 5 to about 10 mg / ml, about 40 to about 60 mg / ml, from about 75 to about 85 mg / ml, or from about 140 to about 160 mg / ml.

In certain exemplary aspects described herein, the anti-LIGHT binding agent is present in the formulation in an amount of about 50 mg / ml to about 170 mg, from about 100 mg / ml to about 160 mg / ml, per example about 150 mg / ml. Alternatively, the anti-LIGHT binding agent is present in an amount of about 50 mg / ml. In another exemplary aspect, a fully human anti-LIGHT IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the ^{amino acid} sequence of ^{SEQ ID NO:} 8 ^{is present in the formulation in an amount of about 150 mg / ml.}

ii. Anti-CXCR5 binding agents, and variants and fragments thereof

In certain aspects described herein, the formulations described herein include an anti-CXCR5 binding agent. The binding agents can be any molecule, such as an antibody, a siRNA, a nucleic acid, an aptamer, a protein or a small molecule organic compound, which binds or binds specifically to CXCR5, or a variant or fragment Of the same. In some aspects described herein, the binding agent is an anti-CXCR5 antibody, or a variant thereof, or an antigen-binding fragment thereof. Anti-CXCR5 antibodies specifically bind to a protein, a polypeptide fragment or a CXCL13 epitope (also known as BLC). The CXCR5 molecule can be of any species. In some aspects described herein, the CXCR5 molecule is from a human, known herein as "hCXCR5". HCXCR5 has the amino acid sequence following, which is identified as SEQ ID NO: 14:

MNYPLTLEMD LENLEDLFWE LDRLDNYNDT SLVENHLCPA TEGPLMASFK AVFVPVAYSL IFLLGVIGNV LVLVILERHR QTRSSTETFL FHLAVADLLL VFILPFAVAE GSVGWVLGTF LCKTVIALHK VNFYCSSLLL ACIAVDRYLA IVHAVHAYRH RRLLSIHITC GTIWLVGFLL ALPEILFAKV SQGHHNNSLP RCTFSQENQA ETHAWFTSRF LYHVAGFLLP MLVMGWCYVG WHRLRQAQR RPQRQKAVRV A V IL TSIFFL CWSPYHIVIF LDTLARLKAV DNTCKLNGSL PVAITMCEFL GLAHCCLNPM LYTFAGVKFR SDLSRLLTKL GCTGPASLCQ LFPSWRRSSL SESENATSLT TF (SEQ ID NO: 14).

In certain exemplary aspects described herein, the anti-CXCR5 antibody is a humanized antibody, a fully human antibody, or a variant thereof or an antigen-binding fragment thereof. In some embodiments, the anti-CXCR5 antibodies prevent the binding of CXCR5 with its ligands and inhibit the biological activity of CXCR5 (for example, the binding of CXCR5 to CXCL13).

In certain specific embodiments, the anti-CXCR5 antibody comprises a heavy chain variable region (VH) that comprises the amino acid sequence of one or more of the following complementarity determining regions (CDRs):

HCDR1 - GFSLIDYGVN ( SEQ ID NO: 15 );

HCDR2 - VIWGDGTTY ( SEQ ID NO: 16 ) or

HCDR3-IVY ( SEQ ID NO: 17 ).

In other specific embodiments, the anti-CXCR5 antibody comprises a light chain variable region (VL) that comprises the amino acid sequence of any one or more of the following complementarity determining regions (CDRs):

LCDR1 - RSSKSLLHSSGKTYLY ( SEQ ID NO: 18 );

LCDR2 - RLSSLA ( SEQ ID NO: 19 ) or

LCDR3 - MQHLEYPYT ( SEQ ID NO: 20 ).

In a specific embodiment, the anti-CXCR5 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequences of SEQ ID NO: 15, 16 and 17.

In another specific embodiment, the anti-CXCR5 antibody comprises a light chain variable region (VL) comprising the amino acid sequences of SEQ ID NO: 18, 19 and 20.

In more specific embodiments, the anti-CXCR5 antibody comprises a heavy chain variable region comprising the amino acid sequences of SEQ ID NO: 15, 16 and 17; and a light chain variable region comprising the amino acid sequences of SEQ ID NO: 18, 19 and 20.

In a specific aspect described herein, the anti-CXCR5 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21:

QVQLKESGPG L V A P S E S L S I T C T V S G FS L I DYGVNWIRQP PGKGLEWLGV IWGDGTTYYN P S L K S R L S IS KDNSKSQVFL KVTSLTTDDT AMYYCARIVY WGQGTLVTVS A (SEQ ID NO:

twenty-one ^).

In another specific aspect described herein, the anti-CXCR5 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22:

DIVMTQAAPS VAVTPGASVS ISCRSSKSLL HSSGKTYLYW FLQRPGQSPQ LLIYRLSSLA SGVPDRFSGS GSGTAFTLRI SRVEAEDVGV YYCMQHLEYP YTFGGGTKLE IK (SE Q ID NO: 22).

In more specific aspects described herein, the anti-CXCR5 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of the SEQ ID NO: 22

In some embodiments, the anti-CXCR5 antibody further comprises a constant region, for example, a constant region of human IgG. In some embodiments, the constant region is a constant region of human IgG4. In further embodiments, the constant region is a constant region of modified human IgG4. In some embodiments, the human IgG4 constant region has the following modifications: S241P (shown below in SEQ ID NO: 23 in bold), L248E (shown below in SEQ ID NO: 23 in bold), and the lack of a terminal lysine in order to avoid heterogeneity. In some embodiments, the constant region of IgG4 comprises the amino acid sequence of SEQ ID NO: 23:

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMISRTPEVT

CWVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG (SEQ ID NO: 23).

In other embodiments, the constant region is a constant region of human Ck. In some embodiments, the constant region of C ^k comprises the amino acid sequence of SEQ ID NO: 24:

RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC (SEQIDNO: 24).

In specific embodiments, the anti-CXCR5 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25:

QVQLKESGPG LVAPSESLSI TCTVSGFSLI DYGVNWIRQP PGKGLEWLGV IWGDGTTYYN PSLKSRLSIS KDNSKSQVFL KVTSLTTDDT AMYYCARIVY WGQGTLVTVS AASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE

s k y g p p c p P p a p e f g g p s VFLFPPKPKD TLMISRTPEV TCWVDVSQE DPEVQFNWYV DGVEVHNAKT

KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLG (SEQ ID NO: 25).

Positions 1-111: variable region of the heavy chain (VH). The CDRs (regions determining complementarity, according to the Kabat definition) are underlined.

Positions 112-432: constant region of human IgG4 (SwissProt IGHG4_HUMAN, including the following modifications: S241P, L248E, and the lack of a terminal lysine to avoid heterogeneity).

In other specific embodiments, the anti-CXCR5 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 26:

DIVMTQAAPS VAVTPGASVS IS C RSSKSLL HSSGKTYLYW FLQRPGQSPQ LLIY RLSSLA SGVPDRFSGS GSGTAFTLRI SRVEAEDVGV YYCMQHLEYP YTFGGGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASWCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC (SEQ IDNO: 26).

Positions 1-112: Variable region of the light chain (VL). The CDRs (regions determining complementarity, according to the Kabat definition) are underlined.

Positions 113-182: constant region of human Ck.

In further embodiments, the anti-CXCR5 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and a light chain comprising the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the anti-CXCR5 antibody further comprises a leader sequence. The leader sequence, in some embodiments, comprises an amino acid sequence of 1-30 amino acids in length, such as 25-25 amino acids, and typically 19 amino acids. The heavy chain, the light chain or both heavy and light chain may comprise a leader sequence. In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 16: MGWSCIILFL VATATGVHS ( SEQ ID NO: 27 ).

In specific aspects described herein, the anti-CXCR5 antibody comprises a heavy chain derived from the amino acid sequence of SEQ ID NO: 28:

MGWSCIILFL VATATGVHSQ VQLKESGPGL VAPSESLSIT CTVSGFSLID YGVNWIRQPP GKGLEWLGVI WGDGTTYYNP SLKSRLSISK DNSKSQVFLK VTSLTTDDTA MYYCARIVYW GQGTLVTVSA ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMISRTPEVT CWVDVSQED

PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG (SEQ ID NO: 28).

Positions 1-19: leader sequence

Positions 20-130: variable region of the heavy chain (VH). The CDRs (regions determining complementarity, according to the Kabat definition) are underlined.

Positions 131-456: constant region of human IgG4 (SwissProt IGHG4_HUMAN, including the following modifications: S241P, L248E, and the lack of a terminal lysine to avoid heterogeneity).

In other specific aspects described herein, the anti-CXCR5 antibody comprises a light chain derived from the amino acid sequence of SEQ ID NO: 29:

MGWSCIILFL VATATGVHSD IVMTQAAPSV AVTPGASVSI SCRSSKSLLH SSGKTYLYWF LQRPGQSPQL L IY RLSSLAS GVPDRFSGSG SGTAFTLRIS RVEAEDVGVY YCMQHLEYPY TFGGGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC (SEQ ID NO: 29).

Positions 1-19: leader sequence

Positions 20-131: variable region of the light chain (VL). The CDRs (regions determining complementarity, according to the Kabat definition) are underlined.

Positions 132-238: constant region of human Ck.

In additional aspects described herein, the anti-CXCR5 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 28, and a light chain comprising the amino acid sequence of SEQ ID NO: 29.

In some aspects described herein, the anti-CXCR5 antibody is a humanized or fully human antibody. Examples of isotypes of humanized and fully human antibodies include IgA, IgD, IgE, IgG and IgM. In some embodiments, the anti-CXCR5 antibody is an IgG antibody. There are four forms of IgG. In some embodiments, the anti-CXCR5 antibody is an IgG4 antibody. In some embodiments of the invention, the anti-CXCR5 antibody is a humanized IgG4 antibody.

In some embodiments of the invention, the anti-CXCR5 antibody is a humanized anti-CXCR5 IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26 (The "Initial CXCR5 Antibody"). In alternative aspects described herein, the anti-CXCR5 antibody is a humanized anti-CXCR5 IgG4 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 3 regions complementarity determinants (CDR) comprising the amino acid sequences of SEQ ID NO: 15, 16 and 17, and wherein the variable region of the light chain comprises 3 CDRs comprising the amino acid sequences of SEQ ID NO : 18, 19 and 20. The identification, isolation, preparation and characterization of anti-CXCR5 antibodies, including the anti-CXCR5 antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO, have been described in detail. : 25 and a light chain amino acid sequence comprising SEQ ID NO: 26, in PCT publication WO 2009/032661.

Certain formulations described herein also include variants of anti-CXCR5 binding agents, such as antibodies. Specifically, the formulations described herein may include variants of the anti-CXCR5 antibody which is a humanized anti-CXCR5 IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26. Variants of anti-CXCR5 antibodies may have similar physicochemical properties based on their high similarity. Variants are defined as antibodies with an amino acid sequence that is at least 95%, at least 97%, for example, at least 98% or 99% homologous to an anti-CXCR5 antibody, and capable of competing for binding to a CXCR5 polypeptide, a CXCR5 polypeptide fragment or a CXCR5 epitope. In some aspects described herein, the variants will enhance, neutralize or otherwise inhibit the biological activity of CXCR5 (eg, binding of CXCL13 to CXCR5). The determination of the competition for binding to the target can be carried out by routine procedures known to those skilled in the art. In some aspects, the variants are human antibodies and, in some aspects described herein, are IgG4 molecules. In some aspects, a variant is at least 95%, 96%, 97%, 98% or 99% identical in its amino acid sequence with the Initial Antibody. The term "variant" refers to an antibody comprising an amino acid sequence that is modified by one or more amino acids in comparison to the amino acid sequences of the anti-CXCR5 antibody. The variant may have conservative sequence modifications, including substitutions, modifications, additions and deletions of amino acids.

Examples of modifications include, but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known blocking / blocking groups, proteolytic cleavage and binding to a cellular ligand or other protein. Amino acid modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis, molecular cloning, oligonucleotide-directed mutagenesis and random PCR-mediated mutagenesis in the nucleic acid encoding the antibodies. Conservative amino acid substitutions include those in which the amino acid residue is replaced by an amino acid residue having similar chemical or structural properties. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acid side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine) and side chains aromatics (for example, tyrosine, phenylalanine, tryptophan). It will be apparent to the expert that classifications of amino acid residue families other than those previously used can also be employed. In addition, a variant may have non-conservative amino acid substitutions, for example, the replacement of an amino acid by an amino acid residue having different structural or chemical properties. Similar secondary variations may also include amino acid deletions or insertions, or both. The guide for determining which amino acid residues can be replaced, modified, inserted or deleted without eliminating the immunological activity can be found using computer programs well known in the art. Computer algorithms, such as, among others, Gap or Bestfit, which are known to those skilled in the art, can be used to optimally align the amino acid sequences to be compared and to define similar or identical amino acid residues. The variants may have equal or different binding affinities, both major and minor, compared to an anti-CXCR5 antibody, but are still capable of specifically binding to CXCR5, and may have a biological activity equal to, greater or less than that of the antibody anti-CXCR5.

The aspects described herein also include antigen-binding fragments of the anti-CXCR5 binding agents, such as antibodies. The term "antigen-binding domain", "antigen-binding region", "antigen-binding fragment" and similar expressions refer to the portion of an antibody that comprises the amino acid residues that interact with an antigen and confer to the binding agent its specificity and its affinity for the antigen (for example, the regions determining complementarity (CDR)). The antigen-binding region can be derived from any animal species, such as rodents (e.g., rabbit, rat or hamster) and humans. In some aspects, the antigen binding region will be of human origin. Non-limiting examples of antigen binding fragments include: Fab fragments, F (ab ') 2 fragments, Fd fragments, Fv fragments, single chain Fv molecules (scFv), dAb fragments and minimal recognition units consisting of amino acid residues that mimic the hypervariable region of the antibody.

In some embodiments of the invention, the anti-CXCR5 binding agents (or a variant thereof or an antigen-binding fragment thereof) will enhance, neutralize or otherwise inhibit the biological activity of CXCR5 in vivo ( for example, the binding of CXCL13 to CXCR5).

In some embodiments of the invention, the anti-CXCR5 binding agents (or a variant thereof or an antigen-binding fragment thereof) are antagonistic binding agents that enhance, neutralize or otherwise inhibit the activity of CXCR5 in vivo ( for example, the binding of CXCL13 to CXCR5).

According to the invention, the anti-CXCR5 binding agent (or a variant thereof or an antigen binding fragment) thereof) is present in the formulations in an amount of 5 mg / ml to 280 mg / ml, for example, from 5 mg / ml to approximately 200 mg / ml, from 5 mg / ml to approximately 125 mg / ml, 5 mg / ml to approximately 75 mg / ml, from 5 mg / ml to approximately 50 mg / ml and from 5 mg / ml to approximately 25 mg / ml. For example, the anti-CXCR5 binding agent may be present in the formulation in an amount of about 5 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, approximately 35 mg / ml, approximately 40 mg / ml, approximately 45 mg / ml, approximately 50 mg / ml, approximately 55 mg / ml, approximately 60 mg / ml, approximately 65 mg / ml, approximately 70 mg / ml ml, approximately 75 mg / ml, approximately 80 mg / ml, approximately 85 mg / ml, approximately 90 mg / ml, approximately 95 mg / ml, approximately 100 mg / ml, approximately 105 mg / ml, approximately 110 mg / ml ml, approximately 115 mg / ml, approximately 120 mg / ml, approximately 125 mg / ml, approximately 130 mg / ml, approximately 135 mg / ml, approximately 140 mg / ml, approximately 145 mg / ml, approximately 150 mg / ml, approximately 155 mg / ml, approximately 160 mg / ml, approximately 165 mg / ml, approximately 170 mg / ml, approximately 175 mg / ml, approximately 180 mg / ml, approximately 185 mg / ml, approximately 190 mg / ml, approximately 195 mg / ml ml, approximately 200 mg / ml, approximately 205 mg / ml, approximately 210 mg / ml, approximately 215 mg / ml, approximately 220 mg / ml, approximately 225 mg / ml, approximately 230 mg / ml, approximately 235 mg / ml, approximately 240 mg / ml, approximately 245 mg / ml, approximately 250 mg / ml, approximately 255 mg / ml, approximately 260 mg / ml, approximately 265 mg / ml, approximately 270 mg / ml, approximately 275 mg / ml, or 280 mg / ml.

In alternative embodiments, the anti-CXCR5 binding agent may be present in the formulation in an amount of 5 to about 25 mg / ml, of about 26 to about 50 mg / ml, of about 51 to about 75 mg / ml , from about 76 to about 100 mg / ml, from about 101 to about 125 mg / ml, from about 126 to about 150 mg / ml, from about 151 to about 175 mg / ml, from about 176 to about 200 mg / ml , from about 201 mg / ml to about 225 mg / ml, from about 226 mg / ml to about 250 mg / ml, from about 251 to 280 mg / ml, from about 5 to about 25 mg / ml, from about 40 to about 60 mg / ml, from about 75 to about 85 mg / ml, or from about 90 to ^about 110 ^{mg / ml.}

In certain exemplary embodiments, the anti-CXCR5 binding agent is present in the formulation in an amount of about 20 mg / ml. Alternatively, the anti-CXCR5 binding agent is present in an amount of about 100 mg / ml. In another exemplary embodiment, a humanized anti-CXCR5 IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26 is present in the ^{formulation in an amount of about} 20 ^{mg / ml or} 100 ^{mg / ml.}

iii. Buffer agents

The formulations of the invention comprise a citrate buffer as a buffering agent. A buffering agent maintains a physiologically adequate pH. In addition, a buffering agent improves the isotonicity and chemical stability of the formulation. The citrate buffer is present in the formulations at a concentration of 5 mM to 50 mM, for example, 5 mM to about 15 mM. For example, the citrate buffer can be present in the ^{formulation at a concentration of about 5 mM, about} 6 ^{mM, about 7 mM, about} 8 ^{mM, about 9 mM, about 10 mM, about 11 mM,} about 12 mM, about mM, approximately 14 mM, approximately 15 mM, approximately 16 mM, approximately 17 mM, approximately 18 mM, approximately 19 mM, approximately 20 mM, approximately 21 mM, approximately 22 mM, approximately 23 mM, approximately 24 mM, approximately 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM approximately 39 mM, approximately 40 mM, approximately 41 mM, approximately 42 mM, approximately 43 mM, approximately 44 mM, approximately 45 mM, approximately 46 mM, approximately 47 mM, approximately 48 mM, approximately 49 mM and 50 mM. In some embodiments, the citrate buffer is present in the formulation at a concentration of about 7 mM to about 13 mM, such as about 9 mM to about 11 mM. In some embodiments, the citrate buffer is present in a concentration of about 10 mM.

^{The formulations of the invention have a pH equal to or less than the smallest of between pH} 6 ^{and the isoelectric point} (pI) of the antibody, and the pH of the formulations ranges from about 5.0 to about 6.0. For example, the pH of the formulations may be about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9 and about 6.0. In some embodiments, the pH of the formulations may vary from about 5.5 to about 6.0. In some embodiments, the pH is about 5.5 or about 6.0. The pH of the formulation can be measured by any means known to those skilled in the art. A means to measure pH is to use a pH meter with a microelectrode. The pH of the formulation can be adjusted using any means known in the art. Some examples of chemicals to alter the pH of the formulations are hydrochloric acid (HCl) and sodium hydroxide (NaOH).

