Glycoengineered Anti-CD20 antibodies

Abstract

Provided herein are methods of producing anti-CD20 antibodies having ofatumumab sequences using host cell cultures such as non-murine host cell cultures (e.g., Chinese hamster ovary (CHO) host cell cultures), e.g., through glycoengineering (e.g., metabolic glycoengineering and / or genetic glycoengineering). CHO host cells expressing such glycoengineered antibodies, and cultures thereof, are also provided. Also provided herein are compositions and pharmaceutical formulations of such antibodies, and methods of treatment using such antibodies (e.g., methods of treating multiple sclerosis).

Description

[0001] GLYCOENGINEERED ANTI-CD20 ANTIBODIES

[0002] BACKGROUND

[0003] Multiple sclerosis (MS) is a chronic, immune-mediated disease of the central nervous system characterized by inflammation, demyelination, and axonal / neuronal destruction, ultimately leading to severe disability. Relapsing MS (RMS) is the most common form of MS.

[0004] Ofatumumab (also known as OMB157, marketed as KESIMPTA® and BONSPRI®) is a fully human type 1 immunoglobulin G1 kappa (IgGl K) monoclonal antibody. Subcutaneous administration of ofatumumab leads to a rapid, frequency- and dose-dependent B cell reduction. Phase III clinical trials demonstrated that disease inflammatory activity in relapsing MS patients was potently suppressed by treatment with ofatumumab. Based on this data, ofatumumab has been approved in over 80 countries for the treatment of adult patients with RMS.

[0005] Ofatumumab reference product is produced in the murine host cell line, NS / 0 cells. However, manufacturing such murine cell-produced ofatumumab can be inefficient. Thus, there is a need in the field for alternative approaches to manufacture ofatumumab that yield a biosimilar ofatumumab that exerts the same or similar functions as the reference ofatumumab product.

[0006] SUMMARY OF THE INVENTION

[0007] Provided herein are alternative methods of producing anti-CD20 antibodies having ofatumumab sequences using host cell cultures such as non-murine host cell cultures (e.g., Chinese hamster ovary (CHO) host cell cultures), e.g., through glycoengineering.

[0008] The invention provides a composition comprising antibodies, wherein the antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated, and wherein:

[0009] (a) less than 1 % of the N-glycans in the antibody composition are N-glycolyl neuraminic acid (NGNA) glycans;

[0010] (b) at least 0.01 % of the heavy chains comprise C-terminal amidation; and / or

[0011] (c) less than 15% of the heavy chains comprise a C-terminal lysine.

[0012] Accordingly, the invention provides a composition comprising antibodies, wherein the antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated, and wherein less than 1 % of the N-glycans in the composition are N- glycolyl neuraminic acid (NGNA) glycans. Also provided herein is a composition comprising antibodies, wherein the antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated, and wherein at least 0.01 % of the heavy chains comprise C-terminal amidation.

[0013] Also provided herein is a composition comprising antibodies, wherein the antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated, and wherein less than 15% of the heavy chains comprise a C-terminal lysine.

[0014] The invention further provides a composition comprising antibodies produced by a process comprising:

[0015] (a) providing non-murine cells comprising nucleic acids encoding the antibodies, wherein the antibodies comprise:

[0016] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 ; and

[0017] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0018] (b) culturing the non-murine cells under conditions which permit expression of the antibodies, wherein the conditions comprise culturing the non-murine cells in the presence of putrescine, L-fucose, dexamethasone, insulin, glucose, galactose, manganese and / or calcium; and

[0019] (c) isolating the antibodies to obtain the composition.

[0020] Also provided herein is a composition comprising antibodies produced by a process comprising:

[0021] (a) providing non-murine cells comprising

[0022] (i) one or more transgenes encoding a fucosyltransferase, a glycoside hydrolase, and / or a GIcNAc transferase; and

[0023] (ii) nucleic acids encoding the antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0024] (b) culturing the non-murine cells under conditions which permit expression of the antibodies; and

[0025] (c) isolating the antibodies to obtain the composition.

[0026] The invention further provides a method of producing a composition comprising antibodies, the method comprising:

[0027] (a) providing non-murine cells comprising nucleic acids encoding the antibodies, wherein the antibodies comprise:

[0028] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and

[0029] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the non-murine cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L-fucose, dexamethasone, insulin, glucose, galactose, manganese and / or calcium, and

[0030] (c) isolating the antibodies to obtain the composition.

[0031] Also provided herein is a method of producing a composition comprising antibodies, comprising,

[0032] (a) providing non-murine cells comprising:

[0033] (i) one or more transgenes encoding a fucosyltransferase, a glycoside hydrolase, and / or a GIcNAc transferase; and

[0034] (ii) nucleic acids encoding the antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0035] (b) culturing the non-murine cells under conditions which permit expression of the antibodies; and

[0036] (c) isolating the antibodies to obtain the composition.

[0037] In one aspect, provided is an antibody composition comprising non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies). In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) each comprise some or all of the amino acid sequences of ofatumumab (e.g., and bind to CD20 and Fc receptors with any one or more characteristics described herein). In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) have one or more light chain complementarity determining regions (LCDRs) having the amino acid sequences of SEQ ID NOs: 4-6 and / or one or more heavy chain complementarity determining regions (HCDRs) having the amino acid sequences of SEQ ID NOs: 8-10. In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) have a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 7 and / or a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 11 . In some embodiments of the non-NS / 0 cell-produced antibodies (e.g., non-murine cell- produced antibodies, such as CHO cell-produced antibodies) having the LCDRs and HCDRs of SEQ ID NOs: 4-6 and 8-10 are lgG1 antibodies, e.g., IgGl K antibodies. In some embodiments, the non-NS / 0 cell- produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) having the LCDRs and HCDRs of SEQ ID NOs: 4-6 and 8-10 (e.g., lgG1 antibodies, e.g., IgGl K antibodies) are human antibodies. In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) having the LCDRs and HCDRs of SEQ ID NOs: 4-6 and 8-10 have a VL having the amino acid sequence of SEQ ID NO: 7 and / or a VH having the amino acid sequence of SEQ ID NO: 11 . In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) having the VL having the amino acid sequence of SEQ ID NO: 7 and / or the VH having the amino acid sequence of SEQ ID NO: 11 further have a light chain having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 ; e.g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1 ; e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1) and / or a heavy chain with at least 95% sequence identity to SEQ ID NO: 2 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2; e.g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 2; e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2). In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) having the VL having the amino acid sequence of SEQ ID NO: 7 and / or the VH having the amino acid sequence of SEQ ID NO: 11 further have a light chain having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 ; e.g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1 ; e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1) and / or a heavy chain with at least 95% sequence identity to SEQ ID NO: 3 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3; e.g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 3; e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3).

[0038] In one aspect, provided is an antibody composition comprising antibodies, wherein the antibodies have a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a heavy chain further comprising a C-terminal lysine, as defined in the amino acid sequence of SEQ ID NO: 3), wherein the CHO cell-produced antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 12.5% of the N-glycans in the composition are afucosylated. In some embodiments, between about 0.1% and about 12.5%, between about 0.5% and about 12%, between about 1% and about 11 %, between about 1 .5% and about 10%, between about 2% and about 9%, between about 3% and about 8%, between about 4% and about 7%, or between about 5% and about 6% of the N-glycans in the composition are afucosylated.

[0039] In one aspect, provided is an antibody composition comprising CHO cell-produced antibodies, wherein the CHO cell-produced antibodies have a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a heavy chain further comprising a C-terminal lysine, as defined in the amino acid sequence of SEQ ID NO: 3), wherein the CHO cell-produced antibodies in the composition comprise an Fc region comprising an N- glycan, wherein less than 10% of the N-glycans in the composition are afucosylated (e.g., 0.1% to 10%, 0.5% to 10%, or 1 % to 10% of the N-glycans in the composition are afucosylated). In some embodiments, less than 7.8% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 7.8%, 0.5% to 7.8%, or 0.5% to 7.8% of the N-glycans in the composition are afucosylated). In some embodiments, 1-6% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 6%, 0.5% to 6%, or 0.5% to 6% of the N- glycans in the composition are afucosylated, e.g., about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6% of the N- glycans in the composition are afucosylated).

[0040] In some embodiments, no more than 4.5% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, no more than 10.3% of the N-glycans in the composition (e.g., N- glycans of the anti-CD20 antibodies) are high mannose glycans, and / or no more than 55% of the N- glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 0.9% to 4.5% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, 1 .8% to 10.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, or 23% to 55% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 0.9% to 4.5% of the N-glycans in the composition are afucosylated, 1 .8% to 10.3% of the N-glycans in the composition are high mannose glycans, and 23% to 55% of the N-glycans in the composition are galactosylated. In some embodiments, the anti-CD20 antibodies of the composition are glycoengineered (e.g., metabolically glycoengineered and / or genetically glycoengineered). In some embodiments, the anti-CD20 antibodies of the composition have a relative antibody dependent cytotoxicity (ADCC) potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab) and / or a relative complement dependent cytotoxicity (CDC) potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, no more than 3.4% of the N-glycans in the composition (e.g., N- glycans of the anti-CD20 antibodies) are afucosylated, no more than 10.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, and / or no more than 55% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 0.9% to 3.4% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, 5.8% to 10.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, or 23% to 55% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 0.9% to 3.4% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, 5.8% to 10.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, and 23% to 55% of the N-glycans in the composition (e.g., N- glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, the anti-CD20 antibodies of the composition are genetically glycoengineered. In some embodiments, the anti-CD20 antibodies of the composition have a relative ADCC potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab) and / or a relative CDC potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell- produced ofatumumab). In some embodiments, no more than 1 .3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, no more than 9.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, and / or no more than 55% of the N- glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 0.9% to 1.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, 5.8% to 9.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, or 38% to 55% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 0.9% to 1 .3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, 5.8% to 9.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, and 38% to 55% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, the anti-CD20 antibodies of the composition are genetically glycoengineered (e.g., produced by a host cell (e.g., a CHO cell) expressing a transgene encoding a fucosyltransferase, e.g., a fucosyltransferase driven by a weak promoter, e.g., ubiquitin C (UBC) promoter). In some embodiments, the anti-CD20 antibodies of the composition have a relative ADCC potency within 30% of a reference murine cell-produced antibody (e.g., NS / O-produced antibody, e.g., NS / 0 cell-produced ofatumumab) and / or a relative CDC potency within 30% of a reference murine cell-produced antibody (e.g., NS / O-produced antibody, e.g., NS / 0 cell-produced ofatumumab).

[0041] In some embodiments, no more than 3.4% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, no more than 10.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, and / or no more than 41 % of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 2.7% to 3.4% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, 6.9% to 10.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, or 23% to 41% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 2.7% to 3.4% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, 6.9% to 10.3% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, and 23% to 41 % of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, the anti-CD20 antibodies of the composition are genetically glycoengineered (e.g., produced by a host cell (e.g., a CHO cell) expressing two or more transgenes that increase fucosylation). In some embodiments, the anti-CD20 antibodies of the composition have a relative ADCC potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab) and / or a relative CDC potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell- produced ofatumumab). In some embodiments, no more than 4.5% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, no more than 5% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, and / or no more than 45% of the N- glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 3.4% and 4.5% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, 1 .8% to 5% of the N-glycans in the composition (e.g., N-glycans of the anti- CD20 antibodies) are high mannose glycans, or 29% to 45% of the N-glycans in the composition (e.g., N- glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, 3.4% and 4.5% of the N- glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are afucosylated, 1 .8% to 5% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are high mannose glycans, and 29% to 45% of the N-glycans in the composition (e.g., N-glycans of the anti-CD20 antibodies) are galactosylated. In some embodiments, the anti-CD20 antibodies of the composition are metabolically glycoengineered (e.g., produced by a host cell (e.g., a CHO cell) culture in conditions suitable for increased fucosylation, e.g., containing media supplements such as putrescine, fucose, insulin, or dexamethasone). In some embodiments, the anti-CD20 antibodies of the composition have a relative ADCC potency within 30% of a reference murine cell-produced antibody (e.g., NS / O-produced antibody, e.g., NS / 0 cell-produced ofatumumab) and / or a relative CDC potency within 30% of a reference murine cell-produced antibody (e.g., NS / O-produced antibody, e.g., NS / 0 cell-produced ofatumumab).

[0042] In some embodiments, less than about 3%, less than about 2%, less than about 1 .5%, less than about 1%, less than about 0.5% or less than about 0.1 % of the N-glycans in the composition are N- glycolyl neuraminic acid (NGNA). In some embodiments, the antibody composition is devoid of murine- associated glycans, such as N-glycolyl neuraminic acid (NGNA), the enzyme for production of which is absent in CHO cells.

[0043] In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N- glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and / or less than 6% of the N-glycans in the composition are mannose-5. In some embodiments, 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N-glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; and 6-13% of the N-glycans in the composition are G2F. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N-glycans in the composition are mannose-5. In another aspect, provided herein is an antibody composition comprising CHO cell-produced antibodies, wherein the CHO cell-produced antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell-produced antibodies in the composition comprise an Fc region comprising an N- glycan, wherein 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and / or less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; and 6-13% of the N-glycans in the composition are G2F. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N-glycans in the composition are mannose-5. The invention further provides antibodies having the same sequences and Fc region N-glycans, produced by other types of non-murine or non-Ns / 0 cells.

[0044] In some embodiments, less than 10% of the N-glycans in the composition are afucosylated (e.g., 0.1% to 10%, 0.5% to 10%, or 1% to 10% of the N-glycans in the composition are afucosylated). In some embodiments, less than 7.8% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 7.8%, 0.5% to 7.8%, or 0.5% to 7.8% of the N-glycans in the composition are afucosylated). In some embodiments, 1-6% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 6%, 0.5% to 6%, or 0.5% to 6% of the N-glycans in the composition are afucosylated, e.g., about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6% of the N-glycans in the composition are afucosylated). In some embodiments, the antibody composition is devoid of murine-associated glycans, such as NGNA.

[0045] In some embodiments, less than 15% of the heavy chains (e.g., heavy chains of the anti-CD20 antibodies) comprise a C-terminal lysine. In some embodiments, less than 10% of the heavy chains (e.g., heavy chains of the anti-CD20 antibodies) comprise a C-terminal lysine. In some embodiments, less than 5% of the heavy chains (e.g., heavy chains of the anti-CD20 antibodies) comprise a C-terminal lysine.

[0046] In some embodiments, at least 0.01 % of the heavy chains comprises C-terminal amidation.

[0047] In another aspect, provided are antibody compositions (e.g., CHO cell-produced antibody compositions) comprising antibodies that each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., SEQ ID NO: 2 and / or SEQ ID NO: 3), wherein less than 15% of the heavy chains comprise C- terminal lysine (e.g., less than 10% of the heavy chains comprise C-terminal lysine, e.g., less than 5% of the heavy chains comprise C-terminal lysine). In some embodiments, less than 1 % of the N-glycans in the composition are NGNA. In some embodiments, the antibody composition is devoid of murine-associated glycans, such as NGNA. In some embodiments, at least 0.01% of the heavy chains comprises C-terminal amidation. In some embodiments, less than 10% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 10%, 0.5% to 10%, or 1% to 10% of the N-glycans in the composition are afucosylated; less than 7.8% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 7.8%, 0.5% to 7.8%, or 0.5% to 7.8% of the N-glycans in the composition are afucosylated); or 1-6% of the N-glycans in the composition are afucosylated (e.g., 0.1% to 6%, 0.5% to 6%, or 0.5% to 6% of the N-glycans in the composition are afucosylated, e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6% of the N-glycans in the composition are afucosylated). In some embodiments, (i) 0.9% to 4.5% of the N-glycans in the composition are afucosylated, 1.8% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 55% of the N-glycans in the composition are galactosylated; (ii) 0.9% to 3.4% of the N-glycans in the composition are afucosylated, 5.8% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 55% of the N-glycans in the composition are galactosylated; (iii) 0.9% to 1 .3% of the N-glycans in the composition are afucosylated, 5.8% to 9.3% of the N-glycans in the composition are high mannose glycans, and / or 38% to 55% of the N-glycans in the composition are galactosylated; (iv) 2.7% to 3.4% of the N-glycans in the composition are afucosylated, 6.9% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 41 % of the N-glycans in the composition are galactosylated; or (v) 3.4% and 4.5% of the N-glycans in the composition are afucosylated, 1.8% to 5% of the N-glycans in the composition are high mannose glycans, and / or 29% to 45% of the N-glycans in the composition are galactosylated.

