Post-translational control of recombinant proteins

Abstract

The invention relates to methods of controlling post-translational modification of a recombinant protein In brief, the methods comprising contacting a recombinant protein and / or a cell capable of producing the recombinant protein with one or more sulfinyl group-containing compounds, thereby controlling post-translational modification. The invention further relates to purified antibodies obtained by the methods as described herein.

Description

[0001] Post-translational control of recombinant proteins

[0002] Field of the invention

[0003] The present invention relates inter alia to methods for controlling post-translational modification of recombinant proteins, including therapeutic antibodies. More particularly, certain methods relate to controlling oxidation and / or glycosylation levels of such proteins during their manufacture.

[0004] Background of the invention

[0005] Eukaryotic cells are often used in the pharmaceutical industry to produce therapeutic proteins, as they can lead to post-translational modifications similar as proteins produced in humans.

[0006] During mammalian cell culture, post-translational modifications such as oxidation of amino acid residues may occur. Amino acid residues such as methionine (Met), cysteine (Cys), histidine (His), tryptophan (Trp) and tyrosine (Tyr) are susceptible to oxidation due to their high reactivity with various reactive oxygen species (ROS). The oxidation profile of a therapeutic protein is often considered a critical quality attribute (CQA) that may need to fall within certain parameters to meet therapeutic requirements. For example, excess oxidation can cause changes in structural integrity and conformational stability, which in turn effects the safety and efficacy of the therapeutic protein.

[0007] The modulation of glycosylation is also of importance during protein production since glycosylation levels (such as mannosylation including the amount of high-mannose glycans) may also impact therapeutic utility and safety. The enrichment of glycan structures such as high-mannose is therefore another challenge during process development.

[0008] Typically, large-scale production of therapeutic proteins relies on expression of recombinant proteins by mammalian cells, often cultivated in large bioreactors. This manufacturing process is often associated with significant ROS production, which may be caused by pH, buffer composition, oxygen inhomogeneity, rich cell culture media, high protein expression, waste product build-up or the like. At commercial scale, the process must maximise protein yield from the cells, while maintaining safety of the protein product, as well as efficiency and cost-effectiveness. However, identifying cell culture parameters that can maintain high quality protein product and yield can be difficult, given the complexity of the cell culturing process.

[0009] There remains a need to control post-translational modifications such as oxidation and glycosylation profiles during the manufacture of therapeutic proteins at commercial scale.

[0010] It is an aim of some embodiments of the present invention to mitigate some of the problems identified in the prior art.

[0011] Summary of certain embodiments of the invention

[0012] The invention relates, in part, to the surprising discovery that contacting cells producing recombinant proteins with one or more sulfinyl-group containing compounds regulates post-translational modifications during their production. Advantageously, the methods described herein allow therapeutic proteins to be produced, for example, with decreased oxidation levels and / or enriched glycan structures, including an increase in the proportion or abundance of high-mannose glycans. Advantageously, post-translational modifications can be controlled using sulf iny l-group containing compounds without needing to modify, for example, the cell line used and / or any other upstream and / or downstream processes during manufacture of the protein. In addition, use of sulf iny l-group containing compounds does not negatively impact other CQAs of the protein.

[0013] Accordingly, the invention provides a method of controlling post-translational modification of a recombinant protein, wherein the method comprises:

[0014] (a) providing a eukaryotic cell capable of recombinant expression of the protein; and

[0015] (b) contacting the cell and / or protein with one or more sulfinyl group-containing compounds.

[0016] In certain embodiments, the cell is capable of recombinant expression and secretion of the protein into a culture medium.

[0017] In certain embodiments, the cell is a mammalian cell such as a Chinese Hamster Ovary (CHO) cell or a murine myeloma cell (Sp2 / 0).

[0018] In certain embodiments, the cell and / or protein is contacted with the sulfinyl group-containing compound(s) by supplementing the culture medium with the compound(s).

[0019] As further described herein, any suitable sulfinyl group-containing compound(s) may be used. In preferred embodiments, the sulfinyl group-containing compound(s) has a structure according to the formula: wherein Ri is a substituent, optionally an alkylamine, C1 -C6 alkylamine or C1 -C4 alkylamine, and wherein R2 is a substituent, optionally OH.

[0020] In even more preferred embodiments, the sulfinyl group-containing compound is hypotaurine or an analogue or precursor thereof.

[0021] In certain embodiments, the cell and / or protein is contacted with a solution such as culture medium comprising the sulfinyl group-containing compound(s) at a concentration between about 1 mM to about 100mM, preferably about 30mM to about 60mM. In certain embodiments, the cell and / or protein is contacted with a solution such as culture medium comprising the sulfinyl group-containing compound(s) at a concentration of at least about 8mM or more, preferably about 10mM, 20mM, 30mM or more.

[0022] In some embodiments, the culture medium is manufactured or prepared so as to contain the sulfinyl group-containing compound(s) before cell culture. In such embodiments, the sulfinyl group-containing compound(s) is present in the culture medium before inoculation with the cells.

[0023] In some embodiments, the culture medium is a production medium. Typically, the production medium is supplemented with the sulfinyl group-containing compound(s) prior to inoculation of the production medium with the cells.

[0024] In some embodiments, the culture medium is a feed medium. Typically, the feed medium is supplemented with the sulfinyl group-containing compound(s) during the cell’s growth or production phase.

[0025] The methods of the invention may be used to control post-translation modification of any recombinant protein as further described herein. In preferred embodiments, the protein is an antibody, an antigenbinding portion of an antibody, a hormone, an Fc-fusion polypeptide, an albumin fusion polypeptide, an enzyme, or a cytokine as further described herein.

[0026] In some embodiments, the antibody is adalimumab, abciximab, alemtuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, brodalumab, certolizumab, cetuximab, daratumumab, daclizumab, denosumab, dupilumab, durvalumab, eculizumab, efalizumab, gemtuzumab, golimumab, guselkumab, ibritumomab, infliximab, ixekizumab, muromonab-CD3, natalizumab, nivolumab, omalizumab, palivizumab, panitumumab, pembrolizumab, ranibizumab, risankizumab, rituximab, secukinumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab or a biosimilar or variant thereof.

[0027] In preferred embodiments, the antibody is guselkumab.

[0028] In even more preferred embodiments, the antibody is secukinumab.

[0029] In some embodiments, the Fc-fusion polypeptide is abatacept, afilbercept, alefacept, belatacept, etarnecept or rilonacept, or wherein the hormone is erythropoietin, parathyroid hormone, growth hormone, insulin, glucagon, follicle stimulating hormone, luteinizing hormone or choriogonadotropin.

[0030] In certain embodiments, the oxidation level of the protein is decreased as compared to a control. Typically, the control is an oxidation level of a protein produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s). Typically, oxidation may be detected on methionine and / or tryptophan residues of the protein. For example, the oxidation level of one or more oxidised species of the protein may be determined by reverse phase-ultra performance liquid chromatography (RP-UPLC) and / or peptide mapping. In some embodiments, the oxidation level of the protein is decreased by about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50% or about 60% or more as compared to the control.

[0031] In some embodiments, the glycosylation level of the protein may be increased as compared to a control. Typically, the control is a glycosylation level of a protein produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s). For example, in the frame of biosimilar compounds, control of the glycosylation profile of a recombinant protein may also be desirable, as the glycosylation profile of said protein may need to be compared to that of the reference (e.g., approved) product.

[0032] In some embodiments, high-mannose glycans of the protein are increased as compared to the control. For example, the high-mannose glycans may be increased by about 10%, about 15%, about 20%, about 30%, about 40%, 50%, about 60%, about 70% about 80%, about 90%, about 100%, about 115%, 120%, 150%, 200% or more as compared to the control. For example, the protein may comprise about 3%, about 4%, about 5%, about 6% or about 7% high-mannose glycans. In some embodiments, the protein may comprise between about 2% to about 4% high-mannose, e.g., about 3% high-mannose.. Typically, the glycosylation level is determined by hydrophilic interaction ultra-high performance liquid chromatography (HILIC-UPLC). In certain embodiments, the protein is harvested from the cell culture to obtain a sample and / or purified preparation of protein as further described herein.

[0033] The invention further provides a method of determining the oxidation and / or glycosylation level of a recombinant protein, wherein the method comprises:

[0034] (a) controlling post-translational modification of the protein according to any method described herein;

[0035] (b) isolating a sample or purified preparation of the protein; and

[0036] (c) determining the level of one or more oxidized and / or glycosylated species at one or more regions of the protein.

[0037] In certain embodiments, oxidation levels of one or more methionine residues of the protein are determined. For example, the oxidation levels may be determined by reverse phase-ultra performance liquid chromatography, peptide mapping and / or any other suitable technique available to the skilled person.

[0038] In preferred embodiments, the therapeutic protein is a monoclonal antibody, and, following step (b), the method further comprises:

[0039] 6) cleaving said antibody into Fc, Fd and light chain fragments, preferably by enzymatic digestion with the enzyme IdeS, more preferably wherein said antibody is treated with carboxypeptidase B and / or dithiothreitol, ii) separating the fragments of said antibody obtained by the enzymatic digestion by reversed phase-ultra performance liquid chromatography (RP-UPLC), iii) subjecting the separated fragments of said immunoglobulin obtained in step c) to UV detection and / or mass spectrometric (MS) analysis.

[0040] In certain embodiments, the proportion or abundance of high-mannose glycans of the protein are determined. For example, % high-mannose glycans may be determined by techniques such as mass spectrometry, glycan profiling, lectin binding assays or the like.

[0041] The invention also provides a protein obtainable by any method as described herein, and pharmaceutical compositions thereof.

[0042] The invention also provides use of any sulfinyl group-containing compounds as described herein for controlling post-translational modification of a recombinant protein, wherein a eukaryotic cell capable of recombinant expression of the protein is contacted with said compound(s).

[0043] Detailed description of certain embodiments

[0044] Embodiments of the invention will now be described by way of example only, and with reference to the accompanying Figures in which:

[0045] Figure 1 A shows the effect of adding potential supplements on oxidation levels (Fc / 2 %) during cell culture. Data is normalised vs. mean of the process controls to highlight potential condition impact. At the highest hypotaurine concentration (A05_Hypotaurine C2, 8mM) an approximate 20% decrease in oxidation is observed as compared to control levels (Figure 1 B), with no significant impact on other CQAs.

[0046] Figure 2A shows the different doses of hypotaurine (8mM, 12mM, 16mM, 20mM and 30mM) tested in subsequent investigations. Figure 2B reveals a dose response with hypotaurine addition, with a decrease in Fc / 2 oxidation of approximately 42% as compared to control levels with the highest dose tested. This dose-response was also observed for LC oxidation levels (Figure 2C). No significant negative impact was observed for other CQAs with tested concentration of hypotaurine in the medium (Figure 2D).

[0047] Figure 3A shows additional doses of hypotaurine (30mM, 40mM, 50mM, 60mM) tested in further investigations. Figure 3B reveals a linear dose-response with hypotaurine addition with decreased Fc / 2 oxidation (%) up to a concentration of approximately 40mM. Figure 3C reveals a dose-response with hypotaurine addition with decreased LC oxidation (%) up to a concentration of approximately 30mM. No significant negative impact was observed for other CQAs with these tested concentrations of hypotaurine in the medium (Figure 3D).

[0048] Figure 4A shows concentrations of hypotaurine (30mM) tested in scale-up experiments. Figure 4B reveals a significant decrease in oxidation (Fc / 2 %) with hypotaurine addition at 3L scale. Figure 4C reveals a significant decrease in oxidation (LC %) with hypotaurine addition at 3L scale. No significant negative impact on productivity or other CQAs observed at 3L scale with 30mM hypotaurine in medium (Figure 4D). Figure 5A shows comparative experiments with hypotaurine (50mM) addition to medium vs. cysteamine (5mM, 10mM) or taurine (10mM, 30mM, 60mM). The cells growth and viability were heavily impacted by the cysteamine concentrations tested, and cultured were stopped early (no results available). The addition of hypotaurine led to dramatically decreased total oxidation (%) as compared to taurine (Figure 5B, Figure 5C), without significant negative impact on productivity or other CQAs observed (Figure 5D).

[0049] Figure 6 shows mass spectrometry analysis to identify and characterize the main oxidized species of secukinumab.

[0050] Sequence listing

[0051] SEQ ID NO: 1 Secukinumab Heavy Chain (full length) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVAAINQDGSEKYYVGSV KGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCVRDYYDILTDYYIHYWYFDLWGRGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0052] SEQ ID NO: 2 Secukinumab Light Chain (full length)

[0053] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQYGSSPCTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

[0054] SEQ ID NO: 3 Secukinumab Heavy Chain (variable) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVAAINQDGSEKYYVGSV KGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCVRDYYDILTDYYIHYWYFDLWGRG

[0055] SEQ ID NO: 4 Secukinumab Light Chain (variable)

[0056] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQYGSSPCTFGQGTRLEIKRTVAA

[0057] SEQ ID NO: 5 Secukinumab Heavy Chain CDR1

[0058] NYWMN

[0059] SEQ ID NO: 6 Secukinumab Heavy Chain CDR2

[0060] AINQDGSEKYYVGSVKGRF

[0061] SEQ ID NO: 7 Secukinumab Heavy Chain CDR3 DYYDILTDYYIHYWYFD

[0062] SEQ ID NO: 8 Secukinumab Light Chain CDR1 RASQSVSSSYLA SEQ ID NO: 9 Secukinumab Light Chain CDR2

[0063] GASS RAT

[0064] SEQ ID NO: 10 Secukinumab Light Chain CDR3 QQYGSSPCTF

[0065] SEQ ID NO: 1 1 Golimumab Heavy Chain lgG1

[0066] QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVK

[0067] GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKG

[0068] PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP

[0069] SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP

[0070] EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK

[0071] VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

[0072] YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0073] SEQ ID NO: 12 Golimumab Light Chain lgG1

[0074] EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

[0075] SEQ ID NO: 13 Denosumab Heavy Chain lgG2

[0076] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITGSGGSTYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPGTTVIMSWFDPWGQGTLVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFG TQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPA PIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0077] SEQ ID NO: 14 Denosumab Light Chain (kappa)

[0078] EIVLTQSPGTLSLSPGERATLSCRASQSVRGRYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG SGTDFTLTISRLEPEDFAVFYCQQYGSSPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC

[0079] SEQ ID NO: 15 Pembrolizumab Heavy chain

[0080] QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG

[0081] INPSNGGTNFNEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSSASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGK

[0082] SEQ ID NO: 16 Pembrolizumab Light chain

[0083] EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

[0084] SEQ ID NO: 17 Guselkumab Heavy Chain

[0085] EVQLVQSGAE VKKPGESLKI SCKGSGYSFS NYWIGWVRQM PGKGLEWMGI IDPSNSYTRY SPSFQGQVTI SADKSISTAY LQWSSLKASD TAMYYCARWY YKPFDVWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

[0086] SEQ ID NO: 18 Guselkumab Light Chain

[0087] QSVLTQPPSV SGAPGQRVTI SCTGSSSNIG SGYDVHWYQQ LPGTAPKLLI YGNSKRPSGV PDRFSGSKSG TSASLAITGL QSEDEADYYC ASWTDGLSLV VFGGGTKLTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV KAGVETTTPS KQSNNKYAAS SYLSLTPEQW KSHRSYSCQV THEGSTVEKT VAPTECS

[0088] SEQ ID NO: 19 Risankizumab A Chain

[0089] QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DQTIHWMRQA PGQGLEWIGY IYPRDDSPKY NENFKGKVTI TADKSTSTAY MELSSLRSED TAVYYCAIPD RSGYAWFIYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG

[0090] SEQ ID NO: 20 Risankizumab B Chain

[0091] QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DQTIHWMRQA PGQGLEWIGY IYPRDDSPKY NENFKGKVTI TADKSTSTAY MELSSLRSED TAVYYCAIPD RSGYAWFIYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG

[0092] SEQ ID NO: 21 Risankizumab C Chain

[0093] DIQMTQSPSS LSASVGDRVT ITCKASRDVA IAVAWYQQKP GKVPKLLIYW ASTRHTGVPS RFSGSGSRTD FTLTISSLQP EDVADYFCHQ YSSYPFTFGS GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

[0094] SEQ ID NO: 22 Risankizumab D Chain

[0095] DIQMTQSPSS LSASVGDRVT ITCKASRDVA IAVAWYQQKP GKVPKLLIYW ASTRHTGVPS RFSGSGSRTD FTLTISSLQP EDVADYFCHQ YSSYPFTFGS GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

[0096] The practice of embodiments of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, pharmaceutical formulation, pharmacology and medicine, which are within the skill of those working in the art.