The isoelectric point is the pH at which a particular molecule or surface does not carry net electrical charge. The pI of an anti-LIGHT or anti-CXCR5 binding agent can be determined by any means known to those skilled in the art. In some embodiments, the pI of an anti-LIGHT or anti-CXCR5 antibody is determined by denatured isoelectric focusing. As shown in Figure 1 , the pI of a completely human anti-LIGHT IgG4 antibody comprising a heavy chain comprising the amino acid sequence of ^{SEQ ID NO: 7 and a light chain comprising the amino acid sequence of the SEQ ID NO:} 8 ^{is 6.8-7.2.} As shown in Figure 11 , the pI of a humanized anti-CXCR5 IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ. ID NO: 26 is 7.6-8.4.

iv. Surfactants

The formulations of the invention further comprise a surfactant, which is also known as a stabilizing agent. Surfactants / stabilizing agents are chemical compounds that interact and stabilize biological molecules and / or general pharmaceutical excipients in a formulation. In certain embodiments, the surfactants can be used together with storage at reduced temperature. Surfactants generally protect the binding agent from the stresses induced by the air / solution interface and the stresses induced by the solution / surface, which may otherwise cause protein aggregation. The surfactants described herein may include, but are not limited to, polysorbates, glycerin, dicarboxylic acids, oxalic acid, succinic acid, fumaric acids, phthalic acids and combinations thereof. Those skilled in the art are aware that other surfactants, for example non-ionic or ionic detergents, may be used, provided they are pharmaceutically acceptable, ie, suitable for administration to subjects. The surfactant is, according to the invention, a polysorbate. Examples of polysorbates include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

In exemplary embodiments, the surfactant is present in the formulations in an amount of about 0.001% to about 0.1% (w / v). For example, the surfactant may be present in ^{the formulations in an amount from about 0, 001% (w / v), about} 0 ^{002% (w / v),} about 0.003% (w / v), about 0.004% (w / v), approximately 0.005% (w / v), approximately 0.006% (w / v), approximately 0.007% (w / v), approximately 0.008% (w / v), approximately 0.009% (w / v), approximately 0.01% (w / v), approximately 0.02% (w / v), approximately 0.03% (w / v), approximately 0.04 % (w / v), approximately 0.05% (w / v), approximately 0.06% (w / v), approximately 0.07% (w / v), approximately 0.08% ( p / v), approximately 0.09% (w / v), and approximately 0.1% (w / v). In particular embodiments, the surfactant is present in formulations from about 0.003% to about 0.05% (w / v), from about 0.004% to about 0.025% (w / v), or about 0.005% to about 0.02% (w / v)), for example about 0.005% (w / v). For example, polysorbate 20 ^{may be present in an amount from about} 0 ^{001% to about 0, 1% (w / v),} about 0.002% to about 0.01% (w / v), from about 0.003% to about 0.008% (w / v) and from about 0.004% to about 0.006% (w / v), for example about 0.005% (w / v). In alternative embodiments, the polysorbate 20 is present in ^{an amount from about 0.001% to about 0, 1% (w / v), about 0.005% to} about 0.05% (w / v) and from about 0.0075% to about 0.025% (w / v), for example about 0.01% (w / v). In additional alternative embodiments, the polysorbate 20 is ^{present in an amount of about} 0 ^{001% to about 0, 1% (w / v), about} 0.005% to about 0.05% (w / v) and about 0.01% to about 0.03% (w / v), for ^{example from about} 0 ^{02% (w / v).}

v. Tonicity agents

The formulations of the invention may optionally also comprise a tonicity agent. Typically, the tonicity agents are used to adjust or maintain the osmolality of the formulations in order to bring it closer to the osmotic pressure of body fluids, such as blood or plasma. The tonicity agents can also maintain the levels of binding agent in a formulation. In part, the tonicity agent contributes to preserving the level, rate or proportion of the therapeutically active binding agent present in the formulation. As used herein, the term "tonicity" refers to the behavior of the biological components in a fluid medium or a fluid solution. The isotonic solutions have the same osmotic pressure as the blood plasma and can be infused intravenously to a subject without changing the osmotic pressure of the subject's blood plasma. In fact, in certain embodiments of the invention, the tonicity agent is present in an amount sufficient to make the formulation suitable for intravenous infusion. Often, the tonicity agent also serves as a bulking agent or as an agent stabilizer. As such, the tonicity agent can allow the binding agent to overcome various stresses, such as freezing and shearing. Tonicity agents may include, but are not limited to, saccharides, sugars, glycerol, sorbitol, mannitol, sodium chloride, potassium chloride, magnesium chloride and other inorganic salts. Those skilled in the art are aware that other tonicity agents may be used as long as they are pharmaceutically acceptable, ie, suitable for administration to subjects.

In certain embodiments, the tonicity agent is present in the formulations in an amount of about 0.1% to 10% (w / v). For example, the tonicity agent may be present in the ^{formulation in an amount from about 0, 1% (w / v), about 0, 2% (w / v), about} 0.3% (w / v), approximately 0.4% (w / v), approximately 0.5% (w / v), approximately 0.6% (w / v), approximately 0.7% (w / v) , approximately 0.8% (w / v), approximately 0.9% (w / v), approximately 1% (w / v), approximately 2% (w / v), approximately 3% ( p / v), approximately 4% (w / v), approximately 4.5% (w / v), approximately 5% (w / v), approximately 5.5% (w / v), approximately 6 ^{% (w / v), approximately 7% (w / v), approximately} 8 ^{% (w / v), approximately 9% (w / v) and} approximately 10% (w / v). Alternatively, the tonicity agent may be present in the formulation in ^{an amount from about 2% to about} 8 ^{% (w / v), from about 3% to about 7% (w / v) and about 4% to approximately} 6 ^{% (p / v). In} additional alternative embodiments, the tonicity agent may be present in the formulation in an amount from about 0.1% to about 1%, from about 0.1% to about 0.5%, of about 0.1 to about 0.3% and about 0.2%.

In certain exemplary embodiments, the tonicity agent is a saccharide. Examples of saccharides include glucose, sucrose, maltose, trehalose, dextrose, xylitol, fructose and mannitol. For example, mannitol ^{may be present in an amount from about} 1 ^{% to about} 10 ^{% (w / v), from about 2% to about} 8 ^{% (w / v), or about 3% at about 5%} (w / v), for example about 4% (w / v). Alternatively, sucrose may be present in an amount of from about 1% to about 10% (w / v), from about 3% to ^about 8 ^{% (w / v), or about 4% at about} 6 ^{% (w / v), for example about 4.5, 5, 5.5 or} 6 ^{% (w / v).}

In certain other exemplary embodiments, the tonicity agent is sodium chloride. For example, ^{sodium chloride may be present in an amount from about 0, 1% (w / v), about} 0.2% (w / v), about 0.3% (w / v), approximately 0.4% (w / v), approximately 0.5% (w / v), approximately 0.6% (w / v), approximately 0.7% (w / v), approximately the 0.8% (w / v), approximately 0.9% (w / v) and approximately 1% (w / v). Alternatively, sodium chloride may be present in the ^{formulation in an amount from about 0, 1% to about 1%, from about} 0 ^1% to about 0.5%, about 0.1 to approximately 0.3% and approximately ^{0, 2%.}

In additional exemplary embodiments, the formulations may comprise one or more tonicity agents. For example, the formulations may comprise one or more of the above tonicity agents in the above concentrations. In certain specific embodiments, the formulations may comprise sucrose and sodium chloride, wherein each of the sucrose and sodium chloride concentrations is between about 0.1% and about 10% (w / v) . In some embodiments, the ^{concentration of sucrose is about} 6 ^{% and the concentration of sodium chloride is} about 0.2%. Alternatively, the sucrose concentration is about 4.5% and the ^{concentration of sodium chloride is about 0, 2%.}

In certain embodiments of the invention, the osmolality of the formulations ranges from about 200 mOsm / kg to about 350 mOsm / kg, from about 270 mOsm / kg to about 330 mOsm / kg, from about 280 mOsm / kg to about 320 mOsm / kg or from about 290 mOsm / kg to about 310 mOsm / kg, being, for example, about 300 mOsm / kg. In other words, the formulations of the invention are, in some embodiments, substantially isotonic, that is, they have substantially the same osmotic pressure as human blood. The osmolality can be measured by any means known to those skilled in the art, such as using pressure-type osmometers or ice-freezing. The osmolality of the formulations of the invention may be regulated, for example, by one or more tonicity agents described herein.

saw. Amino acids

The formulations of the invention may optionally also comprise an amino acid. Examples of amino acids include, but are not limited to, glycine, alanine, aspartic acid, lysine, serine, tyrosine, cysteine, glutamine, methionine, arginine and proline. In exemplary embodiments, the amino acid is present in the formulations in an amount of about 0.1% to 5% (w / v). For example, the amino acid can be present in the ^{formulation in an amount from about 0, 1% (w / v), about 0, 2% (w / v), about} 0.3% (w / v) , approximately 0.4% (w / v), approximately 0.5% (w / v), approximately 0.6% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 0.9% (w / v), about 1.0% (w / v), about the 1.1% (w / v), approximately 1.2% (w / v), approximately 1.3% (w / v), approximately 1.4% (w / v), approximately 1, 5% (w / v), approximately 1.6% (w / v), approximately 1.7% (w / v), approximately 1.8% (w / v), approximately 1.9% (w / v), approximately 2.0% (w / v), approximately 3% (w / v), approximately 4% (w / v) and approximately 5% (w / v). Alternatively, the amino acid is present in the formulation in an amount from about 1.3% to about 1.8% (w / v), or from about 1.4% to about 1.6% (p. / v), for example of approximately 1.5% (w / v). In further alternative embodiments, the amino acid is present in the formulation in an amount of from about 0.5% to about 1.5% (w / v), or from about 0.8% to about 1%, 2% (w / v), for example of approximately 1.0% (w / v). An exemplary amino acid is proline or arginine. For example, proline may be present in the formulation in an amount from about 1% to about 2% (w / v), from about 1.3% to about 1.8% (w / v), from about 1.4% to about 1.6% (w / v), for example of about 1.5% (w / v). Alternatively, arginine may be present in the formulation in an amount from about 0.5% to about 1.5% (w / v), or from about 0.8% to about 1.2% ( w / v), for ^{example about 1, 0% (w / v).}

vii Other excipients

In addition, the formulations of the invention may comprise other excipients including, but not limited to, water for injection, diluents, solubilizing agents, calming agents, additional buffers, inorganic or organic salts, antioxidants or the like. In some embodiments, however, the formulations of the invention do not comprise other excipients, except those described above. Other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may be included in the formulation as long as they do not adversely affect the desired characteristics of the formulation. In a particular embodiment, the formulation is substantially free of preservatives, although, in alternative embodiments, preservatives may be added as necessary. For example, cryoprotectants or lyoprotectants may be included in lyophilized formulations.

viii. Liquid or lyophilized formulations.

The formulations of the invention may be liquid formulations or lyophilized formulations. In some embodiments, the formulations are liquid formulations. In some embodiments, the liquid formulations are ready for injection. Alternatively, the formulations can be lyophilized powders. In some embodiments, the lyophilized powders are ready to be combined with a solvent just prior to administration.

ix Exemplary Formulations

In an exemplary aspect described herein, the specification discloses a stable liquid antibody formulation suitable for subcutaneous administration, the formulation comprising:

a) more than about 80 mg / ml, e.g., about 150 mg / ml, of an anti-LIGHT IgG4 antibody (lymphotoxin type, shows an inducible expression and competes with HSV glycoprotein D for an HVEM, a receptor expressed by T lymphocytes) completely human comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the ^{amino acid} sequence of ^{SEQ ID NO:} 8 ^;

b) approximately 10 mM citrate buffer;

c) about 0.005% (w / v) of polysorbate 20 and

d) about 4% (w / v) mannitol;

the pH of the formulation being approximately pH 5.5.

In certain exemplary aspects, this formulation may be produced:

a) dissolving about 10 mM of sodium citrate dihydrate in water for injection and adjusting the pH of the buffered solution to about pH 5.5, for example, using hydrochloric acid or sodium hydroxide;

b) adding more than about 80 mg / ml, e.g., about 150 mg / ml, of an anti-LIGHT IgG4 antibody (lymphotoxin type, shows an inducible expression and competes with HSV glycoprotein D for an HVEM, an expressed receptor by T lymphocytes) completely human comprising a heavy chain that comprises the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the ^{amino acid} sequence of ^{SEQ ID NO:} 8 ^{, about 4% (w / v) of mannitol and 0.005% (w / v) of polysorbate 20 to the} buffered solution of step a) while stirring in a vessel made of inert material until it is completely dissolved, and then adjusting the pH of the resulting formulation to about pH 5.5 using hydrochloric acid or sodium hydroxide , and then adding buffered solution from step a) to adjust the final weight of the resulting formulation;

c) filtering the formulation of step b) under aseptic conditions using a ^sterilized compatible membrane filter ^{having a nominal pore size of} 0.2 ^{μm, and then sterilizing the formulation by filtration} under aseptic conditions in sterilized containers made of inert material using a ^{sterilized compatible} membrane filter ^{having a nominal pore size of} 0.2 ^μm;

d) filling with the formulation of step c) under aseptic sterile road conditions that are closed with stoppers and hinged covers with a flange; and, optionally,

e) inspecting the containers of stage d) to detect coarse contaminants, that the seal is intact and visible particles.

In another exemplary aspect, the specification discloses a stable liquid antibody formulation suitable for intravenous administration, the formulation comprising:

a) from 5 to about 80 mg / ml, for example, about 50 mg / ml, of an anti-LIGHT IgG4 antibody (lymphotoxin type, shows an inducible expression and competes with HSV glycoprotein D for an HVEM, an expressed receptor by T lymphocytes) completely human comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the ^{amino acid} sequence of ^{SEQ ID NO:} 8 ^;

b) approximately 10 mM citrate buffer and

^{c) about 0, 01% (w / v) of polysorbate 20;}

the pH of the formulation being approximately pH 5.5.

In an alternative exemplary aspect, the specification discloses a stable lyophilized antibody formulation suitable for intravenous administration, the formulation comprising:

a) from 5 to about 80 mg / ml, for example, about 50 mg / ml, of an anti-LIGHT IgG4 antibody (lymphotoxin type, shows an inducible expression and competes with HSV glycoprotein D for an HVEM, an expressed receptor by T lymphocytes) completely human comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the ^{amino acid} sequence of ^{SEQ ID NO:} 8 ^;

b) approximately 10 mM citrate buffer;

^{c) about 0, 01% (w / v) of polysorbate 20;}

d) approximately 5% (w / v) of sucrose and

e) about 1.5% (w / v) proline;

the pH of the formulation being approximately pH 5.5.

In an exemplary embodiment of the invention, the invention provides a stable antibody formulation comprising:

a) 20 mg / ml of a humanized IgG4 anti-CXCR5 antibody (chemokine receptor CXC type 5) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;

b) 10 mM citrate buffer;

^c) 0 ^{02% polysorbate 20;}

^d) 6 ^{% sucrose and}

^{e) 0, 2% sodium chloride;}

the pH of the formulation being pH 6.0.

In an alternative exemplary embodiment of the invention, the invention provides a stable antibody formulation comprising:

a) 100 mg / ml of a humanized IgG4 anti-CXCR5 antibody (chemokine receptor CXC type 5) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26;

b) 10 mM citrate buffer;

^c) 0 ^{01% polysorbate 20;}

d) 4.5% sucrose;

^{e) 0, 2% sodium chloride and}

^f) 1 ^{% arginine;}

the pH of the formulation being pH 6.0.

X. Stability

The formulations described herein are stable at 5 ° C for at least about 1, 2, ^{3, 4, 5,} 6 ^{, 7,} 8 ^{, 9, 10, 11 or 12 months or more, and typically at least about 12 months. , 18 or 24 months or more. In exemplary embodiments, they are stable at 5 ° C for at least about} 6 ^{months or more. In other} exemplary embodiments, they are stable at 5 ° C for at least about 9 months. In further exemplary embodiments, they are stable at 5 ° C for at least about 1 year or more, and ^{typically about} 2 ^years.

C. Modes of administration

In certain embodiments of the invention, the formulations are suitable for parenteral, intravenous, intramuscular, intradermal, subcutaneous or a combination thereof. The formulations of the invention are suitable for administration by means of a variety of techniques.

In some embodiments of the invention, the formulation is administered intravenously. For example, it is desirable that formulations containing 80 mg / ml of IgG4 binding agent, such as an antibody, or less be administered intravenously. Therefore, the formulations are typically sterile. Methods for producing sterile formulations are well known in the art and include, for example, filtration through sterile filtration membranes or autoclaving of the ingredients of the formulation, with the exception of the antibodies, at about 120 ° C during approximately 30 minutes. For example, the specification discloses a stable liquid antibody formulation suitable for intravenous administration, the formulation comprising: a) from about 5 to about 80 mg / ml, eg, about 50 mg / ml, of an anti-LIGHT IgG4 antibody ( lymphotoxin type, shows an inducible expression and competes with the HSV glycoprotein D for a HVEM, a fully human T lymphocyte receptor) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a ^light chain ^{comprising the amino acid sequence of SEQ ID NO:} 8 ^{; b) about 10 mM} citrate ^buffer and c) about 0.01% (w / v) polysorbate 20; the pH of the formulation being approximately pH 5.5. Alternatively, the invention provides a stable antibody formulation comprising: a) about 20 mg / ml of a humanized anti-CXCR5 (humanized CXC chemokine receptor 5) IgG4 antibody comprising a heavy chain comprising the amino acid sequence of the SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26; b) approximately 10 mM ^{citrate buffer; c) about} 0 ^{02% polysorbate 20; d) about} 6 ^{% sucrose and e)} about 0.2% sodium chloride; the pH of the formulation being approximately pH 6.0.

In some embodiments of the invention, the formulation is administered subcutaneously. For example, it is desirable that formulations containing more than 80 mg / ml of IgG4-binding agent, such as an antibody, be administered subcutaneously. In a specific embodiment, it is desirable to subcutaneously administer to subjects a stable liquid antibody formulation comprising: a) about 150 mg / ml of an anti-LIGHT IgG4 antibody (lymphotoxin type, shows an inducible expression and competes with the HSV glycoprotein D by a HVEM, a fully human T lymphocyte receptor) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the ^{amino acid sequence of SEQ ID NO :} 8 ^{; b) approximately 10 mM citrate buffer; c) about} 0.005% (w / v) of polysorbate 20; d) about 4% (w / v) mannitol; being the pH of the formulation approximately pH 5.5. Alternatively, the invention provides a stable antibody formulation comprising: a) about 100 mg / ml of a humanized anti-CXCR5 (humanized CXC chemokine receptor 5) IgG4 antibody comprising a heavy chain comprising the amino acid sequence of the SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26; b) approximately 10 mM citrate buffer; c) about 0.01% polysorbate 20; d) about 4.5% sucrose; e) about 0.2% sodium chloride and f) about 1% arginine; the pH of the formulation being approximately pH 6.0.

D. Dosage and dosage forms

The effective doses of the formulations of the invention vary depending on many different factors, including the means of administration, the target site, the physiological state of the subject, whether the subject is a human being or an animal, other drugs administered and whether the Treatment is prophylactic or therapeutic. Usually, the subject is a human being, but non-human mammals, including transgenic mammals, can also be treated. It may be necessary to assess treatment doses to optimize safety and efficacy.

The formulations of the invention can be administered multiple times. The intervals between individual doses can be daily, weekly, biweekly, monthly or yearly. The intervals can also be irregular. In some methods, the dose is adjusted to achieve a certain concentration of binding agent in the plasma, such as an antibody. The dose and frequency will vary depending on the half-life of the anti-LIGHT or anti-CXCR5 binding agent, such as an antibody, in the subject. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies.

In further embodiments, the invention provides a unit dosage dosage form comprising a therapeutically effective amount of a formulation of the invention for the treatment of one or more diseases in a subject by administering the dosage form to the subject. In some embodiments, the subject is a human being. The human being can be an adult or can be a breast child. The term "dosage unit dosage form" refers to a physically discrete unit suitable as unit doses for the subjects to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic / prophylactic effect in association with the required citrate buffer and pH.

The unit dosage form can be a container comprising the formulation. Suitable containers include, but are not limited to, sealed ampoules, vials, bottles, syringes and test tubes. The containers may be formed of a variety of materials, such as glass or plastic, and may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container is a vial. Generally, the package must maintain the sterility and stability of the formulation.

In specific embodiments, the formulations are packaged in 2 ml vials which are made of colorless transparent type I glass, and are sealed with a stopper (bromobutyl coated with fluoropolymer) sealed with hinged flap covers (polypropylene). The vials, in some embodiments, are filled with 1.2 ml of the ^{formulations, so that the vial has an overfill volume of approximately} 0.2 ^{ml per vial and a} removable volume of 1.0 ml. For example, this means that the dosage strength of the anti-LIGHT antibody (e.g., 150 mg / ml) will be contained within 1 ml of solution.