[0048] In another aspect, provided are antibody compositions (e.g., CHO cell-produced antibody compositions) comprising antibodies that each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., SEQ ID NO: 2 and / or SEQ ID NO: 3), wherein at least 0.01 % of the heavy chains comprises C- terminal amidation. In some embodiments, the antibody composition is devoid of NGNA. In some embodiments, less than 15% of the heavy chains comprise C-terminal lysine (e.g., less than 10% of the heavy chains comprise C-terminal lysine, e.g., less than 5% of the heavy chains comprise C-terminal lysine). In some embodiments, less than 10% of the N-glycans in the composition are afucosylated (e.g., 0.1% to 10%, 0.5% to 10%, or 1% to 10% of the N-glycans in the composition are afucosylated; less than 7.8% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 7.8%, 0.5% to 7.8%, or 0.5% to 7.8% of the N-glycans in the composition are afucosylated); or 1-6% of the N-glycans in the composition are afucosylated (e.g., 0.1% to 6%, 0.5% to 6%, or 0.5% to 6% of the N-glycans in the composition are afucosylated, e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6% of the N-glycans in the composition are afucosylated). In some embodiments, (i) 0.9% to 4.5% of the N-glycans in the composition are afucosylated, 1.8% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 55% of the N-glycans in the composition are galactosylated; (ii) 0.9% to 3.4% of the N-glycans in the composition are afucosylated, 5.8% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 55% of the N-glycans in the composition are galactosylated; (iii) 0.9% to 1 .3% of the N-glycans in the composition are afucosylated, 5.8% to 9.3% of the N-glycans in the composition are high mannose glycans, and / or 38% to 55% of the N-glycans in the composition are galactosylated; (iv) 2.7% to 3.4% of the N-glycans in the composition are afucosylated, 6.9% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 41 % of the N-glycans in the composition are galactosylated; or (v) 3.4% and 4.5% of the N-glycans in the composition are afucosylated, 1 .8% to 5% of the N-glycans in the composition are high mannose glycans, and / or 29% to 45% of the N-glycans in the composition are galactosylated.

[0049] In another aspect, provided are antibody compositions (e.g., CHO cell-produced antibody compositions) comprising antibodies that each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., SEQ ID NO: 2 and / or SEQ ID NO: 3), wherein the antibody composition is devoid of NGNA. In some embodiments, less than 15% of the heavy chains comprise C-terminal lysine (e.g., less than 10% of the heavy chains comprise C-terminal lysine, e.g., less than 5% of the heavy chains comprise C-terminal lysine). In some embodiments, less than 10% of the N-glycans in the composition are afucosylated (e.g., 0.1% to 10%, 0.5% to 10%, or 1% to 10% of the N-glycans in the composition are afucosylated; less than 7.8% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 7.8%, 0.5% to 7.8%, or 0.5% to 7.8% of the N-glycans in the composition are afucosylated); or 1-6% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 6%, 0.5% to 6%, or 0.5% to 6% of the N-glycans in the composition are afucosylated, e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6% of the N-glycans in the composition are afucosylated). In some embodiments, (i) 0.9% to 4.5% of the N-glycans in the composition are afucosylated, 1.8% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 55% of the N-glycans in the composition are galactosylated; (ii) 0.9% to 3.4% of the N-glycans in the composition are afucosylated, 5.8% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 55% of the N-glycans in the composition are galactosylated; (iii) 0.9% to 1 .3% of the N-glycans in the composition are afucosylated, 5.8% to 9.3% of the N-glycans in the composition are high mannose glycans, and / or 38% to 55% of the N-glycans in the composition are galactosylated; (iv) 2.7% to 3.4% of the N-glycans in the composition are afucosylated, 6.9% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 41 % of the N-glycans in the composition are galactosylated; or (v) 3.4% and 4.5% of the N-glycans in the composition are afucosylated, 1 .8% to 5% of the N-glycans in the composition are high mannose glycans, and / or 29% to 45% of the N-glycans in the composition are galactosylated.

[0050] In another aspect, provided herein are antibody compositions comprising antibodies that each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., SEQ ID NO: 2 and / or SEQ ID NO: 3), wherein the antibody composition is devoid of murine-associated glycans, such as NGNA. In some embodiments, the antibodies are CHO cell-produced antibodies. In some embodiments, the antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated, e.g., 1-6% of the N-glycans in the composition are afucosylated. In some embodiments, (a) 32-49% of the N-glycans in the composition are GOF; (b) 28-35% of the N- glycans in the composition are G1 F; (c) 6-13% of the N-glycans in the composition are G2F; and / or (d) less than 6% of the N-glycans in the composition are mannose-5.

[0051] In some embodiments of any of the preceding aspects, the composition comprises between 1.0 and 2.5% N-glycans, as weight / weight of total protein. In some embodiments, the composition comprises 40 to 150 mmol sialic acid (e.g., NeuGc) per mol CHO-produced antibodies. In some embodiments, at least 25% of the N-glycans in the composition are terminally galactosylated. In some embodiments, at least 2% of the N-glycans in the composition are terminally sialylated.

[0052] In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to Fc gamma receptor (FcyR) la from 8 to 11 nM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to Fc gamma receptor (FcyR) la within 30% of a reference murine cell- produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllaHRfrom 15 to 20 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllaHRwithin 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllaLRfrom 2 to 7 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllaLRwithin 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllb from 26 to 37 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllb within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRlllb from 7 to 12 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRlllb within 30% of a reference murine cell-produced antibody (e.g., NS / 0- produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRI I laF158from 2 to 7 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRlllaF158within 30% of a reference murine cell-produced antibody (e.g., NS / O-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRII laV158from 0.1 to 0.6 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRlllaV158within 30% of a reference murine cell-produced antibody (e.g., NS / O-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD at pH 6.0) to FcRn from 0.3 to 0.8 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD at pH 6.0) to FcRn within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab).

[0053] In some embodiments of any of the preceding aspects, the CHO-cell produced antibodies are IgG antibodies. In some embodiments, the CHO-cell produced antibodies are lgG1 antibodies (e.g., IgGl K antibodies).

[0054] In some embodiments of any of the preceding aspects, fewer than 10% of the CHO-cell produced antibodies comprise a heavy chain C-terminal lysine (i.e., position 452 of SEQ ID NO: 3). In some embodiments, at least 0.01% of the CHO-cell produced antibodies comprise heavy chain C-terminal amidation.

[0055] In some embodiments of any of the preceding aspects, the CHO-cell produced antibodies have a relative antibody dependent cytotoxicity (ADCC) potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a relative complement dependent cytotoxicity (CDC) potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell- produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a relative antibody dependent cellular phagocytosis (ADCP) potency within 30% of a reference murine cell- produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a relative C1q binding potency within 10% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab).

[0056] In some embodiments of any of the preceding aspects of the invention, the CHO-cell produced antibodies are glycoengineered antibodies (e.g., metabolically glycoengineered antibodies, genetically glycoengineered antibodies, or a combination of metabolically and genetically glycoengineered antibodies).

[0057] In some aspects of the invention, provided are pharmaceutical formulations containing the antibody composition of any of the previous aspects (e.g., a non-NS / 0 cell-produced anti-CD20 antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation contains an antioxidant (e.g., wherein the anti-CD20 is a CHO cell-produced anti-CD20 antibody). In some embodiments, the antioxidant is methionine (e.g., L- methionine). In some embodiments, the antioxidant (e.g., methionine, e.g., L-methionine) is at a concentration in the pharmaceutical formulation from 0.1 mM to 20 mM (e.g., from 1 mM to 10 mM; e.g., about 0.5 mM, about 1 mM, about 2 mM, about 2.5 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, or about 20 mM). In some embodiments, the pharmaceutical formulation is at a pH from about 5.2 to about 5.8 (e.g., about 5.5 or about 5.6; e.g., pH 5.5 or pH 5.6). In some embodiments, the pharmaceutical formulation comprises arginine, sodium acetate, sodium chloride, EDTA, and / or polysorbate. In some embodiments, the pharmaceutical formulation comprises 1 % (w / v) arginine, 50 mM sodium acetate, 51 mM sodium chloride, 0.05 mM EDTA, and 0.02% (w / v) polysorbate 80. In some embodiments, the antibodies are present in an amount of about 50 mg / mL or about 90 mg / mL.

[0058] In one aspect, provided is a pharmaceutical formulation containing about 50 mg / mL of a CHO cell-produced anti-CD20 antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a CHO cell- produced anti-CD20 antibody having any of the glycosylation profiles, CD20 binding properties, Fc receptor binding properties, ADCC or CDC potencies, etc., described herein) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation containing about 50 mg / mL of the anti-CD20 antibody is in a prefilled syringe or an autoinjector (e.g., formulated for subcutaneous administration, e.g., in a volume from about 0.4 mL to 1 mL, e.g., about 0.4 mL). In some embodiments, the pharmaceutical formulation is for treatment of multiple sclerosis (MS), e.g., relapsing MS (RMS).

[0059] In one aspect, provided is a pharmaceutical formulation containing about 90 mg / mL of a CHO cell-produced anti-CD20 antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a CHO cell- produced anti-CD20 antibody having any of the glycosylation profiles, CD20 binding properties, Fc receptor binding properties, ADCC or CDC potencies, etc., described herein) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation containing about 90 mg / mL of the anti-CD20 antibody is in a prefilled syringe or an autoinjector (e.g., formulated for subcutaneous administration, e.g., in a volume from about 1 .5 mL to 2 mL). In some embodiments, the pharmaceutical formulation is for treatment of multiple sclerosis (MS), e.g., relapsing MS (RMS).

[0060] In one aspect, provided is a pharmaceutical formulation devoid of NGNA containing about 50 mg / mL of an anti-CD20 antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a CHO cell-produced anti-CD20 antibody having any of the glycosylation profiles, CD20 binding properties, Fc receptor binding properties, ADCC or CDC potencies, etc., described herein) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation containing about 50 mg / mL of the anti-CD20 antibody is in a prefilled syringe or an autoinjector (e.g., formulated for subcutaneous administration, e.g., in a volume from about 0.4 mL to 1 mL, e.g., about 0.4 mL). In some embodiments, the pharmaceutical formulation is for treatment of multiple sclerosis (MS), e.g., relapsing MS (RMS).

[0061] In one aspect, provided is a pharmaceutical formulation devoid of NGNA containing about 90 mg / mL of an anti-CD20 antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a CHO cell-produced anti-CD20 antibody having any of the glycosylation profiles, CD20 binding properties, Fc receptor binding properties, ADCC or CDC potencies, etc., described herein) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation containing about 90 mg / mL of the anti-CD20 antibody is in a prefilled syringe or an autoinjector (e.g., formulated for subcutaneous administration, e.g., in a volume from about 1 .5 mL to 2 mL). In some embodiments, the pharmaceutical formulation is for treatment of multiple sclerosis (MS), e.g., relapsing MS (RMS).

[0062] In one aspect, provided is a pharmaceutical formulation containing about 50 mg / mL of an anti- CD20 antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a CHO cell-produced anti-CD20 antibody having any of the glycosylation profiles, CD20 binding properties, Fc receptor binding properties, ADCC or CDC potencies, etc., described herein), wherein less than 15% of the heavy chains comprise C- terminal lysine (e.g., less than 10% of the heavy chains comprise C-terminal lysine), and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation containing about 50 mg / mL of the anti-CD20 antibody is in a prefilled syringe or an autoinjector (e.g., formulated for subcutaneous administration, e.g., in a volume from about 0.4 mL to 1 mL, e.g., about 0.4 mL). In some embodiments, the pharmaceutical formulation is for treatment of multiple sclerosis (MS), e.g., relapsing MS (RMS).

[0063] In one aspect, provided is a pharmaceutical formulation containing about 90 mg / mL of an anti- CD20 antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a CHO cell-produced anti-CD20 antibody having any of the glycosylation profiles, CD20 binding properties, Fc receptor binding properties, ADCC or CDC potencies, etc., described herein), wherein less than 15% of the heavy chains comprise C- terminal lysine (e.g., less than 10% of the heavy chains comprise C-terminal lysine), and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation containing about 90 mg / mL of the anti-CD20 antibody is in a prefilled syringe or an autoinjector (e.g., formulated for subcutaneous administration, e.g., in a volume from about 1 .5 mL to 2 mL). In some embodiments, the pharmaceutical formulation is for treatment of multiple sclerosis (MS), e.g., relapsing MS (RMS).

[0064] In one aspect, provided is a pharmaceutical formulation containing about 50 mg / mL of an anti- CD20 antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a CHO cell-produced anti-CD20 antibody having any of the glycosylation profiles, CD20 binding properties, Fc receptor binding properties, ADCC or CDC potencies, etc., described herein), wherein at least 0.01 % of the heavy chains comprises C-terminal amidation (e.g., 0.01 % to 5% of the heavy chains comprises C-terminal amidation), and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation containing about 50 mg / mL of the anti-CD20 antibody is in a prefilled syringe or an autoinjector (e.g., formulated for subcutaneous administration, e.g., in a volume from about 0.4 mL to 1 mL, e.g., about 0.4 mL). In some embodiments, the pharmaceutical formulation is for treatment of multiple sclerosis (MS), e.g., relapsing MS (RMS).

[0065] In one aspect, provided is a pharmaceutical formulation containing about 90 mg / mL of an anti- CD20 antibody having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a CHO cell-produced anti-CD20 antibody having any of the glycosylation profiles, CD20 binding properties, Fc receptor binding properties, ADCC or CDC potencies, etc., described herein), wherein at least 0.01 % of the heavy chains comprises C-terminal amidation (e.g., 0.01 % to 5% of the heavy chains comprises C-terminal amidation), and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation containing about 90 mg / mL of the anti-CD20 antibody is in a prefilled syringe or an autoinjector (e.g., formulated for subcutaneous administration, e.g., in a volume from about 1 .5 mL to 2 mL). In some embodiments, the pharmaceutical formulation is for treatment of multiple sclerosis (MS), e.g., relapsing MS (RMS).

[0066] In another aspect, provided herein is a prefilled syringe comprising any of the aforementioned pharmaceutical formulations.

[0067] In another aspect, provided herein is an autoinjector comprising the aforementioned prefilled syringe and / or any of the aforementioned pharmaceutical formulations.

[0068] In another aspect, provided herein are host cells that express anti-CD20 antibodies (e.g., any of the anti-CD20 antibodies described herein). In some embodiments, the host cell is not an NS / 0 cell. In some embodiments, the host cell is not a murine cell.

[0069] In some embodiments, the host cell is a CHO cell. In some embodiments, the CHO cell expresses a CHO cell-produced antibody of any of the preceding aspects.

[0070] In some embodiments, the host cell (e.g., the CHO cell) comprises one or more transgenes genes encoding Fut8, Man2a1 , or Mgat2.

[0071] In some embodiments, the host cell (e.g., the CHO cell) comprises a transgene encoding a fucosyltransferase. The expression of the fucosyltransferase may be driven by a weak promoter. In the context of the invention, a weak promoter is one that has a lower rate of transcription in the host cell than a separate promoter that drives expression of the anti-CD20 antibody. Accordingly, in some embodiments, the CHO cell comprises a first transgene encoding the anti-CD20 antibody and a second transgene encoding the fucosyltransferase, wherein expression of the anti-CD20 antibody is driven by a first promoter, and the fucosyltransferase is driven by a second promoter, wherein the first promoter has a higher rate of transcription in the host cell than the second promoter. For example, the first promoter may be CMV, while the second promoter may be a ubiquitin C (UBC) promoter. As a result, the mRNA transcripts for the recombinant anti-CD20 antibody may exceed the number of mRNA transcripts for the recombinant fucosyltransferase. For example, the mRNA transcripts for the recombinant anti-CD20 antibody may exceed the number of the mRNA transcripts for the recombinant fucosyltransferase by a factor of at least two, optionally by a factor of at least four.

[0072] In another aspect, provided is a recombinant host cell expressing an anti-CD20 antibody and a fucosyltransferase, wherein the anti-CD20 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell comprises a first transgene encoding the anti-CD20 antibody and a second transgene encoding the fucosyltransferase, wherein expression of the anti-CD20 antibody is driven by a first promoter, and the fucosyltransferase is driven by a second promoter, wherein the first promoter has a higher rate of transcription in the host cell than the second promoter. In some embodiments, the host cell is a mammalian host cell. In some embodiments, the host cell is not an NS / 0 cell. In some embodiments, the host cell is a CHO cell. In some embodiments, the second promoter is a ubiquitin C (UBC) promoter. In some embodiments, the anti-CD20 antibody is the antibody described in any of the preceding aspects or embodiments.

[0073] In another aspect, provided is a recombinant host cell expressing an anti-CD20 antibody and a fucosyltransferase, wherein the anti-CD20 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the recombinant host cell comprises a first transgene encoding the anti-CD20 antibody and a second transgene encoding the fucosyltransferase, wherein expression of the anti-CD20 antibody is driven by a first promoter, and the fucosyltransferase is driven by a second promoter, wherein the first promoter has a higher rate of transcription in the host cell than the second promoter. In some embodiments, the host cell is a mammalian host cell. In some embodiments, the host cell is not an NS / 0 cell. In some embodiments, the host cell is a CHO cell. In some embodiments, the second promoter is a ubiquitin C (UBC) promoter. In some embodiments, the anti-CD20 antibody is the antibody described in any of the preceding aspects or embodiments.