[0097] Most general chemistry techniques can be found in Comprehensive Heterocyclic Chemistry IF (Katritzky et al., 1996, published by Pergamon Press); Comprehensive Organic Functional Group Transformations (Katritzky et al., 1995, published by Pergamon Press); Comprehensive Organic Synthesis (Trost et al,. 1991 , published by Pergamon); Heterocyclic Chemistry (Joule et al. published by Chapman & Hall); Protective Groups in Organic Synthesis (Greene et al., 1999, published by Wiley-lnterscience); and Protecting Groups (Kocienski et al., 1994).

[0098] Most general molecular biology techniques can be found in Sambrook et al, Molecular Cloning, A Laboratory Manual (2001 ) Cold Harbor-Laboratory Press, Cold Spring Harbor, N.Y. or Ausubel et al., Current Protocols in Molecular Biology (1990) published by John Wiley and Sons, N.Y.

[0099] Most general pharmaceutical formulation techniques can be found in Pharmaceutical Preformulation and Formulation (2nd Edition edited by Mark Gibson) and Pharmaceutical Excipients: Properties, Functionality and Applications in Research and Industry (edited by Otilia M Y Koo, published by Wiley). Most general pharmacological techniques can be found in A Textbook of Clinical Pharmacology and Therapeutics (5th Edition published by Arnold Hodder).

[0100] Most general techniques on the prescribing, dispensing and administering of medicines can be found in the British National Formulary 72 (published jointly by BMJ Publishing Group Ltd and Royal Pharmaceutical Society).

[0101] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pel-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., Academic Press; and the Oxford University Press, provide a person skilled in the art with a general dictionary of many of the terms used in this disclosure. For chemical terms, the skilled person may refer to the International Union of Pure and Applied Chemistry (IUPAC).

[0102] Units, prefixes and symbols are denoted in their Systeme International d’Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range.

[0103] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody" or "a cysteine" is understood to represent one or more antibody or cysteine molecules, respectively. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0104] Post Translational Modifications The invention provides a method of controlling one or more post-translational modification(s) of a recombinant protein.

[0105] As used herein, the term “post-translational modification” may refer to a reaction wherein a chemical moiety is covalently added to, or non-covalently binds to, the protein. Such modifications include, but are not limited to, oxidation, glycosylation, reduction, ubiquitination, phosphorylation, sumoylation, neddylation, ADP-ribosylation, acetylation, S-nitrosylation or nitrosylation, citrullination or deimination, the addition of OCIcNAc, methylation, hydroxylation, fattenylation, ufmylation, prenylation, myristoylation, S-palmitoylation, tyrosine sulfation, formylation, and carboxylation.

[0106] As used herein, the term “controlling” one or more post-translational modification(s) refers to regulation of one or more modifications to the protein after it has been translated in the cell. For example, in the frame of biosimilar compounds, the method of controlling post-translational modification(s) may comprise regulating one or more post-translational modification(s) of the protein so they are within a comparable range to the corresponding post-translational modification(s) of the reference (e.g., approved) product.

[0107] In certain embodiments, the post-translation modification may comprise (or consist of) oxidation of the protein. For example, the method may comprise regulating oxidation of the protein by contacting the cell and / or protein with one or with one or more sulfinyl group-containing compounds as further described herein. Typically, the oxidation level of the protein is decreased to a desirable level. For example, the oxidation level of the protein may be decreased so it falls within an acceptable range.

[0108] In the frame of biosimilar compounds, the “acceptable range” may be a comparable range to the oxidation level of a reference product.

[0109] In certain embodiments, the oxidation levels of the protein are decreased as compared to a control, wherein the control is an oxidation level of a protein produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s).

[0110] In some embodiments, the number of oxidised species of the protein may be reduced. An “oxidised species” refers to variants of the protein formed by oxidation. These variants include isoforms that comprise one or more oxidized amino acid residues. Variants can include oxidation of one or more methionine, tryptophan, lysine, cysteine and / or histidine residues.

[0111] In certain embodiments, the oxidised species of secukinumab comprises oxidation of any one or more methionine residues on the secukinumab heavy chain (SEQ ID NO: 1 ). For example, the oxidised species may comprise oxidation of methionine residues at Met34, Met83, Met262, Met 368 and / or Met438 of the heavy chain. By way of non-limiting example, the oxidised species of secukinumab may comprise oxidation of methionine residues at Met262, Met438 and / or Met368 of the heavy chain (SEQ ID NO:1).

[0112] The oxidation level of a protein can be detected by various methods, including liquid chromatography

[0113] - mass spectrophotometry (LC-MS), reverse phase chromatography (e.g., RP-HPLC or RP-UPLC), normal phase chromatography (NP-HPLC or NP-UPLC), protein A chromatography, ion exchange, for example, WCX-10 HPLC (a weak cation exchange chromatography), IEF (isoelectric focusing), peptide mapping or the like.

[0114] In certain embodiments, the oxidation level of the protein (e.g., total % of one or more oxidized species) is determined by reverse phase-ultra performance liquid chromatography (RP-UPLC) and / or peptide mapping. Typically, oxidation is detected on methionine and / or tryptophan residues of the protein.

[0115] As further described below, the recombinant protein may be an antibody (e.g., a monoclonal antibody). For example, the antibody may be an IgG antibody, preferably lgG1 , lgG2 or lgG4. In such embodiments, the oxidation level may be detected within the Fc, LC and / or Fd regions of the antibody, preferably wherein the oxidation level of at least the Fc region is detected.

[0116] In certain embodiments, the antibody is a diabody, linear antibody, multivalent or multispecific hybrid antibody, a chimeric antibody, humanized antibody or any other type of antibody as further described herein.

[0117] In certain embodiments, the antibody is an antigen-binding portion comprising or consisting of a proteolytic fragment, a Fab, Fab 1 , F(ab)2, single-chain variable fragment (scFv) or any other antigenbinding portion as further described herein.

[0118] In certain embodiments, the oxidation level of M262, M438 and / or M368 residues of secukinumab (or a biosimilar or variant thereof) is decreased as compared to a control, wherein the control is an oxidation level of M262, M438 and / or M368 residues of secukinumab produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s). For example, the oxidation level may be decreased by at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to the control.

[0119] In certain embodiments, the antibody (e.g., secukinumab) has total oxidation of less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1% or less. For example, the antibody (e.g., secukinumab) may have total oxidation (%) of between about 0.14% to about 4.97%. In other words, about 95%, about 96%, about 97%, about 98%, about 99% or more of the harvested antibodies (e.g, secukinumab) may have zero oxidized methionine(s) within the Fc, light chain and Fd regions.

[0120] In certain embodiments, the oxidation level of the Fc region of the antibody (e.g., secukinumab) is decreased as compared to a control, wherein the control is an oxidation level of an Fc region of the antibody produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s). For example, the oxidation level of the Fc region of the antibody may be decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to the control.

[0121] In certain embodiments, the Fc region of the antibody (e.g., secukinumab) may have less than about 1% oxidation, e.g., between about 0.53% to about 0.63% oxidation. In other words, about 99.0% or more of the harvested antibodies may have zero oxidized methionine(s) within the Fc region.

[0122] In certain embodiments, the oxidation level of the LC region of the antibody (e.g., secukinumab) is decreased as compared to a control, wherein the control is an oxidation level of an LC region of the antibody produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s). For example, the oxidation level of the LC region of the antibody may be decreased by at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to the control.

[0123] In certain embodiments, the light chain region of the antibody (e.g., secukinumab) may have less than about 0.5% oxidation, e.g., between about 0.24% to about 0.33% oxidation. In other words, about 99.5% or more of the harvested antibodies may have zero oxidized methionine(s) within the light chain region.

[0124] In certain embodiments, the oxidation level of the Fd region of the antibody (e.g., secukinumab) is decreased as compared to a control, wherein the control is an oxidation level of an Fd region of the antibody produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s). For example, the oxidation level of the Fd region may be decreased by at least about 5%, by at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to the control. Alternatively, there may be no difference in the oxidation level of the Fd region of the antibody (e.g., secukinumab) as compared to the control.

[0125] In certain embodiments, the Fd region of the antibody (e.g., secukinumab) may have less than about 0.5% oxidation, e.g., between about 0.14% to about 0.35% Fd oxidation. In other words, about 99.5% or more of the harvested antibodies may have zero oxidized methionine(s) within the Fd region.

[0126] In certain embodiments, the total oxidation level of the antibody (e.g. secukinumab) may be decreased by at least about 1%, by at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to the control.

[0127] In certain embodiments, the antibody (e.g., guselkumab) has total oxidation of less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1% or less. In other words, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more of the harvested antibodies (e.g, guselkumab) may have zero oxidized methionine(s) within the Fc, light chain and Fd regions.

[0128] In certain embodiments, the total oxidation level of the antibody (e.g. guselkumab) may be decreased by at least about 1%, by at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to the control.

[0129] In certain embodiments, the oxidation level of the Fc region of the antibody (e.g., guselkumab) is decreased as compared to a control, wherein the control is an oxidation level of an Fc region of the antibody produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s). For example, the oxidation level of the Fc region of the antibody may be decreased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to the control.

[0130] In certain embodiments, the Fc region of the antibody (e.g., guselkumab) may have less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% oxidation, In other words, about 95%, about 96%, about 97%, about 98%, about 99.0%, or more of the harvested antibodies may have zero oxidized methionine(s) within the Fc region.

[0131] In certain embodiments, the oxidation level of the LC region of the antibody (e.g., guselkumab) is decreased as compared to a control, wherein the control is an oxidation level of an LC region of the antibody produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s). For example, the oxidation level of the LC region of the antibody may be decreased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to the control.

[0132] In certain embodiments, the light chain region of the antibody (e.g., guselkumab) may have less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, oxidation. In other words, about 95%, about 96%, about 97%, about 98%, about 99% about 99.5% or more of the harvested antibodies may have zero oxidized methionine(s) within the light chain region.

[0133] In certain embodiments, the oxidation level of the Fd region of the antibody (e.g., guselkumab) is decreased as compared to a control, wherein the control is an oxidation level of an Fd region of the antibody produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s). For example, the oxidation level of the Fd region may be decreased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, by at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to the control. Alternatively, there may be no difference in the oxidation level of the Fd region of the antibody (e.g., guselkumab) as compared to the control.

[0134] In certain embodiments, the Fd region of the antibody (e.g., guselkumab) may have less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, oxidation. In other words, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or more of the harvested antibodies may have zero oxidized methionine(s) within the Fd region.

[0135] In certain embodiments, the post-translation modification may comprise (or consists of) glycosylation of the protein. For example, the method may comprise regulating glycosylation of the protein by contacting the cell and / or protein with one or with one or more sulfinyl group-containing compounds as further described herein.

[0136] In certain embodiments, the glycosylation profile of the protein may be controlled such that the type and / or number of polysaccharides which are attached are comparable (e.g., not significantly different) to a reference protein (e.g., approved protein).

[0137] As used herein, the term “glycosylation” refers to the attachment of one or more polysaccharide(s) to a polypeptide. For example, the polysaccharide may comprise 2 to 12 monosaccharides linked together by glycosidic bonds. Glycoproteins can contain N-linked sugar moieties and / or O-linked sugar moieties. The structure and number of sugar moieties attached to a particular glycosylation site can be variable. Such sugar moieties may be, for example, N-acetyl glucosamine, N-acetyl galactosamine, mannose, galactose, glucose, fucose, xylose, glucuronic acid, iduronic acid and / or sialic acids. In certain embodiments, the glycosylation level of the protein is increased as compared to a control, wherein the control is a glycosylation level of a protein produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s). The glycosylation level and / or profile of protein can be detected by various methods, including, for example, mass spectrometry, glycan profiling, lectin binding assays. For example, the proportion or abundance of high-mannose glycans attached to the protein may be determined.

[0138] In certain embodiments, the amount of high-mannose glycans of the protein are increased to a desirable level. For example, the high-mannose glycans may be increased so they fall within an acceptable range. In the frame of biosimilar compounds, the “acceptable range” may be a comparable range to the high-mannose glycans of a reference product.

[0139] As used herein, “high-mannose glycans” refer to N-glycosylation forms of the protein that contains unsubstituted terminal mannose sugars. These glycans typically contain between five and nine mannose residues attached to the chitobiose (GlcNAc2) core. The name abbreviations of high mannose glycans are indicative of the total number of mannose residues in the structure. In certain embodiments, the high mannose glycan species is Mannose 5 (Man 5). In certain embodiments, the high mannose glycan species is Mannose 6 (Man 6), Mannose 7 (Man 7), Mannose 8 (including Mannose 8a (Man 8a) and 8b (Man 8b)), or Mannose 9 (Man 9). In certain embodiments, the high mannose glycan species comprise a mixture of Man 5, Man 6, Man 7, Man 8a, Man 8b, and / or Man 9.

[0140] In certain embodiments, the high mannose glycan content of the protein (e.g., secukinumab) is increased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 115%, at least about 120%, at least about 130%, at least about 140%, at least about 150% or more as compared to the control. The high mannose glycan content of a protein can be measured using any methods known in the art, including, but not limited to, ultra-performance liquid chromatography (UPLC) techniques such as ultrahigh performance liquid chromatography (HILIC-UPLC) as further described herein.

[0141] In certain embodiments, the protein may comprise about 3%, about 4%, about 5%, about 6%, about 7%, or more high-mannose glycans, e.g., as measured by UPLC such as HILIC-UPLC. In some embodiments, the protein may comprise between about 2% to about 4% high-mannose (e.g., about 3% mannose).

[0142] Monitoring critical quality attributes

[0143] In certain embodiments, the invention provides methods of monitoring and / or evaluating samples or purified preparations of recombinant proteins of the invention. For example, samples taken during cell culture may be used to quantitatively and / or qualitatively monitor characteristics of the recombinant protein and the production process. In some embodiments, the real time information can be used to monitor and / or control product and production parameters, including post-translational modifications such as oxidation and / or glycosylation as further described herein. This information may be used to make timely decisions and modify processes as necessary (e.g., to control CQAs within pre-set targets and / or ranges as further described herein).

[0144] In the frame of biosimilar compounds, the “acceptable range” or “pre-set target” may be a comparable range or target as compared to a reference (i.e. , approved) product. The skilled person would understand these ranges and / or targets may vary, for example, depending on the type of reference product, the territory the reference product is approved and / or the manufacturing process being used. The term “critical quality attribute” or “CQA” refers, for example, to one or more of titre, aggregation profile, charge variant analysis, hydrophobic interactions, hydrophilic interactions, and mass analysis (including one or more of amino acid sequence, post-translational modification, higher order analysis, and glycan analysis).

[0145] In certain embodiments, the invention provides a method of determining the oxidation level of a recombinant protein (e.g., monoclonal antibody such as secukinumab), wherein the method comprises:

[0146] (a) obtaining a preparation of the protein according to any method described herein, and

[0147] (b) determining the level of one or more oxidized species at one or more regions of the protein as described herein.

