EP2368123A1

EP2368123A1 - In-process control in a method for producing epo

Info

Publication number: EP2368123A1
Application number: EP09764266A
Authority: EP
Inventors: Franz-Rudolf Kunz; Florian Glaser; Wolfgang Wienand; Rudolf Hanko; Wilfried Eul; Dietmar Reichert
Original assignee: Evonik Degussa GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2008-12-16
Filing date: 2009-12-07
Publication date: 2011-09-28
Also published as: CN102317791A; WO2010076125A1; BRPI0923039A2; SG172203A1; IL213544A0; DE102008054716A1; JP2012512406A; US20120021441A1; CA2747320A1

Abstract

The invention relates to a method for determining the isoform composition of erythropoietin, comprising the following steps: a) isoelectrical focusing of a sample comprising erythropoietin in a gel over a pH range having a lower limit of 2.5 to 3.5 and an upper limit of 5 to 8, wherein the sample comprising erythropoietin originates from a culture supernatant of erythropoietin producing eukaryotic cells; b) transferring of the proteins comprised and separated in the gel to a membrane; c) verifying the erythropoietin bound to the membrane by specific antibodies; and to a method for in-process control of culture supernatants of erythropoietin producing eukaryotic cells during the fermentative production process.

Description

In-process control in a process for the production of EPO

The present invention relates to a method for the detection of erythropoietin, in particular for Inprozesskontrolle of culture supernatants of erythropoietin-producing eukaryotic cells in the context of the fermentative production process. The method is characterized in particular by the fact that the isoform composition of the produced erythropoietin can be determined directly using a special isoelectric focusing (IEF). Together with the data from the erythropoietin content determination (preferably determined by means of ELISA), the quality of the synthesis crude product can be assessed during or immediately after the end of the fermentation, and the subsequent purification process can thus be controlled. This is particularly advantageous in the context of the use of perfusion reactors.

Erythropoietin, EPO for short, is a glycoprotein having a molecular weight of about 34 to 39 kDa. It consists of an unbranched polypeptide chain with 165 amino acids and an O-glycosidic (Ser 126) and three N-glycosidic (Asn 24, Asn 38 and Asn 83) bonded sugar side chains (carbohydrate moiety). The side chains are composed of the monosaccharides mannose, galactose, fucose, N-acetylglycosamine, N-acetylgalactosamine and N-acetylneuraminic acid.

The erythropoietin can occur in various isoforms. These

Variance in the molecular weight of erythropoietin is due to the heterogeneity of sugar chains that are terminally linked to neuraminic acid derivatives. Through different lengths and branching of the chains, a multitude of "sugar branches" can be constructed, which effect the expression of all isoforms of an EPO molecule.

EPO is mainly produced in the kidneys and stimulates the growth factor as the formation of erythrocytes in the bone marrow. In renal insufficiency The damaged kidneys produce too little or no EPO at all, with the result that too few erythrocytes emerge from the stem cells of the bone marrow. This renal anemia can be treated by administering EPO in physiological amounts that stimulate the formation of erythrocytes in the bone marrow. The EPO used for administration can either be obtained from human urine or generated by genetic engineering methods. Since EPO is present in trace amounts in the human body, the isolation of EPO from the natural source for therapeutic applications is virtually impossible. Therefore, genetic engineering methods provide the only economic opportunity to produce this substance in larger quantities.

[0005] The recombinant production of erythropoietin takes place by genetic engineering, above all in so-called CHO cells (Chinese hamster ovary), three basically different methods being used for the cultivation of the eukaryotic host cells (described, inter alia, in EP-AO 148) 605 and EP-A-205,564, BioProcess International 2004, 46; Gorenflo et al., Biotech Bioeng., 2002, 80, 438 and WO9501214).

In a batch process, medium and cells are at the beginning of the

Cultivation introduced into the bioreactor. Nutrients are not added until the end of cultivation and cells are removed from the fermenter, only oxygen is supplied. When one or more substrates are consumed, the process is stopped and the products are harvested from the fermentation supernatant.