[0074] In another aspect, cultures of host cells are provided. In some embodiments, provided is a culture comprising recombinant host cells that express recombinant anti-CD20 antibodies and recombinant fucosyltransferase, wherein the recombinant anti-CD20 antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, and wherein mRNA transcripts for the recombinant anti-CD20 antibodies exceed the number of mRNA transcripts for the recombinant fucosylatransferase. In some embodiments, the mRNA transcripts for the recombinant anti-CD20 antibodies exceed the number of the mRNA transcripts for the recombinant fucosylatransferase by a factor of at least two. In some embodiments, the mRNA transcripts for the recombinant anti-CD20 antibodies exceed the number of the mRNA transcripts for the recombinant fucosylatransferase by a factor of at least four. In some aspects, provided are cultures of host cells (e.g., a CHO cell expressing anti-CD20 antibodies according to embodiments described herein) that include one or more transgenes, e.g., encoding a fucosyltransferase (e.g., Fut8), a glycoside hydrolase (e.g., Man2a1), and / or a GIcNAc transferase (e.g., Mgat2). Additionally, or alternatively, the culture contains putrescine, L-fucose, dexamethasone, insulin, galactose, manganese, and / or glucose (e.g., exogenously supplemented putrescine, L-fucose, dexamethasone, insulin, galactose, manganese, and / or glucose). In some embodiments, the culture comprises putrescine in an amount of about 1 g / L or less, e.g., in an amount of about 1 g / L. In some embodiments, the culture comprises L-fucose in an amount of about 5 g / L or less, e.g., in an amount of about 5 g / L. In some embodiments, the culture comprises dexamethasone in an amount of about 20 pM or less, e.g., in an amount of about 20 pM. In some embodiments, the culture comprises insulin in an amount of about 2 mg / mL or less, e.g., in an amount of about 2 mg / mL. In some embodiments, the culture comprises galactose in an amount of about 15 g / L or less, e.g., in an amount of about 15 g / L. In some embodiments, the culture comprises manganese in an amount of about 50 pM or less, e.g., in an amount from about 1 .5 pM to about 50 pM, e.g., in an amount of about 25.75 pM. In some embodiments, the culture comprises glucose in an amount of about 175 g / L or less, e.g., in an amount from about 100 g / L to about 175 g / L, e.g., in an amount of about 137.5 g / mL. In some embodiments, the culture comprises calcium in an amount greater than 1.2 mM, e.g., 2-3 mM calcium, e.g., about 2.4 mM calcium.

[0075] In another aspect, provided is an antibody composition produced by a process comprising (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., SEQ ID NO: 2 and / or SEQ ID NO: 3); (b) culturing the CHO cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L-fucose, dexamethasone, insulin, galactose, manganese, and / or glucose, and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the CHO cells express a fucosyltransferase (e.g., Fut8), a glycoside hydrolase (e.g., Man2a1), and / or a GIcNAc transferase (e.g., Mgat2). In some embodiments, the CHO cells are cultured in putrescine in an amount of about 1 g / L or less, e.g., in an amount of about 1 g / L. In some embodiments, the CHO cells are cultured in L-fucose in an amount of about 5 g / L or less, e.g., in an amount of about 5 g / L. In some embodiments, the CHO cells are cultured in dexamethasone in an amount of about 20 pM or less, e.g., in an amount of about 20 pM. In some embodiments, the CHO cells are cultured in insulin in an amount of about 2 mg / mL or less, e.g., in an amount of about 2 mg / mL. In some embodiments, the CHO cells are cultured in galactose in an amount of about 15 g / L or less, e.g., in an amount of about 15 g / L. In some embodiments, the CHO cells are cultured in manganese in an amount of about 50 pM or less, e.g., in an amount from about 1 .5 pM to about 50 pM, e.g., in an amount of about 25.75 pM. In some embodiments, the CHO cells are cultured in glucose in an amount of about 175 g / L or less, e.g., in an amount from about 100 g / L to about 175 g / L, e.g., in an amount of about 137.5 g / mL. In some embodiments, the CHO cells are cultured in calcium in an amount greater than 1.2 mM, e.g., 2-3 mM calcium, e.g., about 2.4 mM calcium.

[0076] In another aspect, provided is an antibody composition produced by a process comprising (a) providing CHO cells comprising (i) Fut8, Man2a1 , or Mgat2, and (ii) nucleic acid encoding antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies; and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, insulin, galactose, manganese, and / or galactose. In some embodiments, the CHO cells are cultured in putrescine in an amount of about 1 g / L or less, e.g., in an amount of about 1 g / L. In some embodiments, the CHO cells are cultured in L-fucose in an amount of about 5 g / L or less, e.g., in an amount of about 5 g / L. In some embodiments, the CHO cells are cultured in dexamethasone in an amount of about 20 pM or less, e.g., in an amount of about 20 pM. In some embodiments, the CHO cells are cultured in insulin in an amount of about 2 mg / mL or less, e.g., in an amount of about 2 mg / mL. In some embodiments, the CHO cells are cultured in galactose in an amount of about 15 g / L or less, e.g., in an amount of about 15 g / L. In some embodiments, the CHO cells are cultured in manganese in an amount of about 50 pM or less, e.g., in an amount from about 1 .5 pM to about 50 pM, e.g., in an amount of about 25.75 pM. In some embodiments, the CHO cells are cultured in glucose in an amount of about 175 g / L or less, e.g., in an amount from about 100 g / L to about 175 g / L, e.g., in an amount of about 137.5 g / mL. In some embodiments, the CHO cells are cultured in calcium in an amount greater than 1 .2 mM, e.g., 2-3 mM calcium, e.g., about 2.4 mM calcium.

[0077] In some aspects, an antibody composition of any one of the previous aspects is produced by a process comprising (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies, and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the CHO cells express a fucosyltransferase (e.g., Fut8), a glycoside hydrolase (e.g., Man2a1), and / or a GIcNAc transferase (e.g., Mgat2). In some embodiments, the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, insulin, galactose, manganese, and / or glucose. In some embodiments, the CHO cells are cultured in putrescine in an amount of about 1 g / L or less, e.g., in an amount of about 1 g / L. In some embodiments, the CHO cells are cultured in L-fucose in an amount of about 5 g / L or less, e.g., in an amount of about 5 g / L. In some embodiments, the CHO cells are cultured in dexamethasone in an amount of about 20 pM or less, e.g., in an amount of about 20 pM. In some embodiments, the CHO cells are cultured in insulin in an amount of about 2 mg / mL or less, e.g., in an amount of about 2 mg / mL. In some embodiments, the CHO cells are cultured in galactose in an amount of about 15 g / L or less, e.g., in an amount of about 15 g / L. In some embodiments, the CHO cells are cultured in manganese in an amount of about 50 pM or less, e.g., in an amount from about 1 .5 pM to about 50 pM, e.g., in an amount of about 25.75 pM. In some embodiments, the CHO cells are cultured in glucose in an amount of about 175 g / L or less, e.g., in an amount from about 100 g / L to about 175 g / L, e.g., in an amount of about 137.5 g / mL. In some embodiments, the CHO cells are cultured in calcium in an amount greater than 1 .2 mM, e.g., 2-3 mM calcium, e.g., about 2.4 mM calcium.

[0078] In another aspect, provided is a method of producing an antibody composition, the method comprising:(a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L-fucose, dexamethasone, insulin, galactose, manganese, and / or glucose; and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the CHO cells express a fucosyltransferase (e.g., Fut8), a glycoside hydrolase (e.g., Man2a1), and / or a GIcNAc transferase (e.g., Mgat2). In some embodiments, the CHO cells are cultured in putrescine in an amount of about 1 g / L or less, e.g., in an amount of about 1 g / L. In some embodiments, the CHO cells are cultured in L-fucose in an amount of about 5 g / L or less, e.g., in an amount of about 5 g / L. In some embodiments, the CHO cells are cultured in dexamethasone in an amount of about 20 pM or less, e.g., in an amount of about 20 pM. In some embodiments, the CHO cells are cultured in insulin in an amount of about 2 mg / mL or less, e.g., in an amount of about 2 mg / mL. In some embodiments, the CHO cells are cultured in galactose in an amount of about 15 g / L or less, e.g., in an amount of about 15 g / L. In some embodiments, the CHO cells are cultured in manganese in an amount of about 50 pM or less, e.g., in an amount from about 1 .5 pM to about 50 pM, e.g., in an amount of about 25.75 pM. In some embodiments, the CHO cells are cultured in glucose in an amount of about 175 g / L or less, e.g., in an amount from about 100 g / L to about 175 g / L, e.g., in an amount of about 137.5 g / mL. In some embodiments, the CHO cells are cultured in calcium in an amount greater than 1 .2 mM, e.g., 2-3 mM calcium, e.g., about 2.4 mM calcium.

[0079] In another aspect, provided is a method of producing an antibody composition, the method comprising: (a) providing CHO cells comprising: (i) a fucosyltransferase (e.g., Fut8), a glycoside hydrolase (e.g., Man2a1), and / or a GIcNAc transferase (e.g., Mgat2), and (ii) nucleic acid encoding antibodies (e.g., vector encoding antibodies, e.g., expression vector encoding antibodies), wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies; and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, insulin, galactose, manganese, and / or glucose. In some embodiments, the CHO cells are cultured in putrescine in an amount of about 1 g / L or less, e.g., in an amount of about 1 g / L. In some embodiments, the CHO cells are cultured in L-fucose in an amount of about 5 g / L or less, e.g., in an amount of about 5 g / L. In some embodiments, the CHO cells are cultured in dexamethasone in an amount of about 20 pM or less, e.g., in an amount of about 20 pM. In some embodiments, the CHO cells are cultured in insulin in an amount of about 2 mg / mL or less, e.g., in an amount of about 2 mg / mL. In some embodiments, the CHO cells are cultured in galactose in an amount of about 15 g / L or less, e.g., in an amount of about 15 g / L. In some embodiments, the CHO cells are cultured in manganese in an amount of about 50 pM or less, e.g., in an amount from about 1 .5 pM to about 50 pM, e.g., in an amount of about 25.75 pM. In some embodiments, the CHO cells are cultured in glucose in an amount of about 175 g / L or less, e.g., in an amount from about 100 g / L to about 175 g / L, e.g., in an amount of about 137.5 g / mL. In some embodiments, the CHO cells are cultured in calcium in an amount greater than 1.2 mM, e.g., 2-3 mM calcium, e.g., about 2.4 mM calcium.

[0080] In some aspects, the invention involves a method of producing the antibody composition of any of the previous aspects, the method comprising: (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies, and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the CHO cells express a fucosyltransferase (e.g., Fut8), a glycoside hydrolase (e.g., Man2a1), and / or a GIcNAc transferase (e.g., Mgat2). In some embodiments, the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, insulin, galactose, manganese, and / or glucose. In some embodiments, the CHO cells are cultured in putrescine in an amount of about 1 g / L or less, e.g., in an amount of about 1 g / L. In some embodiments, the CHO cells are cultured in L-fucose in an amount of about 5 g / L or less, e.g., in an amount of about 5 g / L. In some embodiments, the CHO cells are cultured in dexamethasone in an amount of about 20 pM or less, e.g., in an amount of about 20 pM. In some embodiments, the CHO cells are cultured in insulin in an amount of about 2 mg / mL or less, e.g., in an amount of about 2 mg / mL. In some embodiments, the CHO cells are cultured in galactose in an amount of about 15 g / L or less, e.g., in an amount of about 15 g / L. In some embodiments, the CHO cells are cultured in manganese in an amount of about 50 pM or less, e.g., in an amount from about 1.5 pM to about 50 pM, e.g., in an amount of about 25.75 pM. In some embodiments, the CHO cells are cultured in glucose in an amount of about 175 g / L or less, e.g., in an amount from about 100 g / L to about 175 g / L, e.g., in an amount of about 137.5 g / mL. In some embodiments, the CHO cells are cultured in calcium in an amount greater than 1 .2 mM, e.g., 2-3 mM calcium, e.g., about 2.4 mM calcium.

[0081] In any of the preceding aspects of anti-CD20 antibody production, culture conditions may include a pH of about 6.95 or higher, e.g., about 7.1 or higher, e.g., about 7.1 , e.g., pH 7.1 . In some embodiments, the conditions comprise culture in the presence of putrescine. In some embodiments, the putrescine is in an amount from about 0.5 to about 1 .0 mg / mL. In some embodiments, the conditions comprise culture in the presence of fucose. In some embodiments, the fucose is in an amount from about 2.5 mg / mL to about 5.0 mg / mL. In some embodiments, the fucose is in an amount of about 5.0 mg / mL. In some embodiments, the conditions comprise culture in the presence of insulin. In some embodiments, the insulin is in an amount from about 1 ug / mL to about 2 ug / mL. In some embodiments, the conditions comprise culture in the presence of dexamethasone. In some embodiments, the dexamethasone is in an amount from about 10 uM to about 20 uM.

[0082] In some embodiments, any of the aforementioned methods of producing the anti-CD20 antibody, the method further includes preparing a pharmaceutical formulation by adding a pharmaceutically acceptable carrier to the antibody composition. In some embodiments, the pharmaceutical formulation comprises arginine, sodium acetate, sodium chloride, EDTA, and / or polysorbate. In some embodiments, the pharmaceutical formulation comprises 1 % (w / v) arginine, 50 mM sodium acetate, 51 mM sodium chloride, 0.05 mM EDTA, and 0.02% (w / v) polysorbate 80. In some embodiments, the antibodies are present in the pharmaceutical formulation in an amount of about 50 mg / mL or about 90 mg / mL. In some embodiments, the pharmaceutical formulation is at a pH from 5.2-5.8. In some embodiments, the method further includes preparing a pharmaceutical product comprising a container by dispensing the pharmaceutical formulation into the container. In some embodiments, the container is a prefilled syringe or an autoinjector.

[0083] In some aspects, provided herein is a method of treating multiple sclerosis (MS), the method comprising administering (e.g., through subcutaneous injection) an effective amount of any of the antibodies (e.g., CHO cell-produced antibodies) or compositions thereof (e.g., CHO cell-produced antibody compositions, or anti-CD20 antibody compositions devoid of murine-associated glycans) to a patient in need thereof. In some embodiments, the CHO cell-produced antibody composition comprises anti-CD20 antibodies comprising:(a) a light chain comprising the amino acid sequence of SEQ ID NO: 1 ; and (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., SEQ ID NO: 2 and / or SEQ ID NO: 3). In some embodiments, the anti-CD20 antibodies comprise (a) the antibody composition of any of the previous aspects; or (b) the CHO cell-produced antibody of any of the previous aspects. In some embodiments, the CHO cell-produced antibody composition is administered to the patient (e.g., through subcutaneous injection) once per month, once every two months, once every three months, once every four months, once every six months, once every eight months, or once yearly. In some embodiments, the CHO cell-produced antibody composition is administered to the patient (e.g., through subcutaneous injection) once per month, e.g., the CHO cell-produced antibody composition administered to the patient once per month comprises about 20 mg of the anti-CD20 antibodies. In some embodiments, the CHO cell-produced antibody composition is administered to the patient (e.g., through subcutaneous injection) once every two months, e.g., the CHO cell-produced antibody composition administered to the patient once every two months comprises 20 to 200 mg of the anti-CD20 antibodies (e.g., 100 to 150 mg of the anti-CD20 antibodies, e.g., about 110 mg, about 120 mg, about 130 mg, about 135 mg, about 140 mg, or about 150 mg of the anti-CD20 antibodies, e.g., about 135 mg of the anti-CD20 antibodies). In some embodiments, the multiple sclerosis is relapsing multiple sclerosis (RMS). In some embodiments, the multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS), or clinically isolated syndrome (CIS).

[0084] In some aspects, provided herein is a method of treating multiple sclerosis (MS), the method comprising administering (e.g., through subcutaneous injection) an effective amount of a CHO cell- produced antibody composition to a patient in need thereof, wherein the CHO cell-produced antibody composition comprises anti-CD20 antibodies comprising:(a) a light chain comprising the amino acid sequence of SEQ ID NO: 1 ; and (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-CD20 antibodies comprise (a) the antibody composition of any of the previous aspects; or (b) the CHO cell-produced antibody of any of the previous aspects. In some embodiments, the CHO cell-produced antibody composition is administered to the patient (e.g., through subcutaneous injection) once per month, once every two months, once every three months, once every four months, once every six months, once every eight months, or once yearly. In some embodiments, the CHO cell-produced antibody composition is administered to the patient (e.g., through subcutaneous injection) once per month, e.g., the CHO cell-produced antibody composition administered to the patient once per month comprises about 20 mg of the anti-CD20 antibodies. In some embodiments, the CHO cell-produced antibody composition is administered to the patient (e.g., through subcutaneous injection) once every two months, e.g., the CHO cell-produced antibody composition administered to the patient once every two months comprises about 135 mg of the anti-CD20 antibodies. In some embodiments, the multiple sclerosis is relapsing multiple sclerosis (RMS). In some embodiments, the multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS), or clinically isolated syndrome (CIS).

[0085] BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIGS. 1A-1 F are schematic structures of afucosylated glycans that can be found in some of the antibody compositions described herein, e.g., as described in Example 2. N-linkages are on the righthand side of each structure. Squares indicate glucose, circles indicate mannose, and teardrops indicate galactose. FIG. 1A is a schematic structure of GO-N (also known in Oxford nomenclature as A1 , which is herein inclusive of A1 [6] and A1 [3]). FIG. 1 B is a schematic structure of bGO-F (also known as GO or in Oxford nomenclature as A2). FIG. 1C is a schematic structure of M5. FIG. 1 D is a schematic structure of G1 (also known in Oxford nomenclature as A2G[4]1 , which is herein inclusive ofA2[6]G[4]1 and A2[3]G[4] 1). FIG. 1 E is a schematic structure of M6. FIG. 1F is a schematic structure of M7.