[0148] In certain embodiments, the level of one or more oxidized species may be determined to be decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to a control. Typically, the control is a level of one or more oxidized species of the protein (e.g., secukinumab) produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s).

[0149] In certain embodiments, the level of one or more oxidized species may be determined to be decreased by at least about 1%, by at least about 2%, by at least about 3%, by at least about 4%, by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to a control. Typically, the control is a level of one or more oxidized species of the protein (e.g., guselkumab) produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s).

[0150] For example, the recombinant protein (e.g., monoclonal antibody such as secukinumab) may be determined to have total oxidation of less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1% or less. In some embodiments, the antibody may have total oxidation (%) of between about 0.14% to about 4.97%. In other words, about 95%, about 96%, about 97%, about 98%, about 99% or more of the harvested antibodies may have zero oxidized methionine(s) within the Fc, light chain and Fd regions.

[0151] In certain embodiments, the recombinant protein (e.g., monoclonal antibody such as guselkumab) may be determined to have total oxidation of less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1% or less. In other words, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more of the harvested antibodies may have zero oxidized methionine(s) within the Fc, light chain and Fd regions.

[0152] In certain embodiments, the oxidation level of M262, M438 and / or M368 residues of the heavy chain of secukinumab (or a biosimilar or variant thereof) is determined to be decreased as compared to a control, wherein the control is an oxidation level of M262, M438 and / or M368 residues of secukinumab produced by the cell in an equivalent cell culture medium which is not supplemented with the sulfinyl group-containing compound(s).

[0153] In preferred embodiments, the therapeutic protein is a monoclonal antibody (e.g., secukinumab or guselkumab), and, following step (a), the method further comprises: i) cleaving said antibody into fragments by enzymatic digestion with the cysteine endoprotease IdeS (Immunoglobulin degrading enzyme S), optionally wherein said antibody is treated with carboxypeptidase B and / or dithiothreitol, ii) separating the fragments of said antibody obtained by the enzymatic digestion by reversed phase-ultra performance liquid chromatography (RP-UPLC), iii) subjecting the separated fragments of said immunoglobulin obtained in step c) to UV detection and / or mass spectrometric (MS) analysis.

[0154] The level of one or more oxidized species at the Fc, light chain and / or Fd regions of the antibody may be determined as described herein.

[0155] In certain embodiments, the oxidation level of the Fc region, light chain and / or Fd regions of the antibody (e.g., secukinumab) may be determined to be decreased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to a control. Typically, the control is an oxidation level of an Fc region, light chain and / or Fd region of the antibody produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s).

[0156] In certain embodiments, the oxidation level of the Fc region, light chain and / or Fd regions of the antibody (e.g., guselkumab) may be determined to be decreased by at least about 1%, by at least about 2%, by at least about 3%, by at least about 4%, by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more as compared to a control. Typically, the control is an oxidation level of an Fc region, light chain and / or Fd region of the antibody produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s).

[0157] In certain embodiments: the Fc region of the antibody (e.g., secukinumab) may be determined to have less than about 1% oxidation, e.g., between about 0.53% to about 0.63% oxidation. In other words, about 99.0% or more of the harvested antibodies may be determined to have zero oxidized methionine(s) within the Fc region; the light chain region of the antibody (e.g., secukinumab) may be determined to have less than about 0.5% oxidation, e.g., between about 0.24% to about 0.33% oxidation. In other words, about 99.5% or more of the harvested antibodies may be determined to have zero oxidized methionine(s) within the light chain region; and / or the Fd region of the antibody (e.g., secukinumab) may be determined to have less than about 0.5% oxidation, e.g., between about 0.14% to about 0.35% Fd oxidation. In other words, about 99.5% or more of the harvested antibodies may be determined to have zero oxidized methionine(s) within the Fd region.

[0158] In certain embodiments, the invention provides a method of determining the glycosylation level of a therapeutic protein, wherein the method comprises:

[0159] (a) obtaining a preparation of the protein according to any method described herein, and

[0160] (b) determining the level of one or more glycosylated species (e.g., high-mannose glycans) at one or more regions of the protein.

[0161] In certain embodiments the high-mannose glycans of the protein (e.g., secukinumab) may be determined to be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 115%, at least about 120%, at least about 130%, at least about 140%, at least about 150% or more as compared to the control.

[0162] In certain embodiments, the methods of the invention comprise measuring the level of one or more glycosylated species of the protein (e.g., high-mannose glycans) using any suitable technique, including, but not limited to, UPLC (e.g., HILIC-UPLC or the like).

[0163] In certain embodiments, the sulfinyl group-containing compound(s) do not negatively impact one or more other critical quality attributes (CQAs) of the protein as compared to a control, wherein the control is one or more CQAs of a protein produced by an equivalent cell which is not contacted with the sulfinyl group-containing compound(s).

[0164] In certain embodiments, the protein is an antibody (e.g., secukinumab) having at least one or more of the following characteristics, following harvest from the cell culture: a) about 90%, 95%, 99% or more of any unpaired cysteines of the harvested antibody de- cysteinylated; b) about 97%, 98%, 99% or more of the harvested antibody intact; and / or c) a titre of harvested antibody between about 1 g / L to about 10 g / L, preferably about 3 g / L to about 7 g / L. The CQAs can be detected by various methods as further described herein, including, for example, Protein A HPLC (e.g., to determine titre g / L) and / or Intact Mass - methods, such as Cysteinylation by Intact Mass (Cys-IM) (e.g., to determine % de-cysteinylation).

[0165] Recombinant expression of proteins The method of controlling one or more post-translational modification(s) of the recombinant protein is typically performed during upstream processes of cell culture.

[0166] In certain embodiments, the method comprises providing one or more eukaryotic cell(s) capable of recombinant expression of the protein. For example, once an expression vector has been incorporated into an appropriate host cell, the host can be cultured under conditions suitable for expression of the relevant nucleotide coding sequences.

[0167] The term “recombinant protein” refers to a protein produced by recombinant techniques. Typically, the recombinant proteins are produced by mammalian cells. For example, the mammalian cells may grow in suspension or be adherent cells. If an adherent cell is cultured, the culture may also contain a plurality of microcarriers (e.g., microcarriers that contain one or more pores). Because of the relevance of recombinant DNA techniques in the generation of antibodies, one needs not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable domain or constant region. Changes in the constant region will, in general, be made to improve, reduce or alter characteristics, such as complement fixation (e.g., complement dependent cytotoxicity, CDC), interaction with Fc receptors, and other effector functions (e.g., antibody dependent cellular cytotoxicity, ADCC), pharmacokinetic properties (e.g., binding to the neonatal Fc receptor; FcRn). Changes in the variable domain will be made to improve the antigen binding characteristics. In addition to antibodies, immunoglobulins may exist in a variety of other forms including, for example, single-chain or Fv, Fab, and (Fab')2, as well as diabodies, linear antibodies, multivalent or multispecific hybrid antibodies.

[0168] Any cell capable of producing a protein having post-translational may be employed in the present invention. The cell may be a cell line, e.g., an immortalised cell line. The cell may be referred to herein as a “host cell”. It will be understood by the skilled person that a cell of the invention expresses a protein, which may they be post-translationally modified by the cell. Suitable eukaryotic cells may include mammalian cells (e.g. CHO cells, such as CHOZN, CHO-K1 or CHO-K1s cells, or HEK293 cells or HeLa cells), yeast cells (e.g. Saccharomyces cerevisiae or Pichia pastoris) or insect cells (e.g. baculovirus-infected insect cells).

[0169] In certain embodiments, the mammalian cells may be Chinese hamster ovary (CHO) cells (e.g., CHO DG44 cells, CHOZN, CHO-K1 or CHO-K1s cells), murine myeloma cells (Sp2.0), myeloma cells (e.g.,

[0170] NS / 0), B-cells, hybridoma cells, T-cells, human embryonic kidney (HEK) cells (e.g., HEK 293E and HEK 293F), African green monkey kidney epithelial cells (Vero) cells, Madin-Darby Canine (Cocker Spaniel) kidney epithelial cells (MDCK) cells or the like. Preferably, the cells are Chinese Hamster Ovary (CHO) cells.

[0171] The cells are typically engineered with nucleic acid sequences that encode the recombinant protein. The nucleic acids may be comprised in a vector for expression in a host cell. The vectors may comprise a promoter operably linked to the nucleic acids and may further comprise a terminator. In certain embodiments, the vector comprising the nucleic acids that encodes the protein further comprises a nucleic acid encoding a selectable marker. The term “selectable marker” is intended to encompass nucleic acid sequences that, when introduced into the cell, confer a trait suitable for selection of the resulting cell. Nucleic acids encoding selectable markers include, for example, the gene encoding glutamine synthetase, dihydrofolate reductase (DHFR) puromycin N-acetyltransferase, or the like. Two or more vectors may be used where the recombinant protein comprises two or more polypeptide chains (e.g., antibody heavy and light chains).

[0172] The nucleic acid molecules of the invention may be made using any suitable process known in the art. In one embodiment, the nucleic acid molecules may be made using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the invention may be made using molecular biology techniques.

[0173] The DNA construct of the present invention may be designed in silico, and then synthesised by conventional DNA synthesis techniques.

[0174] The above-mentioned nucleic acid sequence information is optionally modified for codon biasing according to the ultimate host cell expression system that is to be employed.

[0175] The terms “nucleotide sequence” and “nucleic acid” are used synonymously herein. Preferably the nucleotide sequence is a DNA sequence.

[0176] In certain embodiments, the recombinant protein is secreted by the eukaryotic cell into the culture medium. For example, a nucleic acid sequence encoding the protein can contain a sequence that encodes a secretion signal peptide at the N- or C-terminus of the antibody, which is cleaved by an enzyme present in the mammalian cell, and subsequently released into the culture medium.

[0177] Cell culture processes

[0178] In certain embodiments, the method further comprises contacting the cell and / or protein with one or with one or more sulfinyl group-containing compounds. Typically, the cells are cultured in a solution such as a culture medium, wherein the solution is supplemented with one or more sulfinyl group- containing compound(s). Typically, the sulfinyl group-containing compound is hypotaurine, or an analogue or precursor thereof, as further described herein.

[0179] In some embodiments, the one or more sulfinyl group-containing compound(s) may be added to a commercially available culture medium before cell culture (e.g., day 0). For example, the culture medium may be manufactured or prepared to contain the sulfinyl group-containing compound(s). In certain embodiments, the cells and / or proteins are contacted with the sulfinyl group-containing compounds before cell culture (e.g., before inoculation with any cells). For example, the cells and / or proteins may be contacted with the sulfinyl group-containing compound prior to production of the protein. In some embodiments, the sulfinyl group-containing compound is added to the culture medium (e.g., production medium) before inoculation with the cells.

[0180] In some embodiments, the cells and / or proteins are contacted with the sulfinyl group-containing compounds during and / or after cell culture. For example, the culture medium (e.g., basal medium or feed medium) may be supplemented with the sulfinyl group-containing compound during production of the protein by the cell. For example, the sulfinyl group-containing compound(s) may be added within the first 10 days of cell culture. In certain embodiments, the sulfinyl group-containing compound(s) is added to the cell culture medium on day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, and / or day 10 of the cell culture. In certain embodiments, the sulfinyl group-containing compound(s) is added to the cell culture medium at later stages, e.g., on day 11 , day 1 , day 13, day 14, day 15, day 16, day 17, day 18, day 19 and / or day 20 of the cell culture. In certain embodiments, the cells are maintained in culture for between about 10 days to about 30 days, preferably between about 14 days to about 17 days. For example, the sulfinyl group-containing compound(s) may be added daily, starting from day 2 of culture with a last addition on day 16.

[0181] In certain embodiments, the sulfinyl group-containing compound(s) is added to the cell culture medium twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every eight days, once every nine days, once every ten days, once every eleven days, once every twelve days, once every thirteen days, once every fourteen days, once every fifteen days, once every sixteen days, once every seventeen days, once every eighteen days, once every nineteen days, or once every twenty days.

[0182] Suitable conditions (such as time) can be determined by the skilled person. For example, optimal conditions can be determined empirically by measuring and comparing oxidation and / or glycosylation levels under different conditions. In one embodiment, a cell may be contacted with the sulfinyl group- containing compound(s) for at least 5, 10 or 15 days.

[0183] As used herein, the terms “culture”, “culturing”, “cell culture” or “cell culturing” refer to the maintenance or proliferation of the cells under a controlled set of physical conditions. For example, the oxygen levels, temperature, pH and / or duration of the cell culture may be monitored and adjusted to maintain or control cell viability, productivity. Typically, a cell culture process has both an expansion phase and a production phase. Typically, the expansion and production phases are separated by a transition or shift phase.

[0184] As used herein, the term “culture medium” refers to any nutrient source suitable for maintaining viability, and preferably further promoting growth and division, of a cell. For example, the nutrient source may contain components required by the cell for growth and / or survival or may contain components that aid in cell growth and / or survival. Typical culture media contains essential ingredients useful for cell metabolism, for instance, amino acids, lipids, carbon source, vitamins and mineral salts. DMEM (Dulbeccosl Modified Eagles Medium), RPMI (Roswell Park Memorial Institute Medium) or medium F12 (Ham's F12 medium) are examples of commercially available culture media. Alternatively, the culture medium may be a “chemically defined medium” or “chemically defined culture medium”, in which all the components can be described in terms of the chemical formulas and are present in known concentrations. The chemically defined medium may be a proprietary medium, developed in-house, or commercially available. The culture medium can be free of proteins and / or free of serum, and can be supplemented by any additional compound(s) such as amino acids, salts and buffers (e.g., calcium, magnesium, and phosphate , nucleosides and bases protein, tissue hydrolysates, etc.), sugars, vitamins, hormones or growth factors (e.g., a mammalian hormone, insulin, transferrin, or epidermal growth factor), depending on the needs of the cells in culture. In preferred embodiments, the culture medium is a chemically defined medium and / or animalcomponent free.

[0185] In certain embodiments, the cell culture medium is supplemented with a concentration of sulfinyl group-containing compounds that leads to no significant effect on cell viability. Typically, the cell culture medium is supplemented with a concentration of sulfinyl group-containing compounds that leads to a decrease in cell viability of no more than 0% to 30% (e.g., a decrease in cell viability of no more than 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30%).

[0186] In some embodiments, a cell is contacted with the sulfinyl group-containing compounds at a concentration that substantially inhibits oxidation. The term “substantially” as used in this context means that the oxidation level of the cell is decreased by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more when compared to the oxidation level of an identical cell that has not been contacted with sulfinyl group- containing compounds. Means of determining oxidation levels of a protein are known in the art and described elsewhere herein.

[0187] In some embodiments, the cell culture medium or any other solution comprises less than about 1 M, less than about 500mM, less than about 250mM, less than about 100mM or less than about 50mM of the sulfinyl group-containing compound. In some embodiments, the cell culture medium or solution comprises the sulfinyl group-containing compound at a concentration of at least about 5mM, at least about 8mM, at least about 10mM, at least about 20mM, at least about 30mM, at least about 40mM, at least about 50mM, at least about 60mM, at least about 70mM, at least about 80mM, at least about 90mM or at least about 100mM or more.

[0188] In some embodiments, the sulfinyl group-containing compound(s) is added to the cell culture medium or solution to a concentration of between about 1 mM to about 10OmM (e.g. about 5mM to about 80 mM), preferably about 30mM to about 60mM.

[0189] In some embodiments, the cell culture medium or solution comprises the sulfinyl group-containing compound at a concentration of about 20mM to about 950mM such as about 30mM to about 750mM or about 30mM to about 250mM. In one embodiment, the culture medium comprises the sulfinyl group-containing compound at a concentration of about 30mM to about 150mM. Preferably, the cell culture medium comprises a sulfinyl group-containing compound at a concentration of about 35mM to about 75mM, more preferably about 40m to about 65mM or about 40mM to about 60mM.