The second known cultivation method is the continuous process in which fresh medium is constantly fed and taken to the same extent fermenter content. Thus, it comes to a constant supply of nutrients, while removing or diluting undesirable metabolic products such as the growth-inhibiting substances ammonium and lactate. Therefore, with this method, higher cell densities can be achieved and over be maintained for a comparatively long period of time. A special case of continuous process management is provided by so-called dialysis reactors in which high molecular weight substances such as proteins are retained in the fermenter, while low molecular weight substances such as substrates can be added or the main waste products ammonium and lactate can be removed from the system. Similarly, the use of perfusion reactors in the microbial production of chemical compounds and proteins with various cell restraint systems is well known and also described for EPO.

Finally, the third possible method is the fed-batch fermentation, in which the culture is started with a fraction of the total fermenter volume and after a short growth phase fresh substrate is added. This enables higher cell densities and longer process times than in the batch process. Another advantage of this method is that the metabolism of the cells can be influenced by the amount of feed, which can lead to a lower production of waste substances. Compared to the continuous process, the product of the cells is accumulated here in the fermenter over a relatively long period of time and thus higher product concentrations are achieved, which facilitates the subsequent work-up.

Coupled to the fermentative process are extensive chromatographic purification methods, so that an EPO can be isolated from the culture supernatants, which is therapeutically applicable and corresponds to the standard defined in the European Pharmacopoeia (Ph.Eur., 01/2002: 1316) of the Guidance on Biosimilar Medicinal Products Containing Recombinant Erythropoietins (EMEA / CHMP / 94256/2005). In this regard, the prior art is in a variety of methods, such as in WO-A-05/121173, EP-AO 228 452, EP-AO 267 678, EP-AO 830 376, EP-A-1 127 063, WO-A-03/045996, EP-AO 428 267 and WO2005121173.

[0010] According to the methods of the prior art, a sample containing erythropoietin is subjected to separation in a polyacrylamide gel, for example, an isoelectric focusing, and the proteins contained are separated by applying an electric field. The electrophoresis is followed by a so-called immunoblot (immunoimprint or immuno-transfer), in which the proteins are transferred to a membrane. In this case, an image of the individual proteins is obtained on the surface of the membrane. The membranes used have the advantage that the proteins are mainly strongly fixed by hydrophobic interactions and the membranes otherwise behave chemically neutral. Because the EPO molecules are located on the membrane surface, they are readily accessible to antibodies that are used to visualize the erythropoietin. Using isoelectric focusing, the isoforms of erythropoietin can be separated.

[0011] To detect the erythropoietin, the membrane is first incubated with a monoclonal anti-EPO antibody which specifically binds to all existing EPO molecules. Other proteins do not react with the antibody. Monoclonal antibodies not bound to EPO can be washed off the membrane because the remainder of the membrane surface has previously been blocked with a non-specific protein.

The binding of the antibody to EPO is reversible because it is based on non-covalent interactions, and may be e.g. reversed by pH changes. In the double blot method, the antibodies bound to erythropoietin are transferred to a second membrane: in an acidic environment, the antibody changes the conformation of its binding domain and upon application of an electric field, the monoclonal antibody dissociates from the EPO molecule and travels in the electric field in the direction Cathode, where it is bound to a second membrane.

The EPO molecules as well as other non-specific proteins remain on the first membrane, since the binding to the first membrane is not affected by pH fluctuations. This is a re-image of the EPO band or the EPO bands obtained. However, there are no erythropoietin molecules on the second membrane, but the specific monoclonal antibodies that previously bound to the erythropoietin molecules fixed on the first membrane.

The visualization of the antibody band (s) is carried out by a second antibody (secondary antibody) which reacts with the anti-EPO monoclonal antibody. This secondary antibody is coupled to specific enzymes (e.g., alkaline phosphatase or peroxidase) that catalyze substrate conversion that undergoes a color reaction.

The need for the second transfer is to reduce the nonspecific signals since the enzyme-labeled secondary antibody can not undergo nonspecific reactions to other components of the sample on the first membrane. In addition, a signal amplification and a concomitant increase in sensitivity by multiple formation of the antibody i5 enzyme conjugates to the first antibody is conceivable. A disadvantage of the double blot method is the significantly increased material and time required.

