WO2019105444A1 - Method for producing protein - Google Patents

Abstract

The present invention falls within the field of biomedicine, and mainly provides a method for preparing and producing an antigen binding protein with regulated glycosylation modification, wherein the method comprises adding amino acids to the culture system during the cell culture cycle. The method can be used to regulate the glycosylation modification for the antigen binding protein, to improve the stability of the protein, to make the function associated with the complement-dependent cytotoxicity effect of the protein change, and to improve the ability to control the production process and the batch-to-batch consistency of the protein.

Description

Protein production method Technical field

The invention belongs to the field of biomedicine, and in particular relates to a preparation method and a production method of a protein, in particular to a preparation method and a production method of an antibody.

Background technique

Glycosylation is an important post-translational modification of therapeutic antibodies. The two most common forms of glycosylation are O-saccharides (oligosaccharides linked to hydroxyl-containing amino acids such as Ser, Thr or Tyr) and N-saccharides (oligosaccharides and Asn-) X-Ser/Thr is linked, X is any amino acid except Pro), and for CHO cells, N-glycosylation is usually located at the Asn297 site of the antibody heavy chain Fc fragment (CH2 region). Protein glycosylation starts in the endoplasmic reticulum and Golgi, and removes Glucose and Mannose residues and binds N-acetylglucosamine (GlcNAc) under the action of glycosyltransferase and glycosidase and substrate. ), sialic acid, fucose, galactose and other sugar groups form complex and diverse glycoforms, which directly affect antibody immunogenicity and biology. Function (Hossler P, Khattak SF, Li ZJ. Optimal and consistent protein glycosylation in mammalian cell culture [J]. Glycobiology, 2009, 19(9): 936–949; Zheng K, Yarmarkovich m, Bantog C, et al. Of glycosylation pattern on the molecular properties of monoclonal antibodies [J]. MAbs, 2014, 6(3), 649-658). Among them, the establishment of galactosylation is the use of galactose as a component of the galactosylation chain reaction, galactose is linked to the adjacent N-acetylglucosamine by galactosyltransferase, galactosylation modification By affecting the spatial conformation of the Fc fragment of the antibody, increasing its ability to bind to the C1q receptor, thereby achieving complement-dependent cytotoxicity (CDC effect) (LIU LM. Antibody Glycosylation and Its Impact on the Pharmacokinetics and Pharmacodynamics of Monoclonal) Antibodies and Fc-Fusion Proteins [J]. Journal of pharmaceutical sciences, 2015, 104: 1866-1884.).

For antibodies with a complement-dependent cytotoxicity (CDC) effect, increased galactosylation modification increases its CDC effect and enhances its killing effect on target cells. It is also important to control the galactosylation modification during the process for antibodies that do not require or require a CDC effect (such as the use of different lgG isoforms or Fc sugar modification sites to engineer their loss of Fc receptor binding function). The galactosylation modification is highly susceptible to various factors such as upstream process parameters, culture scale, reactor material, site change and raw materials. The batch-to-batch consistency of galactosyl modification is also a measure of the controllability and stability of the antibody drug production process. Important indicator. At present, galactosylation can be regulated by regulating pH, pCO2 or metal ion additives in the upstream process, but such regulation is usually accompanied by a decrease in the amount and yield of other proteins. WO2012149197 employs a method of adding manganese or galactose to complete medium to modulate the level of galactosylation of recombinantly expressed antibodies. Low galactosylation of antibodies and batch-to-batch instability often occur in the development of monoclonal antibody drugs, and a method for increasing galactosylation of antibodies is required to enhance galactosylation and enhance the process. Controllability, improve the quality of antibodies.

Summary of the invention

It is an object of the invention to at least provide a method of preparing and/or producing a protein. In one aspect, the invention provides a method of making and/or producing a glycosylated regulated protein. In another aspect, the invention provides a method of making and/or producing a glycosylated regulated antigen binding protein, optionally, the antigen binding protein is an antibody or fragment thereof. It is also an object of the present invention to provide a method of modulating protein glycosylation modification. In one aspect, the invention provides a method of modulating glycosylation modification of a protein. In another aspect, the invention provides a method of modulating a glycosylation modification of an antigen binding protein. In yet another aspect, the invention provides a method of modulating a galactosylation modification of an antibody or fragment thereof.

The object of the invention is at least to provide for the use of amino acids, in particular to provide for the use of amino acids to modulate glycosylation modifications in vitro. In one aspect, the invention provides the use of an amino acid to modulate glycosylation modification of an antigen binding protein in vitro. In another aspect, the invention provides the use of an amino acid to modulate the glycosylation of an antigen binding protein during the preparation and/or production of an antigen binding protein, optionally, the antigen binding protein is an antibody or fragment thereof. It is also an object of the present invention to provide a method of modulating protein glycosylation modifications including, but not limited to, modulation of galactosylation modifications. In yet another aspect, the invention provides the use of a galactosylation modification of an amino acid-modulating antibody or fragment thereof.

The present invention also aims to provide an amino acid application. In one aspect, the present invention provides an amino acid for enhancing the stability of an antigen binding protein. In another aspect, the present invention provides an amino acid for improving an antigen binding protein or antibody drug production process. Application in controllability and/or inter-batch consistency. Alternatively, the antigen binding protein is an antibody or a fragment thereof.