[0190] Typically, the cells are cultured in a bioreactor, holding tank, or a non-bioreactor unit operation vessel. In certain embodiments, unclarified harvest may be obtained from such bioreactor processes as fed- batch, batch, or perfusion (continuous) processes. In certain embodiments, the methods can be performed in a vessel separate from the bioreactor. For example, the methods may be performed within a separate reactor. In preferred embodiments, the cells are cultured in a bioreactor.

[0191] In certain embodiments, the bioreactor may have a volume of, e.g., between about 1 L to about 10,000 L or more. In preferred embodiments, the bioreactor may have a volume of 3,000 - 5,000 L (e.g., 4,000 L). The cell capable of recombinant expression of the protein may be cultured under any suitable conditions that allow production of the protein. Typically, the cell is cultured under conditions that allow production of the protein at commercial scale (e.g., 0.5g / L, 1 g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 10g / L, 20g / L or more).

[0192] In certain embodiments, the cell is cultured in a fed-batch culture system. The term “fed-batch culture” refers to a method of growing cells, where there is a periodic or continuous feed media supplementation to replenish the nutrients which are consumed. In certain embodiment, the cells and culture medium are supplied to the culturing vessel initially, and additional supplements, e.g., nutrients, are fed (e.g., via a feed solution), continuously or in discrete increments to the culture during the culturing process, with or without periodic cell and / or product harvest before termination of culture.

[0193] This cell culture technique has the potential to obtain high cell densities in the order of greater than 10 x 106to 30 x 106cells / ml, depending on the media formulation, cell line, and other cell growth conditions. A biphasic culture condition can be created and sustained by a variety of feed strategies and media formulations that are well-known to the skilled person.

[0194] In certain embodiments wherein the cell is cultured in a fed-batch culture system, culturing of the cells comprises the periodic or continuous addition of a further liquid culture medium and / or cell feed to the first liquid culture medium without substantial or significant removal of the first liquid culture medium or further liquid culture medium from the cell culture. The further liquid culture medium and / or cell feed may be the same as the first cell culture medium. Alternatively, the further liquid culture medium and / or cell feed may not be the same as the first cell culture medium.

[0195] In certain embodiments wherein the cell is cultured in a fed-batch culture system, the cell is contacted with feed media comprising the sulfinyl group-containing compound. In another embodiment, the cell is contacted a plurality of times throughout the production phase with feed media comprising the sulfinyl group-containing compound. In certain embodiments the cell is contacted with the sulfinyl group-containing compound immediately upon being inoculated into a production bioreactor. The term “inoculate” may refer to the process of introducing a cell into a culture vessel. In certain embodiments, a supplemental cell feed and / or a further cell culture medium may be added to the first cell culture medium. For example, the cell culture medium may be supplemented with cell feed and / or further cell culture medium each day of the cell culture from about day 2, 3, 4, 5, 6, 7, 8, 9, 10, or later to the penultimate day or the last day of the cell culture. In certain embodiments, the addition of the supplemental cell feed and / or further cell culture medium may depend on the cell-line, recombinant protein, cell media, process control strategy and / or other conditions selected for the method. In certain embodiments, the cell culture medium is supplemented with cell feed and / or further cell culture medium each day from about day 2, 3 or 4 of the culture to the penultimate day of the culture.

[0196] In certain embodiments, supplemental glucose may be added to the cell culture medium. For example, the supplemental glucose may be added to the cell culture medium to a concentration of between about 2g / L to about 7, g / L, e.g., about 2g / L, about 3g / L, about 4g / L, about 5g / L about 6g / L or about 7g / L. In preferred embodiments, supplemental glucose is added to the cell culture medium to a concentration of about 6g / L. In certain embodiments, the supplemental glucose is added to the cell culture medium when the concentration of glucose in the cell culture medium falls below about 2g / L, about 3g / L or about 4g / L. In certain embodiments, the addition of the supplemental glucose may depend on the cell-line, antibody, cell media, process control strategy and / or other conditions selected for the method.

[0197] The term “batch culture” or “batch process”, refers to a culturing process wherein the culturing of the cells comprises initial inoculation of the cells into a fresh medium and no further nutrient is added until the target product is produced. In certain embodiments wherein the cell is cultured in a batch culture, the cell is contacted with fresh media comprising a sulfinyl group-containing compound.

[0198] In other embodiments, a cell is cultured in a perfusion culture system. The term “perfusion culture” refers to a culture in which the cells and supplements are supplied to the culturing vessel at the start of the culturing process and an additional supplement(s) are fed continuously to the culture, while the product is harvested from the medium during the culturing process. Perfusion culture is one in which the cell culture receives fresh perfusion feed medium while simultaneously removing spent medium. Perfusion can be continuous, stepwise, intermittent, or a combination thereof. Perfusion rates can be less than a working volume to many working volumes per day. Preferably the cells are retained in the culture and the spent medium that is removed is substantially free of cells or has significantly fewer cells than the culture.

[0199] Perfusion can be accomplished by cell retention techniques including centrifugation, sedimentation, or filtration (see for example Voisard et al (2003), Biotechnol Bioteng, 30; 82(7), 751 -65). In accordance with the present invention, the protein may be secreted by the cell into the medium (e.g., growth medium) and extracted from the supernatant throughout the culture period following application of one or more of the aforementioned cell retention techniques. Alternatively, the secreted polypeptide may also be retained during the culture period and subsequently extracted at the end of the culture. In embodiments wherein the cell is cultured in a perfusion culture system, the cell may be contacted continuously throughout the production phase with perfusion feed medium comprising the sulfinyl group-containing compound.

[0200] In certain embodiments, the cell culture medium is supplemented with a mannosidase I inhibitor. In certain embodiments, the mannosidase I inhibitor is Kifunensine. In certain embodiments, the Kifunensine is present in the cell culture medium at a concentration of about 2 pg / kg. In certain embodiments, the kifunensine is present in the cell culture medium at a concentration of less than about 5 pg / kg, e.g., about 4 pg / kg, about 3 pg / kg, about 2 pg / kg, about 1 pg / kg or less.

[0201] In alternative embodiments, the cell culture medium does not contain any mannosidase I inhibitor (e.g., does not contain Kifunensine). Without being bound by theory, contacting the cell and / or protein with the one or more sulfinyl group-containing compounds as defined herein may increase glycosylation such that mannosidase I inhibitors such as Kifunensine are not required in the culture media.

[0202] In certain embodiments, the cell culture medium may be stirred or agitated, e.g., constantly, or intermittently, using any means of stirring or agitating. In certain embodiments, stirring may be axial or radial. In certain embodiments, agitation may be induced by rocking, rotation, or wave-induced agitation. In certain embodiments, the cell culture is constantly stirred or agitated at rate of between about 60-200 rpm (e.g., about 50 rpm, about 60 rpm, about 70 rpm, about 80 rpm, about 90 rpm, about 100 rpm, about 1 10 rpm, about 120 rpm, about 130 rpm, about 140 rpm, about 150 rpm, about 160 rpm, about 170 rpm, about 180 rpm, about 190 rpm, or 200 rpm). In preferred embodiments, the cell culture is stirred or agitated at a rate of about 160 to about 180 rpm (e.g., 170 rpm).

[0203] In certain embodiments, the cell culture may be stirred at a controlled tip speed. The skilled person would understand that appropriate agitation-related shear forces (e.g., spin rates or 30 impeller tip speed) may be selected, for example, on the scale of the culture.

[0204] In certain embodiments, the cells are cultured under a pH of about 6.0 to about 7.5. In certain embodiments, the cells are cultured under a pH of about 6.2 to about 7.6. In certain embodiments, the cells are cultured under a pH of about 6.4 to about 7.4. Typically, the cells are cultured under a pH of about 6.7 to about 7.1. Preferably, the cells are cultured under a pH of about 6.8 to 7.1. More preferably, the cells are cultured under a pH of about 6.95.

[0205] In certain embodiments, the cells are cultured at a first temperature and then actively shifted to a second temperature. For example, the cells are cultured at a first temperature of about 30° to about 40°C (e.g., 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, or 39°C). Typically, the second temperature is about 3°C to about 5°C lower than the first temperature (e.g., about 4°C lower). In preferred embodiments, the cells may be cultured at a first temperature of about 37°C and a second temperature of about 33°C. The temperature may be lowered at any stage of cell culture. In certain embodiments, the cells are cultured at the first temperature for between about 8 days to about 13 days before culturing the cells at the second temperature until the end of the culture period (e.g., about 17 days). Typically, the temperature is lowered during day 10, 11 or 12 of cell culture. In preferred embodiments, the temperature may be lowered during day 10 of the culture. Typically, the temperature may be lowered from any first temperature as described herein to any second temperature as described herein.

[0206] In certain embodiments wherein the protein comprises unpaired cysteines, the cells may be cultured at the second temperature until at least 90% or more of the unpaired cysteines are de-cysteinylated.

[0207] In certain embodiments, gas may be added to the cell culture. Any suitable gas may be used, including, for example, oxygen (O2), carbon dioxide (CO2), nitrogen (N2), and different compositions of mixed gasses, such as 20% 02 / 10% 002 / 70% air or 20% O2 / 5% 002 / 75% air. Any dissolved gas level may be used during cell culture. Once selected, the gas concentration may be optimized according to the cell-line, antibody, cell media, process control strategy and / or other conditions being used. In certain embodiments, the cell culture may comprise about 20% to about 50% dissolved oxygen (DO), typically about 30% to about 40%. Typically, a DO cascade is applied to the cell culture. For example, an automatic two gas mix cascade of Air / O2 may be used. Preferably, the DO cascade includes O2 enrichment. The cascade or other parameters that are selected may typically depend on the desired % DO output.

[0208] The term "dissolved oxygen” or "DO" refer to the amount of oxygen that is dissolved in the medium. It can be measured with an oxygen probe using methods established in the art. Percent oxygen saturation is the amount of oxygen in a solution relative to the total amount of oxygen that the solution can hold at a particular temperature. The levels of dissolved oxygen may be regulated e.g., through gassing (sparge and / or in overlay) of multiple gasses (air, oxygen, carbon dioxide, nitrogen) and / or agitation regulation loop(s).

[0209] In certain embodiments, the cell culture comprises inoculating the cell culture medium with a cell capable of recombinant expression of the protein of interest. In certain embodiments, the cell culture comprises inoculating the cell culture medium at a seeding density of between about 0.2 x 10® cells / ml to about 0.6 x 10® cells / ml (e.g., about 0.2 x 10® cells / ml, about 0.3 x 10® cells / ml, 0.4 x 10® cells / ml, about 0.5 x 10® cells / ml, or 0.6 x 10® cells / ml). In preferred embodiments, the seeding cell density is about 0.4 x 10® cells / ml. In certain embodiments, the target seeding density may be adjusted depending on the cell-line, antibody, cell media, process control strategy and / or other conditions selected for the method. For example, the target seeding density may be about 0.4 x 10® cells per ml. In certain embodiments, the cells are maintained in culture until a designated viable cell density is reached. For example, the viable cell density may be at least about 2.0 x 10® cells per ml. The term “target seeding density” refers to the cell density within the cell culture medium at the initiation of the culture.

[0210] In certain embodiments, the cell culture medium may further comprise an antifoam emulsion. Antifoam emulsion acts to eliminate excessive foaming formed during cell disruption, due to the presence of proteins, lipids and / or carbohydrates in the culture medium. Production of foam is often undesirable and can cause defects on surface coatings and prevent the efficient filling of containers. In certain embodiments, the antifoam emulsion may be a silicone antifoam emulsion. Antifoam emulsions, including silicone antifoam emulsions are commercially available.

[0211] Recombinant Proteins

[0212] Any recombinant protein may be used in the methods described herein. Typically, the proteins are therapeutic proteins, e.g., antibodies, or antigen-binding fragments, variants, biosimilars, or derivatives thereof.

[0213] The term “therapeutic protein” refers to any protein for human or veterinary therapy. For example, in certain embodiments, the therapeutic protein may be indicated for treatment of a disease, such as plaque psoriasis (moderate to severe), psoriatic arthritis, ankylosing spondylitis, axial spondyloarthritis, hidradenitis suppurativa, juvenile idiopathic arthritis (e.g., enthesitis-related arthritis and juvenile psoriatic arthritis), palmoplantar pustular psoriasis (PPP) , generalized pustular psoriasis (GPP), new onset plaque psoriasis, thyroid eye disease (TED), vitiligo, lupus nephritis, or lichen planus.

[0214] In certain embodiments the therapeutic protein may be indicated for treatment of a disease, such as diabetes mellitus, chronic viral infection (e.g., hepatitis), asthma, COPD, or an autoimmune disorder, such as multiple sclerosis (or other demyelinating disorder) or rheumatoid arthritis or clotting or enzyme deficiency. In certain embodiments, the therapeutic protein may be indicated for the treatment of cancer as further described below.

[0215] Preferred therapeutic proteins include antibodies, such as a monoclonal antibody, a diabody, linear antibody, multivalent or multispecific hybrid antibody, a chimeric antibody, humanized antibody or an antigen-binding portion (e.g., a proteolytic fragment, a Fab, Fab 1 , F(ab)2 or single-chain variable fragment (scFv)).

[0216] Other exemplary therapeutic proteins include an interleukin or interferon (e.g., an interferon-alpha, interferon-beta, or interferon-gamma), protein or peptide hormone or growth factor (e.g., insulin, GLP, erythropoietin, GM-CSF, or human growth hormone), clotting factor (e.g., Factor VII, Factor VIII), or enzyme for replacement therapy (e.g., uricase, MYOZYME, phenylalanine hydroxylase, phenylalanine ammonia lyase).

[0217] In certain embodiments, the invention relates to controlling post translational modification of a full- sized antibody or a fragment thereof.

[0218] In preferred embodiments, the present invention relates to controlling post translational modification a monoclonal antibody (or antigen-binding portion thereof) (e.g., secukinumab).

[0219] In preferred embodiments, the present invention relates to controlling post translational modification a monoclonal antibody (or antigen-binding portion thereof) (e.g., guselkumab).

[0220] In certain embodiments, where the protein comprises an antibody or antibody domain, the antibody or domain may be of any human isotype, such as an IgG isotype. Typically, the antibodies are IgG antibodies, e.g., lgG1 , lgG2 or lgG4 isotype, preferably lgG1 isotype. Unless specifically referring to full-sized antibodies such as naturally occurring antibodies, the term “antibody”, and its plural form “antibodies”, includes full-sized antibodies as well as antigen-binding fragments, variants, analogues, biosimilars, or derivatives of such antibodies, e.g., naturally occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules, polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding portions / fragments, such as F(ab')2, Fab proteolytic fragments, and single chain variable region fragments (scFvs).

[0221] The term “antibody portion” or “antibody fragment” refers to a fragment of an intact or a full- length chain or antibody, usually the binding or variable region. Said portions, or fragments, maintain at least one activity of the intact chain / antibody, i.e. they are “functional portions” or “functional fragments”. Preferably the target binding property is maintained. Examples of antibody portions (or antibody fragments) include, but are not limited to, “single-chain Fv”, “single-chain antibodies”, “Fv” or “scFv”. These terms refer to antibody fragments that comprise the variable domains from both the heavy and light chains, but lack the constant regions, all within a single polypeptide chain. In certain embodiments, single-chain antibodies can also be bi-specific and / or humanised.

[0222] In certain embodiments, the therapeutic protein may comprise full length proteins, or functional portions thereof, and may contain modifications known in the art for enhancing activity and / or stability of the molecule.

[0223] In certain embodiments, the antibody is a diabody, linear antibody, multivalent or multispecific hybrid antibody, a chimeric antibody, or humanized antibody.

[0224] In certain embodiments, the antibody is an antigen-binding portion comprising or consisting of a proteolytic fragment, a Fab, Fab 1 , F(ab)2 or single-chain variable fragment (scFv).