Thus, in the prior art methods for the detection of

Erythropoietin known and familiar to the expert, but it is either the detection of erythropoietin described above the significantly more complex doping

20 pel blot method (Chuan et al., Cytotechnology 2006, 51, 67-79, Hoelander et al., Laboratory Practice December 2004, 56-59, Lasne, Journal of Immunological Methods 2003, 276 , 223-226) or only a part of the necessary pH range on the gel when using the electrical focusing, so that the selectivity in the separation of the isoforms for the evaluation of the EPO

25 raw product quality is insufficient (Wimmer et al., Cytotechnology 1994, 16, 137-146). However, the methods of the prior art for the detection of erythropoietin are too complicated and not suitable for in-process control in the feticidal production of erythropoietin.

The object of the present invention is to provide a simplified and improved method for the detection of erythropoietin, which serves the in-process control, wherein the method makes it possible to characterize the culture supernatants from the EPO fermentation processes so that only selected and suitable evaluated fermentation solutions are supplied to each extensive cleaning processes. In particular, the present method should be superior to the prior art methods from the economical point of view.

The technical object is achieved by a method for determining the isoform composition of erythropoietin comprising the following steps: a) isoelectric focusing of a sample containing erythropoietin in a gel over a pH range whose lower limit is from 2.5 to 3.5 and the upper limit thereof is from 5 to 8, wherein the erythropoietin-containing sample is from a culture supernatant of erythropoietin-producing eukaryotic cells; b) transferring the proteins contained in the gel and separated on a membrane; c) Detection of erythropoietin bound to the membrane by specific antibodies.

In a preferred method, first antibodies directed against erythropoietin are bound to the erythropoietin bound to the membrane in step c), whereby the binding of the first antibodies to the erythropoietin bound to the membrane is detected, while the first antibody is detected on the erythropoietin bound erythropoietin bound to the membrane. It is preferred that the detection of the binding of the first antibody to the erythropoietin bound to the membrane by second antibodies, which are directed against the first antibody.

This means that, in the method in step c), first antibodies directed against erythropoietin bind to the erythropoietin bound to the membrane, and second antibodies directed against the first antibodies bind to the first antibodies bound to erythropoietin.

Because, in addition to the determination of the content of EPO, which preferably takes place by means of ELISA, with a special isoelectric focusing (IEF) in combination with a single blotting step, the isoform composition of the fermentation supernatants are determined directly, an advantageous and provided a simplified method which can be used to control the fermentation process and to decide on the selection of the culture supernatants to be purified.

The skilled person who sees himself confronted with the formulated problem, would not have considered in view of the existing state of the art with the prospect of success that the inventive method for the selection of fermentation fractions can be used prior to the purification process. So far, a comparably simple and efficient method has not been described in the literature. It is particularly advantageous that in the isoelectric focusing according to the invention, a pH range whose lower limit is from 2.5 to 3.5 and whose upper limit is from 5 to 8, in particular in a pH range of 3 to 6, on the gel so that the selectivity necessary for the evaluation of the crude EPO product quality in the separation of the isoforms is sufficient. Furthermore, in a preferred embodiment, the method is simplified to such an extent that the detection of the erythropoietin can already take place after a single protein transfer (blot). In the prior art, however, the significantly more expensive double blot method is used for detection. In a preferred method, an enzyme, preferably alkaline phosphatase, is covalently bound to the second antibody which produces a color reaction by catalytically reacting a substrate. Thus, it is preferable to use two antibody solutions on the membrane for colohmetic EPO detection, of which the second antibody contains an alkaline phosphatase, so that then a substrate of this enzyme can be used as a staining reagent. Particular preference is given to using anti-EPO mouse and anti-mouse IgG in combination with BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitrotetrazolium blue).

Furthermore, it is preferred that after incubation of the membrane with a first antibody directed against erythropoietin one or more washing steps and then again an incubation of the membrane with the first antibody directed against erythropoietin antibody is performed. This double or multiple incubation of the membrane with the antibody directed against erythropoietin enables a more complete formation of the antigen-antibody reaction and thus an increase of the specific signal for erythropoietin.