It is an object of the present invention to at least provide a method of preparing and/or producing a protein having altered function of a complement dependent cytotoxicity (CDC effect), which may be a decrease in function or function. Strengthening. In one aspect, the invention provides a protein method for the preparation and/or production of a functional enhancement that causes a CDC effect. In another aspect, the invention provides a method of making and/or producing a functionally enhanced or enhanced antigen binding protein that causes or triggers a CDC effect, optionally, the antigen binding protein is an antibody or fragment thereof.

In some aspects, the adjustment refers to up- or down-regulation. In some specific embodiments, the modulation of glycosylation refers to an increase in galactosylation modification, and in other specific embodiments, the modulation is a reduction in galactosylation modification. In some specific embodiments, the modulation of the CDC effect refers to enhancing the function of the antigen binding protein to elicit a CDC effect, and in other specific embodiments, the modulation of the CDC effect is to reduce the CDC effect of the antigen binding protein. The function.

In some aspects, the method modulates the amount of amino acids in the culture medium during cell culture. In some specific embodiments, the culture medium is a medium.

In some aspects, the amino acid is selected from the group consisting of amino acids that make up a protein. In some aspects, the amino acid is Lysine, optionally the amino acid is L- and/or D-lysine, preferably L-lysine.

Low galactosylation of antibodies and instability between batches often occur in the development of monoclonal antibody drugs. In one aspect, the invention provides a method of making or producing an antibody or fragment thereof, the method comprising adding an additive to a culture system under cell culture conditions. For example, an additive or a feed is added to the culture medium under cell culture conditions. In another aspect, the invention provides a method of increasing galactosylation modification of an antibody or fragment thereof, and in particular to providing a method of increasing galactosylation modification of an antibody or fragment thereof by a medium additive or feed . Alternatively, an amino acid is added to the culture medium in one or more equal or unequal amounts in the culture cycle under cell culture conditions.

In a specific embodiment, the total amount of additional amino acids is from about 0 to 40 g/L of the medium, calculated in w/v, optionally, the total amount of additional amino acids is about 1 g/L, about 2 g/L, about 3 g. /L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L, about 13 g. /L, about 14 g/L, about 15 g/L, about 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L or about 40 g/L.

In a specific embodiment, the total amount of additional amino acids is from about 2 to 12 g/L of medium; preferably, the total amount of additional amino acids is from about 4 to 10 g/L; more preferably, the total amount of additional amino acids It is about 8 g/L of medium.

In some aspects, the additional amino acid is selected from the group consisting of amino acids that make up the protein.

In some aspects, the additional amino acid is selected from the group consisting of lysine, and optionally, the amino acid is L- and/or D-lysine, preferably L-lysine.

In a specific embodiment, the total amount of additional lysine is from about 0 to 40 g/L of the medium, calculated in w/v, and optionally, the total amount of additional lysine is about 1 g/L, about 2 g/ L, about 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/ L, about 13 g/L, about 14 g/L, about 15 g/L, about 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L or about 40 g/L of medium.

In a specific embodiment, the total amount of additional lysine is 4-10 g/L of medium; preferably, the total amount of additional lysine is 4-8 g/L; more preferably, supplemental The total amount of lysine was 8 g/L of medium.

In some embodiments, the amino acid is added 1-8 times, preferably 2 times, 3 times or 4 times during the culture period.

In a specific embodiment, lysine is added at a particular time in the culture cycle. Optionally, add 2, 3, 4, 5 times on any two, three, four, five, six, seven, eight, nine, ten or eleven days of the culture cycle 6, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times or more. In a specific embodiment, the supplementation may be an equal amount of supplementation each time, or an unequal amount of supplementation each time.

In some embodiments, the amino acid is supplemented once every day on the Nth day, the N+2 day, the N+4th day, and the N+6th day of the culture cycle; in a specific embodiment, during the culture cycle Adding amino acids twice on any of the Nth day, the N+2 day, the N+4th day, and the N+6th day; in another specific embodiment, on the Nth day of the culture cycle, Amino acids were added 3 times for any of the three days of N+2 days, N+4 days, and N+6 days. Wherein N is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 3, 4 or 5, more preferably 4. The amino acid is selected from the group consisting of lysine, and optionally the amino acid is L- and/or D-lysine, preferably L-lysine.

In some embodiments, the amino acid is supplemented once every day for each day of the culture medium on the Nth day, the N+2 day, the N+4 day, and the N+6 day of the culture cycle. About 4-10 g of amino acid, the total amount is about 4-10 g / L of the medium, wherein preferably about 8 g of amino acid per liter of the medium is added, the total amount is about 8 g / L of the medium; on the Nth day of the culture period, On any of the N+2 days, the N+4th day, and the N+6th day, the amino acid is added twice, and about 2-5g of amino acid is added per liter of the culture medium, and the total amount is about 4-10g. / L medium, wherein preferably about 4 g of amino acid is added per liter of medium per time, the total amount is about 8 g / L of medium; in another specific embodiment, on the Nth day, N + 2 days of the culture period Adding amino acid 3 times for any three days on the N+4th day and the N+6th day, the total amount is about 4-10g/L medium, preferably the total amount is about 8g/L medium; In an embodiment, the amino acid is supplemented four times on the Nth day, the N+2 day, the N+4th day, and the N+6th day of the culture cycle, and the total amount is about 4-10 g/L of the medium, preferably the total amount. It is about 8 g/L of medium. Wherein N is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 3, 4 or 5, more preferably 4. The amino acid is selected from the group consisting of lysine, and optionally the amino acid is L- and/or D-lysine, preferably L-lysine.