In a specific embodiment, the formulations are packaged secondarily in a container, such as a cardboard box, which protects the vials from light.

E. Treatment procedures

In the present document, there are further provided methods for treating a disease or disorder mediated by LIGHT, the methods comprising administering a formulation of the invention to a subject. The specification further describes a formulation of the invention for use in a method described herein for treating a disease or disorder mediated by LIGHT. In certain aspects described herein, the disease mediated by LIGHT is a chronic intestinal disease or an inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC). In other aspects described herein, the disease mediated by LIGHT is graft-versus-host disease (GVHD).

Methods for treating a disease or disorder mediated by CXCR5 are also provided herein, the methods comprising administering a formulation of the invention to a subject. The specification further describes a formulation of the invention for use in a method described herein for treating a disease or disorder mediated by CXCR-5. In certain aspects, the anti-CXCR5 binding agent is used for the reduction of signs and symptoms, the inhibition of damage progression structure, the induction of a major clinical response, and the prevention of disability in adult patients with moderately to severely active rheumatoid arthritis (RA) who have had an inadequate response to one or more disease-modifying antirheumatic drugs (DMARD) such as methotrexate (MTX) or TNFa antagonists. The anti-CXCR5 binding agent can be used in combination with DMARD or anti-TNFa agonists.

In certain embodiments, the formulations of the invention may be administered in combination with one or more therapies (e.g., therapies other than the formulations of the invention that are currently administered to prevent, treat, manage and / or ameliorate a mediated disease. by LIGHT or a disease mediated by CXCR 5. The use of the expression "in combination" does not restrict the order in which therapies are administered to a subject.A first therapy can be administered before (eg, 1 minute, 45 minutes, 30 minutes , ^{45 minutes, 1 hour, 2 hours, 4 hours,} 6 ^{hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks,} 6 ^weeks, 8 ^{weeks or 12 weeks), simultaneously or afterwards (for example, 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours,} 6 ^{hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks,} 6 ^weeks, 8 ^{weeks or 12 weeks} nas) of the administration of a second therapy to a subject who has suffered, suffers from or is susceptible to suffering from a disease mediated by LIGHT or a disease mediated by CXCR5. Any additional therapy can be administered in any order with the other additional therapies. Non-limiting examples of therapies that can be administered in combination with an antibody of the invention include the approved anti-inflammatory agents listed in the US Pharmacopoeia. UU and / or the Physician's Desk Reference.

F. Kits

Certain aspects described herein include a kit comprising a formulation as described herein. The kit may further comprise one or more containers comprising pharmaceutically acceptable excipients, and include other desirable materials from a commercial and user's point of view, including filters, needles and syringes. Associated with the kits may be instructions that are usually included in commercial packages of therapeutic, prophylactic or diagnostic products, which contain information on, for example, indications, use, dosage, manufacture, administration, contraindications and / or warnings about the use of said therapeutic, prophylactic or diagnostic products. The kit can also be associated with a label that can be any type of data medium (for example, a booklet, a label, a chip, a printed text or a bar code) that includes information. In certain aspects described herein, the instructions, etc., as listed above may be included in or on the label. The kit may further comprise a device for administration of the formulation, and particularly a device containing the formulation, i.e., a pre-filled device such as, for example, a pre-filled syringe or a pre-filled auto-injector. The kit may also comprise a container comprising the formulation, ie, a pre-filled container, such as a vial, a cartridge, a pre-filled envelope or ampoule.

G. Combination of different forms of realization.

In the context of the present invention, any of the embodiments described herein may be combined with one or more of the other embodiments described herein, unless explicitly stated otherwise. In particular, any of the binding agents and antibodies described herein and the formulations thereof described herein may be used in combination with any of the kits, pre-loaded devices or pre-filled containers, or may be used in the methods of treatment or medical uses as described herein in relation to the respective antibody (eg, stable formulations comprising anti-LIGHT antibodies or anti-CXCR5 antibodies can be combined with any of the kits, containers or devices described in this document). Any of the binding agents described herein that specifically bind to an antigen (e.g., a binding agent that specifically binds to LIGHT or a binding agent that specifically binds to CXCR5) can also be used in any of the methods of treatment described herein in relation to the respective antibodies (ie, anti-LIGHT or anti-CXCR5) and vice versa.

EXAMPLES

To help illustrate the invention, the following examples are provided. The examples are not intended to limit the scope of the invention in any way. In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of pharmaceutical formulation, chemistry, molecular biology, recombinant DNA technology, immunology such as antibody technology and standard polypeptide preparation techniques such as those described, for example, by Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), volume 51, Ed .: Paul S., Humana Press (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), Eds .: McCafferty J. et al., Humana Press (1996); Antibodies: A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press (1999); and Current Protocols in Molecular Biology, Eds. Ausubel et al., John Wiley & Sons (1992).

Anti-LIGHT

A fully human anti-LIGHT IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain comprising the amino acid sequence of ^{SEQ ID NO:} 8 ^{(the "Initial LIGHT antibody") ) was used in Examples 1-9 to determine the optimal} formulation ^conditions .

materials

Lot of pharmacological substances

The Initial Antibody, formulated in phosphate buffered saline (PBS) at a concentration of 5.5 mg / ml and at a pH of 7.3 (the "Original Formulation", the "PBS Formulation" or the "Reference Batch") ") was used in the following examples.

Excipients

Table 1 shows the excipients that were used in the following examples, which were chosen according to their acceptability / suitability for use in parenteral administration products.

Table 1 - Excipients used in this study.

Excipients Art No. / load Supplier

Arginine 1.01587 Merck

Citric acid 100241 Merck

HCl 114027 Merck

Sodium Acetate 1.06265 Merck

Sodium Chloride 10158 Riedel de Haen

Sodium hydroxide 114076 Merck

Sodium Citrate 114196 Boehringer Ingelheim KG

Polisorbate 20 139850 Fluka

Trehalose-dihydrate T9531 Sigma-Aldrich

Procedures

The following procedures were used to manufacture the experimental formulations and the formulations containing the Initial LIGHT Antibody.

Manufacture and composition of tampons.

All buffers were made with agitation to dissolve the respective excipients. The pH was adjusted using 0.1 M HCl or 0.1 M NaOH. The overall concentration of all buffers was 10 mM.

Manufacture and composition of excipient stock solutions.

All stock solutions were manufactured with agitation to dissolve the excipients. The concentration was given as weight / weight (w / w).

Sterile filtration of samples

All samples, solutions, buffers, etc., were filtered (0.22 pm) sterile using a Sartopore-2 membrane. The samples were filtered by pouring them into sterile vials or bottles, which were closed under aseptic conditions inside a clean work table to avoid microbiological contamination.

Mechanical stress test

The mechanical stress with a stirring speed of 350 rpm for 2.5 hours at room temperature was carried out using a horizontal laboratory agitation apparatus with a distance of 26 mm (agitation apparatus and incubation bell from Bühler Company). The 2R vials were filled with 1 ml of solution with a head space of approximately 2.5 ml. The mechanical stress test was planned and carried out during the first preformulation studies and during relevant studies for the selection of surfactant.

Thermal stress test

Thermal stress was used as a stress test during all stages of the preformulation program. The samples were stored at 40 ° C for 24 hours or 7 days, depending on the study.

Analytical procedures in formulation filling and finishing

The following analytical procedures were used in filling the formulation and finishing in the following examples.

Appearance

The appearance of the antibody solutions was verified visually and, additionally, documented by taking a photograph with a digital camera.

PH

All pH measurements were made using a pH meter with a microelectrode.

Concentration using UV

The protein concentrations of all antibody solutions were measured against the buffer using a NanoDrop ND1000. Protein concentrations close to or less than 5 mg / ml were diluted 1: 3, while ^{higher concentrations of proteins close to} 20 ^{mg / ml were diluted} 1 ^: 20 ^{, and absorption was measured} at 215 nm and 280 nm.

Dynamic light scattering ( DLS)

The hydrodynamic diameter of the molecule was measured using laser light scattering. The samples were sterile filtered before the analytical if turbidity was observed, so that only soluble aggregates could be detected. Differential scanning calorimetry ( DSC)

Aliquots of most of the preformulation samples were examined by DSC using a Microcal VPCapillary DSC and analyzed in the autosampler at 90 ° C / hour with a filter time of 2 seconds. Samples of 400 pl were placed in 96-well plates and analyzed to determine the unfolding temperature Tm.

Osmolarity

The osmolarity was measured using an automated Knaur osmometer.

Density

The density of the formulations was measured using an Anton Paar DMA4500 downward sphere viscometer.

Analytical procedures in bioanalytical FF

The following analytical procedures were used in the bioanalytical filling and finishing in the following examples.

Size exclusion chromatography ( SEC)

The aggregates, as well as the products of degradation of the Initial Antibody, were quantified using GL exclusion chromatography by size. The assay was carried out by isocratic HPLC with a SUPERDEX 20010/300 column.

Reducing and non-reducing SDS-PAGE

Polyacrylamide gel electrophoresis with sodium dodecylsulfate (SDS-PAGE) was used to analyze molecular integrity (eg, half molecules) and purity. This electrophoretic analysis was performed with gradient gels of 4-12% in reducing and nonreducing conditions. Proteins were visualized with Coomassie staining after electrophoretic separation.

Weak cation exchange ( WCX)

S e u t i l i z o c o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o r o o S y i n f o r m e d s p o r c e n t a n t i n t i n g a n d s a n d s a n d s a n d s a n d s a n d s a n d s T h e n e a n d e a n d e a n d e m e r t h e m e r t a n t e r t h e m e r t a t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t ...

Immunosorbent assay bound to antigen enzyme ( antigen ELISA)

Isoelectric focus ( IEF)

S e r e a l i z o u n IE F. T h e p a t e r i n e s t e r e c e r e c t i n g e s t e r t i n t i n t i c e r t i n e t i n e s t i t i n t i t i t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t L a d e g a n d e a n d e a n d e a n d e r a n d e a n d e a n d a n d e a n d e a n d e a n d e a t e r

Storage

T o d s s s o l a c tio n s, s o u c tio n s s e d e x c i p e n t i n g a n d s a m e r a n c t a n c tio n s 5 ° C (± 3 ° C), s i n o s a n d in c a t i n t a r i o n s.

SUMMARY OF ALL PREPARED AND ANALYZED FORMULATIONS

L a T a b the 2 s ig u ie n t e m e s t r a u r e s s t m a n t e s t h e s o f th e m e m a tio n s t h e s t h e s t h e s t h e s t h e s t h e s t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t. C a d a n d a n c e n t a n t i n t a n t i n t i n t i n t i n t i n t i n a t i n t i n t i n t a n t i n c t i n t i n t i n t i n t i n c t i n t i n a t i n t i n t i n c t i n t i n t i n c t

Table 2 - Summary of all formulations prepared and analyzed

Sample number Buffer pH Concentration Comment

F o rm a tio n 1.1 C i t r a t o 10 m M 5, 0 5, 5 m g / m l

F or r m u la tion 1.2 C i t r a t o 10 m M 5, 5 5, 5 m g / m l

F o rm a tio n 1.3 C i t r a t o 10 m M 6, 0 5, 5 m g / m l

F or r m u la tion 2 P B S 7, 3 <80 5 m g / m l M u y t u r b ia

C i t r a t o 10 m M 5, 0

F o rm a tio n 3.1 P S 20 a l 0, 01% 5 m g / m l

C i t r a t o 10 m M 5, 5

F o rm a tio n 3.2 P S 20 a l 0, 01% 5 m g / m l

C i t r a t o 10 m M 5, 5

F o rm a tion 4 P S 20 a l 0, 01% 80 m g / m l T r a n s p a r e n t

C i t r a t o 10 m M 5, 0

F o rm a tio n 5 P S 20 a l 0, 01% 5 m g / m l

C i t r a t o 10 m M 5, 5

P S 20 to l 0, 01%

P r o l in a a l 1, 5%

F o rm a tion 6.1 S a c a r a s a l 5% 50 m g / m l L y o

C i t r a t o 10 m M 5, 5

P S 20 to l 0, 01%

F o rm a tion 6, 2 S a c a r a s a l 5% 50 m g / m l L y o

F o rm a tion 7 H i s t i n a 10 m M 5, 5 50 m g / m l

H istidine 10 m M 5, 5

Sample number Buffer pH Concentration Comment

Formulation 9 Citrate 10 mM 5.5 50 mg / ml

Citrate 10 mM 5.5

Formulation 10 PS20 0.01% 50 mg / ml

Citrate 10 mM 5.5

Formulation 11 5% sucrose 50 mg / ml lyo

Formulation 12 Citrate 10 mM 7.0 5 mg / ml pDSC

Formulation 13 PBS 5.0 5 mg / ml mdsc

REFERENCE EXAMPLE 1 - Characterization of a formulation in phosphate buffered saline (PBS) and disadvantages associated with it

In this example, the Reference Lot was characterized. As indicated in the Materials section above, the Reference Lot contains the Initial LIGHT Antibody formulated in phosphate buffered saline (PBS) at a concentration of 5.5 mg / ml and a pH of 7.3, and produces in Vitry research solutions (BioSCP).

The isoelectric focus (IEF) was used to determine the isoelectric point (pI) of the Initial Antibody. The pI of the Initial LIGHT Antibody was theoretically calculated as 6.28, and then measured by denatured isoelectric focusing using standard procedures known in the art. As shown in Figure 1, the major bands show that the pI of the Initial LIGHT Antibody was 6.8-7.2.

SDS-PAGE was used to identify the molecular weight of the antibody monomer, potential aggregates or the presence of half molecules. Figure 2 shows an SDS-PAGE gel comparing different batches of reference Lot under reducing and non-reducing conditions. An ELISA was used to determine the antigen-binding activity of the Initial LIGHT Antibody. Figure 3 shows an ELISA plot that was used to determine the antigen binding activity of the first and second batches of Reference Lot.

SEC was used to determine the presence of aggregates, as well as the degradation products of the first batch of Reference Lot. As shown in Figure 4, size exclusion chromatography detected high molecular weight proteins (HMWP), for example, di- / oligomers (RRT0.8) or aggregates, and low molecular weight proteins (LMWP) or degradation products. The first batch of Reference Lot had a purity of 97% monomer content.

WCX was used to monitor the load heterogeneity of the first lot of reference lot. As shown in Figure 5, during the stability studies, rearrangements of acidic, neutral and basic isoforms were produced. The first batch of reference Lot had a distribution of acidic / neutral / basic isoforms of 42.3 / 55.6 / 1.9%.

DSC was used to analyze the unfolding temperature Tm of the first lot of Reference Lot. As ^{shown in Figure 6, all three antibody domains are deployed at} 68 ^{° C, 75 ° C and 78 ° C.}

DLS was used to determine the hydrodynamic diameter of the antibody monomer and possible soluble aggregates. As shown in Figures 7 and 8, a hydrodynamic diameter of approximately 10 nm was detected, but aggregates were observed in PBS. However, the aggregates were not observed in the citrate buffer ( Figure 10 ).

REFERENCE EXAMPLE 2 - Development of formulations buffered with citrate and advantages associated with them

The original buffer, phosphate buffered saline (PBS) at a pH of 7.3, was, in terms of pH, very close to the isoelectric point (pI) of the Initial Antibody (see Example 1). In addition, the original Formulation showed aggregates; half molecules; degradation products; low molecular weight proteins (LMWP); high molecular weight proteins (HMWP) and rearrangements of isoforms of acidic, basic and neutral antibodies (see Example 1). Therefore, there was a need for an improved formulation that did not suffer from these disadvantages. Formulations of the Initial LIGHT Antibody (a fully human anti-LIGHT IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a ^light chain ^{comprising the amino acid sequence of SEQ ID NO:} 8 ^{) containing 10 mM citrate buffer at a pH of 5, 5.5 and} 6 ^{, with and without polysorbate 20. Table 3 shows the analytical results of the first batch of Batch of} reference and the various experimental formulations of the Initial LIGHT Antibody formulated in citrate, at a pH of 5.0 and 5.5 and 6.0, with and without polysorbate 20. Aggregates were found in dynamic light scattering (DLS) measurements for the reference Lot, but not in all the other formulations analyzed. The Tm, measured by differential scanning calorimetry (pDSC), indicated that the higher the pH, the greater the thermodynamic stability that could be assumed. But for concentrated formulations of high concentration of antibodies, the pH had to be chosen below the pl of the antibody.

As shown in Table 4, size exclusion chromatography (SEC) data showed a significantly reduced amount of high molecular weight proteins (HMWP) for the Initial LIGHT Antibody in citrate buffer compared to the Reference Lot. (Phosphate buffer at pH 7.3). On the contrary, no differences could be detected with SDS-PAGE (Table 5).

Table 3 - Analytical results of the formulations

Number of Tm1 Tm2 Tm3 PH Prom. Z Concentration Buffer sample [° C] [° C] [° C] [nm] [mg / ml]

Reference lot 67.94 75.00 77.37 7.3 179.85 5.5 PBS Formulation 1.1 58.39 69.98 75.75 5.0 10.97 5.0 Citrate

10 mM Formulation 1.2 62.02 72.26 76.59 5.5 10.71 5.0 Citrate

10 mM ^{Formulation 1.3 65.46 73.74 77.02} 6.0 ^{10.81 5.0 Citrate}

10 mM Formulation 3.1 58.33 69.93 75.74 5.0 13.14 5.0 Citrate

10 mM PS20 ^{to 0, 01%} Formulation 3.2 61.42 71.97 76.45 5.5 12.79 5.0 Citrate

10 mM PS20 ^{to 0, 01%}

Table 4 - SEC data of formulations

ANTIBODY RRT0.8 LMWP HMWPs

Area Name Area Area Area Area Area Area Area Content shown rel. rel. rel. rel. of monomer mAU * % mAU * % mAU * % mAU * % [mg / ml] min min min

Lot of ref. 255.61 98.00 3.98 1.52 1.50 0.57 0.59 0.23

Formulation ^{223.23 98.07 3.22 1.42} 1.01 ^{0.44 0.16 0.07 45.49} 3.1

Formulation 257.09 98.24 3.74 1.43 0.79 0.30 0.09 0.03 48.92 3.2

Table 5 - SDS-PAGE data of formulations

Size name Size quantity Size size Size size Comment quantity sample kDa rel. % kDa rel. % kDa rel. % kDa rel. %

^{Lot of ref. 172.5 98.4 150.1} _1.4 ^68.4 0.2 Additional ^bands <0.5% ^{Formulation 3.1 166.1 97.7 147.8} 2 ^{71.5 0.3 Standard identical} to Lot of ref.

Size name Size quantity Size size Size size Comment quantity sample kDa rel. % kDa rel. % kDa rel. % kDa rel. %

Formulation 3.2 166.2 96.2 147.2 3.4 71.4 0.4 Standard identical to Lot of ref.

REFERENCE EXAMPLE 3 - Development of high concentration antibody formulations

In view of the improvements provided by the Citrate Buffer Antibody Formulation of Example 2, the components of the citrate buffer were optimized for increased concentrations of Initial LIGHT Antibody. ^Table 6 ^{shows the analytical results of the first batch of high concentration antibody formulations} (approximately 40 mg / ml): high phosphate buffered saline (PBS) at a pH of 7.3 (Formulation 2) or citrate a a pH of 5.5 with polysorbate 20 (Formulation 4).

Table 6 - Analytical results of Formulations 2 and 4

Number of Tm1 Tm2 Tm3 PH Prom. Z Concentration Buffer sample [° C] [° C] [° C] [nm] [mg / ml]

Reference lot 67.94 75.00 77.37 7.3 10.05 5.5 PBS Formulation 2 67.87 74.87 77.28 7.3 12.89 42.1 PBS Formulation 4 61.55 72, 00 76.48 5.5 16.71 39.97 Citrate

10 mM PS20 ^{to 0, 01%}

A slightly reduced monomer content was observed after concentrating the protein solution in citrate buffer. In addition, the dimer concentration was reduced and the high molecular weight proteins (HMWP) could also be significantly reduced (see Table 7). On the contrary, these impurities and by-products increased with increasing concentration in the phosphate buffer. No differences could be detected with the ^{SDS-PAGE analysis (Table} 8 ^).