In a particularly preferred method contains at least one

Solution used in the washing steps following incubation of the membrane with the first antibody directed against erythropoietin, an organic acid in an aqueous medium. This unspecifically bound antibodies are removed from the membrane again and possibly still existing free binding sites on the blotting membrane blocked. Overall, this significantly increases the selectivity of the bands. It is preferred that the solution contains 0.1 to 1, 5 wt .-%, preferably 0.5 to 1 wt .-%, particularly preferably 0.6 to 0.8 wt .-% of the organic acid. In particular, the organic acid is mono-, di- or tricarboxylic acids (such as, for example, acetic acid, propionic acid, lactic acid, succinic acid, ascorbic acid, adipic acid or citric acid), particularly preferably acetic acid. As mentioned above, in a preferred method, the incubation with the first antibody solution is carried out in duplicate and a four-step washing procedure is performed therebetween. In this case, TBST (Tris-Buffered-Saline-Tween) and TBS (Tris-Buffered-Saline) are preferably used as a dilute aqueous organic acid. Dilute aqueous acetic acid and very particularly preferably dilute aqueous acetic acid in the concentration range between 0.5% and 1% are particularly preferably used for this purpose.

In a preferred method, the membrane used is a polyvinylidene fluoride membrane. Preferably, a membrane is used for blotting, which is suitable for binding proteins. Particularly preferred is a microporous polyvinylidene fluoride (PVDF) membrane, and most preferred is the Immobilon-P blotting membrane from Millipore.

In a further preferred method, polyacrylamide gels are used for the isoelectric focusing, which are mounted on an inert Trägerfo- Ne. Preferably, IEF utilizes standardized polyacrylamide gels immobilized on an inert support sheet, e.g. made of polyester. Especially preferred are gels whose pH gradient is formed by free carrier ampholytes in the electric field. Very particular preference is given to Blank PreNets from Serva.

In particular, it is preferred that for adjusting the pH of the

Gels ampholines are used, which adjust a pH range of pH 3 to pH 6 during the isoelectric focusing in the gel. Very particular preference is given to Servalyt ™ 3-6 from Serva.

In another preferred method, the sample containing erythropoietin is from a culture supernatant of erythropoietin-producing eukaryotic cells maintained in perfusion reactors. Preferably, the erythropoietin-containing sample is desalted before isoelectric focusing and optionally concentrated. In a particularly preferred embodiment of the method according to the invention, the determination of the isoform composition of the erythropoietin takes place during the fermentation.

Also preferably, an ELISA test is used for determining the EPO content of the culture supernatants. Particularly preferred is the EPO ELISA test from Roche Diagnostics.

The process according to the invention results in the EPO

Production process a significantly lower expenditure on equipment and personnel and thus enormous time and cost savings. At the latest 24 hours after the presence of the sample of a culture supernatant, e.g. From a perfusion fermentation process, it can be decided whether a purification of the fermentation solution makes sense and how the purification process can be controlled.

The reference material used is a commercially available Erypo®

Preparation (Janssen-Cilag).

[0036] The invention further provides a method for the in vitro control of culture supernatants resulting from the fermentation of erythropoietin-producing eukaryotic cells, comprising the following steps:

a) Determination of the isoform composition of erythropoietin according to the inventive method for the detection of erythropoietin described above; b) determining the content of erythropoietin in the sample, preferably by ELISA; c) Selection of the fermentation solutions for the purification of erythropoietin on the basis of the values and information obtained in steps a) and b), or continue the fermentation. The method is characterized in particular by the fact that the isoform composition of the fermentation supernatants can be determined directly with a special isoelectric focusing (IEF). Together with the data from the EPO content determination (preferably determined by means of ELISA), the EPO quality in the crude product can be directly assessed during or immediately after the end of the fermentation, thus controlling the purification process.

The following example is intended to illustrate the invention without limiting it.

Description of the figure

FIG. 1 shows a blot of various EPO fractions (fractioni to

Fraction 5) on a membrane after isoelectric focusing and development. The sample is from a perfusion fermentation of an erythropoietin-producing CHO cell line over a period of 47 days.

example

EPO is produced by fermentation in CHO cells. The fermentation is carried out according to standard methods, as described in the patent and scientific literature for eukaryotic, in particular CHO cells. Cultivation takes place in the perfusion reactor in culture medium which is free from animal components. The harvest takes place continuously over a period of up to 50 days.