[0087] FIGS. 2A-2C are schematic structures of fucosylated glycans present in relevant quantity in some of the antibody compositions described herein, e.g., as described in Example 2. N-linkages are on the right-hand side of each structure. Squares indicate glucose, circles indicate mannose, teardrops indicate galactose, and triangles indicate fucose. FIG. 2A is a schematic structure of GOF (also known in Oxford nomenclature as F[6]A2). FIG. 2B is a schematic structure of G1 F (also known in Oxford nomenclature as F[6]A2G[4]1 , which is herein inclusive of F[6]A2[6]G[4]1 and F[6]A2[3]G[4]1). FIG. 2C is a schematic structure of G2F (also known in Oxford nomenclature as F[6]A2G[4]2).

[0088] FIGS. 3A-3F are schematic structures of fucosylated glycans that may be detectable in some of the antibody compositions described herein, and which correspond to results in Table 2 of Example 2. N- linkages are on the right-hand side of each structure. Squares indicate glucose, circles indicate mannose, teardrops indicate galactose, triangles indicate fucose, and diamonds indicate sialic acid. FIG. 3A is a schematic structure of GOF-N (also known in Oxford nomenclature as F[6]A1 , which is herein inclusive of F[6]A1 [6] and F[6]A1 [3]). FIG. 3B is a schematic structure of G1 F-N (also known in Oxford nomenclature as F[6]A1 G[4]1 , which is herein inclusive of F[6]A[6]G[4]1 and F[6]A1 [3]G[4]1). FIG. 3C is a schematic structure of G1 FSA-N (also known in Oxford nomenclature as F[6]A1G[4]1S1 , which is inclusive of F[6]A1 [6]G[4]1 S[n]1 and F[6]A1 [3]G[4]1 S[n]1). FIG. 3D is a schematic structure of G1 FSA (also known in Oxford nomenclature as F(6)A2G1 (4)S1 , which is inclusive of F[6]A2[6]G1 [4]S[n]1 and F[6]A2[3]G1 [4]S[n]1). FIG. 3E is a schematic structure of G2FSA (also known in Oxford nomenclature as F[6]A2G[4]2S1 , which is inclusive of F[6]A2[6]G[4]2S1 and F[6]A2[3]G[4]2S1). FIG. 3F is a schematic structure of G2FSA2 (also known in Oxford nomenclature as F[6]A2G[4]2S2, which is herein inclusive of F[6]A2G[4]2S[n,n]2).

[0089] FIG. 4 is an oligosaccharide profile of NS / 0 cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NO: 1 and 2.

[0090] FIG. 5 is a table showing peak retention times, emission units, and relative areas for each peak observed in the oligosaccharide profile shown in FIG. 4.

[0091] FIG. 6 is an oligosaccharide profile of CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NO: 1 and 2.

[0092] FIG. 7 is a table showing peak retention times, emission units, and relative areas for each peak observed in the oligosaccharide profile shown in FIG. 6.

[0093] FIGS. 8A-8C are bar graphs showing glycosylation profiles of NS / 0 cell-produced ofatumumab (left bars) compared to non-glycoengineered CHO cell-produced ofatumumab (right bars). FIG. 8A shows afucosylation levels; FIG. 8B shows high mannose glycan levels; and FIG. 8C shows galactosylation levels.

[0094] FIGS. 9A and 9B are a series of graphs showing results of a Design-of-Experiment (DoE) study assessing the impact of various culture conditions on the glycosylation parameters of CHO cell-produced anti-CD20 antibodies. FIG. 9A shows the impact of pH, putrescin, fucose, insulin, and dexamethasone. FIG. 9B shows the impact of galactose, glucose, harvest day (culture duration), manganese (Mn), and calcium (Ca2+).

[0095] FIG. 10 is a schematic drawing showing two genetic glycoengineering approaches tested for adjusting the glycosylation profile of CHO cell-produced anti-CD20 antibodies to mirror the glycosylation profile of NS / 0 cell-produced anti-CD20 antibodies. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0096] Production of ofatumumab in murine NS / 0 cells can be difficult and inefficient, making it commonly disfavored in the field of commercial monoclonal antibody production. The present invention is based, in part, on a theory that other, non-NS / 0 cells (e.g., non-murine cells, such as Chinese hamster ovary (CHO) cells) could offer a more efficient anti-CD20 antibody manufacturing platform as compared to NS / 0 cells, e.g., by yielding higher antibody titers of antibodies with ofatumumab sequences, reducing manufacturing times, and reducing cost of goods. To test this approach, Applicant engineered CHO cells to express amino acid sequences of ofatumumab, conducted glycosylation analyses of the resulting CHO cell-produced ofatumumab and reference murine cell-produced ofatumumab, and tested various functional attributes of the CHO cell-produced ofatumumab against reference murine cell-produced anti- CD20 antibodies made with the same antibody-encoding vectors. While some variation in post- translational modifications is not uncommon when switching between host cell types, Applicant observed unexpectedly large differences in both glycan structure and the immune cell signaling behavior between the CHO cell-produced ofatumumab and the murine cell-produced ofatumumab. Applicant inferred from these unexpected differences in glycosylation profiles that patients may respond differently to CHO cell- produced ofatumumab, as compared to the NS / 0 cell-produced ofatumumab, which has been shown in multiple clinical trials to be safe and effective. To reduce safety risks associated with these differences, Applicant developed various methods of glycoengineering CHO host cell cultures to express CHO cell- produced ofatumumab having structurally and functionally similar profiles to NS / 0 cell-produced ofatumumab, thereby providing a more efficient process for producing antibodies having the same or similar amino acid sequence profiles of ofatumumab. These methods, the resulting antibodies and antibody compositions thereof, and therapeutic uses of such antibodies and compositions, are described in detail herein.

[0097] I. Definitions

[0098] The term “antibody” as used herein refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that is capable of binding an antigen non-covalently, reversibly and specifically. For example, a naturally occurring “antibody” of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term “antibody” includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, bispecific or multispecific antibodies and anti- idiotypic (anti-ld) antibodies (including, e.g., anti-ld antibodies to antibodies of the disclosure). The antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2).

[0099] As used in this specification, an “anti-CD20 antibody” is an antibody that specifically binds to the human CD20 antigen expressed on B cells. CD20 is expressed on late pre-B cells, mature B cells, and memory B cells, while not expressed on lymphoid stem or plasma cells. Examples of anti-CD20 antibodies include, but are not limited to, ofatumumab, rituximab, tositumomab, ublituximab, ocrelizumab (2H7.vl6), 11 B8 or 7D8 (disclosed in WO 2004 / 035607), an anti-CD20 antibody disclosed in WO 2005 / 103081 , such as C6, an anti-CD20 antibody disclosed in WO 2003 / 68821 such as IMMU-106, an anti-CD20 antibody disclosed in WO 2004 / 103404 such as AME-133 (from Applied Molecular Evolution / Lilly), and anti-CD20 antibody disclosed in US 2003 / 0118592 such as TRU-015 (from Trubion Pharmaceuticals Inc). In some embodiments, anti-CD20 antibodies described herein have sequences of ofatumumab, in which case such antibodies (e.g., CHO cell-produced anti-CD20 antibodies having ofatumumab sequences) can be produced by transfecting a host cell with a vector as described in International Patent Publication No. WO 2004 / 035607, which is incorporated herein by reference in its entirety.

[0100] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0101] Sequence identity between two similar sequences (e.g., antibody variable domains) can be measured by algorithms such as that of Smith, T. F. & Waterman, M. S. (1981) “Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S. B. & Wunsch, CD. (1970) “A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins,” J. Mol. Biol. 48:443 [homology alignment algorithm], Pearson, W. R. & Lipman, D. J. (1988) “Improved Tools For Biological Sequence Comparison,” Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 [search for similarity method]; or Altschul, S. F. et al, (1990) “Basic Local Alignment Search Tool,” J. Mol. Biol. 215:403-10, the “BLAST” algorithm, see blast.ncbi.nlm.nih.gov / Blast.cgi. When using any of the aforementioned algorithms, the default parameters (for Window length, gap penalty, etc.) are used. In one embodiment, sequence identity is done using the BLAST algorithm, using default parameters.

[0102] Optionally, the identity is determined over a region that is at least about 50 nucleotides (or, in the case of a peptide or polypeptide, at least about 10 amino acids) in length, or in some cases over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. In some embodiments, the identity is determined over a defined domain, e.g., the VH or VL of an antibody. Unless specified otherwise, the sequence identity between two sequences is determined over the entire length of the shorter of the two sequences.

[0103] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0104] As used herein, the terms “host cell” or “recombinant host cell” refer to a cell that has been genetically-engineered, e.g., through introduction of a heterologous nucleic acid. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A host cell can carry the heterologous nucleic acid transiently, e.g., on an extrachromosomal heterologous expression vector, or stably, e.g., through integration of the heterologous nucleic acid into the host cell genome. For purposes of expressing an antigen-binding molecule, a host cell can be a cell line of mammalian origin or mammalian-like characteristics, such as Chinese hamster ovary (CHO) cells, murine cells (e.g., NS / 0 cells or SP2 / 0 cells), monkey kidney cells (COS, e.g., COS-1 , COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), PerC6, BSC-1 , human hepatocellular carcinoma cells (e.g., Hep G2), HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives and / or engineered variants thereof. Engineered variants include, e.g., glycan profile modified and / or site-specific integration site derivatives. In some embodiments, host cells of the present invention are not NS / 0 cells (i.e., a cell type other than NS / 0 cells).

[0105] As used herein, the term glycoengineering refers to a change to the glycosylation pattern of a naturally occurring polypeptide or fragment thereof. Glycoengineering includes metabolic engineering of the glycosylation machinery of a cell, including (i) changes in cell environment affecting glycosylation (e.g., nutrient content of media, e.g., presence or increased concentration of putrescine, L-fucose, dexamethasone, and / or insulin) (herein referred to as “metabolic glycoengineering” and (ii) genetic manipulations of the oligosaccharide synthesis pathways to achieve altered glycosylation of glycoproteins expressed in cells (e.g., addition of Fut8, Man2a1 , and / or Mgat2) (herein referred to as “genetic glycoengineering”).

[0106] As used herein, “GO-N” refers to an N-glycan having the structure shown in FIG. 1 Awith the composition of GIcNAcsMam, where GIcNAc is N-acetyl glucosamine and Man is mannose. GO-N is also known in Oxford nomenclature as A1 , which is herein inclusive of A1 [6] and A1 [3]).

[0107] As used herein, “bGO-F” refers to an N-glycan having the structure shown in FIG. 1 B with the composition of GlcNAc4Man3, where GIcNAc is N-acetyl glucosamine and Man is mannose. bGO-F is also known as GO and, in Oxford nomenclature, as A2).

[0108] As used herein, “M5” refers to an N-glycan having the structure shown in FIG. 1 C with the composition of GlcNAc2Mans, where GIcNAc is N-acetyl glucosamine and Man is mannose.

[0109] As used herein, “G1” refers to an N-glycan having the structure shown in FIG. 1 D with the composition of GlcNAc4Ga Man3, where GIcNAc is N-acetyl glucosamine, Gal is galactose, and Man is mannose. G1 is also known as 1 ,6 bG1 F or 1 ,3 bG1 F and, in Oxford nomenclature, asA2G[4]1 , which is herein inclusive of A2[6]G[4]1 and A2[3]G[4]1). As used herein, “M6” refers to an N-glycan having the structure shown in FIG. 1 E with the composition of GlcNAc2Mane, where GIcNAc is N-acetyl glucosamine and Man is mannose.

[0110] As used herein, “M7” refers to an N-glycan having the structure shown in FIG. 1 F with the composition of GlcNAc2Man7, where GIcNAc is N-acetyl glucosamine and Man is mannose.

[0111] High mannose glycans contain terminal mannose sugars. In some embodiments, high mannose glycans contain between five and nine mannose residues attached to a GlcNAc2 core. For example, high mannose glycans may contain five terminal mannose residues (i.e. “M5”), six terminal mannose residues (i.e. “M6”), or seven terminal mannose residues (i.e. “M7”).

[0112] As used herein, “GOF” refers to an N-glycan having the structure shown in FIG. 2A with the composition of GlcNAc4Man3Fuci , where GIcNAc is N-acetyl glucosamine, Man is mannose, and Fuc is fucose. GOF is also known as bGO and, in Oxford nomenclature, as F[6]A2.

[0113] As used herein, “G1 F” refers to an N-glycan having the structure shown in FIG. 2B with the composition of GlcNAc4Ga Man3Fuci, where GIcNAc is N-acetyl glucosamine, Gal is galactose, Man is mannose, and Fuc is fucose. G1 F is also known as 1 ,6 bG1 or 1 ,3 bG1 and, in Oxford nomenclature, as F[6]A2G[4]1 , which is herein inclusive of F[6]A2[6]G[4]1 and F[6]A2[3]G[4]1. As used herein, “G2F” refers to an N-glycan having the structure shown in FIG. 2C with the composition of GlcNAc4Gal2Man3Fuci, where GIcNAc is N-acetyl glucosamine, Gal is galactose, Man is mannose, and Fuc is fucose. G2F is also known as bG2 and, in Oxford nomenclature, as F[6]A2G[4]2.

[0114] As used herein, “GOF-N” refers to an N-glycan having the structure shown in FIG. 3Awith the composition of GIcNAcsMamFuci , where GIcNAc is N-acetyl glucosamine, Gal is galactose, Man is mannose, and Fuc is fucose. GOF-N is also known as 1 ,6 bGO-N or 1 ,3 bGO-N and, in Oxford nomenclature, as F[6]A1 , which is herein inclusive of F[6]A1 [6] and F[6]A1 [3].

[0115] As used herein, “G1 F-N” refers to an N-glycan having the structure shown in FIG. 3B with the composition of GIcNAcsGa MansFuci, where GIcNAc is N-acetyl glucosamine, Gal is galactose, Man is mannose, and Fuc is fucose. G1 F-N is also known as 1 ,6 bG1-N or 1 ,3 bG1-N and, in Oxford nomenclature, as F[6]A1 G[4]1 , which is herein inclusive of F[6]A[6]G[4]1 and F[6]A1 [3]G[4]1 .

[0116] As used herein, “G1 FSA-N” refers to an N-glycan having the structure shown in FIG. 3C with the composition of GIcNAcsGa MansFuciNeuAci , where GIcNAc is N-acetyl glucosamine, Gal is galactose, Man is mannose, Fuc is fucose, and NeuAc is N-acetylneuraminic acid. G1 FSA-N is also known as 1 ,6 bG1 Sc-N or 1 ,3 bG1 Sc-N and, in Oxford nomenclature as F[6]A1 G[4]1 S1 , which is inclusive of F[6]A1 [6]G[4]1 S[n]1 and F[6]A1 [3]G[4]1 S[n]1.

[0117] As used herein, “G1 FSA” refers to an N-glycan having the structure shown in FIG. 3D with the composition of GlcNAc4Ga Man3FuciNeuAci , where GIcNAc is N-acetyl glucosamine, Gal is galactose, Man is mannose, Fuc is fucose, and NeuAc is N-acetylneuraminic acid. G1 FSA is also known as 1 ,6 bG1 SA or 1 ,3 bG1 SA and, in Oxford nomenclature, as F(6)A2G1 (4)S1 , which is inclusive of F[6]A2[6]G1 [4]S[n]1 and F[6]A2[3]G1 [4]S[n]1.

[0118] As used herein, “G2FSA” refers to an N-glycan having the structure shown in FIG. 3E with the composition of GlcNAc4Gal2Man3FuciNeuAci , where GIcNAc is N-acetyl glucosamine, Gal is galactose, Man is mannose, Fuc is fucose, and NeuAc is N-acetylneuraminic acid. G2FSA is also known as 1 ,6 bG2SA or 1 ,3 bG2SA and, in Oxford nomenclature, as F[6]A2G[4]2S1 , which is inclusive of F[6]A2[6]G[4]2S1 and F[6]A2[3]G[4]2S1 .

[0119] As used herein, “G2FSA2” refers to an N-glycan having the structure shown in FIG. 3F with the composition of GlcNAc4Gal2Man3FuciNeuAc2, where GIcNAc is N-acetyl glucosamine, Gal is galactose, Man is mannose, Fuc is fucose, and NeuAc is N-acetylneuraminic acid. G2FSA2 is also known as bG2SA2 and, in Oxford nomenclature, as F[6]A2G[4]2S2, which is herein inclusive of F[6]A2G[4]2S[n,n]2.

[0120] The term “ADCC,” or “antibody dependent cell-mediated cytotoxicity,” as used herein, refers to the cell-mediated reaction where nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is correlated with binding to FcyRllla; increased binding to FcyRllla leads to an increase in ADCC activity.

[0121] The term “ADCP,” or antibody dependent cell-mediated phagocytosis, as used herein, refers to the cell-mediated reaction where nonspecific phagocytic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell. The term “CDC,” or “complement dependent cytotoxicity,” as described herein, refers to the immune response that destroys target cells by activating the complement cascade.