REFERENCE EXAMPLE 4 - Development of lyophilized antibody formulations

To evaluate the viability of lyophilization, different lyophilized experimental formulations were produced and subjected to stability analysis. The concentration of the Initial LIGHT Antibody was increased to 50 mg / ml.

Table 9 shows the lyophilization program that was used in this example.

Table 9 - Freeze-drying program

Lyo program (empty) N ° 8

Charging chamber 5 min / TA / 100%

Freezing 2 h / -45 ° C / 100%

Main drying I 30 min / -45 ° C / 30%

Main drying II 5 h / -20 ° C / 30%

^{Main drying III} 8 ^{h / + 20 ° C / 30%}

Final drying 2 h / + 20 ° C / 3%

Table 10 shows the analytical results of the first batch of Reference Batch and the various experimental lyophilized formulations of the Initial LIGHT Antibody formulated in various combinations of citrate buffer, ^{sucrose, polysorbate} 20 ^{and proline.}

As shown in Table 11, high molecular weight proteins (HMWP) could be significantly reduced using a citrate buffer. No differences were observed in the content of dimers during the storage period at 40 ° C. An increase in low molecular weight proteins (LMWP) was observed after lyophilization. As before, these differences could not be detected with the SDS-PAGE analysis (Table 12 ^).

Table 10 - Analytical data of Formulations 6-6.2 and 11

Number of Tm1 Tm2 Tm3 Time / pH Prom Z Concentration Sample buffer Temp. [nm]

Reference lot 67.94 75.00 77.37 7.3 10.05 5.5 mg / ml PBS

^Formulation 6 ^{Nd Nd Nd N / A 17.46 57.32 5.7} 10 mM ^Citrate PS20 ^{to 0, 01%} 64.30 72.61 77.02 06.01 Formulation T0 59.66 Nd 5.7 10 mM Citrate T1 / 5 ° C 5.7 18.85 Nd PS20 ^{to 0, 01%} T1 / 40 ° C 5.7 19.12 Nd Proline 1.5% T2 / 5 ° C 5.7 Nd Nd

T2 / 40 ° C 5.7 Nd Nd Sucrose at 5% Formulation 6.2 65.45 75.08 79.37 T0 5.7 19.58 Nd Citrate 10 mM T1 / 5 ° C 5.7 31.34 Nd PS20 ^{at 0, 01%} T1 / 40 ° C 5.7 18.1 Nd

T2 / 5 ° C 5.7 Nd Nd Sucrose at 5% T2 / 40 ° C 5.7 Nd Nd

Formulation 11 68.84 75.61 77.91 T0 7.0 98.60 56.49 PBS

^{T1 / 5 ° C 5.7} 20.22 ^{Nd Sucrose} at 5% ^{T1 / 40 ° C 5.7} 22.68 ^Nd

T2 / 5 ° C 5.7 Nd Nd

T2 / 40 ° C 5.7 Nd Nd

REFERENCE EXAMPLE 5 - Accelerated stability study

An accelerated stability study was performed with citrate and histidine buffers. Table 13 shows the analytical results of the first batch of reference Lot and the various experimental formulations of the Initial LIGHT Antibody formulated in various combinations of citrate buffer or histidine buffer. It should be noted that the citrate formulation seemed to work better in all experiments than histidine. In particular, the citrate formulations had a higher monomer content compared to the batch of reference Lot and histidine (Table 13) and the content of low molecular weight proteins (LMWP) and of high molecular weight proteins (Table 13). HMWP) were also significantly lower (Table 14). As before, these differences could not be detected with the SDS-PAGE analysis (Table 15).

Table 13 - Analytical data of Formulations 7, 8, 9 and 10

Number of Tm1 Tm2 Tm3 PH Prom Z Concentration Buffer sample [° C] [° C] [° C] [nm] [mg / ml]

Lot of ref. 67.94 75.00 77.37 7.3 10.05 5.5 PBS

Formulation 7 58.95 68.51 76.20 5.5 12.97 53.65 Histidine 10

mM ^Formulation 8 ^{58.69 68.23 76.12 5.4 13.29 58.72 Histidine 10}

mM

PS20 to ^{0, 01%} Formulation 9 61.67 72.01 76.53 5.6 59.26 55.01 citrate 10

mM Formulation 10 62.24 72.32 76.61 5.6 17.3 55.8 Citrate 10

mM

PS20 at

0 ^, 01 ^%

REFERENCE EXAMPLE 6 - Development of a formulation of high concentration of antibodies for subcutaneous administration

S or b r e a b a s e d e I s r e s u l t e d s e x i t o s o s o f s f o r m u l a tio n s t a m o n a d a s c o n c i t r a t o th e s E j e m p s 2-5, s d e s a r r o l l or u n f o r m u l a tio n d e n t i c h o r p o r a l t a c o n c e n t r a t i o n (150 m g / m l) to d e c u a d a p a r a a d m in i s t r a t i o n s u b c u t a n e a. E l d and s r r o l l o d e f o r m u l a tio n s e r e g r or e n e l A n t i c h o r p or L I G H T I n itia l c o n e l o b j e t i v o r d e s a r r o l l r u n f o r m a d e d s i f i c tio n l í q u i d a c o n u n a v id u t i l a t e p t ab le c u a n d s and lm a c e n to e n t r e 2 and 8 ° C. T h e s t h e s t h e s t h e s t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t th Therefore, these steps have to be monitored during the recording of the formulations using the original assessment, dispersion of the lumen, darkening of the lumen, chromatography and excision, electrophoresis of polyacrylamide, and alcohol chromatography and hatred. íexchangechange cat io n ic od. S e u t i l i n t i n e a n d i n e s t i n e a n t i n g a n t i n e s a n d e a n d e a n d e a n d e a n d e a n d a n d e a n d e a n d e a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n d a n c t S e g u n g h e s, s e s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s T he p H d e f o r m u l a tio n s e e n c u e n t r e n r e g tio n d e e s t a b i l i t y F i s i c a and q uide m ic or p t im to th e s u s t a n c i f r m a c e u t i c a and d e u n a t o le r a b i l i t y f i s i or l or g i c a a c e p t a b l e (p or r e je m p or s m o r i d to d).

T as shown in Table 16, F ormu 14 is a solution for injection and a naïve solution, it is highly transparent and it has the LIGHTI- NIC A lity, sodium citrate (agent agent), polysorbate 20 (stabilizing agent). ) ymanitol (depot agent). S e u t i l i n g a n d a n d o f t h e d i h i d r o d o u r t h e d i c h a n d a n c t h e r t h e r t h e r t h e r t h e r t h e 5 5.

T o d s s e x c i p e n t e s t e n t e s t e r t e n t e n t e r t h e s t h e s t h e s t h e s t e r t e r t e d t h e t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t E u r. and U S P.

Table 16 - Composition

a L o s c o m p o n t e s t a n e m e r a n d s a c e r s a n d s a n d m a m e s a n d e a n d a n d a m e s. S i e x i s t e m a n o n g a n g e a n d, s e d a n t a n d m a n t e n t e r t a n d e a n d e a n d a n d e a n d a n d e a n d e a n d a n d a l a n d e a n d a n d e a n d a n d e a n d a n d e a n d a n d a n d a n d a n d a n d a n d a n d e a t

b S e h a c e r e f e r a n d e c tio n a c t a l a t e a n d a c a c e p e r a n d e a t a t e r.

REFERENCE EXAMPLE 7 - Manufacturing process for the formulation of antibodies for subcutaneous administration

A process of GMP-compatible manufacturing process is developed for the formulation of sub-sectionalization to high-concentration bitumen (Formulation 14) of the E xembly 6. Thefabricationprocedure consists of a lump, adjustment of the H , filtering waste, filling and packaging tapes.

S ellev or just a filter sterilization (according to P h. E ur., And USP) using the food filters of bacteria They have a nominal pore size of 0.2 | jm. An additional filtration procedure was performed before the "filter sterilization" to ensure a low biological load. Sampling of the biological load was carried out before the pre-filtration stage and the sterile filtration.

The preparation and filling of the sterilized solution in the appropriate containers were carried out under aseptic conditions. This was in accordance with pharmacopoeial monographs and related guidelines, which required sterilization by filtration and subsequent aseptic processing. The equipment, which comes into contact with the product after the "sterilization by filtration" stage, was sterilized by heat or steam using a validated procedure (according to Ph. Eur./USP).

The vials were filled to approximately 1.2 ml to ensure a removable volume of 1.0 ml. The 2 ml vials were made of transparent and colorless Type I glass and were closed with a stopper (bromobutyl coated with fluoropolymer) sealed with hinged covers with a flange (polypropylene). The primary packaging materials met the requirements of the Ph. Eur. And USP. The suitability of the primary packaging materials was verified by the results of the stability tests. No incompatibilities were observed with the primary packaging material used. Secondary packaging provides protection to the product against light.

Compatibility with the application syringes was evaluated using 3 different sizes of syringes and needle diameters (between 25 and 28 gauge) in the drug product solution. There were no differences in ^{the quality of the product. Compatibility was evaluated over a period of} 8 ^hours.

The formulation 14 was manufactured in batches of 5 l, the composition of which is shown in table 17. However, the batch size can be adjusted according to clinical requirements.

Table 17 - Batch formula

Components Lot size 5 liters3

[g]

Initial Antibody b 750.00

^Mannitol 200.00

Polysorbate 20 0.25

Sodium citrate dihydrate 14.71

^{Hydrochloric acid, concentrated} 0 ^{qs pH 5.5}

^{Sodium hydroxide} 0 ^{qs pH 5.5}

Water for injection Ad 5285.25 d

Nitrogen Process aid for filtration.

a The vials were filled with a volume of 1.2 ml to ensure a removable volume of 1.0 ml. Therefore, a batch size of 6.0 l results in a theoretical yield of 5000 vials.

b For pH adjustment.

c This was calculated according to the density of the final solution of the pharmacological product (1.05705 mg / ml)

The manufacturing process and process controls for Formulation 14 are shown in the flow chart of Figure 9. Batch manufacturing included the following steps:

I. Sodium citrate dihydrate was dissolved in water for injection while stirring in a vessel made of inert material (e.g., stainless steel or glass), until complete dissolution. The pH value was adjusted to 5.5 using dilute hydrochloric acid (eg 0.1 M hydrochloric acid) and / or sodium hydroxide solution (eg, 0.1 M sodium hydroxide), if necessary .

II. The Initial Antibody, mannitol and polysorbate 20 were diluted in the buffer solution of step 1 while stirring in a vessel made of inert material (eg, stainless steel or glass) until completely dissolved. If necessary, the pH value was adjusted to 5.5 using hydrochloric acid, diluted (e.g., 1 ^{M hydrochloric} acid ^{) and / or sodium hydroxide solution (e.g.,} 1 ^{M sodium hydroxide )). Buffer solution from step} 1 ^{(remnant) was added to adjust the final weight.}

III. a) Prefiltration:

The solution of step II was filtered under aseptic conditions using a compatible and ^sterilized membrane filter ^{(e.g., polyether-sulfone or polyvinylidene difluoride) with a nominal pore size of} 0.2. | jm.

b) Filtering sterilization:

The solution of step IlI.a was sterilized by filtration under aseptic conditions in sterilized containers made of inert material (eg stainless steel or glass) using a sterilized compatible membrane filter (eg, polyether-sulfone or polyvinylidene difluoride) with a ^nominal pore size ^of 0.2 ^jm.

IV. With the solution of stage IlI.b they were filled in aseptic sterile vials, which were closed with stoppers and hinged covers with a flange.

V. Stage IV containers were inspected for coarse contaminants, an intact seal and visible particles.

SAW. The inspected containers of stage V were further packaged in suitable containers (e.g., cartons).

In addition, DLS was used to determine the hydrodynamic diameter of the antibody monomer and the soluble aggregate potentials. As shown in Figure 10, no aggregates were observed in the citrate buffer. However, as shown in Figures 7 and 8, aggregates were observed in PBS. Due to the higher concentration of antibodies, an average Z increase was observed at 28 nm, compared to the sample in PBS.

REFERENCE EXAMPLE 8 - Stability profile for the formulation of antibodies for subcutaneous administration

The stability profile of the clinical lot (lot 2) of Example 7 was evaluated for storage under accelerated and long-term test conditions according to the guidelines of the ICH. Samples were packed and stored ^{in transparent and colorless vials of} 2 ^{ml (type I glass) closed with stoppers (bromobutyl coated with} fluoropolymer) and with hinged covers with a flange (polypropylene).

The following tests were performed during stability: appearance (transparency, color), assay (ELISA antigen, UV), purity (SEC, ^s DS-PAGE under reducing and non-reducing conditions), molecular integrity (SDS-PAGE under non-reducing conditions) ), charge heterogeneity (weak cation exchange chromatography, isoelectric focusing), pH, sterility, bacterial endotoxins, particulate material (visible and sub visible particles) and closure integrity.

The samples were stored in inverted and vertical positions. The results of inverted storage were presented as the strictest condition. The stability data at -20 ° C, 5 ° C and 25 ° C are presented in Tables 18-20, respectively. Investigations into the physical, chemical and biological properties of storage under long-term test conditions confirmed a good stability of the pharmacological product at 5 ° C (see Table 19). Under accelerated test conditions (+25 ° C), only a slight decrease in purity was detected by size exclusion chromatography (see Table 20). Therefore, it was ^{concluded that the medicine should be stored at a temperature of 2 ° C to} +8 ^{° C protected from light.}

Table 18 - Long-term stability at -20 ° C for the pharmacological product

Product Solution for injection Lot N °:

Pharmacological: Antibody LIGHT

Initial

Force of 150 mg / ml Formulation No.: 14

dosage:

Container / closure: 2 ml glass vials

Results 1 3 6 12 18 24

initials month months months months months months Aspect of the

solution

Transparency <I III II> IV IV> IV ^{Color BY7 BY7 BY} 6 ^BY 6 ^BY 6 ^BY7 Identification

IEF

- Pattern Adjusts Is Se Se Se Isoelectric. adjust adjusts adjusts adjusts essay

ELISA antigen

- EC50 value (in ^76% 110 ^{% 76% 103% 96% 105%} compared to the

reference

Total content of 153 mg / ml 148 151 151 149 156 proteins (UV) mg / ml mg / ml mg / ml mg / ml mg / ml Purity

HPLC (SEC)

- Monomer (%

area) 98.2% 97.5% 96.2% 94.5% 94.3% 94.1% - HMWP (% area) 1.3% 2.3% 3.7% 5.4% 5, 5% 5.5% ^{- LMWP (% area) 0.4%} 0 ^, 2 ^% 0 ^, 1 ^% 0 ^, 0 ^% 0 ^, 1 ^{% 0.5%} SDS-PAGE in

conditions no

^{reducers <} 1 ^, 0 ^{% <} 1 ^, 0 ^{% <} 1 ^, 0 ^{% 2,7% <} 1 ^, 0 ^{% <5,0%} - Half of the

molecules

SDS-PAGE in

terms

^{reducing 98%} 100 ^% 100 ^% 100 ^{% 96%} 100 ^% - Relative purity

Integrity

molecular

SDS-PAGE in

conditions no

reducers

- gel pattern Fits No Sees It Is Adjusted adj adjusts adjusts Heterogeneity

load

HPLC (WCX)

- acid 40% 36% 44% 42% 43% 41% - neutral 55% 60% 54% 47% 51% 56% - Basic isoforms ^{5% 4%} 2 ^% 2 ^% 6 ^{% 3%} (% area)

pH 5.5 5.5 5.5 5.5 5.5 5.5 (potentiometry)

Material Complies Complies Complies me particulate

(particles

visible)

Material

particulate

(particles Without Without Without Without subvisibles) 313 analyze analyze analyze 33 analyze Number of 10 ⁵ particles per vial

> 10 ^{| jm}

Number of

particles per vial

> 25 jm

Integrity of Compliant Without Without Without Comply Without Closure Analyze Analyze Analyze Analyze Contamination

microbial

TAMC <1 CFU / 2 ml Without Without Without Without No Analyze Analyze Analyze Analyze Analyte TAnMC <1 CFU / 2 mL Without Without Without Without No Analyze Analyze Analyze Analyze Analyze Table 19 - Long-term stability at 5 ° C for the pharmacological product

Table 20 - Long-term stability at 25 ° C for the pharmacological product

REFERENCE EXAMPLE 9 - Development of an ultra-high antibody concentration formulation for subcutaneous administration

On the basis of the successful results of the citrate-buffered formulations for antibody concentrations of up to 150 mg / ml in Example 7, more concentrated antibody formulations (up to 250 mg / ml) suitable for subcutaneous administration were developed.

Preliminary data showed that in the formulation of antibody concentrations greater than 150 mg / ml higher viscosities can occur which affect the use of the formulation.

Table 21 - Ultra high concentrations with formulation 14

Density Viscosity omatography of tra ^{Concentration} [kg / m-3] [mPa] ^DLS Cr

exclusion by size Mues _{[mg / ml]} Average

_{Z [nm]} HMWP Monomer LMWP Antibody LIGHT

_{Initial_11_30A} ^{237 1,066 42.29 30} _1.3 ^98.6 0.0 LIGHT Antibody

_{Initial_11_30B} 212 ^{1,059 22.58 39} _1.3 ^98.7 0.0 LIGHT Antibody

_{Initial_11_30C} ^{181 1,052 13.57 28 1.3 98.7} 0.1 LIGHT Antibody

_{Initial_11_30D} ^{173 1,046} 8.8 ²⁷ 1.2 ^98.8 0.0 Antibody LIGHT

_{Initial_11_30E} ^{143 1,039 6.16 25} 1.1 ^98.8 0.1

As can be seen in Table 21, the viscosity decreases with lower antibody concentrations, but is still at an acceptable level at the highest concentration formulated with formulation 14. All other parameters appeared to be unaffected or only slightly affected by the ultra high concentrations.

As shown in Table 22, antibody concentrations did not affect the stability of the ^{formulations, which was indicated by long-term identical} 1- ^{month stability data and stress conditions.}

Table 22 - Stability data at 1 month of formulations of Initial Antibodies concentrated ultra-high

Concentration [mg / ml] HMWP Monomer LMWP ^{Antibody LIGHT Initial_11_30A 40 ° C 237 4.7 95.2} 0.2 ^{Antibody LIGHT Initial_11_30B 40 ° C} 212 ^{4.4 95.4} 0.2 ^{Antibody LIGHT Initial_11_30C 40 ° C 181 5, 8 91.7} 2.6 ^{Antibody LIGHT Initial_11_30D 40 ° C 173 3.9 96.0} 0.2 ^{Antibody LIGHT Initial_11_30E 40 ° C 143 4.2 94.7} 1.1 ^{Antibody LIGHT Initial_11_30A 5 ° C 237} _1.4 ^{98 , 6} 0.0 ^{Antibody LIGHT Initial_11_30B 5 ° C} 212 _1.3 ^98.7 0.0 ^{Antibody LIGHT Initial_11_30C 5 ° C 181} _1.3 ^98.7 0.0 ^{Antibody LIGHT Initial_11_30D 5 ° C 173} 1.2 ^98.8 0.0 ^{Antibody LIGHT Initial_11_30E 5 ° C 143} 1.1 ^98.9 0.0

Studies of anti-CXCR5 preformulation (20 mg / ml)

A humanized anti-CXCR5 IgG4 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26 (the "Initial CXCR5 Antibody") was used. ") in Examples 10-12 to determine the conditions of formulation for a formula for a 20 mg / ml formulation.

materials

Pharmacological substance (DS)

The CXCR 5 antibodies, the RSN 0151, were formulated in PBS p H 7, 2 with a concentration of 5.13 mg / ml. Excipients

L a T a b the 23 m u s t r a s e x c i p e n t e s s t u l t i t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t

Table 23 - Excipients used during preformulation

Excipients Art. No. / load Supplier

A c i d o a c a t i c e A 002630 M T P / V W R I n t e r n a tio n a S Y S

A r g i n in a - H C L A 1700 A p p l i C h e m

A r g i n in a 1.01587 M e r c k

A l c o h a n c e r i n c e 113594 I n d u s t r i a l f f a r i / H a r r a n a n & R e im e

A c i d o c t r i c o 100241 M e r c k

D e x t r a n o 40 C L - A O 19 A M e i t o S a n g y o

G l ic i n a 113560 I n d u s t r i a l A f f a i r s 1 / T e s s a n d C h e m e s.