Each fermentation solution to be analyzed is desalted and concentrated prior to isoelectric focusing. For this purpose, initially at a total EPO content of about 14 mg / L (is determined by ELISA test) 15 mL of the sample with the Ultrazentrifugationskit and a molecular weight CutOff of 1 OkDa by centrifugation (60 min at 4000 g and 60 min at 14000 g) to 200 .mu.l concentrated and 12 .mu.l of the concentrate with 28 .mu.l ultrapure water and 10 .mu.L Ethanol mixed and stored for 60 min at -20 ⁰ C. Subsequently, the sample solution is centrifuged in a refrigerated centrifuge (20 min, 16100 g, 0 ⁰ C) and the LJ supernatant of the solution for isoelectric focusing used (IEF sample solution).

Isoelectric focusing starts with a prefocussing of the

Gels (Blank PreNets, Serva, 20 to 60 minutes to approximately 400 Vh) to form the pH gradient from pH 3 to pH 6 (Servalyt ™ 3-6). For this, the voltage and current are set to approximately 300 V and 3.5 mA (cathode buffer: 1 M glycine, anode buffer: 25 mM aspartic acid and glutamic acid in each case). Then 15 μL each of the IEF sample solution and the control sample (Erypo® preparation) are pipetted onto a Sample Application Piece (Serva) and the solutions are isoelectrically focused for approx. 2000 Vh by applying the voltage. Then the focus is briefly interrupted and the Sample Application Pieces are removed before the focusing process continues for a further approx. 2500 Vh. After exceeding 4500 Vh of sample focusing time, isoelectric focusing is stopped and the gel in pre-cooled (4 ° C) blotting buffer (200 mL of 10X Tris / Glycine Buffer (Bio-Rad) diluted to 2 L with ultrapure water and 400 mL of methanol ) Incubated for 15 min.

[0043] In parallel with this, according to the information provided by the supplier, the preparation of the Immobilon P blotting membrane (Millipore) is carried out. Thereafter, the gel is blotted onto the membrane under the following conditions: 50 V constant for 50 min in blotting buffer (1X Tris / Glycine with 20% methanol, Bio-Rad). Immediately after protein transfer, three washes are sequentially performed first in methanol and then twice in water for 30 seconds each. The membrane is then placed in blocking solution (5% skim milk powder solution (Bio-Rad) in IxTBS buffer (Bio-Rad)) and incubated at room temperature for 60 min with gentle shaking. After removing the blocking solution, a washing three times in TBST occurs (0.5% Tween ^® 20 in TBS (Bio-Rad)). This is followed by at least 4 hours incubation in the first antibody solution (1% BSA (Sigma) in 30 ml of IXTBS buffer (Bio-Rad) with 60 μl 500 mM sodium azide solution of the 50 μl anti-EPO mouse (RD system)).

This is followed by another washing procedure with TBST, TBS,

0.7% aqueous acetic acid and then again with TBST, with the memo

5 bran in the acetic acid solution is incubated for 60 min. Then the treatment of the membrane with the first antibody solution is repeated under identical conditions. After washing three times with TBST, the membrane is treated with the second antibody solution (1% BSA (Sigma) in 30 ml of IXTBS buffer (Bio-Rad) with 60 μl of 500 mM sodium azide solution of 35 μl of anti-mouse IgG (Sigma) - lo is added). After washing three times in TBST and rinsing twice with AP buffer (10 ml of 5 M saline solution are diluted to 1 L volume of solution together with 50 ml of 1 M Tris-HCl solution (pH 9.5) and 5 ml of 1 M magnesium chloride solution with ultrapure water) the gel is stained with BCIP / NBT Liquid Substrate System (Sigma) (10 -20 min at room temperature with gentle shaking). By adding AP stop solution (10 ml 0.5 M Na-EDTA solution (pH 8.0) with 20 mL 1 M Tris-HCl solution (pH 8.0) with ultrapure water to 1 L solution volume diluted) is the color reaction finished and washed the membrane again with ultrapure water. After air drying, the membrane can be evaluated visually or densitomethsch (see Figure 1).

20 The EPO content determination of the culture supernatants is carried out by means of

EPO ELISA test from Roche Diagnostics GmbH (photometric enzyme-linked immunosorbent assay for the quantitative in vitro determination of erythropoietin in human serum / plasma for research purposes using antibody precoated microtiter plates).