[0122] The term “effector function” refers to an activity of an antibody molecule that is mediated by binding through a domain of the antibody other than the antigen-binding domain, usually mediated by binding of effector molecules. Effector function includes complement-mediated effector function, which is mediated by, for example, binding of the C1 component of the complement to the antibody. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Effector function also includes Fc receptor (FcR)-mediated effector function, which can be triggered upon binding of the constant domain of an antibody to an Fc receptor (FcR). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, ADCC, ADCP, release of inflammatory mediators, placental transfer and control of immunoglobulin production. An effector function of an antibody can be altered by altering, e.g., enhancing or reducing, the affinity of the antibody for an effector molecule such as an Fc receptor or a complement component. Binding affinity will generally be varied by modifying the effector molecule binding site, and in this case it is appropriate to locate the site of interest and modify at least part of the site in a suitable way. It is also envisaged that an alteration in the binding site on the antibody for the effector molecule need not alter significantly the overall binding affinity but can alter the geometry of the interaction rendering the effector mechanism ineffective as in non-productive binding. It is further envisaged that an effector function can also be altered by modifying a site not directly involved in effector molecule binding, but otherwise involved in performance of the effector function.

[0123] The term "KD,” as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. KD can be measured using surface plasmon resonance (SPR, e.g., using a Biacore system) or bio-layer interferometry (BLI, e.g., using Sartorius OCTET® system). For BLI analysis, experimental data can be evaluated with global fitting using Octet Studio 12.0. software to calculate KD.

[0124] As used herein, a composition is “devoid” of a component when the component is entirely absent from the composition or present in such little amount that it is undetectable, e.g., undetectable by mass spectrometry or by relevant functional readout.

[0125] As used herein, the terms “treating” and “treatment” refer to both therapeutic treatment and prophylactic or preventative therapies, unless specified otherwise.

[0126] In a pharmacological sense, in the context of the present invention, a "therapeutically effective amount" of an antibody refers to an amount effective in the prevention or treatment of a disorder for the treatment of which the antibody is effective.

[0127] As used herein, the terms “subject” and “patient” are used interchangeably and refer to a human. The term “about” in relation to a numerical value x is means x ± 10%, unless the context dictates otherwise.

[0128] In some embodiments, the glycan levels in the antibodies disclosed herein are indicated as % total integrated area.

[0129] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0130] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and.”

[0131] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of those in the art.

[0132] 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 application belongs and as commonly used in the art to which this application belongs. The publications and other reference materials referenced herein to describe the background of the disclosure and to provide additional detail regarding its practice are hereby incorporated by reference.

[0133] II. Anti-CD20 Antibodies

[0134] Provided herein are anti-CD20 antibodies produced in host cells that are not NS / 0 cells (e.g., CHO cell-produced anti-CD20 antibodies), and compositions thereof, that share important structural and functional properties of NS / 0 cell-produced ofatumumab, which has been approved for treatment of multiple sclerosis. The host cells may be non-NS / 0 cells, e.g. non-murine cells. Accordingly, in some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) have one or more light chain complementarity determining regions (LCDRs) having the amino acid sequences of SEQ ID NOs: 4-6 and / or one or more heavy chain complementarity determining regions (HCDRs) having the amino acid sequences of SEQ ID NOs: 8-10. In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) have a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 7 and / or a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 11 . In some embodiments of the non-NS / 0 cell-produced antibodies (e.g., non-murine cell- produced antibodies, such as CHO cell-produced antibodies) having the LCDRs and HCDRs of SEQ ID NOs: 4-6 and 8-10 are lgG1 antibodies, e.g., IgGl K antibodies. In some embodiments, the non-NS / 0 cell- produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) having the LCDRs and HCDRs of SEQ ID NOs: 4-6 and 8-10 (e.g., lgG1 antibodies, e.g., IgGl K antibodies) are human antibodies. In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) having the LCDRs and HCDRs of SEQ ID NOs: 4-6 and 8-10 have a VL having the amino acid sequence of SEQ ID NO: 7 and / or a VH having the amino acid sequence of SEQ ID NO: 11 . In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) having the VL having the amino acid sequence of SEQ ID NO: 7 and / or the VH having the amino acid sequence of SEQ ID NO: 11 further have a light chain having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 ; e.g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1 ; e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1) and / or a heavy chain with at least 95% sequence identity to SEQ ID NO: 2 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2; e.g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 2; e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2). In some embodiments, the non-NS / 0 cell-produced antibodies (e.g., non-murine cell-produced antibodies, such as CHO cell-produced antibodies) having the VL having the amino acid sequence of SEQ ID NO: 7 and / or the VH having the amino acid sequence of SEQ ID NO: 11 further have a light chain having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 ; e.g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 1 ; e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1) and / or a heavy chain with at least 95% sequence identity to SEQ ID NO: 3 (e.g., at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3; e.g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO: 3; e.g., 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3).

[0135] Thus, in some instances, the invention features anti-CD20 antibodies (e.g., IgG antibodies, e.g., lgG1 antibodies, e.g., IgGl K antibodies) having a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., amino acid sequences shared with ofatumumab), and compositions of antibodies thereof (e.g., compositions having a therapeutically effective amount of the anti-CD20 antibodies).

[0136] The type of host cell expressing the anti-CD20 antibodies can confer certain structural features to the antibody, which may differ from a reference antibody produced in another cell type (e.g., NS / 0 cell- produced ofatumumab). Thus, in certain embodiments of the present invention, provided are CHO cell- produced anti-CD20 antibodies that are structurally different from NS / 0 cell-produced antibodies (e.g., due to differences in C-terminal lysine clipping).

[0137] In some instances, compositions of anti-CD20 antibodies (e.g., CHO cell-produced anti-CD20 antibodies) having light chains comprising the amino acid sequence of SEQ ID NO: 1 and heavy chains comprising the amino acid sequence of SEQ ID NO: 2 have a fewer number of C-terminal lysine residues on the antibody heavy chain (e.g., fewer than 15% of the CHO-cell produced antibodies comprise a heavy chain C-terminal lysine (i.e., position 452 of SEQ ID NO: 3), fewer than 10% of the CHO-cell produced antibodies comprise a heavy chain C-terminal lysine (i.e., position 452 of SEQ ID NO: 3) or fewer than 10% of the CHO-cell produced antibodies comprise a heavy chain C-terminal lysine (i.e., position 452 of SEQ ID NO: 3)). C-terminal lysines can be quantified using techniques known in the field, such as mass spectrometry, e.g., as described in Shah et al., Journal of Pharmaceutical Sciences 111 (2022) 2445- 2450, the contents of which are incorporate herein.

[0138] Additionally, or alternatively, C-terminal amidation of the antibody heavy chain can differ between anti-CD20 antibody compositions of the present invention relative to a reference antibody composition. In some instances, compositions of CHO cell-produced anti-CD20 antibodies having light chains comprising the amino acid sequence of SEQ ID NO: 1 and heavy chains comprising the amino acid sequence of SEQ ID NO: 2 have a higher heavy chain amidation than a reference antibody composition (e.g., NS / 0 cell-produced ofatumumab). In some instances, at least 0.01 % of the CHO-cell produced antibodies comprise heavy chain C-terminal amidation. Such heavy chain amidation can be measured using techniques known in the field, such as mass spectrometry, e.g., as described in Shah et al., Journal of Pharmaceutical Sciences 111 (2022) 2445-2450, the contents of which are incorporate herein.

[0139] Thus, in some instances, compositions of anti-CD20 antibodies (e.g., CHO cell-produced anti- CD20 antibodies) having light chains comprising the amino acid sequence of SEQ ID NO: 1 and heavy chains comprising the amino acid sequence of SEQ ID NO: 2 include antibodies having detectable levels of C-terminal amidation (e.g., at least 0.01 % of the heavy chains comprises C-terminal amidation, e.g., from 0.01 % to 5% of the heavy chains comprises C-terminal amidation). C-terminal amidation can be quantified using techniques known in the field, such as mass spectrometry, e.g., as described in Shah et al., Journal of Pharmaceutical Sciences 111 (2022) 2445-2450, the contents of which are incorporate herein.

[0140] In some instances, compositions of anti-CD20 antibodies (e.g., CHO cell-produced anti-CD20 antibodies) having light chains comprising the amino acid sequence of SEQ ID NO: 1 and heavy chains comprising the amino acid sequence of SEQ ID NO: 2 are devoid of NGNA (e.g., have no detectable NG NA).

[0141] Native antibodies produced by mammalian cells are typically glycosylated, e.g., have a branched, biantennary oligosaccharide that is attached by an N-linkage (N-glycan, or N-linked glycan) to Asn302 (Asn297 according to the lgG1 standard numbering) of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GIcNAc), galactose, and sialic acid, as well as a fucose attached to a GIcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made to create antibody variants with certain improved properties through glycoengineering (e.g., metabolic glycoengineering and / or genetic glycoengineering).

[0142] In some instances, the anti-CD20 antibodies provided herein (e.g., CHO cell-produced anti-CD20 antibodies having light chains having the amino acid sequence of SEQ ID NO: 1 and heavy chains having the amino acid sequence of SEQ ID NO: 2) are glycoengineered to achieve functional properties that are suitable for treating MS patients (e.g., functional properties similar to those of NS / O-produced ofatumumab). Such glycoengineered anti-CD20 antibodies may have a different N-glycan profile than their native reference antibody (i.e., non-glycoengineered antibody produced in the unmodified form of the host cell, e.g., unmodified CHO cell).

[0143] Provided herein are antibody compositions (e.g., a CHO cell-produced antibody composition), containing antibodies (e.g., any of the CHO cell-produced anti-CD20 antibodies described herein, e.g., CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2) having an Fc region with an N-glycan (e.g., at Asn302), which are glycoengineered to have a lower percentage of afucosylated N-glycans than the native reference antibody composition.

[0144] Exemplary afucosylated species of N-glycan structures are shown in FIG. 1 .

[0145] In some instances, less than 10% of the N-glycans in the composition are afucosylated (e.g., 0.1% to 10%, 0.5% to 10%, or 1% to 10% of the N-glycans in the composition are afucosylated). In some embodiments, less than 7.8% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 7.8%, 0.5% to 7.8%, or 0.5% to 7.8% of the N-glycans in the composition are afucosylated). In some embodiments, 1-6% of the N-glycans in the composition are afucosylated (e.g., 0.1 % to 6%, 0.5% to 6%, or 0.5% to 6% of the N-glycans in the composition are afucosylated, e.g., about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1 %, about 1 .5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, or about 6% of the N-glycans in the composition are afucosylated).

[0146] In some instances, antibody compositions provided herein (e.g., a CHO cell-produced antibody composition) contain antibodies (e.g., any of the CHO cell-produced anti-CD20 antibodies described herein, e.g., CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2) having an Fc region with an N-glycan (e.g., at Asn302), which are glycoengineered to have a lower percentage of G0F N-glycans, compared the native reference antibody composition. The structure of G0F is shown in FIG. 2A. In some embodiments of an antibody composition comprising glycoengineered CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2, less than about 57% of the N-glycans in the composition are G0F (e.g., 30-57% of the N-glycans in the composition are G0F, e.g., 30-50% of the N-glycans in the composition are G0F, e.g., 32-49% of the N-glycans in the composition are G0F, e.g., 35-45% of the N-glycans in the composition are G0F, e.g., about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41 %, about 42%, about 43%, about 44%, or about 45% of the N-glycans in the composition are G0F). In some instances of the invention in which an antibody composition contains glycoengineered CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2, about 40% of the N-glycans in the composition are G0F.

[0147] In some instances, antibody compositions provided herein (e.g., a CHO cell-produced antibody composition) contain antibodies (e.g., any of the CHO cell-produced anti-CD20 antibodies described herein, e.g., CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2) having an Fc region with an N-glycan (e.g., at Asn302), which are glycoengineered to have a higher percentage of G1 F N-glycans, compared the native reference antibody composition. The structure of GOF is shown in FIG. 2B. In some embodiments of an antibody composition comprising glycoengineered CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2, more than about 17% of the N-glycans in the composition are G1 F (e.g., 17-40% of the N- glycans in the composition are G1 F, e.g., 28-35% of the N-glycans in the composition are GOF, e.g., about 28%, about 29%, about 30%, about 31 %, about 32%, about 33%, about 34%, or about 35% of the N- glycans in the composition are G1 F). In some instances of the invention in which an antibody composition contains glycoengineered CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2, about 31 % of the N-glycans in the composition are G1 F.

[0148] In some instances, antibody compositions provided herein (e.g., a CHO cell-produced antibody composition) contain antibodies (e.g., any of the CHO cell-produced anti-CD20 antibodies described herein, e.g., CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2) having an Fc region with an N-glycan (e.g., at Asn302), which are glycoengineered to have a higher percentage of G2F N-glycans, compared the native reference antibody composition. The structure of G2F is shown in FIG. 2C. In some embodiments of an antibody composition comprising glycoengineered CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2, more than about 2% of the N-glycans in the composition are G2F (e.g., 2-15% of the N-glycans in the composition are G2F, e.g., 6-13% of the N-glycans in the composition are GOF, e.g., about 6%, about 7%, about 8%, about 9%, about 10%, about 11 %, about 12%, or about 13% of the N-glycans in the composition are G2F). In some instances of the invention in which an antibody composition contains glycoengineered CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2, about 8% of the N-glycans in the composition are GOF.

[0149] In some instances, a glycoengineered CHO cell-produced antibody composition of the invention has one, two, or all three of the following properties: (i) a lower percentage of GOF N-glycans compared to the native reference antibody (non-engineered CHO cell-produced) composition (e.g., lower than about 57% of the N-glycans are GOF), (ii) a higher percentage of G1 F N-glycans compared to the native reference antibody (non-engineered CHO cell-produced) composition (e.g., higher than about 17% of the N-glycans are G1 F), and (iii) a higher percentage of G2F N-glycans compared to the native reference antibody (non-engineered CHO cell-produced) composition (e.g., higher than about 2% of the N-glycans are GOF).

[0150] In some instances, antibody compositions provided herein (e.g., a CHO cell-produced antibody composition) contain antibodies (e.g., any of the CHO cell-produced anti-CD20 antibodies described herein, (e.g., CHO cell-produced anti-CD20 antibodies having amino acid sequences of SEQ ID NOs: 1 and 2) having an Fc region with an N-glycan (e.g., atAsn302)) having a mannose-5 content of less than about 6% of the N-glycans in the composition.

[0151] In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N- glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and / or less than 6% of the N-glycans in the composition are mannose-5. In some embodiments, 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N-glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; and 6-13% of the N-glycans in the composition are G2F. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N-glycans in the composition are mannose-5.

[0152] In another aspect, provided herein is an antibody composition comprising CHO cell-produced antibodies, wherein the CHO cell-produced antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell-produced antibodies in the composition comprise an Fc region comprising an N- glycan, wherein 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and / or less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; and less than 6% of the N- glycans in the composition are mannose-5. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; and 6-13% of the N-glycans in the composition are G2F. In some embodiments, 32-49% of the N-glycans in the composition are GOF; 28-35% of the N-glycans in the composition are G1 F; 6-13% of the N-glycans in the composition are G2F; and less than 6% of the N-glycans in the composition are mannose-5.

[0153] In some embodiments of any of the preceding aspects, the composition comprises between 1 .0 and 2.5% N-glycans, as weight / weight of total protein.

[0154] In some embodiments, the composition comprises 40 to 150 mmol sialic acid (e.g., NeuGc) per mol CHO-produced antibodies.

[0155] In some embodiments, at least 25% of the N-glycans in the composition are terminally galactosylated.

[0156] In some embodiments, at least 2% of the N-glycans in the composition are terminally sialylated.

[0157] In some embodiments, at least 1 % of the N-glycans in the composition are terminally sialylated. Functional properties conferred by the preceding structural properties of anti-CD20 antibodies, and compositions thereof, include binding of the antibody to Fc receptors (e.g., Fc receptors present in a patient, e.g., Fc gamma receptors (FcyR), e.g., FcyRla, FcyRllaHR, FcyRllaLR, FcyRllb, FcyRlllb, FcyRI I laF158, and / or FcyRI I laV158). Fc receptor binding (e.g., binding affinity, as can be measured by dissociation constant KD) can mediate biological responses to a therapeutic antibody. For instance, ADCC and CDC are biological responses through which ofatumumab has been reported to exert its therapeutic effects and safety profile. The glycan profiles described above can influence whether Fc receptor binding leads to therapeutic effects through ADCC and / or CDC. In addition to the glycan profiles described above, provided herein further are Fc receptor binding properties characteristic of anti-CD20 antibodies (e.g., CHO cell-produced anti-CD20 antibodies described herein) that can be associated with therapeutic effects and safety of these antibodies and compositions thereof, e.g., in the treatment of MS.

[0158] In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to Fc gamma receptor (FcyR) la from 8 to 11 nM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllaHRfrom 15 to 20 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllaLRfrom 2 to 7 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllb from 26 to 37 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRlllb from 7 to 12 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRI I laF158from 2 to 7 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRI I laV158from 0.1 to 0.6 pM. In some embodiments, the CHO-cell produced antibodies have a mean binding affinity (KD at pH 6.0) to FcRn from 0.3 to 0.8 pM.

[0159] Binding affinity can be measured using a Biacore, surface plasmon resonance (SPR) or BLI assay.