[0225] A “Fab fragment” is comprised of one light chain and the variable and CH1 domains of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0226] A “Fab1 fragment” that contains one light chain and one heavy chain and contains more of the constant region, between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between two heavy chains is called a F(ab')2 molecule.

[0227] A “F(ab')2” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between two heavy chains.

[0228] In accordance with the present invention, the antibody or antigen-binding portion thereof may belong to any Ig type, for example, lgG1 , lgG2, lgG3 or lgG4. In certain embodiments where the therapeutic protein is an antibody, the therapeutic protein is an IgG 1 antibody or an lgG2 antibody.

[0229] In certain embodiments, the invention relates to controlling post translational modifications of an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof may be adalimumab or a biosimilar thereof, abciximab or a biosimilar thereof, alemtuzumab or a biosimilar thereof, atezolizumab or a biosimilar thereof, avelumab or a biosimilar thereof, basiliximab or a biosimilar thereof, bevacizumab or a biosimilar thereof, brodalumab or a biosimilar thereof, certolizumab or a biosimilar thereof, cetuximab or a biosimilar thereof, daratumumab or a biosimilar thereof, daclizumab or a biosimilar thereof, denosumab or a biosimilar thereof, dupilumab or a biosimilar thereof, durvalumab or a biosimilar thereof, eculizumab or a biosimilar thereof, efalizumab or a biosimilar thereof, gemtuzumab or a biosimilar thereof, golimumab or a biosimilar thereof, guselkumab or a biosimilar thereof, ibritumomab or a biosimilar thereof, infliximab or a biosimilar thereof, ixekizumab or a biosimilar thereof, muromonab-CD3 or a biosimilar thereof, natalizumab or a biosimilar thereof, nivolumab or a biosimilar thereof, omalizumab or a biosimilar thereof, palivizumab or a biosimilar thereof, panitumumab or a biosimilar thereof, pembrolizumab or a biosimilars thereof, ranibizumab or a biosimilar thereof, risankizumab or biosimilar thereof, rituximab or a biosimilar thereof, secukinumab or a biosimilar thereof, tildrakizumab or a biosimilar thereof, tocilizumab or a biosimilar thereof, tositumomab or a biosimilar thereof, trastuzumab or a biosimilar thereof, ustekinumab or a biosimilar thereof, or vedolizumab or a biosimilar thereof.

[0230] In certain embodiments, the antibody or antigen-binding portion thereof binds to an antigen having a known or potential therapeutic significance, such as a disease-related antigen. By way of non-limiting example, the antibody or antigen-binding portion thereof may bind an antigen that is involved in the initiation, development, progression or worsening of a disease for example, cancer, inflammatory disease, autoimmune disease, cardiovascular disease or ophthalmologic disease.

[0231] In certain embodiments, the antibody or antigen-binding portion thereof is one that binds to a cytokine or receptor thereof, for example an antibody or antigen-binding portion thereof that binds to one or more of interleukin-6 (IL-6), an IL-6 receptor (e.g. tocilizumab described in WO 2019 / 043096), tumour necrosis factor alpha (TNFa), a TNFa receptor, interleukin 12 (IL-12), an IL-12 receptor, interleukin 23 (IL-23), an IL-23 receptor, interleukin 17 (IL-17), an IL-17 receptor, interleukin 17A (IL-17A) or an IL- 17A receptor.

[0232] In preferred embodiments, the antibody comprises an unpaired cysteine (e.g., secukinumab). The term ‘unpaired cysteine’ refers to a cysteine that is not involved in conserved antibody disulfide bonding. The status of an unpaired cysteine may be free or non-bonded with any other molecule (e.g., un-cysteinylated and / or un-glutathionylated). Alternatively, the status of an unpaired cysteine may be non-free or bonded with another molecule (e.g., cysteinylated, glutathionylated, reacted with any other component and / or oxidized to sulfinic or sulfonic acid).

[0233] Any antibody containing one or more unpaired cysteines may be used in the methods of the invention. Typically, the antibodies that are used contain one or more cysteine(s) in one or more CDRs. Typically, the antibody requires the unpaired cysteine to be free (i.e., un-cysteinylated) for correct structure and / or function (e.g., to recognize antigen). Typically, the cysteine is surface accessible, e.g., not sterically protected from disulfide bond formation as part of a folded region of the antibody. Typically, the cysteine is naturally occurring, e.g., the antibody has not been artificially engineered with any additional cysteine residue to facilitate attachment of any other molecule. Advantageously, the methods provided herein do not substantially effect unpaired cysteines on an antibody, antibody binding fragment or variant thereof comprising unpaired cysteines (e.g., secukinumab).

[0234] In certain embodiments the antibody or antigen-binding portion thereof is an anti-IL-17 antibody or an anti-IL-17 receptor antibody. For example, the anti-IL-17 antibody may be secukinumab or ixekizumab and the anti-IL-17 receptor antibody may be brodalumab and also biosimilars thereof.

[0235] In preferred embodiments, the antibodies are anti-IL-17 (i.e., IL-17A) antibodies. As used herein, “anti-IL-17 antibodies” include any antibodies (or antigen-binding fragments thereof) capable of specifically binding to IL-17.

[0236] In certain embodiments, the anti-IL17 antibody comprises:

[0237] (a) a full-length heavy chain having at least about 85%, 90%, 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 ; and / or

[0238] (b) a full-length light chain having at least about 85%, 90%, 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0239] In certain embodiments, the anti-IL17 antibody comprises:

[0240] (a) a full-length heavy light chain comprising SEQ ID NO:1 ; and / or

[0241] (b) a full-length light chain comprising SEQ ID NO:2.

[0242] In certain embodiments, the anti-IL17 antibody comprises:

[0243] (a) a VH sequence having at least about 85%, 90%, 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 3; and / or

[0244] (b) a VL sequence having at least about 85%, 90%, 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0245] As used herein, “sequence identity” refers to a sequence having the specified percentage of amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government’s National Center for Biotechnology Information BLAST web site.

[0246] In certain embodiments, the anti-IL17 antibody comprises:

[0247] (a) a VH sequence comprising SEQ ID NO:3; and / or

[0248] (b) a VL sequence comprising SEQ ID NO:4.

[0249] In certain embodiments, the anti-IL17 antibody comprises the following CDRs: (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 5;

[0250] (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 6;

[0251] (c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 7;

[0252] (d) LCDR1 comprising the amino acid sequence of SEQ ID NO: 8;

[0253] (e) LCDR2 comprising the amino acid sequence of SEQ ID NO: 9; and

[0254] (f) LCDR3 comprising the amino acid sequence of SEQ ID NO: 10.

[0255] As used herein, the term "Complementarity Determining Regions" (CDRs) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1 , CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat (i.e., about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Further information regarding CDR sequences of specific antibodies is available, for example, from the IMGT® database for therapeutic monoclonal antibodies, (Poiron C., Wu Y., Ginestoux C., Ehrenmann, Duroux P. and Lefranc M-P. JOBIM 2010, Paper 13 (2010).

[0256] The VH or VL typically contains three CDRs and four framework regions (FRs), arranged from aminoterminus to carboxyl-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The amino acids that make up the CDRs and the FRs (and thus the variable regions), respectively, can be readily identified for any given heavy or light chain sequence by one of ordinary skill in the art (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983))

[0257] In certain embodiments, the invention provides a method for selectively reducing cysteine in the light chain CDR3 of an anti-IL-17 antibody. For example, the light chain CDR3 may have one unpaired cysteine. In this scenario, the disclosed method is capable of reducing this cysteine in both light chains of the anti-IL17 antibody.

[0258] In preferred embodiments, the anti-IL-17 antibody is secukinumab, or a biosimilar or variant thereof as described herein.

[0259] In preferred embodiments, the invention provides a method of controlling post translational modification of secukinumab without masking of the -SH group of the cysteine at position 97 of the light chain (LC) of secukinumab. The invention also encompasses controlling post translational modification of secukinumab without masking of the -SH group of equivalent unpaired cysteine(s) in variants of secukinumab. For example, an “equivalent” unpaired cysteine may be located at a different position of secukinumab, e.g., due to deletions and / or substitutions between secukinumab and the variant antibody.

[0260] References herein to "secukinumab" include the originator drug substance (as commercially available), secukinumab as defined in W02006 / 013107 A1 (Novartis) (particularly AIN457 therein) and elsewhere in the art, and also biosimilars thereof.

[0261] As used herein, "biosimilar" refers to an antibody that is similar to an approved reference antibody (e.g., secukinumab) based upon data derived from (a) analytical studies that demonstrate that the antibody is highly similar to the reference antibody notwithstanding minor differences in clinically inactive components; (b) animal studies (including the assessment of toxicity); and / or (c) clinical studies (including the assessment of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, purity, and potency in one or more appropriate conditions of use for which the reference antibody is licensed and intended to be used.

[0262] In certain embodiments, the biosimilar and reference antibody (e.g., secukinumab or guselkumab) utilize the same mechanism(s) of action for the condition(s) of use prescribed, recommended, or suggested in the proposed labeling, but only to the extent the mechanism(s) of action are known for the reference antibody. Typically, the condition(s) of use prescribed, recommended, or suggested in the labeling proposed for the biosimilar have been previously approved for the reference antibody. Typically, the route of administration, the dosage form, and / or the strength of the biosimilar antibody are the same as those of the reference product.

[0263] In certain embodiments, the facility in which the biosimilar is manufactured, processed, packed, or held meets standards designed to assure that the antibody continues to be safe, pure, and potent. The reference antibody may be approved in at least one of the U.S., Europe, or Japan. In certain embodiments the antibody or antigen-binding portion thereof is an anti-IL-12 and / or anti-IL-23 antibody. For example, the anti-IL-12 and / or anti-IL-23 antibody or antigen-binding portion thereof may be ustekinumab, guselkumab, tildrakizumab or risankizumab. In preferred embodiments, the anti-IL-12 and / or anti-IL-23 antibody or antigen-binding portion thereof is guselkumab.

[0264] In certain embodiments the antibody or antigen-binding portion thereof is an anti-TNFa antibody. For example, the anti-TNFa antibody or antigen-binding portion thereof may be golimumab, adalimumab, etanercept or certolizumab. In certain embodiments, the anti- TNFa antibody or antigen-binding portion thereof is golimumab.

[0265] In certain embodiments the anti-TNFa antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 11 . For example, the anti- TNFa antibody or antigen-binding portion thereof may comprise a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11 . Preferably, the anti-TNFa antibody or antigen-binding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 1 1.

[0266] In certain embodiments the anti-TNFa antibody or antigen-binding portion thereof comprises a light chain having at least 70% sequence identity to SEQ ID NO: 12. For example, the anti- TNFa antibody or antigen-binding portion thereof may comprise a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 . Preferably, the anti-TNFaantibody or antigenbinding portion thereof comprises a light chain that comprises (more preferably consists of) SEQ ID NO: 12.

[0267] In certain embodiments the anti-TNFa antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 11 and a light chain having at least 70% sequence identity to SEQ ID NO: 12. Preferably, the anti-TNFa antibody or antigen-binding portion thereof comprises a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11 and a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12. Even more preferably, the anti-TNFa antibody or antigenbinding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 11 and a light chain that comprises (more preferably consists of) SEQ ID NO: 12.

[0268] In certain embodiments the anti-TNFa antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) having at least 70% identity to the corresponding VH sequence of SEQ ID NO: 11 . Preferably, the anti-TNFa antibody or antigen-binding portion thereof comprises a VH having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VH sequence of SEQ ID NO: 11 . Even more preferably, the anti- TNFa antibody or antigen-binding portion thereof comprises a VH that comprises (more preferably consists of) the corresponding VH sequence of SEQ ID NO: 11 .

[0269] In certain embodiments the anti-TNFa antibody or antigen-binding portion thereof comprises a light chain variable region (VL) having at least 70% sequence identity to the corresponding VL sequence of SEQ ID NO: 12. Preferably, the anti-TNFa antibody or antigen-binding portion thereof comprises a VL having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VL sequence of SEQ ID NO: 12. Even more preferably, the anti- TNFa antibody or antigen-binding portion thereof comprises a VL that comprises (more preferably consists of) the corresponding VL sequence of SEQ ID NO: 12.

[0270] In certain embodiments the anti-TNFa antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least 70% sequence identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 11 . Preferably, the anti-TNFa antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least sequence 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 11 . Even more preferably, the anti-TNFa antibody or antigen binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 that comprises (more preferably consists of) the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 11 . In certain embodiments the anti-TNFa antibody or antigen-binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 70% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence defined by SEQ ID NO: 12. Preferably, the anti-TNFaantibody or antigen binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 12. Even more preferably, the anti-TNFa antibody or antigenbinding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 that comprises (more preferably consists of) the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 12.

[0271] In certain embodiments the anti-TNFa antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence that consists of the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 1 1 and a light chain CDR1 , light chain CDR2 and light chain CDR3 that consists of the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 12.

[0272] In certain embodiments the antibody or antigen-binding portion thereof is an anti-IL-23 antibody. For example, the anti-IL-23 antibody or antigen-binding portion thereof may be guselkumab.

[0273] In certain embodiments the anti-IL-23 antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 17. For example, the anti- IL-23 antibody or antigen-binding portion thereof may comprise a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 17.

[0274] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a light chain having at least 70% sequence identity to SEQ ID NO: 18. For example, the anti- IL-23 antibody or antigen-binding portion thereof may comprise a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a light chain that comprises (more preferably consists of) SEQ ID NO: 18.

[0275] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 17 and a light chain having at least 70% sequence identity to SEQ ID NO: 18. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17 and a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18. Even more preferably, the anti- IL-23 antibody or antigen- binding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 17 and a light chain that comprises (more preferably consists of) SEQ ID NO: 18.

[0276] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) having at least 70% identity to the corresponding VH sequence of SEQ ID NO: 17. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a VH having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VH sequence of SEQ ID NO: 17. Even more preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a VH that comprises (more preferably consists of) the corresponding VH sequence of SEQ ID NO: 17.

[0277] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a light chain variable region (VL) having at least 70% sequence identity to the corresponding VL sequence of SEQ ID NO: 18. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a VL having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VL sequence of SEQ ID NO: 18. Even more preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a VL that comprises (more preferably consists of) the corresponding VL sequence of SEQ ID NO: 18.

[0278] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least 70% sequence identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 17. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least sequence 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 17. Even more preferably, the anti- IL-23 antibody or antigen binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 that comprises (more preferably consists of) the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 17. In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 70% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence defined by SEQ ID NO: 18. Preferably, the anti- IL-23 antibody or antigen binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 18. Even more preferably, the anti- IL-23 antibody or antigenbinding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 that comprises (more preferably consists of) the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 18.

[0279] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence that consists of the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 17 and a light chain CDR1 , light chain CDR2 and light chain CDR3 that consists of the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 18.

[0280] In certain embodiments the antibody or antigen-binding portion thereof is an anti-IL-23 antibody. For example, the anti-IL-23 antibody or antigen-binding portion thereof may be Risankizumab.

[0281] In certain embodiments the anti-IL-23 antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 19 or SEQ ID NO: 20. For example, the anti- IL-23 antibody or antigen-binding portion thereof may comprise a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19 or SEQ ID NO: 20. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 19 or SEQ ID NO: 20.

[0282] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a light chain having at least 70% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 22. For example, the anti- IL-23 antibody or antigen-binding portion thereof may comprise a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 22. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a light chain that comprises (more preferably consists of) SEQ ID NO: 21 or SEQ ID NO: 22.

[0283] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 19 or SEQ ID NO: 20 and a light chain having at least 70% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 22. Preferably, the anti- IL- 23 antibody or antigen-binding portion thereof comprises a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19 or SEQ ID NO: 20 and a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 21 or SEQ ID NO: 22. Even more preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 19 or SEQ ID NO: 20 and a light chain that comprises (more preferably consists of) SEQ ID NO: 21 or SEQ ID NO: 22.