25 For the individual fractions from the perfusion reactor (total fermentation time 47 days), EPO contents are determined in the following ranges: Fraction 1 (fermentation until day 5): approx. 80 mg / L

Fraction 2 (fermentation until day 13): about 75 mg / L

Fraction 3 (fermentation until day 20): about 140 mg / L

Fraction 4 (fermentation until day 25): approx. 80 mg / L Fraction 5 (fermentation until day 32): approx. 150 mg / L

The evaluation of the results according to Figure 1 shows that in particular the purification of the fractions 2 and 4 and optionally still the fraction 3 is useful. In these fractions, the proportion of therapeutically useful isoforms in relation to the total EPO content is highest. In contrast, although the highest EPO content is detected for fraction 5 according to the ELISA test, the desired isoforms compared to the Erypo® reference material are only present in traces.

Claims

claims

1. A method for determining the isoform composition of erythropoietin comprising the following steps: a) isoelectric focusing of a sample containing erythropoietin in a gel over a pH range, the lower limit of 2.5 to 3.5 and the upper Limit of 5 to 8, wherein the erythropoietin-containing sample is from a culture supernatant of erythropoietin-producing eukaryotic cells; b) transferring the proteins contained in the gel and separated on a lo membrane; c) Detection of erythropoietin bound to the membrane by specific antibodies.

2. The method according to claim 1, wherein in step c) first antibodies directed against erythropoietin bind to the erythropoietin bonded to the membrane, and the binding of the first antibodies to the erythropoietin bound to the membrane is detected, while the first antibody is bound to the erythropoietin bound to the membrane.

3. The method according to claim 2, characterized in that the detection of the binding of the first antibody to the bound to the membrane

Erythropoietin is carried out by second antibodies directed against the first antibodies.

4. The method according to claim 1, characterized in that in step c) directed against erythropoietin first antibody bind to the erythropoietin bound to the membrane, and that directed against the first antibody

25 second antibodies bind to the first antibody bound to erythropoietin.

5. The method according to claim 3 or 4, characterized in that an enzyme, preferably alkaline phosphatase, is covalently bound to the second antibody, which generates a color reaction by catalytic reaction of a substrate.

5. The method according to any one of claims 1 to 5, characterized in that after incubation of the membrane with a first antibody directed against erythropoietin one or more washing steps and then carried out again incubation of the membrane with the first antibody directed against erythropoietin becomes.

The method of any one of claims 1 to 6, characterized in that at least one solution used in the washing steps following incubation of the membrane with the first antibody directed against erythropoietin contains an organic acid in an aqueous medium.

8. The method according to claim 7, characterized in that the solution is 0.1 to 1.5% by weight, preferably 0.5 to 1% by weight, particularly preferably

Contains 0.6 to 0.8 wt .-% of the organic acid.

9. The method according to claim 7 or 8, characterized in that the organic acid is a mono-, di- or tricarboxylic acid, preferably selected from the group consisting of acetic acid, propionic acid, lactic acid,

Succinic acid, ascorbic acid, adipic acid or citric acid, and most preferably acetic acid.

10. The method according to any one of claims 1 to 9, characterized in that a polyvinylidene fluoride membrane is used as the membrane.

11. The method according to any one of claims 1 to 10, characterized in that for the isoelectric focusing polyacrylamide gels are used, which are mounted on an inert carrier film.

12. The method according to any one of claims 1 to 11, characterized in that are used to adjust the pH of the gel ampholines, which adjust a pH range of pH 3 to pH 6 during the isoelectric focusing in the gel.

The method of any one of claims 1 to 12, characterized in that the erythropoietin-containing sample is from a culture supernatant of erythropoietin-producing eukaryotic cells maintained in perfusion reactors.

14. The method according to claim 13, characterized in that the lo erythropoietin-containing sample is desalted before the isoelectric focusing and optionally concentrated.

15. The method according to any one of claims 1 to 14, characterized in that the determination of the isoform composition of the erythropoietin takes place during the fermentation.

i5 16. A method of in-process control of culture supernatants, in particular from perfusion reactors derived from the fermentation of erythropoietin-producing eukaryotic cells, comprising the steps of: a) determining the isoform composition of erythropoietin according to the method of any one of claims 1 to 15;

20 b) Determination of the content of erythropoietin in the sample, preferably by ELISA; c) Selection of the fermentation solutions for the purification of erythropoietin on the basis of the values obtained in steps a) and b) and information, or continuation of the fermentation.

25