[0160] In some embodiments of any of the preceding aspects, the CHO-cell produced antibodies have a relative antibody dependent cytotoxicity (ADCC) potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a relative complement dependent cytotoxicity (CDC) potency within 30% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell- produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a relative antibody dependent cellular phagocytosis (ADCP) potency within 30% of a reference murine cell- produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). In some embodiments, the CHO-cell produced antibodies have a relative C1q binding potency within 10% of a reference murine cell-produced antibody (e.g., NS / 0-produced antibody, e.g., NS / 0 cell-produced ofatumumab). III. Production and Glycoengineering

[0161] Methods of producing antibodies having the amino acid sequences of ofatumumab are provided herein. In some embodiments, the methods include production by glycoengineering to make glycoengineered anti-CD20 antibodies having the amino acid sequences of SEQ ID NO: 1 and 2 (e.g., glycoengineered CHO cell-produced anti-CD20 antibodies having the amino acid sequences of SEQ ID NO: 1 and 2 having any of the structural and / or functional properties described in Section II, above). Any such glycoengineered antibodies can be produced in non-Ns / 0 cells, e.g. non-murine cells, suitable for the production of antibodies for use in therapy in humans. In some embodiments, the non-Ns / 0 cells are non-Ns / 0 mammalian cells. In some embodiments, the non-murine cells are non-murine mammalian cells. For example, any of such glycoengineered antibodies can be produced in CHO cells.

[0162] Provided herein are methods of producing anti-CD20 antibodies having the amino acid sequences of SEQ ID NO: 1 and 2 in CHO cells. CHO cells are epithelial cells which are derived from the ovary of the Chinese hamster ovary (Tijo and Puck (1958) J. Exp. Med. 108: 259- 271). From the original CHO cell line, several other cell lines have been obtained, including CHO-KI, CHO-Toronto, CHO-DXBI I, CHO-DG44 and CHO KI SV. All these cell lines can be used to produce the composition comprising anti- CD20 antibodies according to the present invention. In some embodiments, a CHO-KI cell line or cells derived therefrom is used to produce the composition comprising antiCD20 antibodies of the present invention. The CHO-KI cell line has been obtained from a single clone of the original CHO cells (Kao and Puck (1968) Proc. Nat. Acad. Sci. USA 60(4): 1275-1281). The CHO-KI cell line can be adapted to suspension growth and / or to a chemically defined medium (see, e.g., Bort et al. (2010) Biotechnol. J. 5(10): 1090-1097). In some instances of the present invention, CHO-KI cells or cells derived therefrom are used. The cells which are derived from the CHO-KI cells are cells which originate from the CHO-KI cells, but have been subjected to one or more adaptation processes, such as adaptation to serum-free medium or suspension growth, or further modifications.

[0163] CHO cells suitable for use as part of the present invention include, e.g., any CHO cell lines available at American Type Culture Collection (ATCC) or European Collection of Cell Cultures (ECACC). Exemplary CHO cell lines include e.g., CHO-K1 cell, CHO-DUXB1 1 , CHO-DG44, CHO-S cells, or derivatives thereof, e.g., as described further herein.

[0164] For instance, CHO-C8TD is derived from a CHO K1 cell line and further modified by deleting telomeric region of chromosome, so this CHO-C8TD cell line has a chromosome 8 telomere deletion, high productivity, and good cell growth. In some instances of any of the anti-CD20 antibodies described herein, or processes of producing such anti-CD20 antibodies, the antibodies are produced in CHO-C8TD cells.

[0165] Alternatively, CHO-HPT3 is a CHO K1 cell line showing reduced proteolytic activity, so this cell line has lower growth, productivity and cloning efficiency than CHO-C8TD. In some instances of any of the anti-CD20 antibodies described herein, or processes of producing such anti-CD20 antibodies, the antibodies are produced in CHO-HPT3 cells. CHO cells used to produce the anti-CD20 antibodies of the invention are genetically modified to express the anti-CD20 antibodies. The term "genetically modified CHO cells" as used herein means that CHO cells have been modified or altered by any suitable genetic means and methods known to the skilled person such that they express the anti-CD20 antibodies. In one embodiment, the genetic modification to express the anti-CD20 antibodies is the only genetic modification of the CHO cells. In another embodiment the CHO cells are genetically modified to increase or decrease the expression of one or more enzymes which have an impact on the glycosylation profile of expressed anti-CD20 antibodies, such as a fucosyltransferase (e.g., Fut8), a glycoside hydrolase (e.g., Man2a1), and / or a GIcNAc transferase (e.g., Mgat2). In one embodiment, the CHO cells are genetically modified to decrease the expression of afucosylated N-glycans (e.g., increasing fucosylated N-glycans). In another embodiment. In some instances, the CHO cells do not comprise a genetic modification other than the genetic modification to express the anti-CD20 antibody. Methods for genetically modifying CHO cells are known to the skilled person and particularly include the transfection of the CHO cells with one expression vector encoding the heavy and the light chain of the antibody or with a first expression vector encoding the heavy chain of the antibody and a second expression vector encoding the light chain of the antibody. In one embodiment, the recombinant antibody is produced from one recombinant nucleic acid molecule which encodes both the heavy and the light chain of the antibody. In a more preferred embodiment, the recombinant antibody is produced from two recombinant nucleic acid molecules having the same or different promoters. In an even more preferred embodiment, the recombinant antibody is produced from two recombinant nucleic acid molecules having the same promoter. In a most preferred embodiment, the recombinant antibody is produced from two recombinant nucleic acid molecules which differ from each other only by the encoded gene and the selection marker used to select the transfected cells.

[0166] The elements and methods needed to construct expression vectors which are suitable for expressing an antibody in CHO cells are well-known to the skilled person and described for example in Makrides et al. (1999) Protein Expr. Purif 17: 183-202 and Kaufman (2000) Mol. Biotechnol. 16: 151-161 . Further, the skilled person is aware of methods for introducing the expression vectors into the CHO cells. These methods include the use of commercially available transfection kits such as Lipofectamine® of ThermoFisher, PEImax of Polyplus Sciences) or Freestyle Max of Invitrogen.

[0167] Further suitable methods include electroporation, calcium phosphate-mediated transfection and DEAE-dextrane transfection. After transfection the cells are subjected to selection by treatment with a suitable agent based on the selection marker(s) encoded by the expression vector(s) to identify the stably transfected cells. To produce the anti-CD20 antibody of the invention, the genetically modified CHO cells are cultured in a suitable culture medium. The terms "medium", "cell culture medium" and "culture medium" are interchangeably used herein and refer to a solution containing nutrients which are required for growing mammalian cells. Typically, a cell culture medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and / or survival. In some instances, the medium is chemically defined in that all its components and their concentration are known. In some instances, the medium is serum- free and hydrolysate-free and does not contain any components derived from animals. In some instances, the medium is chemically defined, serum- free, animal- component-free and hydrolysate-free.

[0168] Media for growing CHO cells are commercially available and include PowerCHO-2 CD available from Lonza, CD OptiCHO™ Medium available from ThermoFisher and EX-CELL® CD CHO Serum-Free Medium available from Sigma- Aldrich. These media may be supplemented with further reagents such as recombinant insulin, lipids, ferric citrate, PEG20000, extra amounts of some sugar types and extra amounts of some or all amino acids. CHO cells can be cultured in suspension, i.e. in a non-adherent state.

[0169] For culturing the CHO cells different strategies are available, including batch culture, continuous culture and fed-batch culture. Within the present invention, preferably a fed-batch culture process is used to produce anti-CD20 antibodies described herein. In fed-batch culture the culturing process is started with a certain volume of the medium and one or more nutrients are fed at later time-point(s) of the culture process to prevent nutrient depletion while no product is removed from the cell culture broth.

[0170] The method of the present invention may be used to produce anti-CD20 antibodies in large scale, i.e., in a production volume of at least 50 L to 100 L (e.g., at least 500 L, at least 1 .000 L, at least 5,000 L, or at least 10,000 L).

[0171] After the antibodies are produced by the CHO cells, they are harvested. Since recombinant proteins, in particular antibodies, expressed from mammalian cells are typically secreted into the cell culture fluid during the cultivation process, the product harvest at the end of the cultivation process occurs by separating cell culture fluid comprising the antibody from the cells. The cell separation method should be gentle to minimize cell disruption to avoid the increase of cell debris and release of proteases and other molecules that could affect the quality of the immunoglobulin product. Usually, the harvesting of the cell culture fluid comprising the involves centrifugation and / or filtration, whereby the recombinant protein is present in the supernatant and the filtrate, respectively. Expanded bed adsorption chromatography is an alternative method to avoid centrifugation / filtration methods.

[0172] After harvesting the cell culture fluid comprising the antibody, the antibody is purified from the cell culture fluid. The purification of recombinant proteins and in particular recombinant antibodies is usually accomplished by a series of chromatographic steps such as anion exchange chromatography, cation exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, mixed mode chromatography and size exclusion chromatography. The purification of recombinant antibodies usually starts with a protein A affinity chromatography to capture the antibody and is followed by one or more additional chromatographic steps such as cation exchange chromatography and mixed mode chromatography. Further, the purification process may comprise one or more ultra-, nano- or diafiltration steps.

[0173] In some instances, provided is an antibody composition produced by a process comprising (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L- fucose, dexamethasone, and / or insulin, and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the CHO cells express a fucosyltransferase (e.g., Fut8), a glycoside hydrolase (e.g., Man2a1), and / or a GIcNAc transferase (e.g., Mgat2).

[0174] In another embodiment, provided is an antibody composition produced by a process comprising (a) providing CHO cells comprising (i) Fut8, Man2a1 , or Mgat2, and (ii) nucleic acid encoding antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies; and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, and / or insulin.

[0175] In some embodiments, an antibody composition of any one of the previous aspects is produced by a process comprising (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies, and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the CHO cells express Fut8, Man2a1 , or Mgat2. In some embodiments, the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, and / or insulin.

[0176] In another instance, provided is a method of producing an antibody composition, the method comprising: (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L-fucose, dexamethasone, and / or insulin; and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the CHO cells express Fut8, Man2a1 , or Mgat2.

[0177] In another instance, provided is a method of producing an antibody composition, the method comprising: (a) providing CHO cells comprising: (i) Fut8, Man2a1 , or Mgat2, and (ii) nucleic acid encoding antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies; and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, and / or insulin. In some embodiments, the invention involves a method of producing the antibody composition of any of the previous aspects, the method comprising: (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise: (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2; (b) culturing the CHO cells under conditions which permit expression of the antibodies, and (c) isolating the antibodies to obtain the antibody composition. In some embodiments, the CHO cells express Fut8, Man2a1 , or Mgat2. In some embodiments, the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, and / or insulin.

[0178] In some embodiments, the conditions which permit expression of the antibodies comprise culturing the non-murine cells (e.g. CHO cells) in the presence of 1 g / L putrescine, 5 g / L fucose, 1 mg / L insulin, 15 g / L galactose, 1.5 pM manganese, 175 g / L glucose (e.g. glucosexH2O concentration in feed). The conditions may further comprise culturing at pH 7.1 . The culturing may be carried out for a duration of 12 days.

[0179] In some embodiments, the conditions which permit expression of the antibodies comprise culturing the non-murine cells (e.g. CHO cells) in the presence of 1 g / L putrescine, 5 g / L fucose, 2 mg / L insulin, 10 pM dexamethasone, 50 pM manganese, 175 g / L glucose (e.g. glucosexH2O concentration in feed). The conditions may further comprise culturing at pH 7.1 . The culturing may be carried out for a duration of 14 days.

[0180] In some embodiments, the conditions which permit expression of the antibodies comprise culturing the non-murine cells (e.g. CHO cells) in the presence of 0.5 g / L putrescine, 2.5 g / L fucose, 10 pM dexamethasone, 1 .5 pM manganese, 175 g / L glucose (e.g. glucosexH2O concentration in feed). The conditions may further comprise culturing at pH 7.1 . The culturing may be carried out for a duration of 14 days.

[0181] In some embodiments, the conditions which permit expression of the antibodies comprise culturing the non-murine cells (e.g. CHO cells) in the presence of 1 g / L putrescine, 2.5 g / L fucose, 1 mg / L insulin, 25.8 pM manganese, 137.5 g / L glucose (e.g. glucosexH2O concentration in feed). The conditions may further comprise culturing at pH 7.1. The culturing may be carried out for a duration of 13 days.

[0182] In some embodiments, the conditions which permit expression of the antibodies comprise culturing the non-murine cells (e.g. CHO cells) in the presence of 0.5 g / L putrescine, 2.5 g / L fucose, 1 mg / L insulin, 10 pM dexamethasone, 50 pM manganese, 100 g / L glucose (e.g. glucosexH2O concentration in feed). The conditions may further comprise culturing at pH 6.95. The culturing may be carried out for a duration of 13 days.

[0183] In some embodiments, the conditions which permit expression of the antibodies comprise culturing the non-murine cells (e.g. CHO cells) in the presence of 1 g / L putrescine, 1 mg / L insulin, 20 pM dexamethasone, 50 pM manganese, 137.5 g / L glucose (e.g. glucosexH2O concentration in feed). The conditions may further comprise culturing at pH 7.1 . The culturing may be carried out for a duration of 14 days. In some embodiments, the conditions which permit expression of the antibodies comprise culturing the non-murine cells (e.g. CHO cells) in the presence of 1 .5 pM manganese, 160.4 g / L glucose (e.g. glucosexfW concentration in feed) and 1 .2 mM calcium. The conditions may further comprise culturing at pH 6.95. The culturing may be carried out for a duration of 14 days.

[0184] In some embodiments, the conditions which permit expression of the antibodies comprise culturing the non-murine cells (e.g. CHO cells) in the presence of 15 g / L galactose, 25.8 pM manganese, 160.4 g / L glucose (e.g. glucosexH2O concentration in feed) and 9.1 mM calcium. The conditions may further comprise culturing at pH 7.05. The culturing may be carried out for a duration of 14 days. These conditions may be particularly suitable for methods combining metabolic and genetic glycoengineering (e.g. Example 5).

[0185] The invention also provides a cell culture comprising any of the cells disclosed herein (e.g. a non-murine cell expressing an anti-CD20 antibody, e.g. a CHO cell expressing an anti-CD20 antibody) and any of the above conditions. The invention also provides a composition comprising anti-CD20 antibodies produced by a process comprising any of the above conditions.

[0186] IV. Pharmaceutical Formulations

[0187] The present disclosure provides pharmaceutical formulations comprising any of the anti-CD20 antibodies described herein (e.g., glycoengineered anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2, e.g., CHO cell-produced anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2) in combination with one or more pharmaceutically acceptable carriers or excipients. Similarly, pharmaceutical formulations provided herein can include any of the anti- CD20 antibody compositions described herein (e.g., glycoengineered anti-CD20 antibody compositions having antibodies with amino acid sequences of SEQ ID NOs: 1 and 2, e.g., antibody compositions comprising CHO cell-produced anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2) in combination with one or more pharmaceutically acceptable carriers or excipients. Any of the antibodies described herein can be formulated, as applicable, as described in International Patent Publication No. WO 2009 / 009407, which is incorporated herein by reference in its entirety.

[0188] In some instances, pharmaceutical formulations include a therapeutically effective amount of anti- CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2), wherein the formulation further comprises 10 to 100 mM sodium acetate, 25 to 100 mM sodium chloride, 0.5 to 5% arginine free base, 0.02 to 0.2 mM EDTA, 0.01 to 0.2% polysorbate 80 and adjusted to pH 5.0 to 7.0.

[0189] In some instances, pharmaceutical formulations include a therapeutically effective amount of anti- CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2) in the concentration range of 20-300 mg / mL, wherein the formulation further comprises 50 mM sodium acetate, 51 mM sodium chloride, 1 % arginine free base, 0.05 mM EDTA, 0.02% polysorbate 80, and adjusted to pH 5.5. In one embodiment, the invention relates to a pharmaceutical formulation comprising a therapeutically effective amount of anti-CD20 antibodies described herein (e.g., CHO cell-produced anti- CD20 antibodies having SEQ ID NOs: 1 and 2), wherein the formulation further comprises 10 to 100 mM sodium acetate, 25 to 100 mM sodium chloride, 0.5 to 5% arginine free base, 0.02 to 0.2 mM EDTA, 0.01 to 0.2 % polysorbate 80 and adjusted to pH 5.0 to 7.0.

[0190] In one embodiment, the invention relates to a pharmaceutical formulation comprising anti-CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2) in the concentration range of 20-300 mg / mL, e.g., wherein the formulation further comprises 50 mM sodium acetate, 51 mM sodium chloride, 1 % arginine free base, 0.05 mM EDTA, 0.02% polysorbate 80, and adjusted to pH 5.5.

[0191] In another embodiment, the invention relates to a pharmaceutical formulation comprising anti- CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2), wherein the CHO cell-produced anti-CD20 antibodies are present in an amount of about 20-300 mg / mL, 50-300 mg / mL, 100-300 mg / mL, 150-300 mg / mL, 200-300 mg / mL, or 250-300 mg / mL. In some embodiments, the CHO cell-produced anti-CD20 antibodies are present in an amount of about 50 mg / mL. In some embodiments, the CHO cell-produced anti-CD20 antibodies are present in an amount of about 90 mg / mL.

[0192] In another embodiment, the invention relates to a pharmaceutical formulation comprising anti- CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2), wherein sodium acetate is present in an amount of about 50 mM, 40 mM, 45 mM, 55 mM, or 60 mM. In other embodiments, the sodium acetate may be present in an amount of 10 to 100 mM, 20 to 100 mM, 30 to 100 mM, 40 to 100 mM, 50 to 100 mM, 60 to 100 mM, 70 to 100 mM, 25 to 80 mM, or 30 to 70 mM.