H C l 114027 I n d u s t r i a l A f f a i r s 1 / M e r c k

H i s t i d in a 1.04352 M e r c k

D ih id r o n o f o f f o f t o f t o f t o r t 1.04871 M e r c k

L i s in a 62840 F lu k a

C o r u r o d e m a g e s i 814733 M e r c k

M a l t o s a 105911 M e r c k

M a n i t o l A 000780 M T P / R o q u e t e F r e r e s

A c e t t o s d o s 1.06265 M e r c k

C o r u r o d e d o u r 10158 I n d u s t r i a l f f a r i 1 / R e d o f H e n e

H i d r o x id o d s o d d 114076 I n d u s t r i a l A f f a i r s 1 / M e r c k

C i t t a r d i o n d 114196 I n d u s t r i a l f f a i s i / B o e r in g e r i n g e l h e r i g h i d o f o f o f o f o f o f o f t o d d i t h e r 1.06586 M e r c k

a n h id r o

P o l i s o r b a t 20 139850 I n d u s t r i a l A f f a i r s 1 / F lu k a

S ucc in ato / á c id osucc í ic ic 14079 F lu ka

Excipients Art. No. / load Supplier

Sucrose S3929 Sigma-Aldrich

Trehalose-dihydrate T9531 Sigma-Aldrich

Trometamol 114011 Industrial Affairs / Merck

Procedures

The following procedures were used to make the experimental formulations and formulations of the invention containing the Initial CXCR5 Antibody.

Manufacture and composition of tampons.

All buffers were produced by constantly stirring to dissolve the respective excipients. The pH was adjusted using 0.1 M HCl or 0.1 M NaOH. The overall concentration of all buffers was 10 mM.

Manufacture and composition of excipient stock solutions.

All stock solutions were produced by constantly stirring to dissolve the excipients. The concentrations were given as weight / weight (w / w).

UF / DF - small scale

The UF / DF experiments were performed using Vivaspin units (Sartorius Stedim, Gottingen, Germany) with a Hydrosart membrane and a cutoff limit of 30 kDa to remove the phosphate buffer and replace it with the appropriate buffers and increase the concentration. These units were placed in a common laboratory centrifuge (Multifuge 3S, Haereus, Germany) and centrifuged at 2000 rpm (860 G, rotor radius 192 mm) at 5 ° C.

UF / DF - larger scale

The UF / DF experiments were performed using Vivaflow units (Sartorius Stedim, Gottingen, Germany) with a Hydrosart membrane and a cutoff limit of 30 kDa to remove the phosphate buffer and replace it with the appropriate buffers and increase the concentration. The equipment was placed inside a clean workbench under aseptic conditions and the process was carried out at room temperature.

Sterile filtration of samples.

All samples, solutions, buffers, etc. they were filtered sterile (0.22 pm) using a Sartopore-2 membrane. The samples were filtered by pouring them into sterile vials or vials and they were closed in aseptic conditions inside a clean workbench to avoid microbiological contamination.

Mechanical stress test

Mechanical stress with a stirring speed of 350 rpm / min for 2.5 hours at room temperature was carried out using a horizontal laboratory agitation apparatus with a distance of 26 mm (agitation apparatus and incubation bell from Bühler Company). The 2R vials were filled with 1 ml of solution with a head space of approximately 2.5 cm3.

A mechanical stress test was planned and conducted during the first preformulation studies. As the analytical results did not show any additional information compared to the thermal stress tests, during the selection of the buffer or the selection of the pH, this test was only used during the selection of the surfactant.

Thermal stress test

Thermal stress was used as an accelerated stress test during all stages of the preformulation program. The samples were stored at 40 ° C for 24 h, 7 days or 3 months, according to the study.

Analytical procedures in filling the formulation and finishing

The following analytical procedures were used in the following examples.

Appearance

The appearance of the antibody solutions was visually verified and, in addition, documented by taking a photograph with a camera.

PH

All pH measurements were made using a pH meter with a microelectrode.

Concentration using UV

The protein concentration of all antibody solutions was measured against the buffer using a Nanodrop ND1000. Protein concentrations close to or below 5 mg / ml were diluted 1: 3, and higher protein concentrations close to 20 mg / ml were diluted 1:20, before measuring the absorption at 215 nm and 280 nm.

Dynamic light scattering ( DLS)

The hydrodynamic diameter of the molecule was measured using laser light scattering. The samples were sterile filtered before the analytical if turbidity was observed, so that only soluble aggregates could be detected.

Thioflavin assay

The fluorescence measurements of some preformulation samples were carried out using a Tecan GENios Plus, XFLOUR4. The samples were subjected to mechanical stress (4 h at 37 ° C, agitation speed 300 units / min and 26 mm distance in an agitation apparatus and incubator hood from the Bühler company). The fluorescence spectra of thioflavin-T were measured at room temperature. 10 pl of thioflavin-T solution (10.1 mM in ultrapure water) was added to 1 ml of the formulations and mixed gently. The mixture was then distributed into a black V-shaped cup of Eppendorf and then into a 96 well plate (100 pl per well).

The thioflavin-T assay was used at the beginning of the pre-formulation activities to detect insoluble aggregates. But, since these aggregates can be observed visually as a turbidity of the solution, this procedure was not used for the entire preformulation program.

Differential scanning calorimetry ( DSC)

Aliquots of the preformulation samples were examined by DSC using a DSC VPCapillary from Microcal and analyzed in the automatic sampling instrument at 90 ° C / h with a filter time of 2 seconds. Samples of 400 pl were placed in 96-well plates and analyzed to determine the unfolding temperature Tm.

Osmolarity

The osmolarity was measured using an automated Knaur osmometer.

Density

The density of the formulations was measured using an Anton Paar DMA4500 downward sphere viscometer.

Analytical procedures in bioanalytical FF.

Size exclusion chromatography ( SEC)

The oligomers / dimers of the antibodies were quantified using size exclusion chromatography. The assay was carried out by isocratic HPLC with a SUPERDEX 20010/300 column.

SDS-PAGE, reducer and non-reducer

Polyacrylamide gel electrophoresis with sodium dodecylsulfate (SDS-PAGE) was used to analyze the molecular integrity (eg, half molecules) and appearance of degradation products. This analysis by electrophoresis was performed with homogeneous gels at 15% in reducing and non-reducing conditions. Proteins were visualized with silver staining after separation by electrophoresis.

WCX

Weak cation exchange chromatography (WCX) was used to control the charge heterogeneity of the antibody. The percentage of basic, neutral and acid isoforms was reported. The assay was carried out by discontinuous HPLC with a ProPac WCX10 column.

Antigen ELISA

The antigen ELISA was performed to determine the functionality of the antibody. The binding property to a 28-mer peptide of the CXCR5 antigen was controlled in comparison with the current antibody pattern. This power was reported as the relative EC50.

Isoelectric focus ( IEF)

An IEF was performed.

Storage

All buffer solutions, excipient solutions and samples were stored at 5 ° C, unless otherwise mentioned.

SUMMARY OF ALL PREPARED AND ANALYZED FORMULATIONS

Table 24 below shows a summary of all the formulations that were prepared and analyzed in Examples 10-12. Each of the formulations contained the Initial CXCR5 Antibody. PBS stands for phosphate buffered saline. PB means phosphate buffer. PS means polysorbate. LA means the Initial CXCR5 Antibody.

Table 24 - Summary of all formulations prepared and analyzed.

Sample number Buffer pH LA_09_05-1 PBS 155 mM 7.5

LA _09_05-2 PBS 155 mM 7.0 LA_09_05-3 PBS 155 mM 6.5 LA_09_06-1 PB 5 mM 7.5 LA_09_06-2 PB 5 mM 7.0 LA_09_06-3 PB 5 mM 6.5 LA_09_07-1 PB 10 mM 7.5 LA_09_07-2 PB 10 mM 7.0 LA_09_07-3 PB 10 mM 6.5 LA_09_08-1 Citrate 10 mM 7.0 LA_09_08-2 Citrate 10 mM 6.5 ^{LA_09_08-3 Citrate 10 mM} 6.0 LA_09_08-4 Citrate 10 mM 5.5 LA_09_08-5 Citrate 10 mM 5.0 ^{LA_09_09-1 Saline solution 150 mM} 6.0 LA_09_10-1 Acetate 10 mM 5.5 LA_09_10-2 Acetate 10 mM 5.0 ^{LA_09_11-1 Succinate 10 mM} 6.0 LA_09_11-2 Succinate 10 mM 5.5 LA_09_11-3 Succinate 10 mM 5.0 LA_09_12-1 Histidine 10 mM 6.5 ^{LA_09_12-2 Histidine 10 mM} 6.0 LA_09_12-3 Histidine 10 mM 5.5 Sample number Buffer PH ^{LA_09_13-1 Glycine 10 mM} 8.0 LA_09_13-2 Glycine 10 mM 7.0 ^{LA_09_14-1 Arginine 10 mM} 8.0 ^{LA_09_14-2 Arginine 10 mM} 6.0 LA_09_15-1 TRIS 10 mM 8.5 LA_09_15-2 TRIS 10 mM 7.5 ^{LA_09_16 Citrate 10 mM} 6.0 ^{LA_09_16_1 Citrate 10 mM / PS20} 6.0 ^{LA_09_16_2 Citrate 10 mM / PS80} 6.0 ^{LA_09_16_3 Citrate 10 mM / LutrolF} 68 6.0 ^{LA_09_16_4 Citrate 10 mM / Cremophor} 6.0 ^{LA_09_16_5 Citrate 10 mM / Solutol HS15} 6.0 ^{LA_09_16_6 Citrate 10mM / SDS} 6.0 LA_09_17 Acetate 10 mM 5.5 LA_09_17_1 Acetate 10 mM PS20 5.5 LA_09_17_2 Acetate 10 mM PS80 5.5 ^{LA_09_17_3 Acetate 10 mM Lutrol F} 68 ^5.5 LA_09_17_4 Acetate 10 mM Cremophor R40 5,5 LA_09_17_5 Acetate 10 mM Solutol HS15 5,5 LA_09_17_6 Acetate 10 mM SDS 5,5 LA_09_18 Succinate 10 mM 5,0 LA_09_18_1 Succinate 10 mM PS20 5,0 LA_09_18_2 Succinate 10 mM PS80 5 , 0 ^{LA_09_18_3 Succinate 10 mM Lutrol F} 68 ^5,0 LA_09_18_4 Succinate 10 mM Cremophor 5.0 LA_09_18_5 Succinate 10 mM Solutol HS15 5,0 LA_09_19 Histidine 10 mM 5.5 LA_09_19_1 Histidine 10 mM PS20 5.5 LA_09_ 19_2 Histidine 10 mM PS80 5.5 ^{LA_09_19_3 Histidine 10 mM Lutrol F} 68 ^5.5 LA_09_19_4 Histidine 10 mM Cremophor 5.5 LA_09_19_5 Histidine 10 mM Solutol ^HS15 5.5 ^{LA_09_20 Arginine 10 mM} 6.0 ^{LA_09_20_1 Arginine 10 mM PS20} 6.0 ^{LA_09_20_2 Arginine 10 mM PS80} 6.0 ^{LA_09_20_3 Arginine 10 mM Lutrol F} 68 6.0 ^{LA_09_20_4 Arginine 10 mM Cremophor} 6.0 ^{LA_09_20_5 Arginine 10 mM Solutol} HS15 6.0 LA_09_21 Histidine 10 mM PS20 5.5 LA_09_22 PBS 155 mM 7.2 Sample number Buffer PH LA_09_22_1 PBS 155 mM 7.2 LA_09_22_2 PBS 155 mM NaCl 7.2 LA_09_22_3 PBS 155 mM MgCh 7.2 LA_09_22_4 PBS 155 mM CaCh 7.2 LA_09_22_5 PBS 155 mM mannitol 7.2 LA_09_22_6 PBS 155 mM maltose 7 , 2 LA_09_22_7 PBS 155 mM trehalose 7.2 LA_09_22_8 PBS 155 mM sucrose 7.2 LA_09_22_9 PBS 155 mM Dextran40 7.2 LA_09_22_10 PBS 155 mM Benzyl alcohol 7.2 LA_09_22_11 PBS 155 mM arginine 7.2 LA_09_22_12 PBS 155 mM lysine 7, 2 ^{LA_09_23 Citrate 10 mM (= LA_09_16)} 6.0 ^{LA_09_23_1 Citrate 10 mM} 6.0 ^{LA_09_23_2 Citrate 10 mM NaCl} 6.0 ^{LA_09_23_3 Citrate 10 mM MgCh} 6.0 ^{LA_09_23_4 Citrate 10 mM mannitol} 6.0 ^{LA_09_23_5 Citrate 10 mM maltose} 6, 0 ^{LA_09_23_6 Citrate 10 mM trehalose} 6.0 ^{LA_09_23_7 Citrate 10 mM sucrose} 6.0 ^{LA_09_23_8 Citrate 10 mM Benzyl alcohol} 6.0 ^{LA_09_23_9 Citrate 10 mM arginine} 6.0 ^{LA_09_23_10 Citrate 10 mM Lysine} 6.0 LA_09_24 Acetate 10 mM (= LA_09_17) 5.5 LA_09_24_1 Acetate 10 mM 5.5 LA_09_24_2 Acetate 10 mM NaCl 5.5 LA_09_24_3 Acetate 10 mM MgCh 5.5 LA_09_24_4 Ac 10 mM mannitol 5,5 LA_09_24_5 Acetate 10 mM maltose 5,5 LA_09_24_6 Acetate 10 mM trehalose 5,5 LA_09_24_7 Acetate 10 mM sucrose 5,5 LA_09_24_8 Acetate 10 mM Benzyl alcohol 5,5 LA_09_24_9 Acetate 10 mM Arginine 5,5 LA_09_24_10 Acetate 10 mM Lysine 5.5 LA_09_25 10 mM Histidine (= LA_09_19) 5.5 LA_09_25_1 10 mM Histidine 50 mM NaCl 5.5 LA_09_25_2 10 mM Histidine MgC ^ 50 mM 5.5 LA_09_25_3 10 mM Histidine 5% Mannitol 5.5 LA_09_25_4 Histidine 10 mM 10% maltose 5.5 LA_09_26_1 Histidine 10 mM PS20 (= LA_09_21) 5.5 Sample number Buffer pH LA_09_26_2 Histidine 10 mM PS20 NaCl 50 mM 5.5 LA_09_26_3 Histidine 10 mM PS20 MgCh 50 mM 5.5 LA_09_26_4 Histidine PS20 5% mannitol 5.5 LA_09_26_5 Histidine PS20 10% maltose 5.5 ^{LA_09_26_6 Histidine PS20} 6 ^{% trehalose 5.5} LA_09_26_7 Histidine PS20 5% sucrose 5.5 LA_09_26_8 Histidine PS20 9 mg benzyl alcohol 5.5 LA_09_26_9 Histidine PS20 arginine-HCl 20 mM 5.5 LA_09_26_10 Histidine PS20 lysine 20 mM 5.5 ^{LA_09_27 Citrate 10 mM PS20} 6.0 ^{LA_09_27_A Citrate 10 mM Prototype formulation PS20} 6.0 ^{LA_09_27_B Citrate 10 mM Prototype formulation PS20} 6.0 ^{LA_09_27_C Citrate 10 mM Prototype formulation PS20} 6.0 ^{LA_09_27_D Citrate 10 mM Prototype formulation PS20} 6.0 LA_09_28 Acetate 10 mM PS20 5.5 LA_09_28_A Acetate 10 mM prototype formulation PS20 5.5 LA_09_28_B Acetate 10 mM prototype formulation PS20 5.5 LA_09_28_C Acetate 10 mM prototype formulation PS20 5.5 LA_09_28_D Acetate 10 mM prototype formulation PS20 5.5 LA_09_29 Histidine 10 mM P S20 5.0 LA_09_29_A Prototype formulation of 10 mM histidine PS20 5.0 LA_09_29_B Prototype formulation of 10 mM histidine PS20 5.0 LA_09_29_C Prototype formulation of 10 mM histidine PS20 5.0 LA_09_29_D Prototype formulation of histidine 10 mM PS20 5.0

Example 10 - Phosphate buffer formulation

The following is a general description of the results of the characterization of the pharmacological substance Initial CXCR5 antibody in a phosphate buffer.

IEF

The pI (isoelectric point) of the Initial CXCR5 Antibody was theoretically calculated as 7.6 and was confirmed by denatured isoelectric focusing (pI from 7.6 to 8.4). See figure 11.

SDS-PAGE

SDS-PAGE was used to determine the molecular weight of the antibody monomer, potential aggregates or the presence of half molecules. Figure 12 showed an example of an SDS-PAGE gel for comparing different batches of pharmacological substances under reducing and non-reducing conditions.

ELISA

Figure 13 shows an example of an ELISA graph for determining the antigen binding activity of the Initial Antibody.

SEC

As shown in Figure 14, size exclusion chromatography detected high molecular weight proteins (HMWP), for example, di- / oligomers or aggregates and low molecular weight proteins (LMWP) or degradation products. The initial CXCR5 Antibody batch had a purity of 99% monomer content.

WCX

The weak cation exchange chromatography for the lead antibody is shown in Figure 15, which represents charge heterogeneity. During the stability studies, the arrangement of the acid peaks changed and the percentage of basic isoforms increased. The Initial CXCR5 Antibody had a distribution of acidic / neutral / basic isoforms of 14/85/1%.

Dynamic scattering of light

As shown in Figure 16, DLS was used to determine the hydrodynamic diameter of the antibody monomer and possible soluble aggregates.

In conclusion, the Initial Antibody could be stable in PBS, but the formation of aggregates is easy to generate by cutting forces or light stress.

In addition, the pH of PBS is close to the pI of the Initial CXCR5 Antibody. Therefore, the formulation must be formulated at least one pH degree below the pI.

Table 25 shows the results of a three month stability study for the Initial CXCR5 Antibody. The Initial Antibody was stored at different temperatures and analyzed after one and three months.

Table 25 - Analytical results of a 3-month DS stability study

Stability data at 3 months with the CXCR5 Initial Antibody buffered in PBS indicated that there were no relevant changes at 5 ° C and -20 ° C storage. After 3 months under accelerated conditions (+25 ° C), significant changes could be observed. Additional bands, analyzed by SDS-PAGE and Western blot analysis, showed an increase in basic isoforms and a decrease in acid isoforms, suggesting degradation products.

Example 11 - Optimization of buffer and pH

PBS pH 7.2 showed aggregation and degradation after the freeze / thaw cycles and after freezing storage. Therefore, it was necessary to find another buffer and a better pH range. In addition, PBS is not suitable for freezing solutions, since a pH change occurs.

30 different buffers with various pH and buffer systems were used to select the best pH range. These experiments were performed on a very small scale and were analyzed intensively.

Screening of selection of the best buffer and pH - small scale (yield 5 ml)

The analytical results are summarized in Table 40, Table 41, Table 42 and Table 43.

In figure 17 and figure 18, the appearance of two suitable buffer systems (acetate and histidine) after thermal stress is shown. PH 5.5 in acetate and pH 5.0 in histidine were chosen for further evaluation. By way of comparison, in Figure 19, the appearance of an incompatible buffer system (TRIS buffer) is shown.