In some embodiments, the amino acid is added once every day on the 4th, 6th, 8th, and 10th day of the culture cycle, and about 4-10 g of amino acid is added per liter of the culture medium. The total amount is about 4-10 g/L of the medium, wherein preferably about 8 g of amino acid is added per liter of the medium, and the total amount is about 8 g/L of the medium; on the 4th, 6th, and 8th of the culture period. On any two days of the day and the 10th day, the amino acid is added twice, and about 2-5 g of amino acid is added per liter of the culture medium, and the total amount is about 4-10 g/L of the medium, and preferably, the culture is carried out per liter. About 4 g of amino acid is added to the base, and the total amount is about 8 g/L of the medium; in another specific embodiment, any three days of the fourth, sixth, eighth, and tenth days of the culture period are supplemented. Adding amino acid 3 times, the total amount is about 4-10 g / L medium, preferably a total amount of about 8 g / L medium; in another specific embodiment, on the 4th day, 6th day, 8th of the culture period Amino acids were added 4 times a day and on day 10, for a total amount of about 4-10 g/L of medium, preferably a total amount of about 8 g/L of medium. The amino acid is selected from the group consisting of lysine, and optionally the amino acid is L- and/or D-lysine, preferably L-lysine.

In a specific embodiment, on the 4th, 6th, 8th, and 10th day of the culture cycle, lysine is added in equal amounts, that is, about 2 g per liter of the medium is added each time, and the total amount is Approximately 8 g/L of medium.

In some aspects, the amino acids of the invention may be added to the culture medium or culture system in the form of a solid or a prepared concentrate.

In some aspects, the cell culture medium can comprise a product or component that is serum free and/or animal free. In some embodiments, the cell culture medium can be chemically defined, wherein all chemical components are known. As will be appreciated by those skilled in the art, an animal or mammalian cell can be cultured in a defined medium without undue experimentation by those skilled in the art, as appropriate to the particular cell being cultured. Commercially available media can be utilized including, but not limited to, CD OptiCHO, Hycell, CD CHO, Growth A, Dynamis, Iscove's Modified Dulbecco's Medium, RPMI 1640 and Minimum basal medium-α (MEM-α), Dulbecco's Modification of Eagle's Medium (DMEM), DME/F12, αMEM, Earl's basic culture with Earl BSS Basal Medium Eagle with Earle's BSS, high glucose DMEM with glutamine, high glucose DMEM without glutamine, low glucose DMEM without glutamine, DMEM with glutamine: F12 1:1, GMEM (Glasgow's MEM), GMEM with glutamine, Grace's Complete Insect Medium, Grace Insect Medium without FBS, Ham's F-10 with glutamine, with Ham's F-12 for glutamine, IMDM with HEPES and glutamine, IMDM with HEPES and no glutamine, 15 (Leibovitz) (2X) without glutamine or phenol red, no glutamine 15 (Leibovitz), McCoy's 5A Modified Medium, medium 199, Eagle's MEM without glutamine or phenol red (2X), Eagle with glutamine MEM-Ear BSS, glutamine-free Eagle's MEM-Ear BSS, glutamine-free Eagle's MEM-Hankes BSS, glutamine-containing NCTC-109, with glutamine Pharmacy of Richter's CM Medium (Richter's CM Medium), RPMI 1640 with HEPES, glutamine and/or penicillin-streptomycin, RPMI 1640 with glutamine, RPMI 1640 without glutamine, Schneider Schneider's Insect Medium or any other medium known to those skilled in the art that is formulated for a particular cell type. Appropriate concentrations or amounts of supplemental components or ingredients, including optional components, may be added to the above exemplary media as needed or as needed and as known and practiced by one of ordinary skill in the art.

In some aspects, the cell culture can also supplement the feed of specific nutrients that are difficult to formulate or that are rapidly consumed in cell culture. Such nutrients may be amino acids such as tyrosine, cysteine and/or cystine. For example, the concentrated tyrosine solution can be fed separately to cell cultures grown in cell culture medium containing tyrosine. Concentrated solutions of tyrosine and cystine can also be fed separately to cell cultures grown in cell culture medium lacking tyrosine, cystine and/or cysteine. Independent feeding can begin before the preparation or production period or at the beginning of the preparation or production period. The cell culture medium can be treated using a method or apparatus to sterilize or sterilize the medium prior to addition to the bioreactor and/or cell culture.

In some aspects, the cell lines or host cells used in the invention are genetically engineered to express a protein of commercial or scientific concern. Cell lines are typically derived from lineages produced by original cultures that can maintain culture for an indefinite period of time. The cells may contain, for example, an expression vector (construct) introduced by transformation, transfection, infection or injection, such as a plasmid or the like, which has a coding sequence encoding a protein expressed and produced in a culture method, or a portion thereof. Such expression vectors contain the elements necessary for insertion of the coding sequence for transcription and translation. Sequences containing the proteins and polypeptides produced by the encoding, as well as expression vectors suitable for transcriptional and translational control elements, can be constructed using well known and readily practiced methods. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in J. Sambrook et al, 2012, Molecular Cloning, A Laboratory Manual, 4th edition Cold Spring Harbor Press, Plainview, NY or any prior version; FMAusubel et al, 2013, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY or any prior version; Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, all incorporated herein by reference for all purposes.