[0193] In yet another embodiment, the invention relates to a pharmaceutical formulation comprising anti- CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2), wherein acetic acid is present (e.g., about 100 mM acetic acid) to adjust the formulation to about pH 5.5. In other embodiments, the pH may be adjusted to pH 5.0, 5.5, 6.0, 6.5 or 7.0. In yet other embodiments of the invention, NaOH or HCI is used to adjust the pH to 5.0, 5.5, 6.0, 6.5 or 7.0.

[0194] In yet another embodiment, the invention relates to a pharmaceutical formulation comprising anti- CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2), wherein sodium chloride is present in an amount of about 51 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 52 mM, 53 mM, 54 mM, or 55 mM. In other embodiments, the sodium chloride may be present in an amount of 25 to 100 mM, 35 to 90 mM, 45 to 80 mM, 25 to 70 mM, or 45 to 70 mM.

[0195] In another embodiment, the invention relates to a pharmaceutical formulation comprising anti- CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2), wherein arginine free base is present in an amount of about 1%, 0.7%, 1.3%, or 2.0%. In other embodiments, the arginine free base may be between 0.5 and 5.0%, 0.5 to 2.0%, 0.5 to 2.5%, 0.5 to 3.0%, 0.5 to 3.5%, 0.5 to 4.0%, or 0.5 to 4.5%. In another embodiment, the invention relates to a pharmaceutical formulation comprising CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2, wherein EDTA is present in an amount of about 0.05 mM, 0.03 mM, 0.04 mM, or 0.06 mM. In other embodiments, the EDTA may be present in an amount of 0.02 mM - 0.2 mM, 0.02 mM - 0.1 mM, 0.02 mM - 0.15 mM, 0.04 mM - 0.1 mM, 0.03 mM - 0.15 mM, or 0.03 mM - 0.2 mM.

[0196] In another embodiment, the invention relates to a pharmaceutical formulation comprising anti- CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2), wherein polysorbate 80 is present in an amount of about 0.02%, 0.015%, or 0.025%. In other embodiments, the polysorbate 80 may be present in an amount of 0.01 - 0.2%, 0.01 - 0.15%, 0.02 - 0.2%, 0.02 - 0.15%, 0.01 - 0.25%, or 0.01 - 0.05%.

[0197] In another embodiment, the invention relates to a pharmaceutical formulation comprising anti- CD20 antibodies described herein (e.g., CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2), wherein an antioxidant, e.g., methionine (e.g., L-methionine), is present in the formulation. Such antioxidants can be particularly useful when used with a composition of CHO cell-produced antibodies, which can experience higher oxidative stress as compared to murine (e.g., NS / 0 cell)-produced antibodies.

[0198] V. Therapeutic Methods and Uses

[0199] Provided herein are methods of treating multiple sclerosis (MS) in a subject in need thereof, wherein the method comprises subcutaneously administering a glycoengineered anti-CD20 antibody to the subject (e.g., as part of any of the antibody compositions or pharmaceutical formulations described herein). Also provided are uses of glycoengineered anti-CD20 antibodies in the manufacture of a medicament for treating MS in a subject in need thereof. Compositions for use in the treatment of MS are also provided herein, where the compositions for use include any of the antibodies, compositions, or pharmaceutical formulations described herein.

[0200] In an embodiment, the MS is RMS. In an embodiment, the MS is RRMS. In an embodiment, the MS is PPMS. In an embodiment, the MS is SPMS. In an embodiment, the MS is CIS. Therapeutic methods and uses that can be readily applied to treatment with the anti-CD20 antibodies described herein (e.g., as part of any of the antibody compositions or pharmaceutical formulations described herein) are described in International Patent Publication No. WO 2018 / 033841 and PCT Application No. PCT / EP2024 / 078440, each of which is incorporated herein by reference in its entirety.

[0201] In one embodiment, provided herein is a method of treating multiple sclerosis (MS), the method comprising administering (e.g., through subcutaneous injection) an effective amount of an antibody composition (e.g., a CHO cell-produced antibody composition) to a patient in need thereof, wherein the antibody composition (e.g., CHO cell-produced antibody composition) comprises anti-CD20 antibodies comprising: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 1 ; and (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 (e.g., a light chain comprising the amino acid sequence of SEQ ID NO: 3). In some embodiments, the anti-CD20 antibodies comprise (a) the antibody composition of any of the previous aspects; or (b) the CHO cell-produced antibody of any of the previous aspects. In some embodiments, the CHO cell-produced antibody composition is administered to the patient (e.g., through subcutaneous injection) once per month, once every two months, once every three months, once every four months, once every six months, once every eight months, or once yearly. In some embodiments, the CHO cell-produced antibody composition is administered to the patient (e.g., through subcutaneous injection) once per month, e.g., the CHO cell-produced antibody composition administered to the patient once per month comprises about 20 mg of the anti-CD20 antibodies. In some embodiments, the CHO cell-produced antibody composition is administered to the patient (e.g., through subcutaneous injection) once every two months, e.g., the CHO cell-produced antibody composition administered to the patient once every two months comprises about 135 mg of the anti-CD20 antibodies. In some embodiments, the multiple sclerosis is relapsing multiple sclerosis (RMS). In some embodiments, the multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS), or clinically isolated syndrome (CIS).

[0202] In some embodiments, provided herein is a method of treating multiple sclerosis (MS), the method comprising subcutaneously administering a CHO cell-produced antibody of any of the previous aspects to the subject once every about 2 months, e.g., at a dose of about 100-600 mg (e.g., about 600 mg, about 200 mg or about 135 mg). Also provided herein is a method of treating multiple sclerosis in a subject in need thereof, the method comprising subcutaneously administering the CHO cell-produced antibody of any of the previous aspects to the subject at a dose of about 100-600 mg once every about 2- 6 months. In some embodiments, the CHO cell-produced antibody of any of the previous aspects is subcutaneously administered to the subject once every 2 months (Q2M). In some embodiments, the CHO cell-produced antibody composition administered to the patient once every two months comprises 20 to 200 mg of the anti-CD20 antibodies (e.g., 100 to 150 mg of the anti-CD20 antibodies, e.g., about 110 mg, about 120 mg, about 130 mg, about 135 mg, about 140 mg, or about 150 mg of the anti-CD20 antibodies, e.g., about 135 mg of the anti-CD20 antibodies). In some embodiments, the CHO cell-produced antibody of any of the previous aspects is subcutaneously administered to the subject at a dose of about 135 mg.

[0203] With reference to the embodiments described herein, the skilled person would understand that the term “4 weeks” is equivalent to “1 month”, the term “12 weeks” is equivalent to “3 months”, the term “24 weeks” is equivalent to “6 months”, unless specifically stated otherwise or obvious from context. The skilled person would also understand that in clinical practice, “about 2 months” can mean between 6 and 10 weeks, and “2 months” can mean between 7 and 9 weeks, typically 8 weeks. Similarly, the skilled person would understand that in clinical practice, “about 6 months” can mean between 20 and 28 weeks and “6 months” can mean between 22 and 26 weeks, typically 24 weeks. The skilled person would also understand that the term “every 2 months” means “once every 2 months”, and “every about 2 months” means “once every about 2 months”, and so on, unless specifically stated otherwise or obvious from the context. Also provided herein is a method of treating MS in a subject in need thereof, the method comprising subcutaneously administering a CHO cell-produced antibody described herein to the subject during an initial dosing regimen, followed by a subsequent dosing regimen as disclosed herein. The disclosure therefore encompasses subcutaneously administering one or more doses of the CHO cell- produced antibody described herein to the subject at specific time intervals during an initial dosing regimen (e.g., a loading dosing regimen), followed by subcutaneously administering the CHO cell- produced antibody described herein to the subject at specific time intervals during a subsequent dosing regimen (e.g., a maintenance dosing regimen). In some embodiments, the initial dosing regimen comprises administering three doses of the CHO cell-produced antibody described herein at weeks 0, 1 and 2. In some embodiments, the subsequent dosing of the CHO cell-produced antibody described herein is higher than the initial dosing of the CHO cell-produced antibody described herein. For example, the initial dose of the CHO cell-produced antibody described herein may be about 15-25 mg, 16-24 mg, 18-22 mg, 19-21 mg, e.g., about 20 mg, e.g., 20 mg. One or more of these initial doses may be administered. In the examples, 3 initial doses are administered. The initial dose interval is typically more frequent than the subsequent dose interval. Weekly initial doses are used in the examples. A specific embodiment of an initial regimen for use in the disclosed methods is therefore 3 weekly doses (at weeks 0, 1 and 2) of about 20 mg of the CHO cell-produced antibody described herein.

[0204] In one embodiment, the initial (e.g., loading) dose(s) of the CHO cell-produced antibody is / are administered in a pharmaceutical composition that comprises the CHO cell-produced antibody at a concentration of 50 mg / ml, and the subsequent (e.g., maintenance) dose(s) of the CHO cell-produced antibody is / are administered in a pharmaceutical composition that comprises the CHO cell-produced antibody at a concentration of about 50 mg / mL (e.g., 50 mg / mL).

[0205] In an exemplary embodiment, the CHO cell-produced antibody described herein is administered during a subsequent (e.g., maintenance) dosing regimen at a concentration of about 65-115 mg / mL, 70- 110 mg / mL, 80-100 mg / mL, 85-95 mg / mL, preferably 90 mg / mL (e.g., about 135 mg antibody in about 1.5 mL (e.g., 1 .5 mL) total volume), and during the initial (e.g., loading) dosing regimen at a concentration of about 35-65 mg / mL, 40-60 mg / mL, 45-55 mg / mL, 47-53 mg / mL, preferably 50 mg / mL (e.g., about 20 mg antibody in 0.4 mL total volume). In a specific embodiment, the initial (e.g., loading) dose(s) of the CHO cell-produced antibody is / are administered in a pharmaceutical composition that comprises the CHO cell- produced antibody at a concentration of 50 mg / ml, and the subsequent (e.g., maintenance) dose(s) of the CHO cell-produced antibody is / are administered in a pharmaceutical composition that comprises the CHO cell-produced antibody at a concentration of about 90 mg / mL (e.g., 90 mg / mL).

[0206] Any reference to a method of treatment herein also discloses the antibody or composition for use in said method of treatment, the use of the antibody or composition in said method of treatment, and the use of the antibody or composition in the manufacture of a medicament for said treatment.

[0207] Any reference to a method of treatment herein also discloses a pharmaceutical composition for use in said treatment, comprising the antibody or antibody composition. Delivery

[0208] Any antibody or composition thereof (e.g., CHO cell-produced antibody) disclosed herein may be administered via any suitable route (e.g., intravenous injection or infusion), but is typically administered subcutaneously (s.c.), typically via injection.

[0209] In some embodiments, the CHO cell-produced antibody is self-administered by subcutaneous injection. In some embodiments, the initial doses and / or subsequent doses of the CHO cell-produced antibody are self-administered by subcutaneous injection. In some embodiments, the anti-CD20 antibody is administered in an outpatient setting.

[0210] In some embodiments, the CHO cell-produced antibody is administered in the abdomen, thigh, or upper outer arm of the subject.

[0211] In one embodiment, subcutaneous administration of the CHO cell-produced antibody is achieved using a pre-filled syringe. In one embodiment, subcutaneous administration of the CHO cell-produced antibody is achieved using a pre-filled pen, i.e., autoinjector. A non-limiting example of an autoinjector suitable for use according to the present disclosure is the Sensoready® pen. A pre-filled syringe consists of a bulk pre-filled syringe assembled into a needle safety device. A pre-filled pen consists of a bulk prefilled syringe assembled into an auto-injector (Al).

[0212] Thus, provided herein is a pre-filled syringe or a pre-filled pen, i.e., autoinjector, (e.g., an autoinjector containing a pre-filled syringe) containing any of the CHO cell-produced antibodies described herein. In some embodiments, the pre-filled syringe or the pre-filled pen, i.e., autoinjector, contains a single unit dose of about 20 mg of the CHO cell-produced antibody. In some embodiments, the pre-filled syringe or the pre-filled pen, i.e., autoinjector, contains a single unit dose of between about 110-160 mg of the CHO cell-produced antibody. In some embodiments, the pre-filled syringe or the pre-filled pen, i.e., autoinjector, contains a single unit dose of between about 120-150 mg of the CHO cell-produced antibody. In a preferred embodiment, the pre-filled syringe or the pre-filled pen, i.e., autoinjector, contains a single unit dose of between about 130-140 mg of the anti- CHO cell-produced antibody. In another preferred embodiment, the pre-filled syringe or the pre-filled pen, i.e., autoinjector, contains a single unit dose of about 135 mg of the anti-CD20 antibody.

[0213] In a specific embodiment, ofatumumab is provided in a prefilled-pen (autoinjector) containing a single unit dose of about 130-140 mg (e.g., about 135 mg, e.g., 135 mg) of the CHO cell-produced antibody. The single unit dose is intended for use in the subsequent regimen, as described herein. In some embodiments, the CHO cell-produced antibody is administered to the subject in a pharmaceutical formulation, as described herein. In one embodiment, the pharmaceutical formulation comprising the CHO cell-produced antibody is provided in a pre-filled syringe. In a preferred embodiment, the pharmaceutical composition comprising the CHO cell-produced antibody is provided in a pre-filled pen, i.e., autoinjector. A non-limiting example of an autoinjector suitable for use according to the present disclosure is the Sensoready® pen. In some embodiments, the CHO cell-produced antibody may be administered in a volume of about 0.4 mL to about 1 .5 mL. In some embodiments, the initial dose of the CHO cell-produced antibody is administered in a volume of about 0.4 mL. In some embodiments, the subsequent dose of the CHO cell-produced antibody is administered in a volume of about 1.5 mL. In some embodiment, the CHO cell- produced antibody is provided in a pre-filled syringe or a pre-filled pen, i.e., autoinjector, containing about 0.4 mL solution for injection wherein the solution for injection comprises the CHO cell-produced antibody in a pharmaceutical formulation as disclosed herein. In some embodiments, the CHO cell-produced antibody is provided in a pre-filled syringe or a pre-filled pen, i.e., autoinjector, containing about 0.4 mL solution for injection, wherein the solution comprises the CHO cell-produced antibody at a concentration of about 50 mg / mL. In some embodiments, the initial doses of the CHO cell-produced antibody are administered in a pre-filled syringe or a pre-filled pen, i.e., autoinjector, containing about 0.4 mL solution for injection, wherein the solution comprises the CHO cell-produced antibody at a concentration of about 50 mg / mL.

[0214] In some embodiments, CHO cell-produced antibody is provided in a pre-filled syringe or a prefilled pen, i.e., autoinjector, containing about 1 .5 mL solution for injection wherein the solution for injection comprises CHO cell-produced antibody at a concentration of between about 80 and about 100 mg / mL. In some embodiments, the subsequent dose of CHO cell-produced antibody is provided in a prefilled syringe or a pre-filled pen, i.e., autoinjector, containing about 1 .5 mL solution for injection, wherein the solution comprises CHO cell-produced antibody at a concentration of between about 80 and about 100 mg / mL.

[0215] In some embodiments, CHO cell-produced antibody is provided in an injection device as described in U.S. Provisional Patent Application No. 63 / 705,387, which is incorporated herein by reference in its entirety.

[0216] Monitoring MS disease progression

[0217] Methods for monitoring disease progression in MS are described below and can be combined with the methods of treatment disclosed herein. Additional methods useful in monitoring patients having MS and treated according to the methods described herein are described in International Patent Publication Nos. WO 2004 / 035607 and WO 2018 / 033841 , each of which is incorporated herein by reference in its entirety.

[0218] Disease progression in a subject with MS may also be assessed using the Expanded Disability Status Scale (EDSS), the Symbol Digit Modalities Test (SDMT), the Rey Auditory Verbal Learning Test (RAVLT), Brief Visuospatial Memory Test-Revised (BVMT-R), the timed 25-foot walk test (T25-FW) and the 9-hole pegboard test (9-HPT).

[0219] Another method for monitoring disease progression in a subject with MS is oculomotor assessment. Oculomotor assessment may involve measuring eye movement parameters obtained from fixation, pro-saccade, anti-saccade, smooth pursuit visual and optokinetic nystagmus tasks. Oculomotor assessment may serve as an eye movement biomarker for disease progression, optionally a digital eye movement biomarker.

[0220] The method for monitoring disease progression in a subject with MS using oculomotor assessment may comprise tracking eye movement. Suitable methods and systems for tracking eye movement have been described in WO2019161503, WO2022232935 (Innodem Neurosciences), US20170276934 (ICSPI Corp.), US20180342066 (Sony Interactive Entertainment), WO2021028858, W02007076479 (Alcon), all of which are incorporated herein in full by reference.

[0221] Thus, disclosed herein is a method wherein the subject may have been treated or is currently being treated with a CHO cell-produced anti-CD20 antibody having sequences of ofatumumab (e.g., amino acid sequences of SEQ ID NOs: 1 and 2), and the method comprises oculomotor assessment as described above.

[0222] In some embodiments, a target level of B cell depletion is achieved following treatment according to the methods disclosed herein. Preferably, the methods provided herein deplete and / or maintain the subjects B cell counts to less than about 10 cells / pL (e.g., 10 cells / pL).