The following buffers were selected for analysis on a larger UF / DF scale:

^{• 10 mM Citrate, pH} 6

• Acetate 10 mM, pH 5.5

• 10 mM Succinate, pH 5

• 10 mM histidine, pH 5

^{• 10 mM Arginine, pH} 6

Screening of selection of the best buffer and pH - large scale (yield ~ 20 g)

After selecting the best buffers and pH, a larger quantity of Initial CXCR5 Antibody was prepared in each buffer system using the Sartorius Vivaflow system. Each batch was analyzed analytically and the results are described below.

10 mM citrate buffer, pH 6 ( LA_09_016)

The UF / DF stage worked well and only a slightly cloudy solution was obtained; no difficulties were encountered during the sterile filtration. No hydrodynamic diameter increase was observed, analyzed by DLS.

The analytical results indicated that there is no increase in the dimers and there are no changes in the basic or acidic isoforms compared to the material of the initial CXCR5 Antibody batch. See Table 26 and Figure 20.

^{Table 26 - Analytical results of the Initial Antibody in citrate buffer pH} 6

Acetate Buffer 10 mM, pH 5.5 ( LA 09 017)

The UF / DF stage worked well, but a cloudy solution was obtained; filter blockage during sterile filtration. The analytical results indicated that there is no increase in the dimers and there are no changes in the basic or acidic isoforms compared to the material of the initial CXCR5 Antibody batch. See Table 27 and Figure 21.

Table 27 - Analytical results of the Initial Antibody in an acetate buffer pH 5.5

10 mM Succinate Buffer, pH 5 ( LA_09_018)

Sterile filtration after UF / DF was difficult to perform due to filter clogging. The yield of 12 g was very low.

The analytical results indicated a slight decrease in the dimers and no change in the basic or acidic isotherms compared to the material of the initial CXCR5 Antibody batch. After mechanical stress, the concentration of the dimer increased slightly and the peak of acidic isotherms in WCX decreased as the basic isoforms increased. See Table 28 and Figure 22.

Table 28 - Analytical results of the Initial Antibody in succinate buffer pH 5 Sample pH Appearance UV Conc Yield DLS Tm LA_09_018 4.9 Slightly cloudy after UF / DF, ^{22.4 mg / ml} 12 ^{g 12.82 nm 73, 3 ° C} Transparent after filtration

Histidine buffer 10 mM, pH 5 ( LA 09 019)

Sterile filtration after UF / DF was very difficult to perform due to blocking of the filter. The yield of 10.5 g was very low.

The analytical results indicated a slight decrease in the dimers and no change in the basic or acidic isoforms compared to the batch material of the Initial CXCR5 Antibody. After mechanical stress, the concentration of the dimer increased slightly and the peak of acidic isoforms in WCX decreased as the basic isoforms increased. See Table 29 and Figure 23.

Table 29 - Analytical results of the Initial Antibody in histidine buffer pH 5 Sample pH Appearance Conc. UV Yield DLS Tm LA_09_019 5.4 Slightly cloudy after UF / DF, 23.4 mg / ml 10.5 g 11.32 nm nd Transparent after filtration

10 mM Arginine Buffer, pH 6 ( LA 09 020)

Sterile filtration after UF / DF was very difficult to perform. The DLS showed a peak presented with a ^hydrodynamic diameter ^of 21.08 ^{nm, which could indicate the formation of dimers.}

The analytical results indicated a slight increase in the dimers of 0.29% in the batch of Initial CXCR5 Antibody to 0.49% in arginine. After mechanical stress, 0.61% of dimers were found and an increase in the basic isoforms was detected in WCX. See Table 30 and Figure 24.

Table 30 - Analytical Results of the Initial Antibody in the Arginine Buffer pH 5 Sample pH Appearance Conc. UV Yield DLS Tm ^LA_09_020 6.2 ^{Slightly cloudy after UF / DF,} 22.5 mg / ml 15.3 g 21.08 nm nd Transparent after filtration

In conclusion, three of the five batches are compatible with the Initial CXCR5 Antibody at a concentration of 20 mg / ml:

• Citrate pH 6.0

• Acetate pH 5.5

• Histidine pH 5.0

These lots were characterized in terms of compatibility and stability in more detail.

Example 12 - Compatibility with excipients

All the batches mentioned above were used for compatibility studies with surfactants. The Compatibility studies were performed with the Initial CXCR5 Antibody and four selected buffers. The succinate pH 5.0 and the arginine pH 6.0 were not already analyzed with excipients, since these buffers were not compatible with the Initial CXCR5 Antibody. The excipients were classified as follows:

• Surfactants

• Sugars

• You go out

• Others (amino acids, preservative)

Mechanical stress (stirring speed 350 / min, 2.5 h, room temperature) was applied to analyze the effect of surfactants, and thermal stress (+ 40 ° C, one week) was used to analyze all other excipients . Surfactants

The guidance studies on the selection of the type of surfactants (LA_08_001) and the surfactant concentration (LA_09_003, 0.01%, 0.05% and 0.1%) indicated that a concentration of 0.01% was sufficient to prevent visible aggregates. The following surfactants were not suitable for the Initial CXCR5 Antibody: PVP K12 and K17, since both showed turbidity before applying mechanical stress. In addition, it was demonstrated that ionic surfactants such as sodium dodecylsulfate were not compatible with protein solutions of the Initial CXCR5 Antibody.

As an example, Figure 25 shows the appearance of different solutions buffered with citrate with various surfactants after mechanical stress, and compared to a solution without any surfactant. The analytical results are shown in Table 44 and Table 45.

Other excipients

After a thermal stress of 40 ° C for one week and the analytical determination, it was possible to provide a selection of excipients compatible with the Initial CXCR5 Antibody in different buffer systems.

Some excipients could not be analyzed in the four buffer systems, since there was only a small volume of sample available.

After reviewing all the analytical data, it was identified that the excipients in Table 31 are compatible with the Initial CXCR5 Antibody. These excipients did not show a significant increase in dimers, HMWP or basic isoforms analyzed by SEC and WCX.

All hydrodynamic diameter measurements indicated an acute monomer peak and the Tm of the appropriate excipients did not decrease compared to the Initial CXCR5 Antibody in the respective buffer system. All the analytical data were summarized in Table 46, Table 47, Table 48 and Table 49.

Table 31 - Compatibility of all the excipients analyzed in the different buffer systems

In conclusion, compatibility studies with surfactants clearly show that polysorbate 20 is suitable for all selected buffers in combination with the Initial CXCR5 Antibody at 20 mg / ml. The surfactant prevents the formation of particles during mechanical stress. Almost all other surfactants led to an increase in HMWP.

The following excipients were selected to formulate the different prototype formulations: NaCl, trehalose (sucrose is more or less comparable to trehalose in terms of stability) and Arginine-HCl.

STUDY OF STABILITY OF THE 3-MONTH PROTOTYPE

To support the development of the formulation of the Initial CXCR5 Antibody, twelve different prototype formulations were manufactured and placed under different conditions (-20 ° C, 5 ° C and 40 ° C) for three months.

Three different buffer systems were selected based on the buffer, the pH and the screening of excipients described above.

Citrate pH 6.0 (formulation number LA_09_027), acetate pH 5.5 (LA_09_028) and histidine pH 5.0 (LA_09_029) were used as 10 mM buffer solutions with 20 mg / ml Initial Antibody and four different combinations of excipients (Table 32).

These four excipients showed promising results after the screening of the excipient. NaCl was selected to adjust the osmolarity, trehalose was chosen for the adjustment of the tonicity and to have a sugar for a lyophilization option, if necessary. Additionally, trehalose can stabilize the antibody, and arginine-HCl was also selected as a stabilizer. It was found that polysorbate 20 was useful to prevent aggregation during mechanical stress.

The following paragraphs show selected data that were compiled during the stability study to select the best buffer system and the best excipients for the development of the formulation. Table 33 lists all storage conditions, time points and analytical procedures.

Table 32 - Compositions of four different formulation options

^{Formulation NaCl a, a-trehalose * H} 2 ^{O Arginine Polysorbate 20}

^A _{3 mg} ^{25 mg 20 mM} 10 ^mg

^{B 50 mg} 10 ^mg

^C 6 mg ^{20 mM} 10 ^mg

^{D 50 mg 20 mM} 10 ^mg

Table 33 - Storage conditions and time points for analytical tests

Conditions of T0 T 21 days T 6 weeks T 3 months storage

SEC, WCX, SDS-PAGE, ELISA, Tm, pH,

DLS, UV, appearance,

HIAC, osmolarity

-80 ° C SEC, WCX, SDS-PAGE *, Tm, pH,

DLS, UV

-20 ° C SEC, WCX, SDS-SEC, WCX, SDS-SEC, WCX, SDS-PAGE *, Tm, pH, DLS, PAGE *, Tm, pH, PAGE *, Tm, pH, DLS, UV DLS, UV UV, appearance 5 ° C SEC, WCX, SDS-SEC, WCX, SDS-SEC, WCX, SDS-PAGE *, Tm, pH, DLS, PAGE *, Tm, pH, PAGE *, Tm, pH, DLS, UV, HIAC DLS, UV UV, appearance 40 ° C SEC, WCX, SDS-PAGE SEC, WCX, SDS-SEC, WCX, SDS- *, Tm, pH, DLS, UV PAGE *, Tm, pH, PAGE *, Tm, pH,

DLS, UV DLS, UV

* if it is reasonable

Unfortunately, the stability data at 3 months of the Initial CXCR5 Antibody in the PBS buffer were not comparable with the stability of the prototype due to batch differences and due to the different accelerated conditions.

Size exclusion chromatography (SEC)

In Figure 26, an increase in the formation of dimers of up to 10% can be clearly observed after three months of storage in the four histidine formulations. The acetate formulations showed an ^{increase in the content of dimers of up to} 6 ^{%. In the four citrate formulations, the} dimer ^{concentration} was less than 2%, even after three months at 40 ° C.

Weak cation exchange chromatography (WCX)

As the determination of neutral, basic and acid isoforms is a good indicator of the stability of different formulations, these procedures were used to amend the SEC data.

In Figure 27 it can again be observed that histidine is worse for the stability of the Initial CXCR5 Antibody under accelerated conditions. A slight increase in the basic isoforms can be observed for the four acetate formulations, but curiously for the citrate formulations, discrimination between the four formulations in this respect is not possible. Additionally, Figure 28 shows a strong decrease of the neutral isoforms for the histidine formulations, and a slight decrease in the acetate. Again, the Initial CXCR5 Antibody in citrate is the least affected.

SDS-PAGE

The results of the SDS-PAGE measurements can be found in the results tables in the appendix. See Tables 37-61.

Deployment temperature (Tm)

The unfolding temperature can be used to predict the stability of different formulations and was measured in this case with the Microcal equipment. The higher the Tm, the more promising the formulations will be. The accuracy of the measurements of Tm was / - 0.4 ° C.

Among the formulations of citrate and acetate, almost no differences were noted between Tm in T0. In addition, formulations A, B and D had a slightly higher Tm, compared to C. Formulations A, B and D all contain trehalose.

The histidine formulations had a significantly lower Tm in all cases.

^{Table 34 - Deployment of temperatures at T} 0

Formulation LA_09_027 LA_09_028 LA_09_29

At 81.4 ° C 81.1 ° C 79.4 ° C

B 81.5 ° C 81.6 ° C 81.0 ° C

C 80.7 ° C 80.5 ° C 78.9 ° C

D 81.6 ° C 81.7 ° C 80.7 ° C

pH

Since pH is of great interest and importance for the stability of an antibody solution, the pH was monitored. The following figures show the delta of pH between T0, T1, T2 and T3 under conditions of accelerated storage. The formulations that most stabilize the pH are those buffered with citrate, and especially the formulations B and D (figure 29). In buffered solutions with CXCR5 Initial Antibody acetate, the pH moved to higher values (Figure 30). In the solutions buffered with histidine, the pH decreased slightly (FIG. 31).

DLS

The hydrodynamic diameter of the monomer and possible soluble aggregates were measured using the dynamic scattering of light.

Only after storage under accelerated conditions (+40 ° C), soluble aggregates could be observed <200 nm. These aggregates were produced mainly in the histidine-buffered formulation LA_09_029 A, C, D after 3 weeks of storage.

The citrate-buffered formulations showed only slight aggregates (figure 32) after three weeks in formulation C and after six weeks of storage in formulation A. Some aggregates could also be detected after three months in formulation B. But, in comparison with the formulations buffered with acetate, the amount was very small.

The formulation LA_09_028 C buffered with acetate showed some aggregates <200 nm after three weeks, and after three months also in the formulation A. See figure 33.

UV

When tracking the protein concentration by UV measurements, no significant differences were noted between all time points, samples and formulations. As the volume of the sample was very small, the concentration was measured with a Nanodrop equipment. The results varied / - 5%. For detailed information, see Tables 50-61.

Appearance

After the storage period of three months, all the samples remained clear and colorless without any turbidity, even in histidine. This observation also indicates that all the aggregates measured in DLS were soluble. The insoluble and subvisible aggregates could be detected by measuring the blocking of light by HIAC.

HIAC

Particles subvisible at T0 were detected and after three weeks of storage at 5 ° C. It can be clearly seen from Table 36 that particle formation was observed mainly in an acetate buffer. Interestingly, histidine showed good results for the four different formulations. In the citrate formulations A, B and C are also good. Since the particle level> 10 μm and> 25 μm and the values in all formulations are well below the limits defined in Ph. Eur. And USP, the formation of particles is not a cause for concern.

Osmolarity

The quantification of the excipients to adjust the osmolarity was carried out before the samples were manufactured by calculation, since a volume of samples was not available to guide the experiments. Therefore, the osmolarity was lower than it should be (ideally between 280 and 320 mOsmol / kg). Table 35. Additional studies will be carried out for a better fit during the optimization studies of the formulation.

Table 35 - Osmolarity at T0 for all prototype formulations.

Formulation LA_09_027 LA_09_028 LA_09_29

^{A 241} 221 220

B 181 160 165

^{C 238} 220 220

D 214 192 197

Ċ

conclusion

In conclusion, citrate buffer, acetate buffer and histidine buffer showed no changes after storage at 5 ° C and -20 ° C, and only a secondary increase in degradation products was observed with acetate buffer after 3 months

Storage of the Initial CXCR5 Antibody under accelerated conditions led to significant changes in the DS. Although minor changes were observed in the citrate buffer, the acetate buffer showed a significant increase in the products of degradation and aggregation and a decrease in neutral isoforms to give acidic or basic isoforms.

A tremendous effect was observed in the Initial CXCR5 Antibody under accelerated conditions (up to 29.6% of high molecular weight proteins and up to 8.2% of di- / oligomer and up to 1.3% of low weight proteins molecular). In addition, cation exchange chromatography revealed a decrease of 50% neutral isoforms.

The target concentration of 20 mg / ml could be achieved with all the buffers analyzed, for example, citrate, acetate and histidine.

The pH range of a stable DP could be defined as pH 5-6.5.

Two extension steps (4 ml ^ 100 ml - 1000 ml UF / DF) were successfully carried out with three selected buffers.

The reduction of the formation of aggregates with 0.01% polysorbate 20 in all the selected buffers after the mechanical stress (speed of the agitation apparatus 350 / min, 2.5 h, TA) was evaluated and confirmed analytically.

One could observe the absence or decreased HMWP, increasing the filterability (0.22 pm) by ^{addition of} 0. ^{01% polysorbate 20.}

The amount of dimers / oligomers was very dependent on the buffer and the pH and was analyzed using SEC, SDS-PAGE and DLS.

Characterization of the pharmacological substance.

The CXCR5 Initial Antibody molecule is very stable in terms of degradation or formation of half molecules, but it turned out during the pre-formulation activities, that the Initial CXCR5 Antibody dissolved in PBS at pH 7.2 has a tendency to aggregation. Therefore, this buffer is not suitable for long-term stability. The formation of visible and subvisible particles during storage or freeze / thaw cycles should be carefully controlled during formulation development and stability studies.

Selection of best buffer and pH

After selecting the best buffer and pH, it was identified that 10 mM citrate buffer at pH 6.0 was suitable for 20 mg / ml solutions of Initial CXCR5 Antibody. 10 mM histidine buffer pH 5 or 10 mM acetate buffer pH 5.5 could serve as backup options.

Compatibility study with excipients.

The following excipients are recommended for prototype formulations:

• Polysorbate 20

• Trehalose / sucrose

• NaCl

• Arginine-HCl

The following excipients are not recommended for development:

• Benzyl alcohol

• Maltose

• Mannitol

• Dextran

• Lysine-HCl

3-month stability study of the prototype formulation.

An excellent stability of 20 mg / ml of Initial CXCR5 Antibody in citrate buffer pH 6.0, acetate buffer pH 5.5 and histidine buffer pH 5.0 at 5 ° C and -20 ° C after three months was observed. storage. A slight degradation was observed at 40 ° C (<5% reduction of the monomer content) with citrate buffer, while the acetate buffer showed an increase, but significant, and the histidine buffer a strong increase of the product.

All the formulations analyzed showed a significant reduction in particle formation during storage compared to the generic discovery formulation in PBS pH 7.2.

^{Therefore, the recommendation of this preformulation study is to use a buffer of 10 mM citrate pH} 6 ^for DS and DP of the Initial CXCR5 Antibody. A filtered and sterile buffered solution containing 20 mg / ml of Initial CXCR5 Antibody and excipients that increase stability should be feasible with a storage recommendation at 5 ° C in vials.

For tonicity adjustment, trehalose and NaCl could be used and polysorbate 20 should be used to prevent the formation of aggregates.

The feasibility of the UF / DF experiments to change the buffer system and / or to increase the mAB concentration from 5 mg / ml to 20 mg / ml can be shown at different scales.

^X LU>

Studies of anti-CXCR5 formulation (20 mg / ml)

The data in Examples 13-16 were collected during the formulation studies for the Initial CXCR5 Antibody and its pharmacological product for intravenous and subcutaneous administration. The objective of the formulation studies was to provide an injection solution of CXCR5 Initial Antibody lacking visual particles, stable, transparent or slightly opalescent and colorless or slightly yellow, for phase I.

MATERIALS

Pharmacological substance (DS)

Two batches of pharmacological substances were used for these formulation studies. One was formulated in phosphate buffered saline (PBS) and the other was formulated in citrate buffer. See table 62.

Table 62 - Lots of pharmacological substances available

Lot No. Amount Initial Ab Concentration Buffer pH value [-]

^RSN0151 10 ^{g 5.0 mg / ml PBS 155 mM 7.2}

^SCB0001 20 ^{g 20.30 mg / ml citrate 10 mM} 6.0

Excipients

Table 63 shows the excipients that were used during the formulation studies.

Table 63 - Excipients

Excipient No. of material Supplier

Arginine 1.01587 Merck

Citric acid 100241 Merck

Histidine 1.04352 Merck

Hydrochloric acid 114027 H600

Sucrose S3929 Sigma-Aldrich

Sodium Acetate 1.06265 Merck

Sodium Chloride 10158 H600

Sodium citrate 114196 H600

Sodium hydroxide 114076 H600

Polisorbate 20 139850 H600

a, a-Trehalose T9531 Sigma-Aldrich

PROCEDURES

Preparation of the sample

Ultrafiltration / diafiltration was performed on a small scale using VivaSpin devices with a Hydrosart membrane and a cutoff limit of 30 kDa. The RSN material was concentrated from 5 mg / ml to 20 mg / ml, and the phosphate buffer (PBS) was exchanged for 10 mM citrate buffer, pH 6.0, acetate buffer, pH 5.5, or buffer. histidine, pH 5.0. The VivaSpin units were placed at room temperature (RT) in a common laboratory centrifuge and centrifuged at 2000 rpm. The solution was filtered on a Minisart of 0.2 μm before the ^{analytical tests. All samples were stored between 2 ° and} +8 ^{° C, well closed and protected from light,} until analytical tests at T0 and after a week of thermal stress at 40 ° C or after mechanical stress for 2.5 hours , 300 rpm at room temperature (only for evaluation of the ^polysorbate 20 concentration ^).