In the present invention, animal cells, mammalian cells, cultured cells, animal or mammalian host cells, host cells, recombinant cells, recombinant host cells and the like are all terms of cells which can be cultured according to the method of the present invention. These cells are cell lines obtained or derived from mammals and are capable of growing and surviving when subjected to monolayer culture or suspension culture in a medium containing appropriate nutrients and/or other factors. Proteins are typically selected for expression and secretion, or can be engineered at the molecular level to express and secrete large amounts of specific proteins, more particularly related glycoproteins, to cells in the culture medium. It will be appreciated that the protein produced by the host cell may be endogenous to the host cell or homologous thereto, or the protein may be heterologous to the host cell (ie, foreign), for example, the human protein may be from the Chinese hamster ovary (CHO) Host cell production and secretion. In addition, mammalian proteins or proteins originally obtained or derived from mammalian organisms can be obtained by the methods of the invention and in some protocols can be secreted by the cells into the culture medium.

The methods of the invention can be used to culture a variety of cells. In one embodiment, the cultured cells are eukaryotic cells, such as plant and/or animal cells. The cells can be mammalian cells, fish cells, insect cells, amphibian cells or avian cells. A variety of mammalian cell lines suitable for culture growth are available from storage facilities as well as commercial suppliers. Cells that can be used in the methods of the invention include, but are not limited to, Chinese hamster ovary cells (CHO), CHO-S cells, CHO-DG44 cells, or any other cell type known to those skilled in the art.

The invention can be used for cultivating micro-reaction systems, and can also be used in pilot-scale small bioreactors, such as bioreactors for culturing 1L, 2L and 3L, and large bioreactors for production, such as bioreactors such as 10L and 50L. .

In one aspect, the invention provides a method of making and/or producing a glycosylated regulated antigen binding protein, in another aspect, the invention provides a method of modulating a glycosylation modification of an antigen binding protein, In one aspect, the invention provides a method of modulating a galactosylation modification of an antibody, or a fragment thereof, including but not limited to the following antibodies or fragments thereof: Abagovomab, abciximab (Abciximab), Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomabpentetate , Amatuximab, Anatumomabmafenatox, Anifrolumab, Anrukinzumab, Apolizumab, Asimo Monoclonal antibody (Arcitumomab), azeizumab (Aselizumab) Atumumab, Atelizumab, Atorolimumab, Bapineuzumab, Basiliximab, Batilizumab (Bavituximab), Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bay Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab mertansine, Blinatumomab, Bruzinzumab (Blosozumab), Brentuximabvedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Mo Cantuzumabravtansine, Caplacizumab, Capromabpendetide, Carlumab, Catusmaxomab, cBR96-soft Doxorubicinimoconjugate, ceclizumab, Peizhuzhu Certolizumabpegol, Cetuximab, Citatuzumabbogatox, Cixutumumab, Clazakizumab, Cleriximab ), Clivatuzumabtetraxetan, Conatumumab, Concizumab, Crenezumab, Dacetuzumab, Da Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Dimobizumab (Detumomab), Dorlimomabaritox, Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Emetrex Monoclonal antibody (Ecromeximab), Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eide Eldelumab, Elotuzumab, Elsilimomab, Evanvatuzuma b), Enlimomab pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evozumab (Evolocumab), Exbivirumab, Fanolesomab, Faralimomab, Farartuzumab, Fasimumab, FBTA05, non-dimensional Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fantolizumab ), Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Caliximab Anti-Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gitutobe Gemtuzumabozogamicin, Gemtuzumab, Gevokizumab, Girentuximab, Glembatumumabvedotin, Golimumab (Golimumab), Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, IMAB362, Imciromab, Ingtaluzumab, Inclacumab, Indatuximabravtansine, Infliximab (Infliximab), Inolimomab, Inotuzumab ozogamicin, Intetumumab, Ipilimumab, Intolimumab ( Iratumumab), Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Ram Lamalizumab, Lebrikizumab, Lemalesomab Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirimumab, Rodehizumab (Lodelcizumab) ), Louvotuzumabmertansine, Lucatumumab, Lumiliximab, Mapatumumab, Margetuximab, Mars Maslimomab, Matuzumab, Mavrilimumab, Mepolizumab, Metelimumab, Mirabuzumab (Milatuzumab) ), Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Movi Metavizumab, Moxetumomabpasudotox, Moromonab-CD3, Nacolomabtafenatox, Namilumab, Tanamo Anti-Naptumomabestafenatox, Narnatumab, Natalizumab, Nebakuzumab (Nebacumab), Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, 巯Nofetumomabmerpentan, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olatimumab (Olaratumab), Orukakiumab, Omalizumab, Onartuzumab, Ontuxizumab, Oportuzumabmonatox , Oregovomab, Orticumab, Otelixizumab, Otletuzumab, Oxelumab, Ozambiz Anti-(Ozanezumab), Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Panikomab, Panor Panobacumab, Parsa attuzumab, Pascolizumab, Patelizumab (Pateclizumab) ), Patritumab, pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pegzumab (Pexelizumab), Pidilizumab, Pinatuzumabvedotin, Pintumomab, Placulumab, Plalatuzumabvedotin ), Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, Rekutu Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Riga Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab , Romosozumab, Rontalizumab, Rovelizumab, Ruclizumab (Ruplizum) Ab), Samalizumab, Sarulumab, Satumomabpendetide, Secukinumab, Seribantumab, Stu Setoxaximab, Sevirumab, SGN-CD19A, SGN-CD33A, Sibrotuzumab, Sifalimumab, Siltuximab, West Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab , Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacituzumab Tetraxetan), Tadocizumab, Talizumab, Tanezumab, Taplitumomabpaptox, Tefibazumab, Ate Telimomabaritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN1412, Ticilimumab (tremelimumab), Tigatuzumab Tildrakizumab, TNX-650, Tocilizumab (atlizumab) ), Toliizumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, TRBS07, Qu Tregalizumab, Tremelimumab, Tucotuzumabcelmoleukin, Tuvirumab, Ublituximab, Urelumab (Urelumab) ), Urtoxazumab, Ustekinumab, Vantictumab, Vapaliximab, Vatelizumab, Vidozhu Monoclonal antibody (Vedolizumab), Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab Vortexuzumabmafodotin, Votumumab, and Zalamumum Ab), Zanolimumab, Zatuximab, Ziralimumab, Zolimomab aritox, and inventions of PCT/US2015/043723 and CN201580042278.8 Any antibody or fragment thereof disclosed in the patent application, including but not limited to: humanized hu13C5-hIgG1, hu13C5-hIgG4, hu5G11-hIgG1, hu5G11-hIgG4, and/or chimeric ch13C5-hIgG1, ch13C5-hIgG4, ch5G11 -hIgG1, ch5G11-hIgG4, and/or hybridoma antibodies 13C5, 5G9, 5G11, 8C6, 7B4, 4D1, 4A8, 8H4, 8H3, 15F1, etc. (see WO2016022630 and CN107001463A). The antigen binding protein may further comprise at least 80% (eg, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the antigen binding protein: the heavy chain CDR1 sequence is Thr-Tyr-Gly-Val- His (SEQ ID NO: 1), CDR2 sequence is Val-Ile-Trp-Arg-Gly-Val-Thr-Thr-Asp-Tyr-Asn-Ala-Ala-Phe-Met-Ser (SEQ ID NO: 2) , the CDR3 sequence is Leu-Gly-Phe-Tyr-Ala-Met-Asp-Tyr (SEQ ID NO: 3), and/or the light chain CDR1 sequence is Lys-Ala-Ser-Gln-Ser-Val-Ser-Asn -Asp-Val-Ala (SEQ ID NO: 4), the CDR2 sequence is Tyr-Ala-Ala-Asn-Arg-Tyr-Thr (SEQ ID NO: 5), and the CDR3 sequence is Gln-Gln-Asp-Tyr-Thr -Ser-Pro-Tyr-Thr (SEQ ID NO: 6) antibody or fragment of antibody.