[0284] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) having at least 70% identity to the corresponding VH sequence of SEQ ID NO: 19 or SEQ ID NO: 20. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a VH having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VH sequence of SEQ ID NO: 19 or SEQ ID NO: 20. Even more preferably, the anti- IL- 23 antibody or antigen-binding portion thereof comprises a VH that comprises (more preferably consists of) the corresponding VH sequence of SEQ ID NO: 19 or SEQ ID NO: 20.

[0285] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a light chain variable region (VL) having at least 70% sequence identity to the corresponding VL sequence of SEQ ID NO: 21 or SEQ ID NO: 22. Preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a VL having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VL sequence of SEQ ID NO: 21 or SEQ ID NO: 22. Even more preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a VL that comprises (more preferably consists of) the corresponding VL sequence of SEQ ID NO: 21 or SEQ ID NO: 22.

[0286] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least 70% sequence identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 19 or SEQ ID NO: 20. Preferably, the anti- IL-23 antibody or antigenbinding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least sequence 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 19 or SEQ ID NO: 20. Even more preferably, the anti- IL-23 antibody or antigen binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 that comprises (more preferably consists of) the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 70% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence defined by SEQ ID NO: 21 or SEQ ID NO: 22. Preferably, the anti- IL-23 antibody or antigen binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 21 or SEQ ID NO: 22. Even more preferably, the anti- IL-23 antibody or antigen-binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 that comprises (more preferably consists of) the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 21 or SEQ ID NO: 22.

[0287] In certain embodiments the anti- IL-23 antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence that consists of the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 19 or SEQ ID NO: 20 and a light chain CDR1 , light chain CDR2 and light chain CDR3 that consists of the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 21 or SEQ ID NO: 22. In other embodiments, an antibody or antigen-binding portion thereof is one that binds to receptor activator of nuclear factor-kappa B ligand (RANKL), receptor tyrosine-protein kinase erbB-2 (HER2), receptor tyrosine-protein kinase erbB-3 (HER3), vascular endothelial growth factor (VEGF), VEGF-A, B-lymphocyte antigen CD20 (CD20), programmed cell death protein 1 (PD-1), or programmed death-ligand 1 (PD-L1 ).

[0288] In certain embodiments the antibody or antigen-binding portion thereof is an anti-RANKL antibody or antigen-binding portion thereof. An exemplary anti-RANKL antibody or antigen binding portion thereof is denosumab.

[0289] In certain embodiments the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 13. For example, the anti- RANKL antibody or antigen-binding portion thereof may comprise a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13. Preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 13. In certain embodiments the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain having at least 70% sequence identity to SEQ ID NO: 14. For example, the anti- RANKL antibody or antigen-binding portion thereof may comprise a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. Preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain that comprises (more preferably consists of) SEQ ID NO: 14.

[0290] In certain embodiments the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 13 and a light chain having at least 70% sequence identity to SEQ ID NO: 14. Preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13 and a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. Even more preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 13 and a light chain that comprises (more preferably consists of) SEQ ID NO: 14.

[0291] In certain embodiments the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) having at least 70% identity to the corresponding VH sequence of SEQ ID NO: 13. Preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a VH having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VH sequence of SEQ ID NO: 13. Even more preferably, the anti- RANKL antibody or antigen-binding portion thereof comprises a VH that comprises (more preferably consists of) the corresponding VH sequence of SEQ ID NO: 13.

[0292] In certain embodiments the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain variable region (VL) having at least 70% sequence identity to the corresponding VL sequence of SEQ ID NO: 14. Preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a VL having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VL sequence of SEQ ID NO: 14. Even more preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a VL that comprises (more preferably consists of) the corresponding VL sequence of SEQ ID NO: 14.

[0293] In certain embodiments the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least 70% sequence identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 13. Preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least sequence 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 13. Even more preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 that comprises (more preferably consists of) the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 13.

[0294] In certain embodiments the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 70% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence defined by SEQ ID NO: 14. Preferably, the anti-RANKL antibody or antigen binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 14. Even more preferably, the anti-RANKL antibody or antigen-binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 that comprises (more preferably consists of) the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 14.

[0295] In certain embodiments the anti-RANKL antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence that consists of the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 13 and a light chain CDR1 , light chain CDR2 and light chain CDR3 that consists of the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 14.

[0296] In certain embodiments, the antibody or antigen binding portion thereof is an anti-PD-1 antibody. For example, the anti-PD-1 antibody or antigen binding portion thereof may be pembrolizumab.

[0297] In certain embodiments the anti-PD-1 antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 15. For example, the anti-PD-1 antibody or antigen-binding portion thereof may comprise a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15. Preferably, the anti-PD-1 antibody or antigen-binding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 15.

[0298] In certain embodiments the anti-PD-1 antibody or antigen-binding portion thereof comprises a light chain having at least 70% sequence identity to SEQ ID NO: 16. For example, the anti-PD-1 antibody or antigen-binding portion thereof may comprise a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16. Preferably, the anti- PD-1 antibody or antigen-binding portion thereof comprises a light chain that comprises (more preferably consists of) SEQ ID NO: 16.

[0299] In certain embodiments the anti- PD-1 antibody or antigen-binding portion thereof comprises a heavy chain having at least 70% sequence identity to SEQ ID NO: 15 and a light chain having at least 70% sequence identity to SEQ ID NO: 16. Preferably, the anti- PD-1 antibody or antigen-binding portion thereof comprises a heavy chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15 and a light chain having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16. Even more preferably, the anti- PD-1 antibody or antigenbinding portion thereof comprises a heavy chain that comprises (more preferably consists of) SEQ ID NO: 15 and a light chain that comprises (more preferably consists of) SEQ ID NO: 16.

[0300] In certain embodiments the anti- PD-1 antibody or antigen-binding portion thereof comprises a heavy chain variable region (VH) having at least 70% identity to the corresponding VH sequence of SEQ ID NO: 15. Preferably, the anti- PD-1 antibody or antigen-binding portion thereof comprises a VH having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VH sequence of SEQ ID NO: 15. Even more preferably, the anti- PD-1 antibody or antigen-binding portion thereof comprises a VH that comprises (more preferably consists of) the corresponding VH sequence of SEQ ID NO: 15.

[0301] In certain embodiments the anti- PD-1 antibody or antigen-binding portion thereof comprises a light chain variable region (VL) having at least 70% sequence identity to the corresponding VL sequence of SEQ ID NO: 16. Preferably, the anti- PD-1 antibody or antigen-binding portion thereof comprises a VL having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding VL sequence of SEQ ID NO: 16. Even more preferably, the anti- PD-1 antibody or antigen-binding portion thereof comprises a VL that comprises (more preferably consists of) the corresponding VL sequence of SEQ ID NO: 16.

[0302] In certain embodiments the anti- PD-1 antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least 70% sequence identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 15. Preferably, the anti- PD-1 antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence having at least sequence 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 15. Even more preferably, the anti- PD-1 antibody or antigen binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 that comprises (more preferably consists of) the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 15. In certain embodiments the anti- PD-1 antibody or antigen-binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 70% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence defined by SEQ ID NO: 16. Preferably, the anti- PD-1 antibody or antigen binding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 16. Even more preferably, the anti- PD-1 antibody or antigenbinding portion thereof comprises a light chain CDR1 , light chain CDR2 and light chain CDR3 that comprises (more preferably consists of) the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 16. In certain embodiments the anti- PD-1 antibody or antigen-binding portion thereof comprises a heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence that consists of the corresponding heavy chain CDR1 , heavy chain CDR2 and heavy chain CDR3 sequence of SEQ ID NO: 15 and a light chain CDR1 , light chain CDR2 and light chain CDR3 that consists of the corresponding light chain CDR1 , light chain CDR2 and light chain CDR3 sequence of SEQ ID NO: 16.

[0303] In embodiments where the therapeutic is a cytokine, the cytokine may be any cytokine with known or potential therapeutic applications. In certain embodiments the cytokine is a human cytokine. In one embodiment the cytokine is an interferon (IFN), for example, IFN alpha 2a, IFN alpha 2b, IFN beta 1 a, IFN beta 1b, IFN gamma 1 b.

[0304] In certain embodiments where the therapeutic protein is a hormone, the hormone may be any hormone with known or potential therapeutic applications. In certain embodiments, the hormone is a human hormone. In certain embodiments the hormone is erythropoietin (EPO), parathyroid hormone, growth hormone, insulin, glucagon, follicle stimulating hormone, luteinizing hormone or choriogonadotropin. In one embodiment, the protein is a hormone which regulates erythropoiesis (e.g., EPO).

[0305] Sulfinyl qroup-containinq compounds

[0306] Any sulfinyl group-containing compound may be used in the methods of the invention. The compound may comprise at least or up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sulfinyl groups.

[0307] In certain embodiments, the sulfinyl group-containing compound(s) has a molecular mass of less than 1000 Da (e.g., 900 Da or less, 800 Da or less, 700 Da or less, 600 Da or less, 500 Da or less, 400 Da or less, 300 Da or less, 200 Da or less, or 100 Da or less).

[0308] In certain embodiments, the sulfinyl group-containing compound(s) further comprises an amine (e.g., a primary, secondary, tertiary or quaternary amine). Preferably, the sulfinyl group-containing compound(s) further comprises a primary amine.

[0309] In certain embodiments, the sulfinyl group-containing compound can have the structure: wherein Ri is a substituent, and R2 is a substituent.

[0310] In certain embodiments, the sulfinyl group-containing compound can comprise a sulfinic acid group. Thus, in certain embodiments, the sulfinyl group-containing compound can be a sulfinic acid group- containing compound. In certain embodiments, the sulfinyl group-containing compound moiety can be a compound comprising a sulfinic acid group. The compound can comprise at least or up to 1 sulfinic acid group, at least or up to 2 sulfinic acid groups, at least or up to 3 sulfinic acid groups, at least or up to 4 sulfinic acid groups, at least or up to 5 sulfinic acid groups, at least or up to 6 sulfinic acid groups, at least or up to 7 sulfinic acid groups, at least or up to 8 sulfinic acid groups, at least or up to 9 sulfinic acid groups, or at least or up to 10 sulfinic acid groups.

[0311] In certain embodiments, the sulfinyl group-containing compound can have the structure: wherein Ri is a substituent. Non-limiting examples of substituents include hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alky I groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, ureido groups, epoxy groups, and / or ester groups.

[0312] In certain embodiments, Ri is an alkylamine. In certain embodiments, Ri is a C1-C20 alkylamine (e.g., a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkylamine). In certain embodiments, Ri is a C1 -C10 alkylamine (e.g., a C1 , C2, C3, C4, C5, C6, C7, C8, C9, or C10 alkylamine). In certain embodiments, Ri is a C1-C6 alkylamine (e.g., a C1 , C2, C3, C4, C5 or C6 alkylamine). In certain embodiments, Ri is a C1 -C4 alkylamine (e.g., a C1 , C2, C3, or C4 alkylamine).

[0313] In certain embodiments, Ri is a cycloalkylamine. In certain embodiments, Ri is a C3-C20 cycloalkylamine (e.g., a C3, C4, C5, C6, C7, C8, C9, C10, C11 , C12, C13, C14, C15, C16, C17, C18, C19, or C20 cycloalkylamine). In certain embodiments, Ri is a C3-C10 cycloalkylamine (e.g., a C3, C4, C5, C6, C7, C8, C9, or C10 alkylamine). In certain embodiments, Ri is a C3-C6 cycloalkylamine (e.g., a C3, C4, C5 or C6 alkylamine).

[0314] In certain embodiments, the sulfinyl group-containing compound can be an intermediate of taurine synthesis (e.g., biosynthesis from cysteine).

[0315] In certain embodiments, the sulfinyl group-containing compound can be hypotaurine. In certain embodiments, the sulfinyl group-containing compound has the structure:

[0316] In certain embodiments, the sulfinyl group-containing compound is homohypotaurine.

[0317] Additional examples of a sulfinyl group-containing compound include, but are not limited to: n- octylsulfinic acid; propanesulfinic acid; 2-furansulfinic acid; cysteinesulfinic acid; F-octanesulfinic acid; 2-propanesulfinic acid; purine-6-sulfinic acid; 1 -heptanesulfinic acid; 1 -pentanesulfinic acid; L- cysteinesulfinic acid; 2-naphthy Isulfinic acid; piperidinesulfinic acid; 3-pyridinesulfinic acid; 4- pyridinesulfinic acid; 1 -naphthylsu Ifinic acid; w-D-camphorsulfinic acid; chromone-3-sulfinic acid; cyclohexanesulfinic acid; but-2-ene-2-sulfinic acid; thiophene-2-sulfinic acid; 4-morpholinesulfinic acid; thiophene-2-sulfinic acid; pyrimidine-2-sulfinic acid; 2-chloroethylsulfinic acid; 3- aminopropanesulfinic acid; 3-aminopropane-1 -sulfinic acid; l-homocysteinesulfinic acid; norkhelline-6- sulfinic acid; indan-1-one-6-sulfinic acid; ethyl sulfinic acid chloride; 2-benzothiazolesulfinic acid; 4- chlorobenzenesulfinic acid; acenaphthene-3-sulfinic acid; perfluorobutanesulfinic acid; 2- amino(H)ethanesulfinic acid; 2-aminoethanesulfinic acid; 2-imidazolin-2-ylsulfinic acid; ethanesulfinic acid sodium salt; 1 -methylpyrrole-2-sulfinic acid; 1 -methylpyrrole-3-sulfinic acid; 3-methyl-butane-1 - sulfinic acid; 2-methyl-1 -propanesulfinicacid; 4-amino-toluene-2-sulfinic acid; 3-nitro-toluene-4-sulfinic acid; 2-methylpropane-2-sulfinic acid; 2-ethylhex-1 -ene-1 -sulfinic acid; 2-butylnon-1-ene-1 -sulfinic acid; sodium, 7H-purine-6-sulfinic acid; 1 -octanesulfonic-2-sulfinic acid; 1 H- benzimidazole-2-sulfinic acid; chloro sulfinic acid methyl ester; P-toluene sulfinic acid zinc salt; toluene-4-sulfinic acid- anhydride; 1 -methylimidazole-2-sulfinic acid; 2-naphty Isulfinic acid sodium salt; butane-1 -sulfinic acid ethyl ester; 2'-hydroxybiphenyl-2-sulfinic acid; toluene-4-sulfinic acid butyl ester; 2-py ridinesulfinic acid sodium salt; 5-methylselenophene-2-sulfinic acid; 8-nitro-naphthalene-1 -sulfinic acid; 6- methylnaphthalene-2-sulfinicacid; furan-2-sulfinicacid, lithium-salt; propane-2-sulfinic acid methyl ester; toluene-4-sulfinic acid methyl ester; propane-1 -sulfinic acid methyl ester; toluene-4-sulfinic acid phenyl ester; toluene-4-sulfinic acid benzyl ester; 4-chlorobenzene sulfinic acid sodium; 2-chloro-5- nitrobenzenesulfinic acid; 5-chloro-naphthalene-1 -sulfinic acid; 2-propene-1 -sulfinic acid, ethyl ester; 3-oxo-3-phenylpropane-1 -sulfinic acid; 4,6-diaminopyrimidine-2-sulfinic acid; 6-acetylamino-toluene- 3-sulfinic acid; 6-methyl-4-oxochromene-3-sulfinic acid; 2,1 ,3-benzothiadiazole-4-sulfinic acid; toluene-4-sulfinic acid cyclohexylamide; toluene-4-sulfinic acid, ammonium salt; benzofuran-2-sulfinic acid lithium salt; benzo-2,1 ,3-thiadiazole-4-sulfinic acid; toluene-4-sulfinic acid benzhydryl ester; naphthalene-2-sulfinic acid methyl ester; naphthalene-1 -sulfinic acid methyl ester; 3- chlorobenzenesulfinic acid sodium salt; 3,5-dimethyl-1 ,2-oxazole-4-sulfinic acid; 2-chloro-5-nitro- toluene-4-sulfinic acid; 1 H-purine-6-sulfinic acid, monosodium salt; 2-acetamidoanisole-4-sulfinic acid, hydrate; 5-dimethylaminonaphthalene-1 -sulfinic acid; benzothiazole-2-sulfinic acid dimethylamide; 2-methyl-8-nitro-naphthalene-1 -sulfinic acid; 2-methyl-5-nitro-naphthalene-1 -sulfinic acid; 3-oxo-3-thiophen-2-ylpropane-1 -sulfinic acid; 4-acetamido-2,6-dimethylbenzenesulfinic acid; 3- (tert-butoxy)-3-oxopropane-1 -sulfinic acid; 2-methyl-propane-1 -sulfinic acid methyl ester; 2-methyl- propane-2-sulfinic acid methyl ester; p-toluenesulfinicacid; (R)-(+)-2-methyl-propane-2-sulfinic acid amide; 4-bromo-2,1 ,3-benzothiadiazole-7-sulfinic acid; toluene-4-sulfinic acid-(1-methyl-heptyl ester); 2-butene-1 -sulfinic acid, 4-phenyl-, methyl ester; 3-formyl-1 H-indole-2-sulfinic acid methyl ester; 3- oxo-3-(phenethylamino)propane-l-sulfinic acid; sodium, 2-acetamido-1 ,3-thiazole-5-sulfinic acid; 3-(4- methoxyphenyl)-3-oxopropane-1 -sulfinic acid; 2,5-dichlorothiophene-3-sulfinic acid sodium salt; 3- trifluoromethylphenyl sulfinic acid sodium salt; 2,5-dichlorothiophene-3-sulfinic acid sodium salt; sulfinic acid, 2-chloro-5-nitrobenzene-, sodium salt; 7-Octene-1 -sulfinic acid, 2-oxo-2-phenylethyl ester; 9,10-dioxo-9,10-dihydro-anthracene-1 -sulfinic acid; 4-amino-7H-pyrrolo[2,3-d]pyrimidine-2- sulfinic acid; 2-hydroxy-tridecane-1 -sulfinic acid 4-methyl-anilide; 2-methyl-propane-2-sulfinic acid cyclohexylideneamide; 1 ,1 ,2,2,3,3,4,4,4-nonafluoro-butane-1 -sulfinicacid amide; 4-chloro-1 ,1 ,2,2,3,3,4,4-octafluorobutane-1 -sulfinic acid; (1 R)-2-methyl-propane-2-sulfinic acid 4-fluoro- benzylideneamide; 2-methyl-propane-2-sulfinic acid 1 -p-tolyl-meth-(E)-ylideneamide; (R,R)-2- methylpropane-2-sulfinic acid 1-(naphthalen-1 -yl)ethylamide; and toluene-sulfinic acid-(4)-(1 -phenylethyl ester).