Analytical procedures

The following techniques were used for the analysis of samples:

Table 64 - Analytical techniques used

Technique (Company) Parameter to be investigated

Organoleptic (-) Appearance

Nephelometer (Hach Lange) Turbidity

PH meter (WTW) pH value

UV (Perkin Elmer) Concentration of mAB

Density meter (Paar) Density

Osmometer (Knauer) Osmolality

Viscosimeter (Paar) Viscosity

Dynamic light scattering (Malvern) Hydrodynamic diameter

SEC (N / A) Mono / di- / oligomer and high molecular weight protein (HMWP) / low molecular weight (LMW)

WCX (N / A) Isoforms (acidic / basic / neutral)

ELISA * (N / A) Power (agglutination)

SDS-Page (network.) ** (N / A) HC / LC, fragments of mAB

SDS-Page (not network.) ** (N / A) Aggregation and degradation products.

HIAC * (N / A) Particulate material

* Some samples will be analyzed.

** SDS-Page will be carried out in case SEC shows unusual results.

Example 13 - Optimization of additional pH

The preformulation studies identified a buffer of 10 mM citrate at pH 6.0 as the best buffer with less tendency to aggregation of the Initial CXCR5 Antibody. To obtain a pH profile in a citrate buffer, the pH-dependent stability was evaluated by steps of pH 5.0 to 7.0. Due to the limited availability of the drug substance, the depth detection of the pH was performed only with 10 mM citrate buffer. Samples were taken at T0 and after one week of thermal stress at 40 ° C. See Tables 65-69.

Table 65 - Sample summary

Lot No. Target pH value ( -) Measured pH value ( -) LA_09_030 5.0 5.1

LA_09_031 5.3 5.4

LA_09_032 5.5 5.6

LA_09_033 5.7 5.8

LA_09_034 6.0 6.1

LA_09_035 6.3 6.4

LA_09_036 6.5 6.6

LA_09_037 6.7 6.8

LA_09_038 7.0 7.1

^Table 66 ^{-results T0}

Lot No. Appearance pH value mAB conc. Measured hydrodynamic diameter ( -) ( mglml) ( nm)

LA_09_030 Transparent 5.1 23.35 15.42 + agr.

LA_09_031 Transparent 5.4 21.95 12.85

Lot No. Appearance pH value mAB conc. Measured hydrodynamic diameter ( -) ( mglml) ( nm) LA_09_032 Transparent 5,6 23,00 12,98 LA_09_033 Transparent 5,8 21,21 12,99 LA_09_034 Transparent 6,1 22,77 12,83 LA_09_035 Transparent 6,4 23,87 13,22 LA_09_036 Transparent 6,6 23 , 74 13,04 LA_09_037 Transparent 6,8 22,85 13,00 LA_09_038 Transparent 7,1 21,96 13,32

Table 67 - results T1 week 40 ° C

Lot No. Appearance pH value mAB conc. Measured hydrodynamic diameter ( -) [mglml] ( nm) LA_09_030 Transparent 5.2 15.62 * 15.27 agr.

LA_09_031 Transparent 5.5 21.91 16.74 agr.

LA_09_032 Transparent 5,6 24,32 13,59 LA_09_033 Transparent 5,8 24,74 13,83 LA_09_034 Transparent 6.1 24.18 13.25 LA_09_035 Transparent 6.5 N / A 13.41 LA_09_036 Transparent 6.6 23 , 03 13.37 LA_09_037 Transparent 6.9 22.68 13.24 LA_09_038 Transparent 7.2 23.33 14.40

* Unusual result due to dilution error.

^Table 68 ^{- Results of ASD T0}

Lot N °% Monomer% Di-lOligomer% HMWP%%%% basic

LA_09_030 99.63 0.37 - - 13.60 85.18 1.22

LA_09_031 99.57 0.43 - - 13.52 85.24 1.24

LA_09_032 99.48 0.52 - - 13.71 85.04 1.25

LA_09_033 99.51 0.49 - - 13.99 84.61 1.40

LA_09_034 99.41 0.59 - - 13.62 85.17 1.21

LA_09_035 99.24 0.76 - - 13.72 84.64 1.64

LA_09_036 98.72 1.28 - - 13.72 84.45 1.83

LA_09_037 98.95 1.05 - - 13.60 84.73 1.67

LA_09_038 98.58 1.42 - - 13.84 84.13 2.03

Table 69 - results T1 week 40 ° C

Lot No.% Monomer% Di- l Oligomer% HMWP% LMW% acid% neutral% basic LA_09_030 95.19 0.97 3.29 0.55 11.90 83.92 4.18 LA_09_031 96.47 0.89 2.14 0.50 12.19 84.70 3.11 LA_09_032 96.82 0.92 1.69 0.57 12.14 85.48 2.38 LA_09_033 97.13 0.94 1.48 0.45 12.41 85.04 2.55 Lot N °% Monomer ^% Di- / Oligomer% HMWP% LMW% acid% neutral% basic LA_09_034 97,73 0,97 0,82 0,48 12,35 85,69 1,96 LA_09_035 97,58 1,12 0,89 0,41 11,93 85,74 2,33 LA_09_036 97,47 1, 32 0.86 0.35 12.01 85.46 2.53 LA_09_037 97.35 1.41 0.87 0.37 12.08 85.28 2.64 LA_09_038 96.97 1.62 0.97 0, 44 11.65 85.10 3.25

In conclusion, the data confirm the results already generated during the preformulation studies: the increase in pH causes the monomer content to decrease and the dimer rate to increase. Samples at 40 ° C showed at a lower pH value a reduction in HMW up to pH 6.0 and then an increase up to pH 5.0.

Example 14 - Optimization of additional buffer

Next, the citrate, acetate and histidine buffers (as buffer) were screened at 5/10/25/50 mM at the selected pH values. See Tables 70-84.

Table 70 - Sample Summary - Citrate Buffer pH 6.0

Lot No. Conc. Of citrate buffer [mM]

LA_09_040 5

LA_09_034 10

LA_09_041 25

LA_09_042 50

Table 71 - results after T0

Lot No. Appearance pH value Conc of mAB Measured hydrodynamic diameter ( -) [mg / ml] ( nm)

LA_09_040 Transparent 6.1 20.05 13.47

LA_09_034 Transparent 6.1 22.77 12.83

LA_09_041 Transparent 6.2 20.48 11.91

LA_09_042 Transparent 6.1 22.19 11.87

Table 72- results after T1 week 40 ° C

Lot No. Appearance pH value Conc of mAB Measured hydrodynamic diameter ( -) [mg / ml] ( nm)

LA_09_040 Transparent 6.3 21.62 13.78

LA_09_034 Transparent 6.1 24.18 13.25

LA_09_041 Transparent 6.2 18.37 12.50

LA_09_042 Transparent 6.2 20.59 12.07

Table 73 - T0 results

Lot N °% Monomer% Di- / Oligomer% HMWP%%%% basic

( RRT 0.84) ( RRT 0.68)

LA_09_040 99.49 0.51 - - 13.19 85.81 1.00

LA_09_034 99.41 0.59 - - 13.62 85.17 1.21

LA_09_041 99.55 0.42 0.03 - 13.24 85.67 1.09

Lot N °% Monomer% Di- / Oligomer% HMWP%%%% basic

( RRT 0.84) ( RRT 0.68)

LA_09_042 99.60 0.39 0.01 - 13.41 85.48 1.11

Table 74 - results after thermal stress 1 week / + 40 ° C

Lot N °% Monomer% Di- / Oligomer% HMWP%%%% basic

( RRT 0.84) ( RRT 0.68)

LA_09_040 98.58 0.86 0.17 0.39 12.52 85.95 1.53

LA_09_034 97.73 0.97 0.82 0.48 12.35 85.69 1.96

LA_09_041 98.81 0.65 0.21 0.33 12.54 86.07 1.38

LA_09_042 98.87 0.59 0.14 0.40 12.45 86.10 1.45

Table 75 - Sample summary - histidine tampon pH 5.0

Lot No. of histidine buffer [mM] LA_09_043 5

LA_09_044 10

LA_09_045 25

LA_09_046 50

Table 76 - results after T0

Lot No. Appearance pH value Conc of mAB measured hydrodynamic diameter ( -) [mg / ml] ( nm) LA_09_043 Transparent 5.5 21.89 8.40 agr. LA_09_044 Transparent N / A 6.95 * 11.34 LA_09_045 Transparent 5.2 21.78 11.86 agr. LA_09_046 Transparent 5.1 20.04 4.18 * Data reduced due to dilution error of the sample.

Table 77 - results after week T1 40 ° C

Lot No. Appearance pH value measured Conc. Of mAB Diameter

( -) [mg / ml] hydrodynamic LA_09_043 Transparent

LA_09_044 Transparent 5.5 21.34 8.81 LA_09_045 Transparent 5.5 24.18 13.25 LA_09_046 Transparent 5.2 23.62 11.88

5.1 21.41 12.50 agr.

Table 78 - Results of ASD T0

Lot N °%% Di-% HMWP%% acid% basic%

LA_09_043 99.55 0.45 - - 13.69 85.16 1.15 LA_09_044 * N / AN / AN / AN / AN / AN / AN / A LA_09_045 99.68 0.32 - - 13.73 85.00 1,27 Lot N °%% Di-% HMWP%% acid% basic%

LA_09_046 99.70 0.30 - - 13.43 85.49 1.08 * Not analyzed due to dilution error.

Table 79 - results after thermal stress 1 week / + 40 ° C

Lot N ° %% Di-% HMWP%% acid% basic%

LA_09_043 98.72 0.82 - 0.46 13.63 84.60 1.75 LA_09_044 * N / AN / AN / AN / AN / AN / AN / A LA_09_045 98.30 0.80 0.44 0.56 12.79 85.19 2.02 LA_09_046 97.79 0.68 1.07 0.46 12.61 84.75 2.64 * Not analyzed due to dilution error.

Table 80 - Summary of the samples - Acetate buffer pH 5.5

Lot No. of acetate buffer. [mM] LA113244_09_053 5

LA113244_09_054 10

LA113244_09_055 25

LA113244_09_056 50

Table 81 - results after T0

Lot No. Appearance pH value Conc of mAB Diameter measured ( -) [mg / ml] hydrodynamic LA_09_053 Transparent 5.88 25.41 10.44 LA_09_054 Transparent 5.68 21.91 13.21 agr. LA_09_055 Transparent 5.56 21.53 14,06 LA_09_056 Transparent 5.56 22.08 13.54

Table 82 - results after T0

Lot No. Appearance pH value Conc of mAB Diameter measured ( -) [mg / ml] hydrodynamic LA_09_053 Transparent 5.88 26.43 11.48

LA_09_054 Transparent 5.69 23.40 13.44

LA_09_055 Transparent 5.61 21.53 14.46

LA_09_056 Transparent 5.56 21.68 13.71

Table 83 - T0 results

Lot N%% D i-% HMWP%% acid% basic%

LA_09_053 98.89 0.99 0.02 0.10 14.44 83.67 1.89

LA_09_054 98.84 1.09 0.07 - 11.30 86.80 1.90

LA_09_055 98.91 0.99 0.07 0.03 11.30 86.77 1.93

LA_09_056 98.97 0.87 0.10 0.06 11.27 86.90 1.83 Table 84 - results after thermal stress 1 week / + 40 ° C

Lot N °% Monomer% Di- / Oligomer% HMWP%% acid%% basic

( RRT 0.84) ( RRT 0.68)

LA_09_053 97.68 1.96 0.04 0.32 14.53 81.50 3.91

LA_09_054 97.83 1.99 0.09 0.09 11.13 85.79 3.08

LA_09_055 97.88 2.00 0.09 0.09 11.09 85.76 3.15

LA_09_056 98.10 1.22 0.59 0.09 10.92 86.24 2.74

In conclusion, the data confirm the results generated during the preformulation studies. When using citrate as the buffering agent, the monomer content is slightly higher than with the acetate buffer and the histidine buffer. With histidine, a high aggregation behavior is observed, even at T0, which causes difficulties in the preparation of analytical samples. A significant difference between the buffer concentrations analyzed can not be measured, so the three citrate, histidine and acetate buffers will be used at a concentration of 10 mM.

Example 15 - Additional optimization of surfactant

On the basis of the preformulation tests, the addition of nonionic surfactant polysorbate 20 (0.01%) showed beneficial effects on the stability, for which reason an additional evaluation of its concentration was carried out ^{adding the following concentrations of polysorbate} 20 ^{to the respective buffers:} 0.0025% / 0.005% / 0.01% / 0.02%. See Tables 85-94.

Table 85 - Summary of samples in acetate buffer

Lot No. Polysorbate 20 in [mg / ml] Concentration as a percentage [%] LA_09_058 0.2 0.02

LA_09_059 0.1 0.01

LA_09_060 0.05 0.005

LA_09_061 0.025 0.0025

^Table 86 ^{- results after T0}

Lot No. Appearance pH value Conc of mAB Measured hydrodynamic diameter ( -) [mg / ml] ( nm)

LA_09_058 Transparent 5.61 23.71 12.50

LA_09_059 Transparent 5.64 22.76 12.94

LA_09_060 Transparent 5.63 23.89 12.83

LA_09_061 Transparent 5.64 25.79 12.88

Table 87 - results after mechanical stress 300 rpm / 150 min

Lot No. Appearance pH value Conc of mAB Measured hydrodynamic diameter ( -) [mg / ml] ( nm)

LA_09_058 Transparent 5.61 22.82 12.48

LA_09_059 Transparent 5.67 22.47 12.73

LA_09_060 Transparent 5.55 22.90 12.59

LA_09_061 Transparent 5.65 25.19 12.76

^Table 88 ^{- T0 results}

Lot N °%% Di-% HMWP%% acid% basic%

LA_09_058 99.17 0.80 0.03 - 11.25 86.94 1.81 LA_09_059 99.16 0.81 0.03 - 11.27 86.94 1.79 LA_09_060 99.17 0.81 0.03 - 11.44 86.80 1.76 LA_09_061 99.13 0.84 0.03 - 11.31 86.91 1.78

Table 89 - results after mechanical stress 300 rpm / 150 min

Lot N °% Monomer% Di- / Oligomer% HMWP%% acid%% basic

( RRT 0.84) ( RRT 0.68)

LA_09_058 99.16 0.81 0.03 - 11.27 86.95 1.78 LA_09_059 99.16 0.82 0.02 - 11.24 86.91 1.85 LA_09_060 99.17 0.81 0.03 - 11.46 86.77 1.77 LA_09_061 99.16 0.81 0.02 11.22 86.97 1.79

Table 90 - Summary of samples in citrate buffer

Lot No. Polysorbate 20 in ( mg / ml) Concentration as a percentage ( %) LA_09_062 0.2 0.02

LA_09_063 0.1 0.01

LA_09_064 0.05 0.005

LA_09_065 0.025 0.0025

Table 91 - results after T0

Lot No. Appearance pH value Conc of mAB Measured hydrodynamic diameter ( -) [mg / ml] ( nm) LA_09_062 Transparent 6.05 23.72 12.67 LA_09_063 Transparent 6.03 25.18 12.73 LA_09_064 Transparent 6.04 23.85 12.47 LA_09_065 Transparent 6.04 22.65 12.46

Table 92 - results after T1 week 40 ° C

Lot No. Appearance pH value Conc of mAB Measured hydrodynamic diameter ( -) [mg / ml] ( nm) LA_09_062 Transparent 6,07 23,44 12,99 LA_09_063 Transparent 6,03 24,39 12,59 LA_09_064 Transparent 6,04 23,93 12,39 LA_09_065 Transparent 6,04 22,27 12,37

Table 93 - T0 results

Lot N °% Monomer% Di- / Oligomer% HMWP%% acid%% basic LA_09_062 99.25 0.70 0.05 - 11.33 86.24 2.43 LA_09_063 99.28 0.68 0.04 - 22.00 86.36 2.64 LA_09_064 99.23 0.74 0.03 - 10.93 86.45 2.62 Lot N °% Monomer% Di- / Oligomer% HMWP%% acid%% basic

LA_09_065 99.28 0.69 0.03 - 10.97 86.25 2.77

Table 94 - results after mechanical stress 300 rpm / 150 min

Lot N °% Monomer% Di- / Oligomer% HMWP ^%% % basic% acid

LA_09_062 99.27 0.69 0.04 - 11.25 86.27 2.48

LA_09_063 99.32 0.65 0.03 - 10.87 86.59 2.54

LA_09_064 99.19 0.78 0.03 - 10.91 86.56 2.53

LA_09_065 99.16 0.80 0.04 - 10.79 86.51 2.70

In conclusion, no significant differences could be measured in the samples containing acetate or citrate buffer with various concentrations of polysorbate. To ensure the prevention of mAb against mechanical stress for a period of time longer than that analyzed for 150 min, the polysorbate concentration was set at 0.2 mg / ml. This amount was also proposed based on preformulation studies.

Example 16 - Optimization of additional isotonicity

During preformulation studies, NaCl, trehalose and arginine-HCl were identified as additives for isotonicity and stability purposes. The arginine-HCl was removed due to less stability effects of the mAb. According to the buffer concentration and the pH value, the amount of isotonicity agent / stabilizer is adapted to achieve an osmolality of at least 240 mOsmol / kg according to Ph.Eur.

The use of trehalose was questioned since it is not a compendium excipient and has a high price. During preformulation studies, sucrose caused a little more aggregation, but was not followed up or verified in subsequent studies. Therefore, a new short-term stability study was designed for four weeks, which includes trehalose and sucrose in both citrate buffer and 10 mM acetate with storage temperatures of 5 °, 25 ° and 40 ° C. See Tables 95-103.

The fine adjustment of the osmolality of at least 240 mOsmol / kg was carried out with NaCl.

Table 95 - Sample summary

Lot No. Buffer Target pH value [-] Polysorbate 20 NaCl Stabilizing agent LA_09_051A Citrate 10 mM 6.0 0.2 mg / ml 2 mg / ml Sucrose 60 mg / ml LA_09_051B Citrate 10 mM 6.0 0.2 mg / ml 2 mg / ml Trehalose 60 mg / ml LA_09_0524 Citrate 10 mM 5, 5 0.2 mg / ml 2 mg / ml Sucrose 60 mg / ml LA_09_052B Citrate 10 mM 5.5 0.2 mg / ml 2 mg / ml Trehalose 60 mg / ml

Table 96 - T0 results

Lot No. Appearance pH value measured ( -) Conc of mAB ( mg / ml) Osmolality ( mOsmol / kg) LA_09_051A Transparent 5.89 21.46 289 LA_09_051B Transparent 5.94 21.46 268 LA_09_052A Transparent 5.82 22.07 273 LA_09_052B Transparent 5.80 22.07 256

Table 97 - results T 4 weeks, 5 ° C

Lot No. Measured pH value ( -) Conc of mAB ( mg / ml) Hydrodynamic diameter ( nm) LA_09_051A 6.02 21.81 13.58

LA_09_051B 5.95 22.10 13.35

LA_09_052A 5.86 21.70 15.12

Lot No. Measured pH value ( -) Conc of mAB ( mg / ml) Hydrodynamic diameter ( nm) LA_09_052B N / A N / A N / A

Table 98 - results T 4 weeks, 25 ° C

Lot No. Measured pH value ( -) Conc of mAB ( mg / ml) Hydrodynamic diameter ( nm) LA_09_051A 6.06 22.30 13.57

LA_09_051B 6.02 22.09 13.41

LA_09_052A 5.91 21.98 15.14

LA_09_052B N / A N / A N / A

Table 99 - results T 4 weeks, 40 ° C

Lot No. Measured pH value ( -) Conc of mAB ( mg / ml) Hydrodynamic diameter ( nm) LA_09_051A 6,04 22,21 14,73 LA_09_051B 5,95 21,86 14,11 LA_09_052A 5,91 22,23 16,37 LA_09_052B 5,89 22,84 16,02

Table 100 - T0 results

Lot N%% D i-%%% Conc of mAB ( mg / ml)

LA_09_051A 99.53 0.47 13.78 83.85 2.37 23.45

LA_09_051B 99.54 0.46 13.73 84.83 1.94 22.91

LA_09_052A 99.44 0.56 13.83 83.99 2.18 22.54

LA_09_052B 99.44 0.56 14.39 83.38 2.23 23.19

Table 101 - Results after thermal stress 4 weeks / + 5 ° C

Lot N%% Di-% HMWP%%%% Conc. Of mAB ( RRT 0.84) ( RRT 0.68) ( mg / ml) LA_09_051A 99.21 0.38 - 0.41 11.34 87.11 1.25 24.24 LA_09_051B 98.97 0.46 - 0.57 11.26 87.17 1.78 23.71 LA_09_052A 98.81 0.54 - 0.65 11.47 86.86 1.67 22.63 LA_09_052B 99.00 0.55 - 0.45 11.46 86.90 1.64 23.17

Table 102 - results after thermal stress 4 weeks / + 25 ° C

Lot N%% Di-%% LMW%%% Conc. Of mAB ( mg / ml)

LA_09_051A 98.87 0.46 - 0.67 11.01 87.31 1.69 26.16

LA_09_051B 98.72 0.53 - 0.75 11.02 87.29 1.70 23.63

LA_09_052A 98.25 0.83 - 0.92 11.69 86.35 1.96 24.27

LA_09_052B 98.52 0.74 - 0.74 11.46 86.62 1.92 23.52 Table 103 - results after thermal stress 4 weeks / + 40 ° C

Lot N%% Di-%% LMW%%% Conc. Of mAB ( mg / ml)

LA_09_051A 96.84 0.95 1.06 1.15 10.16 87.05 2.79 25.04

LA_09_051B 96.96 0.97 1.01 1.06 10.01 87.05 2.83 23.60

LA_09_052A 96.00 1.63 1.17 1.20 11.32 84.89 3.79 24.11

LA_09_052B 96.23 1.49 1.21 1.07 11.01 85.30 3.69 24.29

In conclusion, no significant differences were measured between the citrate and acetate buffer, and no difference was seen in accelerated conditions between trehalose and sucrose. Citrate buffer with sucrose was selected for further studies.