Published data of PCT International Application WO2016022630 discloses a new class of anti-PD-L1 antibodies with high affinity for PD-L1, which can significantly inhibit the interaction of PD-L1 and PD-1 on the cell surface and significantly promote T The cells secrete IL-2 and IFN-[gamma], the entire contents of which are hereby incorporated by reference in its entirety, the entire contents of the entire disclosure of the disclosure of the disclosure of

Galactosylation modification is an important post-translational modification of monoclonal antibodies, for example, affecting drug drug controllability, stability, batch-to-batch consistency, and in addition, it can increase antibody CDC effect, whereas galactosylation is more in culture process. Difficult regulation, different culture scales and subtle differences in process parameters have a significant impact on the level of galactosylation of antibodies. Surprisingly, the inventors found that the galactosylation modification can be regulated by the medium additive, so that the galactosylation modification is significantly improved, and the controllability and reproducibility of the process are enhanced, and the protein batch is ensured. Second-order consistency; while achieving galactosylation modification, other antibody masses such as acid region variants and polymer content were also significantly reduced, improving the quality of the antibody.

Explanation of terms:

The term "antibody" as used herein refers to a binding protein having at least one antigen binding domain. The antibodies and fragments thereof of the invention may be the entire antibody or any fragment thereof. Thus, antibodies and fragments of the invention include monoclonal antibodies or fragments thereof and antibody variants or fragments thereof, as well as immunoconjugates. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab)' fragments, Fv fragments, isolated CDR regions, single chain Fv molecules (scFv), and other antibody fragments known in the art. Antibodies and fragments thereof can also include recombinant polypeptides, fusion proteins, and bispecific antibodies. The antibodies and fragments thereof disclosed herein can be of the IgGl, IgG2, IgG3 or IgG4 isotype. The term "isotype" refers to the type of antibody encoded by the heavy chain constant region gene. The antibodies and fragments thereof of the invention can be derived from any species including, but not limited to, mice, rats, rabbits, primates, llamas, and humans. The antibody and fragments thereof can be chimeric antibodies, humanized antibodies or intact human antibodies. In one embodiment, the antibody is an antibody produced by a mouse-derived hybridoma cell line. In one embodiment, the antibody is a murine antibody. In another embodiment, the antibody is a chimeric antibody. In another embodiment, the chimeric antibody is a mouse-human chimeric antibody. In another embodiment, the antibody is a humanized antibody. In another embodiment, the antibody is derived from a murine antibody and is humanized.

The term "chimeric antibody" as used herein is an antibody having at least a portion of a heavy chain variable region derived from one species and at least a portion of a light chain variable region; and a constant derived from another species At least part of the district. For example, in one embodiment, a chimeric antibody can comprise a murine variable region and a human constant region.