[0318] In certain embodiments, the method comprises culturing the cell in a cell culture medium, wherein the cell culture medium comprises a sulfinyl group-containing compound (preferably a sulfinic acid group- containing compound) and / or a thiosulfonic acid group-containing compound.

[0319] Thiosulfonic acid group-containing compounds have the formula R-S2O2H.

[0320] In certain embodiments, the thiosulfonic acid group-containing compound further comprises an amine. In certain embodiments, the amine is a primary, secondary, tertiary or quaternary amine. Preferably, the amine is a primary amine.

[0321] In certain embodiments, the thiosulfonic acid group-containing compound has a molecular mass of less than 1000 Da (e.g., 900 Da or less, 800 Da or less, 700 Da or less, 600 Da or less, 500 Da or less, 400 Da or less, 300 Da or less, 200 Da or less, or 100 Da or less).

[0322] In certain embodiments, the R group of the thiosulfonic acid group-containing compound is an alkylamine. In certain embodiments, the R group of the thiosulfonic acid group-containing compound is a C1 -C10 alkylamine (e.g., a C1 , C2, C3, C4, C5, C6, C7, C8, C9, or C10 alkylamine). In certain embodiments, the R group of the thiosulfonic acid group-containing compound is a C1 -C6 alkylamine (e.g., a C1 , C2, C3, C4, C5 or C6 alkylamine). In certain embodiments, the R group of the thiosulfonic acid group-containing compound is a C1 -C4 alkylamine (e.g., a C1, C2, C3, or C4 alkylamine).

[0323] In certain embodiments, the thiosulfonic acid group-containing compound is thiotaurine.

[0324] In certain embodiments, the sulfinyl group-containing compound (e.g., sulfinic acid group-containing compound) and / or thiosulfonic acid group containing-compound can be replaced by or supplemented with at least one of the following compounds: Methionine, Hypotaurine, EGCG, ketoglutarate, tocopherol (e.g., alpha-tocopherol), resveratrol, baicalein, selenium / selenocysteine, NAG and / or pyruvate. In certain preferred embodiments, the sulfinyl group-containing compound (e.g., sulfinic acid group-containing compound) and / or thiosulfonic acid group containing-compound can be replaced by or supplemented with at least one of Alpha-tocopherol, Baicalein, Selenium / selenocysteine, and / or Pyruvate.

[0325] Any compound herein can be purified. A compound herein can be least 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more pure. All compounds as disclosed herein can include analogues or precursor thereof, stereoisomers, enantiomers, diastereomers, mixtures, racemates, atropisomers, and tautomers thereof.

[0326] Any compound described herein can be provided in the form of a salt (e.g., a pharmaceutically- acceptable salt). The acceptable salts include, for example, acid-addition salts and base-addition salts. The acid that is added to the compound to form an acid-addition salt can be an organic acid or an inorganic acid. A base that is added to the compound to form a base-addition salt can be an organic base or an inorganic base. In certain embodiments, an acceptable salt is a metal salt. In certain embodiments, an acceptable salt is an ammonium salt.

[0327] Metal salts can arise from the addition of an inorganic base to a compound of the invention. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In certain embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.

[0328] Harvesting and / or isolation of proteins

[0329] The protein of the invention may be harvested to provide a sample and / or purified preparation of protein. In certain embodiments, the protein may be harvested from the cell culture by centrifugation, flocculation, depth filtration and / or tangential flow filtration. Such techniques are well established in the art.

[0330] The term “harvested” refers to the separation of the protein from the cells and cell debris of the cell culture. In certain embodiments, the harvested protein (e.g., clarified cell culture fluid) is subjected to any one or more additional downstream processing steps. In certain embodiments, the method comprises passing the harvested protein through one or more further chromatography materials to further purify the protein for therapeutic uses.

[0331] In one embodiment, a protein produced by the cell may be secreted by the cell into the culture medium and thus the protein may be isolated by harvesting the culture medium with or without filtration to remove cells and other solid material. Alternatively, the protein may be retained by the cell (for example, intracellularly or bound to the surface of the cell) and the protein may be isolated by lysis of the cell, for example, through physical disruption by glass beads and / or exposure to high pH conditions and subsequent filtration.

[0332] In certain embodiments, a recombinant protein produced according to the invention may be isolated. Methods of isolating recombinant proteins produced by cells are known in the art. Thus, in one embodiment, a use or method may comprise a step of isolating the recombinant protein. An isolated protein may be free (or substantially free) from alternative polypeptides or cellular matter. In other words, a protein may be considered “isolated” when the protein of the invention constitutes at least about 90% or more of the total polypeptides present, preferably when the protein of the invention constitutes at least 95%, 98% or 99% (more preferably at least 99.9%) of the total polypeptides present. Isolating can be achieved using any suitable methods known in the art such as any suitable purification methods, e.g., chromatographic methods. Suitable methods may include affinity chromatography, ion exchange (e.g., cation or anion exchange) chromatography and immunoaffinity chromatography. In some embodiments the protein of the invention may further comprise a tag to aid in purification, such as a His-tag, which may be subsequently removed, e.g., by way of a cleavage site, such as a TEV cleavage site, engineered between the tag and polypeptide.

[0333] Pharmaceutical composition

[0334] After purifying the recombinant protein, it can be used to prepare a pharmaceutical composition. In addition to the purified protein as active agent, the composition typically contains at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are substances which do not interfere with the physiological activity of the recombinant protein, and which stabilize the pharmaceutical composition and / or enhance solubility or decrease viscosity of the pharmaceutical composition. Typical pharmaceutically acceptable excipients for recombinant proteins include buffers, salts, sugars or sugar alcohols, amino acids and surface-active agents.

[0335] In certain embodiments, the invention provides a pharmaceutical composition comprising a protein of the invention; and a pharmaceutically acceptable carrier, excipient, and / or salt.

[0336] Oral formulations may include pharmaceutically acceptable carriers known in the art in dosages suitable for oral administration. Such carriers enable the compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like suitable for ingestion by the subject.

[0337] Formulation for oral use can be obtained through combination of active compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds if desired to obtain tablets or dragee cores. Suitable excipients include carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilising agents may be added, such as cross linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof.

[0338] Dragee cores can be provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterise the quantity of active compound.

[0339] Formulations for oral use include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally stabilisers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilisers. Formulations for parenteral administration include aqueous solutions of active compounds. For injection, the formulations of the invention may take the form of aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous suspension injections can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension can also contain suitable stabilisers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0340] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated may be used in the formulation.

[0341] In one aspect, the invention provides a protein or pharmaceutical composition of the invention for use in medicine. The invention also provides use of the protein or pharmaceutical composition of the invention in the manufacture of a medicament. The invention also provides a method of treatment comprising administering a protein or pharmaceutical composition of the invention to a subject.

[0342] In one aspect the invention provides a protein or pharmaceutical composition of the invention for use in the treatment of a cancer, an inflammatory disorder, an autoimmune disorder, cardiovascular disorder or an ophthalmologic disorder. In a related aspect, there is provided use of the protein or pharmaceutical composition of the invention in the manufacture of a medicament for treating a cancer, an inflammatory disorder, an autoimmune disorder, cardiovascular disorder or an ophthalmologic disorder. Likewise, there is provided a method of treating a cancer, an inflammatory disorder, an autoimmune disorder, cardiovascular disorder or an ophthalmologic disorder, the method comprising administering a glycosylated polypeptide or a pharmaceutical composition of the invention to a subject.

[0343] A “subject” may be a mammal, such as a human or other animal. Preferably “subject” means a human subject.

[0344] The term “disorder” as used herein also encompasses a “disease”. In one embodiment the disorder is a disease.

[0345] The term “treat” or “treating” as used herein encompasses prophylactic treatment (e.g. to prevent onset of a disorder) as well as corrective treatment (treatment of a subject already suffering from a disorder). Preferably “treat” or “treating” as used herein means corrective treatment.

[0346] The term “treat” or “treating” as used herein refers to the disorder and / or a symptom thereof. Therefore a protein or pharmaceutical composition of the invention of the invention may be administered to a subject in a therapeutically effective amount or a prophy lactically effective amount. A “therapeutically effective amount” is any amount of the protein or pharmaceutical composition of the invention, which when administered alone or in combination to a subject for treating said disorder (or a symptom thereof) is sufficient to effect such treatment of the disorder, or symptom thereof.

[0347] A “prophylactically effective amount” is any amount of the protein or pharmaceutical composition of the invention that, when administered alone or in combination to a subject inhibits or delays the onset or reoccurrence of a disorder (or a symptom thereof). In some embodiments, the prophylactically effective amount prevents the onset or reoccurrence of a disorder entirely. “Inhibiting” the onset means either lessening the likelihood of a disorder’s onset (or symptom thereof) or preventing the onset entirely.

[0348] Administration of the protein or pharmaceutical composition of the invention may be accomplished orally or parenterally.

[0349] In a particularly preferred embodiment, the formulation is administered parenterally. Methods of parenteral delivery include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intra-ventricular, intravenous, intraperitoneal, or intranasal administration.

[0350] The optimal dosage will be determined by the clinician. The precise dosage to be administered may be varied depending on such factors as the age, sex and weight of the subject, the method and formulation of administration, as well as the nature and severity of the disorder to be treated. Other factors such as diet, time of administration, condition of the subject, drug combinations, and reaction sensitivity may be taken into account. An effective treatment regimen may be determined by the clinician responsible for the treatment. One or more administrations may be given, and typically the benefits are observed after a series of at least three, five, or more administrations. Repeated administration may be desirable to maintain the beneficial effects of the composition.

[0351] The treatment may be administered by any effective route, such as by subcutaneous injection, although alternative routes which may be used include intravenous, intramuscular or intra-lesional injection, oral, aerosol, parenteral, topical or via a suppository.

[0352] The treatment may be administered as a liquid formulation, although other formulations may be used. For example, the treatment may be mixed with suitable pharmaceutically acceptable carriers, and may be formulated as solids (tablets, pills, capsules, granules, etc) in a suitable composition for oral, topical or parenteral administration. Most preferably, the formulation is administered subcutaneously.

[0353] Embodiments related to the various uses of the invention are intended to be applied equally to the methods, glycosylated polypeptides, pharmaceutical compositions, therapeutic uses / methods, and vice versa.

[0354] Examples The methods of the present invention are supported and illustrated by reference to the following nonlimiting examples.

[0355] Example 1 - Antibody production

[0356] All experiments were performed with a CHO cell line expressing secukinumab. The antibody was produced in a fed-batch bioreactor process, with experiments performed using ambr250TM(100-250 mL working volume) or in a 3L bioreactor (Eppendorf Bioflo®).

[0357] The appropriate amount of cell suspension is added to pre-warmed equilibrated production medium to reach the target seeding density (TSD). Feed media were added daily from day 2 until day 16 of culture included. The glucose was added on demand from day 5 to day 16 included. To control foam build-up, antifoam was added on demand, several times a day if required. The pH was controlled by sparging CO2 if acidification is required and by adding base if the culture is too acidic. The dissolved oxygen level was controlled by using a multi-stage aeration cascade.

[0358] Example 2 - Analysis of the recombinant protein

[0359] 1.1. Protein A HPLC

[0360] IgG concentration was determined by affinity chromatography. Following culture, cell culture media samples were centrifuged for a minimum of 2 min at 2000 g and the resulting supernatant was filtered using 0.22 pm PES (polyethylene sulfone) membranes. A chromatography system, equipped with a Protein-A column and a UV detector set at 280 nm, was employed to inject 20 pL of the sample. The material bound to the column was eluted at pH 2.5, and the peak area was recorded. The IgG concentration was determined against a calibration curve established on the same day using a reference standard.

[0361] 1.2. Cys-IM and / or HIC HPLC

[0362] The level of cysteinylation was analysed by HIC-HPLC or Cysteinylation by Intact Mass (CysIM) methods as known in the art and previously described in WO2022117773A1 .

[0363] 1.3. Glycans HILIC UPLC

[0364] N-glycan analysis (including the analysis of M5, 6, 7, 8 and 9) was performed by HILIC-UPLC. N- glycans were cleaved from antibodies using an N-glycosidase F approach. The released glycans were then fluorescently labelled with RapiFlour-MS reagent from Waters and analysed using HILIC- UPLC. The chromatographic conditions employed are described in Table 1 below:

[0365] Table 1 - chromatographic conditions employed for high mannose monitoring using HILIC- UPLC.

[0366] Automated data analysis of separated glycans was performed using Chromelean console (Thermo Fisher Scientific).