Determination of the parameters of the DP manufacturing process

A batch of DS in citrate buffer was used to determine the parameters of the manufacturing process. The preformulation studies indicated that DS was not so susceptible to oxidation, and that light protection or blanketing or nitrogen purge was required during fabrication. Standard glass equipment and silicone tubes (SaniTech65) were used.

Order of addition

The experiments that evaluated the order of addition of the excipients were limited due to the small volume of dilution of DS.

The DS was weighed in a glass bottle, the polysorbate 20 as the first excipient, the sucrose as the second excipient and the NaCl as the third excipient were added and rinsed with 10 mM citrate buffer pH 6.0 to dilute the DS content to 20 mg / ml.

Agitation speed and time

The stirring speed was adjusted to 100 rpm to reduce the mechanical stress for the DS. Due to the fact that all the excipients were very soluble in water, the stirring time was adjusted to 5 minutes.

Supervision parameters and IPC

Supervision parameters such as appearance, turbidity, density and viscosity, and IPCs such as pH value and osmolality were routinely checked during sample production according to the following Table 104:

Table 104

Before filtration After filtering After filling Appearance colorless to slightly colorless to slightly colorless to slightly yellow yellow yellow

Density 1006 mg / ml Not measured Not measured

Transparent Transparent Turbidity Viscosity Unmeasured No measurement <5 mPa s

PH value 6.0 ± 0.2 (20-25 ° C) 6.0 ± 0.2 (20-25 ° C) Not measured Osmolality 290 ± 40 mOsmol / kg 290 ± 40 mOsmol / kg Not measured

No problems were observed during manufacturing. The limits for osmolality were established on the basis of the measured data.

Filtration process

According to the preformulation studies, polyethersulfone was a suitable membrane for sterile filtration (Sartorius, 0.22 pm). No potential pH changes could be observed after filtration, since the filtration rate and time showed standard values for the filtration of an aqueous solution. The integrity tests of the filter were carried out routinely without problems.

Filling process

Standard dosing equipment made of stainless steel, such as the filling pump and the filling needle, were investigated. Likewise, the duration and filling speed were monitored. The extractable volume of filled DP was determined. An overfill of 0.2 ml was required to ensure a removable volume of 1.5 ml.

Compatibility of materials

All the preformulation and formulation studies were performed on glass as standard manufacturing equipment, which is also a recommendation for the equipment that will be used for the manufacture of GMP.

Cleaning agents

The cleaning of the manufacturing equipment was carried out according to the respective standard operating procedures using the dishwasher with the standard Neodisher® cleaning agent. Previously, a manual cleaning with water for injection was carried out. No harmful effects of the cleaning agents were observed.

Summary of additional formulation studies for the Initial CXCR5 Antibody (20 mg / ml)

For the selection of the DP formulation of the Initial Phase I CXCR5 Antibody, 10 mM citrate was selected at pH 6.0 as the buffer before histidine and acetate. The pH value of the solution was set at 6.0, since increasing or decreasing the pH value means a reduction in the monomer content. The buffer concentration was adjusted to an average concentration of 10 mM, although there were no significant differences between the concentrations of 5-50 mM.

The polysorbate 20 was chosen as the surfactant with 0.2 mg / ml (0.02%), sufficient to stabilize the DS against mechanical stress.

Sucrose was selected as the stabilizer against thermal stress ahead of trehalose. The ^{sucrose concentration was set at 60 mg / ml (} 6 ^%).

NaCl will be used as an isotonizing agent at a concentration of 2.0 mg / ml (0.2%) to achieve a DP osmolality of approximately 300 mOsmol / kg.

STUDIES OF FORMULATION ANTI-CXCR5 (100 mg / ml)

The data of Examples 17-21 were collected during the formulation studies for the Initial CXCR5 Antibody and its pharmacological product for intravenous and subcutaneous administration. The objective of the formulation studies was to provide an injection solution of CXCR5 antibody free of visual particles stable, transparent or slightly opalescent and colorless or slightly yellow, for phase I.

Procedures

Preparation of the sample

A small-scale UF / DF was performed using VivaSpin devices with a Hydrosart membrane and a cut-off value of 30 kDa. The RSN material was concentrated from about 20 mg / ml to 100 mg / ml. All solutions were already in the final formulation buffer (10 mM citrate buffer at pH 6.0).

The VivaSpin units were placed at room temperature in a common laboratory centrifuge and centrifuged at 2000 rpm. The solution was filtered over Minisart of 0.2 μm before the analytical tests.

^{All samples were stored between 2 ° and} +8 ^{° C, well closed and protected from light, until} analytical ^tests at T0 and after a week of thermal stress at 40 ° C or after mechanical stress.

Analytical procedures

The following techniques were used for the analysis of samples:

Table 105 - Analytical techniques used

Parameter technique to be investigated

Organoleptic Appearance

Turbiedad Nephelometer

Parameter technique to be investigated

PH meter pH value

UV Concentration of mAB

Density Density

Osmolality Osmometer

Viscosity Viscosity

DLS Hydrodynamic diameter

DSC * Deployment temperature

SEC Content of Mono- / Di- / Oligomer / HMW

WCX Isoforms (acidic / basic / neutral)

ELISA * Power (agglutination)

SDS-Page (network.) ** HC / LC, fragments mAB

SDS-Page (not network.) ** Aggregation and degradation products.

HIAC * Subvisible particles

* Some samples will be analyzed.

** SDS-Page will be carried out in case SEC shows unusual results.

Example 17 - Screening of excipients

The preformulation studies identified a buffer of 10 mM citrate at pH 6.0 as the best buffer with less tendency to aggregation of the Initial CXCR5 Antibody. In previous studies at 20 mg / ml, a ^{formulation containing 10 mM citrate buffer, 60 mg / ml (} 6 ^{%) sucrose, 2 mg / ml (0.2%) NaCl and 0.2} mg / ml was selected. ml (0.02%) of polysorbate 20. These excipients and some alternatives were analyzed to confirm the suitability ^{of the selected formulation at a higher concentration} (100 ^{mg / ml).}

The different formulations were subjected to thermal stress at 40 ° C for 7 days and mechanically at 100 rpm for 5 hours. Additionally, the unfolding temperature for the different formulations was screened at 100 mg / ml using DSC (differential scanning calorimetry).

The following excipients were tested:

Sucrose ^ 60 mg / ml

Trehalose ^ 60 mg / ml

Arginine ^ 30 mg / ml

Lysine ^ 30 mg / ml

Glycine ^ 30 mg / ml

NaCl or mannitol was added as an isotonicity agent. No salts were needed for viscosity reduction (around 2.1 cP).

The results of days T0 and T7 are shown in Table 106.

Table 106 - Screening of excipients

Thermal stress

None of the samples showed turbidity before or after stress.

The lysine showed: a pH change to 9.8, a very high tendency to aggregation, a very high increase of acidic and basic isotherms, and high molecular weight bands on SDS-PAGE. As a result, it was excluded from ^further consideration ^.

Mannitol formulations showed poor binding in an ELISA assay after stress. As a result, NaCl is the favored isotonicity agent.

Sucrose showed slightly better chemical stability than trehalose, but additional bands were observed on SDS-PAGE after stress (for both).

Arginine (especially in the presence of NaCl) and glycine had a similar SEC profile, but no additional bands were observed on SDS-PAGE after stress.

Protein-associated formation measured by dynamic light scattering (DLS)

The Initial CXCR5 Antibody showed a significant increase in the hydrodynamic diameter (Z average) with increasing concentration (Figure 34). This behavior was completely reversible after a dilution. For further investigation of this effect, the different concentrations of Initial CXCR5 Antibody were measured by ultra-analytical centrifugation (AUC) and aggregation was excluded. The conclusion of the AUC study was that this behavior was due to the formation of associated proteins.

The effect of the excipient mentioned above on this behavior was studied and the results are shown in Figure 35. The average Z was measured before and after thermal stress. The stabilizing effect was similar to all the excipients tested, but the increase in average Z was generally reduced using amino acids as stabilizers (arginine, lysine or glycine). Lysine was excluded due to a higher content of aggregates after stress. Arginine showed a better effect than glycine. Both amino acids were considered for the final design of the experiment to choose the best combination of excipients.

Mechanical stress

Lysine formulations, as well as all formulations containing mannitol, were excluded. The SEC data showed no effect of stress on the samples analyzed. See Table 107.

Table 107 - Mechanical stress

SEC before stress SEC after stress Mechanical mechanical formulation Composition of the HMW monomer HMW

formulation (mg / ml) monomer [%] [%] [%] [%]

^{Sucrose (60) NaCl (2)} 2.6 ^{97.3 2.7 97.2 080_A Trehalose (60) NaCl (2) 2.7 97.2} 2.6 ^{97.3 080_B}

Arginine (30) 080_C1 2.5 97.5 2.3 97.6 Arginine (20) NaCl (2) 080_C2 2.5 97.4 2.5 97.4

Glycine (20) 080_D1 2.5 97.5 2.4 97.5 Glycine (20) NaCl (2) 080_D2 2.5 97.5 2.4 97.5

The same reduction was observed in average Z in the presence of amino acids. Sucrose had a better protective effect than trehalose against mechanical stress. Arginine and glycine performed better in combination with NaCl. See figure 36.

Screening by differential scanning calorimetry (DSC)

A screening study was performed to determine the unfolding temperature of the Initial CXCR5 Antibody using Differential Scanning Calorimetry (DSC). Sucrose, trehalose, argenine and glycine were selected.

The results of Tm are listed in Table 108.

Table 108: Effect of different excipients on the Tm values of the Initial CXCR5 Antibody. All ^{formulations were in 10 mM citrate buffer at pH} 6

Excipient screening Tm1 Tm2 Tm3

Sucrose NaCl 65.3 73.6 83.8

Excipient screening Tm1 Tm2 Tm3 Trehalose NaCl 65.5 73.9 83.9

Arginine 63.8 72.2 82.6

Arginine NaCl 64.3 72.8 82.6

Glycine 64.8 74.1 84.2

Glycine NaCl 64.9 73.6 83.8

On the basis of Tm1, sucrose and trehalose showed the highest values. Arginine performed better in combination with NaCl.

In conclusion, the data collected suggest that the final formulation of the 100 mg / ml Initial CXCR5 Antibody would contain a combination of sugar (in some embodiments, sucrose) and an amino acid (in some embodiments, arginine or glycine) in the presence of NaCl as the isotonicity agent.

Example 18 - Surfactant Screening

The polysorbate as a stabilizer was evaluated for the protection of the Initial CXCR5 Antibody against thermal and mechanical stresses.

Polysorbate 20 and 80 were tested in two different concentrations: 0.1 and 0.2 mg / ml.

Thermal stress

The DLS showed no effect by the addition of polysorbate after thermal stress. The formation of HMW and fragments after 7 days of storage at 40 ° C was observed in all samples, detected by SEC. No additional bands were detected on SDS-PAGE. Slight changes were observed after thermal stress, but no differences were observed between PS20 and PS80, as well as between the 2 concentrations (data not shown).

Mechanical stress

The DLS showed no changes after mechanical stress. Polysorbate 20 did not show aggregations after mechanical stress. Polysorbate 80 showed aggregate formation after mechanical stress. No additional bands were observed on SDS-PAGE (data not shown).

In conclusion, polysorbate 20 was the desired surfactant due to the superiority in mechanical stabilization of the Initial CXCR5 Antibody.

Example 19 - Preselection of prototype formulation using DSC

On the basis of excipient screening and surfactant detection studies, 12 different combinations of excipients were suggested (see tables 109 and 110)

The unfolding temperature for all formulations was determined by DSC and the resulting Tm, as well as the osmolality for each formulation, are listed in Tables 109 and 110.

Table 109: Combinations of excipients for the pre-selection study of prototype formulations (arginine) with DSC. The values of Tm and osmolality are also listed

Table 110: Combinations of excipients for the study of pre-selection of prototype formulations (glycine) with DSC. The values of Tm and osmolality are also listed

The formulations did not show large differences in Tm, but the osmolality varied greatly. The pre-selection of the prototype formulations was based on Tm and osmolality. Consequently, in each group of excipients (arginine and glycine), the highest Tm (regardless of osmolality) was selected. In addition, the highest Tm in the isotonic region was also selected.

Example 20 - Exploratory stability study of the prototype

The selection of the above prototype resulted in 4 prototype formulations, which are listed in Table 111. Said formulations were analyzed to determine mechanical stress (100 rpm for 5 hours), 5 freeze / thaw cycles and isothermal stress at 5, 20 and 40 ° C.

Table 111: Prototype formulations for the formulation of the Initial CXCR5 Antibody of 100 mg / ml

Composition

Formulation Osmo (mosmol / kg) Sucrose Arginine Glycine NaCl PS 20

^{LA_10_102_A 60} 20 2 0.1 ⁵¹⁸

^{LA_10_102_B 45} 10 2 0.1 ³⁷⁴

^{LA_10_102_C 60 15} 2 0.1 ⁵⁵⁰

^{LA_10_102_D 30 7,5} 2 0,1 ³²⁵

Mechanical stability

^{The Initial CXCR5 Antibody in 10 mM citrate buffer at pH} 6 ^{, without addition of any excipient (DS formulation), was} also subjected to stress in parallel with the prototype formulations. A species of high molecular weight was measured by DLS after the mechanical stress of DS (figure 37), stress in which no changes have been observed in all the formulations analyzed after mechanical stress. The formation of aggregates after mechanical stress was measured by size exclusion chromatography (SEC) and the results are shown in Table 112. In general, the 4 formulations were equally stable to mechanical stress, except Formulation A, where the SEC found more HMW after mechanical stress. See figure 38.

Table 112: Results of size exclusion chromatography (SEC) of prototype formulations before and after mechanical stress

SEC

For_A For_B For_C For_D

% Mon. % Mon. % Mon. % Mon.

-20 ° C

Before 99.6 99.5 99.5 99.5

After 98.1 99.5 99.3 99.4

Freeze / thaw stability

No significant differences were detected, neither in DS nor in DP, after 5 cycles of freezing / thawing.

Therefore, there should be no problems of instability due to freezing and thawing (the data is not shown).

Stability study of the exploratory prototype

The prototype formulations were stored at -20, 5, 20 and 40 ° C. They were analyzed at the beginning of the study, after 1 ^{month, after 3 months and after} 6 ^{months. The formulations were selected based on the results of 3} months (Tables 113-115). The results showed that formulation B performed better with respect to SEC, WCX and subvisible particles, especially at 40 ° C.

Table 113: Results of size exclusion chromatography (SEC) of the prototype formulations after 3 months

Table 114: Results of weak cation exchange chromatography (WCX) of the propotype formulations after 3 months

Table 115: Subvisible particles measured by blocking light at zero T and after 3 months (5 ° C)

In conclusion, the studies showed better results for the formulation LA_10_102_B. This formulation had ^{a concentration of 100 mg / ml of Initial CXCR5 Antibody in 10 mM citrate buffer at pH} 6 ^{and contained the} following excipients:

Sucrose 45 mg / ml (4.5%);

^Arginine 10 ^{mg / ml (} 1 ^%);

^NaCl 2 ^{mg / ml (0, 2%); Y}

^{Polysorbate 20} 0.1 ^{mg / ml (0, 01%).}

Example 21 - Support stability data for the formulation of 100 mg / ml

Additional stability studies were performed on the formulation of the 100 mg / ml Initial CXCR5 Antibody identified in Example 20. Additional studies were performed at -20, 5 and 25 ° C. The results are shown in Tables 116-118.

Table 116 - Stability data for 100 mg / ml formulation of Initial CXCR5 Antibody at -20 ° C

Table 117 - Stability data for 100 mg / ml formulation of Initial CXCR5 Antibody at 5 ° C

Table 118 - Stability data for 100 mg / ml formulation of Initial CXCR5 Antibody at 25 ° C

Claims (10)

1. A formulation comprising: -from 5 mg / ml to 280 mg / ml of an IgG4 antibody, wherein the antibody binds to the chemokine receptor CXC type 5 (CXCR5) and comprises a heavy chain variable region and a light chain variable region, wherein the antibody is a humanized anti-CXCR5 IgG4 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26, -5 to 50 mM citrate as buffer agent; ^{- 0, 001% to 0, 1% w / v of polysorbate 20; the pH of the formulation being from pH 5 to pH} 6 ^{; Y wherein the monomeric antibody has a purity of 90% after storage for at least} 6 months at 5 ° C. 2. The formulation of claim 1, further comprising 0.1 to 10% w / v of a tonicity agent. 3. The formulation of claim 1 or 2, further comprising from 0.1% to 5% w / v amino acid. 4. The formulation of any one of claims 1-3, wherein the formulation is a liquid formulation. The formulation of any one of claims 1-3, wherein the formulation is a lyophilized formulation. ^{6. The formulation of any one of claims 1-3, wherein the antibody monomer has a} purity of 90% after storage for at least 9 months at 5 ° C. ^{The formulation of any one of claims 1 to} 6 ^{, which formulation shows a reduced amount of} at least one by-product selected from the group consisting of aggregates, half molecules, degradation products, low molecular weight proteins, high weight proteins molecular and rearrangements of acidic / basic / neutral isoforms of the antibody compared to an anti-CXCR5 reference formulation comprising an anti-CXCR5 antibody in phosphate buffered saline at pH 7.3. ^{8. The formulation of claim 1, comprising:} a) 20 mg / ml of a humanized IgG4 anti-CXCR5 antibody (chemokine receptor CXC type 5) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26; b) 10 mM citrate buffer; ^c) 0 ^{02% polysorbate 20; Y which further comprises d)} 6 ^{% sucrose; Y e) 0, 2% sodium chloride;} the pH of the formulation being pH 6.0. The formulation of claim 1, comprising: a) 100 mg / ml of a humanized IgG4 anti-CXCR5 antibody (chemokine receptor CXC type 5) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 and a light chain comprising the amino acid sequence of SEQ ID NO: 26; b) 10 mM citrate buffer; ^c) 0 ^{01% polysorbate 20; Y} which further comprises d) 4.5% sucrose; ^{e) 0, 2% sodium chloride; Y f)} 1 ^{% arginine;} the pH of the formulation being pH 6.0. 10. A pre-filled device or a pre-filled container, such as a syringe, cartridge, vial, ampoule or auto-injector comprising the formulation of any one of claims 1-9.