The term "humanized antibody" as used herein is an antibody comprising a complementarity determining region (CDR) derived from a non-human antibody; and a framework region derived from a human antibody and a constant region.

The term "derived" as used herein, when used to refer to a molecule or polypeptide relative to a reference antibody or other binding protein, means a molecule or polypeptide that is capable of specifically binding to the same epitope as a reference antibody or other binding protein.

The term "cell culture medium" or "medium medium" as used herein refers to a nutrient solution for maintaining, growing, proliferating or amplifying a cell in an artificial environment (outside of a multicellular organism or tissue). For example, a basal medium prepared to support cell growth, a cell for cultivating specific cells, a production medium prepared to promote monoclonal antibody production, and a concentrated medium prepared by concentrating nutrients at a high concentration. . Nutrients and medium components refer to the components constituting the cell culture medium, which are used interchangeably in the present invention.

The term "cell cycle" as used herein refers to the period in which cells are seeded to the reactor culture, and the day 0 of the culture of the reactor is the start date of the culture, and can be recorded as the first day of the culture period.

The term "antigen-binding protein producing cell" as used herein refers to a cell for producing an antigen-binding protein.

The terms "feed medium" and "feed medium" as used herein may refer to a medium consisting of a specific nutrient or a plurality of nutrients, which are both concentrated components of the base medium. Different feed medium components and concentrations can be prepared depending on the cells to be cultured.

The term "complement-dependent cytotoxicity (CDC)" as used herein refers to a cytotoxic effect in which complement is involved, that is, by binding a specific antibody to a corresponding antigen on the surface of a cell membrane to form a complex and activating the classical pathway of complement, the resulting attack membrane The complex exerts a cleavage effect on the target cells.

DRAWINGS

Figure 1 shows the trend of cell density during culture. The abscissa indicates the number of days of culture period (for example, D1 indicates the first day of the culture period), and the ordinate VCD indicates the cell density per ml of culture medium (×10 ⁶ cells);

Figure 2 shows the trend of cell viability during the culture process, the abscissa indicates the number of days of the culture period (for example, D1 indicates the first day of the culture period), and the ordinate VA indicates the percentage of cell viability (%);

Figure 3 protein expression amount;

Figure 4 shows the glycoform distribution of the purified antibody, the abscissa indicates different glycoforms, and the ordinate indicates the percentage (%) of each glycoform;

Figure 5 model analysis: model of fit (Summary of fit);

Figure 6 Model Analysis: Scaled&Centered Coefficients;

Figure 7 Model Analysis: Contour map (4D Contour of G0F, G1Fa, G1Fb and G2F).

Detailed ways

The present invention provides a method of producing an antigen binding protein having increased galactosylation modification, comprising adding an amino acid at a specific time in a culture cycle of a production cell of the antigen-binding protein. The amino acid may be selected from the group consisting of lysine, lysine concentrate or lysine-containing medium. Alternatively, the amino acid is L- and/or D-lysine, preferably L-lysine. The specific time in the production cell culture cycle may be 2 times, 3 days, any 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days of the culture period. Amino acid, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times or more. Amino acid can be added once every day, 2 days, 3 days or 4 days on the Nth day, the N+2 day, the N+4 day, and the N+6 day in the production cell culture cycle. A third, third or fourth amino acid, wherein N is any integer from 1 to 10, preferably 3, 4 or 5, more preferably 4. The total amount of additional amino acids may be from about 0 to 40 g/L of medium, optionally, the total amount of additional amino acids is about 1 g/L, about 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L. About 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L, about 13 g/L, about 14 g/L, about 15 g/L. Approximately 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L, or about 40 g/L of medium, optionally, the additions are added in equal portions.

The present invention provides a method for increasing the stability of an antigen binding protein comprising adding an amino acid at a specific time in a culture cycle of a production cell of the antigen binding protein. The amino acid may be selected from the group consisting of lysine, lysine concentrate or lysine-containing medium. Alternatively, the amino acid is L- and/or D-lysine, preferably L-lysine. The specific time in the production cell culture cycle may be 2 times, 3 days, any 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days of the culture period. Amino acid, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times or more. Amino acid can be added once every day, 2 days, 3 days or 4 days on the Nth day, the N+2 day, the N+4 day, and the N+6 day in the production cell culture cycle. A third, third or fourth amino acid, wherein N is any integer from 1 to 10, preferably 3, 4 or 5, more preferably 4. The total amount of additional amino acids may be from about 0 to 40 g/L of medium, optionally, the total amount of additional amino acids is about 1 g/L, about 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L. About 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L, about 13 g/L, about 14 g/L, about 15 g/L. Approximately 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L, or about 40 g/L of medium, optionally, the additions are added in equal portions.

In the present invention, an amino acid can be used to modulate glycosylation modification of an antigen binding protein in vitro, including adding an appropriate amount of amino acid at a specific time in a culture cycle of the antigen-binding protein producing cell. The amino acid may be selected from the group consisting of lysine, optionally the L- and/or D-lysine, preferably L-lysine, which is a galactosylation of the protein.

In the present invention, an amino acid can also be used to increase the stability of an antigen binding protein, including adding an appropriate amount of amino acid at a specific time in the culture cycle of the antigen-binding protein producing cell. The amino acid may be selected from the group consisting of lysine, optionally the L- and/or D-lysine, preferably L-lysine, which is a galactosylation of the protein.