[0367] 1.4. RP-UPLC

[0368] A subunit mass analysis method was developed to monitor antibody methionine and tryptophan oxidation. Samples are treated with FabRICATOR® (IdeS), Carboxypeptidase B (cpB) and dithiothreitol (DTT) to generate three individual IgG subunits (light chain (LC), Fd’ and single chain Fc / 2). These subunits are then analyzed by reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled with UV detector and the levels of oxidation on each subunit quantified.

[0369] The chromatographic conditions for oxidation monitoring by RP-UPLC of secukinumab are described in Table 2 below:

[0370] | Chromatographic System | Vanquish Flex UHPLC / Thermo Fisher Scientific |

[0371] Table 2 - chromatographic conditions for oxidation monitoring by RP-UPLC

[0372] 1.8 Peak identification for Secukinumab

[0373] A reference sample of secukinumab, as well as forced oxidized and de-amidated samples, were analyzed by mass spectrometry to identify and characterize the main oxidized species of this antibody. Samples were subjected to forced oxidation with H2O2 during 2h, 4h, 8h or 2 days or forced deamidation at pH 9.0 for 7 days. Most of the peaks obtained, for untreated or stressed samples were identified with RP-LC-MS. Major peaks for unstressed samples were identified as Fc / 2 with lysine clipping and GOF glycosylation, Fd’ and LC. For minor peaks linked to the Fc / 2 part, pre-peaks were identified as oxidized species, post peaks were identified as a succinimide variant of the main peak and the un-glycosylated Fc / 2. Without being bound by theory, high levels of oxidation may occur in the following sites: M262, M438 and M368. For minor peaks linked to the LC part, two pre-peaks were identified as deamidated LC for the first one and oxidized LC for the second one. Post peak was identified as the LC with a cyclization of the glutamic acid at the N-terminus. For minor peaks linked to the Fd’ part, pre-peaks were identified as one unknown peak, one fragment (fragment G55-G248), and oxidized species. The post-peak was identified as the Fd’ part with a cyclization of the glutamic acid at the N-terminus. Final naming is described in Figure 6.

[0374] The extracted ion chromatogram (EIC) of the main peaks and oxidized species is used to determine the % oxidation using the following formula:

[0375] [peak area of EIC of the oxidized species] / [peak area of EIC of the oxidized species + peak area of EIC of the non-oxidized species] x 100

[0376] The total % oxidation refers to the % sum of Fc / 2 oxidation, LC oxidation and Fd oxidation.

[0377] Following peak identification using MS, % Fc / 2 oxidation, % LC oxidation, % Fd oxidation or % total oxidation may be quantified using the well-established principles of UV detection (based on Beer- Lambert’s law).

[0378] Example 3 - Screening of antioxidants

[0379] The effect of adding various potential antioxidants on oxidation levels during secukinumab production was tested. Antibodies were produced as described in Example 1 , but with any one of the following compounds added to the production medium: Methionine, Hypotaurine, EGCG, ketoglutarate, tocopherol, resveratrol, baicalein, selenium / selenocysteine, NAC or pyruvate. Any potential effects of these compounds in decreasing oxidation is shown in Figure 1A.

[0380] Of these compounds, hypotaurine addition showed the most significant decrease in oxidation with a dose-response trend (=20% decrease at d17 with the highest hypotaurine concentration tested with no significant negative impact on other CQAs (Figure 1 B). These data are normalized vs mean of the process controls to highlight potential condition impact.

[0381] Example 4 - optimization of hypotaurine dosage

[0382] The effect of hypotaurine in reducing oxidation was investigated at a range of different concentrations (8mM, 12mM, 16mM, 20mM, 30mM, Figure 2A). A dose response was confirmed with hypotaurine addition, with a decrease of 42% in Fc / 2 oxidation levels as compared to the control value at the highest dose tested (Figure 2B). This dose response was also confirmed for LC oxidation levels (Figure 2C). No significant negative impact was observed on other CQAs with tested concentrations of hypotaurine in medium (Figure 2D). For example, no significant changes in the titer of the antibody (g / L) or impact on % de-cysteinylation of the antibody was observed upon addition of hypotaurine. In addition, hypotaurine supplementation was also shown to increase high-mannose glycan levels in a dose-dependent manner, with the 30mM dose leading to a 18.7% increase in high mannose compared to the control (A01 / A08 average).

[0383] As hypotaurine addition showed a significant improvement for Fc / 2 and LC oxidation levels with a dose-response trend and no significant negative impact on other CQAs, higher hypotaurine concentrations were also tested (30mM, 40mM, 50mM and 60mM, Figure 3A). The positive impact was confirmed with hypotaurine addition, with linearity of dose response verified up to 40mM (Figure 3B). This dose response at higher concentrations was also confirmed for LC oxidation levels, up to 30mM (Figure 3C). No significant negative impact was observed on other CQAs with tested concentrations of hypotaurine in medium (Figure 3D). Again, no significant changes in the titer of the antibody (g / L) or impact on % de-cysteinylation of the antibody was observed upon addition of hypotaurine. Advantageously, the amount of high-mannose glycans was increased at high concentrations. In addition, hypotaurine supplementation was also shown to increase high-mannose glycan levels in a dose-dependent manner, with the 60mM dose leading to a 109.4% increase in high mannose compared to the control (A01 ).

[0384] As hypotaurine addition showed a significant improvement for Fc / 2 and LC oxidation at ambr scale (Figures 1 -3), it was next tested at 3L scale (Figure 4A). The positive impact was confirmed with hypotaurine addition at 3L scale for both Fc / 2 oxidation (Figure 4B) and LC oxidation (Figure 4C). No significant negative impact on productivity or other CQAs observed at 3L scale with 30mM hTau (C6) in medium (Figure 4D). Again, no significant changes in the titer of the antibody (g / L) or impact on % de-cysteinylation of the antibody was observed upon addition of hypotaurine. In addition, hypotaurine supplementation was also shown to increase high-mannose glycan levels in a dose-dependent manner, with the 30mM dose leading to a 9.6% increase in high mannose compared to the control (B10).

[0385] Example 5 - Comparison of hypotaurine with other antioxidants

[0386] The addition of hypotaurine was next compared with cysteamine and taurine at 3L scale (Figure 5). The cells growth and viability were heavily impacted by the cysteamine concentration tested and both cultures were stopped early on (=no results available). The addition of hypotaurine led to a dramatic reduction in oxidation as compared to taurine (Figure 5B, Figure 5C) without any significant negative impact on other CQA’s (Figure 5D). Again, no significant changes in the titer of the antibody (g / L) or impact on % de-cysteinylation of the antibody was observed upon addition of hypotaurine.

[0387] Example 6 - Effect of hypotaurine on Guselkumab oxidation

[0388] The effect of adding hypotaurine on oxidation levels during guselkumab production was also tested. Antibodies were produced and assessed essentially as described above, but with 8mM hypotaurine added to the production medium.

[0389] In Example 4, a dose-response is observed, with higher % decreases in oxidation as compared to control at higher doses of 12mM, 16mM, 20mM, 30mM, 40mM or more. Even at a low dose of 8mM, however, hypotaurine was found to decrease total oxidation of Guselkumab by at least approximately 5% (see Table 3 below) without any significant impact on antibody titer (data not shown).

Claims

CLAIMS1. A method of controlling post-translational modification of a recombinant protein, wherein the method comprises:(a) providing a eukaryotic cell capable of recombinant expression of the protein; and(b) contacting the cell and / or protein with one or more sulfinyl group-containing compounds.

2. The method according to claim 1 , wherein the sulfinyl group-containing compound(s) has a structure according to the formula:wherein Ri is a substituent, optionally an alkylamine, C1 -C6 alkylamine or C1 -C4 alkylamine, and wherein R2 is a substituent, optionally OH.

3. The method of claim 1 or 2, wherein the sulfinyl group-containing compound(s) is hypotaurine or an analogue or precursor thereof.

4. The method according to any one of claims 1 to 3, wherein the cell and / or protein is contacted with a solution comprising the sulfinyl group-containing compound(s) at a concentration of about 30mM to about 60mM.

5. The method according to any one of claims 1 to 4, wherein:(i) the cell is cultured in a production medium, preferably wherein the production medium is supplemented with the sulfinyl group-containing compound(s) prior to inoculation of the production medium with the cells; and / or(ii) the cell is cultured in a feed medium, preferably wherein the feed medium is supplemented with the sulfinyl group-containing compound(s) during the cell’s growth or production phase.

6. The method according to claim 5, wherein:(i) the cell and / or protein is contacted with the sulfinyl group-containing compound(s) within the first 10 days of cell culture; and / or(ii) the cells are maintained in a culture for between about 14 days to about 17 days.

7. The method according to any one of claims 1 to 6, wherein the protein is an antibody, an antigenbinding portion of an antibody, a hormone, an Fc-fusion polypeptide, an albumin fusion polypeptide, an enzyme, or a cytokine.

8. The method according to claim 7, wherein the Fc-fusion polypeptide is abatacept, afilbercept, alefacept, belatacept, etarnecept or rilonacept, or wherein the hormone is erythropoietin, parathyroid hormone, growth hormone, insulin, glucagon, follicle stimulating hormone, luteinizing hormone or choriogonadotropin.

9. The method according to any one of claims 1 to 6, wherein the protein is a monoclonal antibody, preferably wherein the antibody is an IgG.

10. The method according to claim 9, wherein the antibody is adalimumab, abciximab, alemtuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, brodalumab, certolizumab, cetuximab, daratumumab, daclizumab, denosumab, dupilumab, durvalumab, eculizumab, efalizumab, gemtuzumab, golimumab, guselkumab, ibritumomab, infliximab, ixekizumab, muromonab-CD3, natalizumab, nivolumab, omalizumab, palivizumab, panitumumab, pembrolizumab, ranibizumab, risankizumab, rituximab, secukinumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab or a biosimilar or variant thereof.11 . The method according to claim 9 or 10, wherein the monoclonal antibody comprises one or more unpaired cysteines, preferably wherein the one or more unpaired cysteines are in the complementary determining region (CDR) of the antibody.

12. The method according to any one of claims 9 to 11 , wherein the antibody is secukinumab, or a biosimilar or variant thereof.

13. The method according to claim 9 or 10, wherein the antibody is guselkumab or a biosimilar or variant thereof.

14. The method according to any preceding claim, wherein the post-translational modification comprises oxidation of the recombinant protein, optionally wherein the oxidation level of the protein is decreased as compared to a control, wherein the control is an oxidation level of a protein produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s).

15. The method according to claim 14, wherein the oxidation level of the protein is decreased by about 10% or more.

16. The method according to claim 14 or 15, wherein oxidation is detected on methionine and / or tryptophan residues of the protein.

17. The method according to any one of claims 14-16, wherein the oxidation level of one or more oxidised species of the protein is determined by reverse phase-ultra performance liquid chromatography (RP-UPLC) and / or peptide mapping.

18. The method according to any one of claims 14 to 17, wherein the protein is an antibody, and further wherein:(i) the oxidation level of the Fc region of the antibody is determined to be decreased by at least about 15% or more as compared to a control, wherein the control is an oxidation level of an Fc region of the antibody produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s); and / or(II) the oxidation level of the light chain region of the antibody is determined to be decreased by at least about 10% or more as compared to a control, wherein the control is an oxidation level of a light chain region of the antibody produced by an equivalent cell not contacted with the sulfinyl group- containing compound(s).

19. The method according to any preceding claim, wherein the post-translational modification comprises glycosylation of the recombinant protein.

20. The method according to claim 19, wherein the glycosylation level of the protein is increased as compared to a control, wherein the control is a glycosylation level of a protein produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s).

21. The method of claim 19 or 20, wherein high-mannose glycans are determined to be increased as compared to a control, wherein the control is the amount of high-mannose glycans of a protein produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s).

22. The method of any one of claims 19-21 , wherein:(i) the high mannose glycan content of the protein is determined to be increased by at least 5% or more as compared to the control; and / or(II) the protein comprises between about 2% to about 4% high-mannose.

23. The method according to any one of claims 19-22, wherein the glycosylation level is determined by hydrophilic interaction ultra-high performance liquid chromatography (HILIC-UPLC).

24. The method according to any one of the preceding claims, wherein the method further comprises isolating a sample or purified preparation of the protein.

25. A method of determining the oxidation and / or glycosylation level of a recombinant protein, wherein the method comprises:(a) controlling post-translational modification of the protein according to the method of any one of claims 1 to 24,(b) isolating a sample or purified preparation of the protein; and(c) determining the level of one or more oxidized and / or glycosylated species at one or more regions of the protein.

26. The method of claim 25, wherein the protein is a monoclonal antibody, preferable wherein the antibody is an IgG.

27. The method according to claim 26, wherein the antibody is adalimumab, abciximab, alemtuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, brodalumab, certolizumab, cetuximab, daratumumab, daclizumab, denosumab, dupilumab, durvalumab, eculizumab, efalizumab, gemtuzumab, golimumab, guselkumab, ibritumomab, infliximab, ixekizumab, muromonab-CD3, natalizumab, nivolumab, omalizumab, palivizumab, panitumumab, pembrolizumab, ranibizumab, risankizumab, rituximab, secukinumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab or a biosimilar or variant thereof.

28. The method according to claim 27, wherein the antibody is secukinumab, or a biosimilar or variant thereof, or guselkumab, or a biosimilar or variant thereof.

29. The method according to any one of claims 25 to 28, wherein the oxidation level of one or more methionine residues of the protein is determined, preferably by reverse phase-ultra performance liquid chromatography and / or peptide mapping.

30. The method according to any one of claims 26 to 29, wherein the method further comprises: i) cleaving said antibody into Fc, light chain and / or Fd fragments preferably by enzymatic digestion with the enzyme IdeS, more preferably wherein said antibody is also treated with carboxypeptidase B and / or dithiothreitol, ii) separating the fragments of said antibody obtained by the enzymatic digestion by reversed phase-ultra performance liquid chromatography (RP-UPLC), iii) subjecting the separated fragments of said immunoglobulin obtained in step c) to UV detection and / or mass spectrometric (MS) analysis.31 . The method of any one of claims 26 to 29, wherein:(i) the oxidation level of the Fc region of the antibody is determined to be decreased by at least about 15% or more as compared to a control, wherein the control is an oxidation level of an Fc region of the antibody produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s); and / or(ii) the oxidation level of the light chain region of the antibody is determined to be decreased by at least about 10% or more as compared to a control, wherein the control is anoxidation level of a light chain region of the antibody produced by an equivalent cell not contacted with the sulfinyl group-containing compound(s);32. The method of any one of 25 to 31 , wherein(i) the high-mannose glycan content of the protein is determined to be increased by at least 5% or more as compared to the control; and / or(II) the protein has between about 2% to about 4% high-mannose.

33. A protein obtainable by the method of any one of the preceding claims.

34. A pharmaceutical composition comprising a protein of claim 33 and a pharmaceutically acceptable carrier.

35. A protein according to claim 33 or a pharmaceutical composition according to claim 34 for use in medicine.

36. Use of a sulfinyl group-containing compound(s) for controlling post-translational modification of a recombinant protein, wherein a eukaryotic cell capable of recombinant expression of the protein is contacted with the sulfinyl group-containing compound(s).

37. Use according to claim 36, wherein:(i) the sulfinyl group-containing compound(s) has a structure according to the formula:wherein Ri is a substituent, optionally an alkylamine, C1 -C6 alkylamine or C1 -C4 alkylamine, and wherein R2 is a substituent, optionally OH, preferably wherein the sulfinyl group-containing compound(s) is hypotaurine or an analogue or precursor thereof;(II) the oxidation level of the protein is decreased as compared to a control, wherein the control is an oxidation level of a protein produced by an equivalent cell not contacted with the sulfinyl group- containing compound(s); and / or(iii) the glycosylation level of the protein is increased as compared to a control, wherein the control is a glycosylation level of a protein produced by an equivalent cell not contacted with the sulfinyl group- containing compound(s), optionally wherein the glycosylation level is high mannose glycan content of the protein.

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