In the present invention, lysine may be used for preparing an antigen-binding protein which causes a change in function of complement-dependent cytotoxicity, including, modulating the content of lysine in a culture medium of an antigen-binding protein-producing cell to reduce or Enhances the function of antigen-binding proteins that cause complement-dependent cytotoxicity.

In some aspects, the culture period of the production cell of the antigen-binding protein may be a reactor culture period, and the 0th day of the reactor culture is the start date of the culture, which may be recorded as the first day of the culture period.

In some aspects, the antigen binding protein includes, but is not limited to, an antibody or derivative thereof, preferably a monoclonal antibody or a derivative thereof.

The invention is further described in conjunction with the specific embodiments, which are intended to illustrate and not to limit the scope of the invention. Again, the invention is not limited to any of the specific preferred embodiments described herein. It should be understood by those skilled in the art that equivalent substitutions or corresponding improvements to the technical features of the present invention are still within the scope of the present invention. Unless otherwise stated, the reagents used in the following examples are all commercially available products, and the solution can be formulated using conventional techniques in the art. The anti-PD-L1 humanized monoclonal antibody in the examples was obtained according to the method described in WO2016022630.

Example 1 seed solution preparation

PD-L1 humanized monoclonal antibody cryopreserved working cell bank (GS CHO cell line) in a liquid nitrogen tank (1 ml), thawed in a 37 ° C water bath, and transferred to Dynamis (Thermofisher) containing seed medium. The cells were cultured in shake flasks and placed in a carbon dioxide incubator (Thermo). Subculture was carried out at a cell density of about 3.0 to 4.0 × 10 ⁶ cells/ml. After passage, the density was about 0.8 ± 0.2 × 10 ⁶ cells/ml, and the passage medium was Dynamis (containing 100 μg/ml MSX (Sigma)). The culture conditions were: 36.5 ° C, 8% CO ₂ , 130 rpm. A culture solution which is in a logarithmic growth phase and has a good cell state is obtained as a seed liquid.

Example 2 reactor culture

The seed solution was inoculated into an AMBR reactor (Sartorius-stedim, model Ambr15-24), the base medium was Dynamis (Thermofisher), the initial temperature was set to 36.5 ° C, the rotation speed was set to 900 rpm, and DO (Dissolved Oxygen; dissolved) Oxygen) associated O ₂ self-control initial setting is 40%; pH value associated with CO ₂ and 0.5mol / L sodium bicarbonate solution, the initial setting is 7.00 ± 0.20; air Sparger continues to Hengtong, ventilation is 0.02cm ³ / min (ccm); culture period 11 days. The DOE experimental design (MODDE software) was used to optimize the culture parameters pH, DO, cooling temperature and feed concentration. The screening factors were pH, DO, Arg (Sigma), Lys (Sigma) and Feed2 (Irvine). Scientific), pH setting is 6.7, 7.2 and other levels, factor type is Quantitative; DO is set to 20%, 90% and other levels, factor type is Quantitative; Arg (arginine; arginine) The set value is 0, 8g, etc., and the factor type is Quantitative; Lys (lysine; lysine) is set to a total value of 0, 8g per liter of medium. The factor type is Quantitative; the feed concentration is set to 18%, 30%, and the factor type is Quantitative; the response values are G0F, G1Fa, G1Fb, and G2F, and the Frac Fac Res V+ design model is used. The experimental design is shown in Table 1. Arg, Lys and Feed2 are added in equal amounts on D4, D6, D8 and D10, respectively. For example, lysine is added in equal amounts, that is, about 2 g per liter of medium per lysine is added. The amount was about 8 g/L of medium, and both pH and DO were adjusted on D5 days.

Table 1 experimental design (a total of 17 groups of experiments)

Example 3 Antibody Glycoform Detection Method

The sample was digested with PNGase-F to remove N-glycoside, and the protein was precipitated by adding ice ethanol. After centrifugation, the supernatant containing N-glycoside was taken, dried and labeled with 2-AB (Sigma), and the labeled 2-AB Glycan passed the super High performance liquid chromatography (UPLC) detection.

results and analysis:

In this experiment, conditions such as pH, DO, Arg addition concentration, Lys and Feed2 feed concentration were screened. The cell density and cell viability during the culture process are shown in Fig. 1 and Fig. 2. The protein expression after harvest is shown in Fig. 3. It is seen that the cell growth state is good during the culture, and the lysine-added groups are in the plateau. The cell density was maintained above 8×10 ⁶ cells/ml, and the cell viability was maintained above 96%, which was equivalent to the cell density and cell viability of the unadded group. The protein expression of each group of lysine added was greater than 2.5. g/L.

The cell harvesting solution was subjected to one-step purification to determine the glycoform distribution of the antibody. As shown in Fig. 4, the contents of galactosylation modified G0F, G1Fa, G1Fb and G2F at the central points CS2-5, CS2-6 and CS2-7 were respectively maintained stably. At about 33%, 30%, 13%, and 17%, the difference between batches is small.

Model analysis:

Model of fit: In Figure 5, the response values G0F, G1Fa, G2Fb, and G2F have higher R2 values, Q2 values, Model Validity, and Reproducibility, indicating that the model and data fit, model predictability, and model validity. Both sex and repeatability are good.

Scaled&Centered Coefficients: As can be seen from Fig. 6, Lys, pH and DO are significant values of response value G0F, and both are negatively correlated with G0F; Lys and pH are significant items of response value G1Fa, and both are negatively correlated with G1Fa. Lys and pH were significant values of G1Fb, and all were positively correlated with G1Fb; Lys, pH, Feed2 and DO were significant G2F responses, Lys, pH and Feed2 were positively correlated with G2F, and DO was negatively correlated with G2F; In addition, there are interactions such as pH*Lys, DO*Feed2 and Arg*Feed2.

G0F, G1Fa, G1Fb and G2F contour maps: As can be seen from Figure 7, Lys and pH are the significant values of the response values G0F, G1Fa, G1Fb and G2F, and the interaction between the remaining factors and factors is not considered, and the amount of Lys is added. Increase from 0 to 8g / L, G0F can be reduced by 20%; pH from 7.25 to 6.75, G0F can be reduced by 30%; G0F and G1Fa, G1Fb and G2F are mutually converted, reducing G0F can increase antibody galactosylation level .

Conclusion: The addition of Lys can effectively increase the galactosylation modification. The total amount is increased to 8g/L medium. The G0F content is reduced from 45% without added Lys to 25%, and the G1Fa, G1Fb and G2F contents are increased. In addition, from the galactosylation modification of the central points CS2-5, CS2-6 and CS2-7, it can be seen that the addition of Lys can increase the batch-to-batch consistency of galactosylation modification, thereby increasing the stability and controllability of the process. Sex.

Example 4 rituximab and trastuzumab cell culture

According to the corresponding methods of Examples 1-3, seed solution preparation, reactor culture, antibody glycoform detection, and result analysis and model analysis of rituximab and trastuzumab-producing cells were sequentially performed. The results showed that the addition of Lys during the culture process could effectively increase the galactosylation modification of the monoclonal antibody. After the total amount of Lys was 8 g/L, the G0F content of the antibody was significantly decreased.

Example 5 Chemically defined medium culture

According to the corresponding method of Examples 1-3, the medium was replaced with a chemically determined typical CHO medium (the determined chemical composition is the same as that of CN103773732B, claim 1), and the PD-L1 humanized monoclonal was sequentially performed. The antibody was cryopreserved into a seed cell of a working cell bank (GS CHO cell line), reactor culture, antibody glycoform detection, and analysis of results and model analysis. The results showed that the addition of Lys during the culture process could effectively increase the galactosylation modification of the monoclonal antibody. After the total amount of Lys was 8 g/L, the G0F content of the antibody was significantly decreased.

In view of the present disclosure, while the compositions and methods of the present invention have been described in accordance with the preferred embodiments, those skilled in the art may, without departing from the concept, the spirit and the scope of the present invention, The compositions and/or methods described herein, as well as the order of the steps or steps of the methods, are varied.

The disclosures of all of the documents cited herein are hereby incorporated by reference in their entirety to the extent of the extent of the disclosure of the disclosure of the disclosure of the disclosure.

Claims (10)

A method for producing an antigen-binding protein having increased galactosylation modification, characterized in that an amino acid is added at a specific time in a culture period of a production cell of an antigen-binding protein. The method according to claim 1, wherein said amino acid is selected from the group consisting of lysine, lysine concentrate or lysine-containing medium, and optionally, said amino acid is L- and/or D-lysine. Acid, preferably L-lysine. The method according to any one of claims 1 to 2, wherein the specific time in the production cell culture cycle is any 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 in the culture period. Add 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acids on days, 9 days, 10 days or 11 days. The method according to any one of claims 1 to 3, wherein any one of day N, day 2, day 2, day N+4, and day N+6 in the cell culture cycle is produced, 2 Amino acid 1 or 2, 3 or 4 amino acids are added to days, 3 or 4 days, wherein N is any integer from 1 to 10, preferably 3, 4 or 5, more preferably 4. A method according to any one of claims 1 to 4, wherein the total amount of additional amino acids is from about 0 to 40 g/L of the medium, optionally, the total amount of additional amino acids is about 1 g/L, about 2 g/L, About 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L, About 13 g/L, about 14 g/L, about 15 g/L, about 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L or about 40 g/L of the medium, optionally, the supplement Add in equal parts. The use of an amino acid for the in vitro regulation of glycosylation modification of an antigen binding protein is characterized in that an appropriate amount of amino acid is added at a specific time in the culture cycle of the antigen-binding protein producing cell. The use according to claim 6, wherein the amino acid is selected from the group consisting of lysine, optionally the amino acid is L- and/or D-lysine, preferably L-lysine, said glycosylation It is the galactosylation of proteins. The use of lysine in the preparation of an antigen-binding protein which changes the function of complement-dependent cytotoxicity, characterized in that the content of lysine in the culture medium of the antigen-binding protein-producing cell is regulated to reduce or enhance the antigen The function of the binding protein to cause complement-dependent cytotoxicity. The method according to any one of claims 1 to 8, wherein the culture cycle of the antigen-binding protein producing cell is a reactor culture cycle, and the 0th day of the reactor culture is the start date of the culture, and is recorded as the first of the culture cycle. one day. The method according to any one of claims 1-8, wherein the antigen binding protein is an antibody or a derivative thereof, preferably a monoclonal antibody or a derivative thereof.

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