[0223] Low B cell counts have been observed to lead to inhibition of lesions and a decrease in risk of relapses. Thus, in some embodiments, a target level of Gd+T1 lesions rates per MRI scan is achieved.

[0224] In some embodiments, the methods provided herein result in one or more of: a) reduction in number of Gd+T1 lesions relative to baseline; b) reduction in number of new or enlarging T2 lesions relative to baselines; and / or c) reduction in annualized relapse rate (ARR) relative to baseline.

[0225] In some embodiments, the methods provided herein achieve Gd+T1 lesion rates of - 0.25 or less. In some embodiments, the methods provided herein achieve Gd+T1 lesion rates of < 0.1. In some embodiments, the methods provided herein achieve Gd+T1 lesion rates of 0.01-0.05. In some embodiments, the methods provided herein Gd+T1 lesion rates of 0.005-0.05. In some embodiments, the methods provided herein achieve Gd+T1 lesion rates < 0.005. In some embodiments, the methods provided herein achieve Gd+T1 lesion rates of < 0.02. In a preferred embodiment, the methods provided herein achieve Gd+T1 lesion rates of < 0.03.

[0226] In some embodiments, the methods provided herein achieve annualized new or enlarging T2 lesions rates of 4 or less. In some embodiments, the methods provided herein achieve annualized new or enlarging T2 lesions rates of less than 2. In some embodiments, the methods provided herein achieve annualized new or enlarging T2 lesions rates of less than 1 . In some embodiments, the methods provided herein achieve annualized new or enlarging T2 lesions rates of less than 0.9. In some embodiments, the methods provided herein achieve annualized new or enlarging T2 lesions rates of less than 0.8. In a preferred embodiment, the methods provided herein achieve annualized new or enlarging T2 lesions rates of < 0.72.

[0227] In some embodiments, the method provided herein achieved annual relapse rates (ARR) of less than 0.25. In some embodiments, the method provided herein achieve ARR of less than 0.22, less than 0.20, less than 0.15. or less than 0.12. In a preferred embodiment, the methods provided herein achieve an ARR of < 0.11.

[0228] Accordingly, the methods provided herein (e.g., using ofatumumab as the anti-CD20 antibody) may result in one or more of: a) Gd+ T1 lesion rates of < 0.03; b) annualized rates of new or enlarging T2 lesions of < 0.72; and / or c) annualized relapse rates (ARR) of < 0.11 .

[0229] The invention has been described above with reference to CHO cells. However, as the skilled person would understand from the disclosure herein any non-Ns / 0 or non-murine cell types suitable for the production of monoclonal antibodies for use in therapy in humans may be used. Accordingly, the invention also provides antibodies and antibody compositions produced in non-Ns / 0 (e.g., non-murine) cells having the properties disclosed herein for CHO cell-produced antibodies (e.g., N-glycan profiles, functional properties and / or C-terminal modification properties). Accordingly, the invention further provides methods for producing antibody compositions disclosed herein comprising providing non-Ns / 0 (e.g., non- murine) cells and culturing said cells according to conditions disclosed herein, and also provides the cell cultures described herein for culturing non-Ns / 0 (e.g., non-murine) cells. The skilled person would understand that the cell types disclosed herein are those producing antibodies suitable for use in human therapy.

[0230] VI. Examples

[0231] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0232] Example 1 : Effect of host cell line on in vitro ADCC and CDC potency of ofatumumab

[0233] Anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2 were produced in NS / 0 cells (NS / 0-cell produced anti-CD20 antibodies) and in CHO cells (CHO cell-produced anti-CD20 antibodies) using conventional methods (i.e., neither antibody was glycoengineered). Antibodies were produced in each cell type by transfection of coding genes in the expression vector. After transfection and selection, cell lines with stable, randomly integrated genes were generated. Generated cell lines expressed anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2.

[0234] To determine whether the host cell type influenced effector function of the antibodies expressed, each antibody was tested in a panel of in vitro functional assays designed to assess ADCC or CDC function. Relative potency were expressed as EC50 fold ratios (potency ratios) between CHO and NS / 0 as a reference sample (i.e., a 4.8 value for CHO refers to 480% CHO vs 100% NS / 0). Table 1 : Effector function potency comparison between NS / 0 cell-produced anti-CD20 antibody and non-glycoengineered, CHO cell-produced anti-CD20 antibody (relative differences in CHO cells relative to NS / 0 cells)

[0235] Potency Readout Host Cell Assay NS / 0 CHO

[0236] ADCC BjabADCC 1.00 4.8

[0237] ADCC hFcyR3A reporter 1.00 2.45

[0238] CDC Bjab CDC 1.00 0.47

[0239] CDC PBMC 1.00 0.52

[0240] CDC Ri1 CDC 1.00 0.93

[0241] These results show an increase in ADCC potency and a decrease in CDC potency by CHO cell-produced anti-CD20 antibodies having ofatumumab sequences, relative to NS / 0 cell-produced anti-CD20 antibodies expressed by the same protein-encoding nucleic acid sequences.

[0242] Example 2: Glycosylation profiles of NS / 0 cell-produced ofatumumab and CHO cell-produced ofatumumab

[0243] To better understand the structural features associated with the striking change in ADCC and CDC effector function between NS / 0 and CHO cell-produced anti-CD20 antibodies observed in Example 1 , glycosylation profiles of the two antibodies were analyzed and compared.

[0244] IgG antibodies contain N-linked glycosylation sites in the Fc domain, which are predominantly made up of complex biantennary sugars of varying structures. To detect and quantify individual structures, the glycan moiety is released from the protein and labelled with a fluorescence dye. The labelled glycans are separated based on their varying hydrophilicity using normal phase high-performance liquid chromatography (NP-HPLC) and subsequently identified and quantified by fluorescence detector (FLD) detection.

[0245] The anti-CD20 antibodies produced in Example 1 contain a single N-linked glycosylation site at Asn302 on each of the heavy chains. Changes in the carbohydrate structure could potentially affect Fc- mediated effector functions, as well as the stability of an antibody. To monitor N-glycosylation, the N- linked glycans were released from the antibody using a N-glycanase (PNGase F). The N-linked glycans were then labelled with InstantPC and analyzed for quantification. The carbohydrate map provides a “fingerprint,” a characteristic of glycan distribution, and it can be used as an indication for identity and relative abundance of different glycan variants presented in an antibody.

[0246] The neutral oligosaccharide profiles were generated by NP-HPLC of fluorescently labelled glycans released using PNGase F. FIGS. 4 and 5 show the graph and corresponding tabulated results for NS / 0 cell-produced antibody, respectively. FIGS. 6 and 7 show the graph and corresponding tabulated results for CHO cell-produced antibody, respectively. Comparative results between the two antibodies for a selection of glycan species is summarized in Table 2, below. Structures of each glycan are shown in FIGS. 1A-1 F, 2A-2C, and 3A-3F.

[0247] Table 2: Glycan composition (% total integrated area) of NS / 0 cell-produced anti-CD20 antibodies compared to CHO cell-produced anti-CD20 antibodies

[0248] Glycan NS / 0 CHO

[0249] GO-N 0.7 1.9

[0250] GOF-N 2.7 4 bGO-F 0.2 5.4

[0251] GOF 39 57

[0252] M5 2.4 4

[0253] G1 F-N / G1 2.3 2.2

[0254] G1 0.1 0.5

[0255] G1 F 31 18

[0256] M6 1.0 0.7

[0257] G1 FSA-N 1.2 0.6

[0258] G2F 8.4 1.7

[0259] G1 FSA 0.2 0.2

[0260] M7 1.0 0.4

[0261] G2FSA 1.4 0.5 bG2FSA0.1 0.2

[0262] Graphs showing the percentages of afucosylation, high mannose glycans, and terminal galactosylation in NS / 0 cell-produced anti-CD20 antibodies compared to CHO cell-produced anti-CD20 antibodies are shown in FIGS. 8A, 8B, and 8C, respectively.

[0263] Anti-CD20 antibodies produced in CHO cells showed a significantly different glycosylation pattern compared to anti-CD20 antibodies produced in NS / 0 cells. In particular, the CHO cell-produced anti-CD20 antibodies exhibited higher levels of afucosylation (FIG. 8A) and lower levels of terminal galactosylation (FIG. 8C).

[0264] Based on these results, set against historical data, Applicant estimated that CHO cell-produced anti-CD20 antibody could exert meaningfully higher ADCC potency, as compared to NS / O-produced anti- CD20 antibody. The CDC potency of CHO cell-produced anti-CD20 antibody may also be reduced.

[0265] Example 3: Metabolic Glycoengineering of CHO cell-produced anti-CD20 antibodies

[0266] Significant adjustment of the glycosylation pattern of CHO cell-produced anti-CD20 antibodies is required in order to yield CHO cell-produced anti-CD20 antibodies which achieve ADCC and CDC potencies within 30% of reference NS / 0 cell-produced ofatumumab. ADCC and CDC potencies within 30% of reference NS / 0 cell-produced ofatumumab optimally align the safety and efficacy of the CHO cell- produced anti-CD20 antibodies with the safety and efficacy of the murine-produced anti-CD20 antibodies.

[0267] This example describes methods of metabolic glycoengineering CHO cell-produced anti-CD20 antibodies to mirror the glycan structure of NS / 0 cell-produced anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2, thereby mirroring the effector function (e.g., ADCC and CDC) of NS / 0 cell-produced anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2. Success in achieving these objectives offers improved methods of manufacturing anti-CD20 antibodies, such as ofatumumab, which can be costly and inefficient to produce, whilst retaining desirable properties of the anti-CD20 antibodies that arise from production in NS / 0 cells. Pools of CHO cell-produced anti- CD20 antibodies having reduced afucosylation and increased galactosylation, relative to unmodified CHO cell-produced antibodies, were produced using metabolic glycoengineering methods described below.

[0268] The metabolic engineering study was conducted to identify bioprocess parameters and media additives using micro bioreactors. The following factors and ranges, shown in Table 3, were tested in various combinations in a Design-of-Experiment (DoE) mode (72 experimental conditions altogether).

[0269] Table 3: Metabolic glycoengineering bioprocess factors

[0270] Parameter Unit Low level Center point High level pH / 6.80 6.9517.10

[0271] Putrescin2g / L 010.5 1

[0272] L-fucose2g / L 012.5 5

[0273] Insulin2mg / L 011 2

[0274] Dexamethasone2pM 0110 20

[0275] Galactose2g / L 017.5 15

[0276] Manganese2pM 1.5125.75 50

[0277] GlucosexH2O concentration in feed g / L 100 137.5 1751

[0278] Process duration days 12 13 1411platform settings

[0279] 2cumulative concentration (in medium or added as feed)

[0280] DoE results showing the relationship of each metabolic parameter to relevant glycosylation structure are shown in FIGS. 9A and 9B.

[0281] In addition to these systematic DoE tests, several additional metabolic glycoengineering runs were carried out to test parameters listed in Table 4, below. Table 4: Additional metabolic glycoengineering experiments

[0282] Experiment Test condition (reference)

[0283] Control / USP platform v2.1 /

[0284] Early harvest Process duration 12 days (14)

[0285] Increased calcium 2.4 mM (1 .2)

[0286] Increased magnesium 5.2 mM (2.6)

[0287] Increased temperature after shift 34.5 °C (33.0)

[0288] Multi-parameter estimates Temperature after shift 34.5 °C (33.0); putrescine

[0289] 1 g / L (0); L-fucose 5 g / L (0); insulin 2 mg / L (0); dexamethasone 20 pM (0); galactose 15 g / L (0); Mn2+ 25.75 pM (0); Glucose*H2O concentration in feed 137.5 g / L (175); seeding VCD 0.7E6 cells / mL (0.4); Feed start time 72 h (48 h); process duration 12 days (14)

[0290] For each of the metabolic glycoengineering parameter sets tested, glycosylation profiles were quantified. To predict the functional impact of glycosylation profiles on ADCC and CDC potencies, historical models based on observed correlation between glycosylation and function in IgG 1 anti-CD20 antibodies were used. In these models, afucosylation impacted ADCC potency to the greatest extent, followed by galactosylation and high-mannose, each of which impacted ADCC potency to a greater degree than all other glycosylation parameters. CDC potency was impacted to the greatest extent by galactosylation, followed by high mannose, followed by all other glycosylation parameters.

[0291] Six metabolic engineering processes were identified as producing CHO cell-produced ofatumumab having glycosylation profiles suitable to achieve ADCC and CDC potencies within 30% of reference NS / 0 cell-produced ofatumumab based on these models. Bioprocess parameters of these six sets of metabolic engineering processes are described in Table 5. Glycosylation parameters for each of the five CHO cell-produced ofatumumab batches are shown in Table 6. Percent change in ADCC and CDC potencies, relative to NS / 0 cell-produced ofatumumab, for each of the five CHO cell-produced ofatumumab batches are shown in Table 7.

[0292] Table 5: Metabolic glycoengineering bioprocesses for CHO cell-production of ofatumumab batches modeled to achieve 70% to 130% ADCC and CDC potencies compared to NS / 0 cell- produced ofatumumab. Units are as provided in Table 3.

[0293] Batch No: 12 13 18 40 42 48 pH 7.1 7.1 7.1 7.1 6.95 7.1

[0294] Putrescin 1 1 0.5 1 0.5 1

[0295] Fucose 5 5 2.5 2.5 2.5 0

[0296] Insulin 1 2 0 1 1 1

[0297] Dexamethasone 0 10 10 0 10 20

[0298] Galactose 15 0 0 0 0 0

[0299] Manganese 1.5 50 1.5 25.8 50 50

[0300] GlucosexH2O concentration in175 175 175 137 5 100 137 5feed Process duration 12 14 14 13 13 14

[0301] Table 6: Glycan levels (% total integrated area) observed for metabolically engineered CHO cell- produced ofatumumab batches modeled to achieve 70% to 130% ADCC and CDC potencies compared to NS / 0 cell-produced ofatumumab.

[0302] Batch No: 12 13 18 40 42 48

[0303] Afucosylation % 3.4 3.4 3.9 4.3 4.4 4.5

[0304] High mannose % 5.0 4.6 4.8 3.2 1.8 3.3

[0305] Galactosylation % 44.5 35.8 31.5 26 30.6 29.1

[0306] Terminal sialylation % 3.1 2.1 1.8 1.8 1.5 1.6

[0307] Table 7: Relative potency of metabolically engineered CHO cell-produced ofatumumab batches modeled to achieve 70% to 130% ADCC and CDC potencies, shown as % increase compared to NS / 0 cell-produced ofatumumab.

[0308] Batch No: 12 13 18 40 42 48

[0309] ADCC +29.3% +21.8% +24.3% +23.2% +28.0% +28.6%

[0310] CDC +2.43% -7.53% -12.0% -18.4% -12.2% -14.2%

[0311] These results demonstrate several approaches to metabolic glycoengineering of CHO cell-produced anti- CD20 antibodies having SEQ ID NOs: 1 and 2, based, at least in part, on restoring afucosylated N- glycans back to the level of NS / 0 cell-produced reference anti-CD20 antibodies. Six of the metabolically engineered antibody batches exhibited reduced levels of afucosylation that more closely resemble NS / 0 cell-produced ofatumumab, which predictive modelling projected to exhibit relative ADCC and CDC potencies within 30% of NS / 0 cell-produced ofatumumab. Example 4: Genetic Glycoengineering of CHO cell-produced anti-CD20 antibodies

[0312] This example describes methods of genetic glycoengineering CHO cell-produced anti-CD20 antibodies to mirror the glycan structure of NS / 0 cell-produced anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2, thereby mirroring the effector function (e.g., ADCC and CDC) of NS / 0 cell-produced anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2. Pools of CHO cell-produced anti-CD20 antibodies having reduced afucosylation and increased galactosylation, relative to NS / 0 cell-produced antibodies, were produced using glycoengineering methods described below.

[0313] This genetic glycoengineering study produced genetically modified pools of CHO cell-produced cells expressing ofatumumab amino acid sequences SEQ ID NOs: 1 and 2 and tested the pools against negative controls in micro bioreactors.

[0314] Transfection and CHO Stable Pool generation:

[0315] CHO-C8TD cells expressing SEQ ID NOs: 1 and 2 were cultured in growth medium + 10 nM MTX at 36.5°C, 10% CO2, 150 RPM.

[0316] Cells were transfected by Nucleofector® (Amaxa) using 5 x 106cells and linear plasmid. Cells were transfected with 0.5 pg of one of the following two linearized vectors, as shown in FIG. 10:

[0317] GE-1) pBW_UBC_Fut8_MG118 (Fut8 gene under UBC promotor)

[0318] GE-2) pBW_Fut8_Man2a1 _Mgat2_MG119 (all genes under SV40 promotors)

[0319] In GE-2, alpha-mannosidase 2 (Man2a1) and alpha-1 ,6-mannosyl-glycoprotein 2-beta-N- acetylglycosaminyltransferase (Mgat2) genes were included in GE-2 vector to facilitate reduction of high mannose, thereby reducing detrimental impact on high mannose prevalence in the antibody composition.

[0320] UBC (ubiquitin-C) promoter was selected to drive fucosyltransferase 8 (Fut8) expression in vector GE-1. UBC promoter was selected based on Applicant’s observation that SV40-driven Fut8 transgene overexpression resulted in an unexpected, and undesirable, increase in high mannose species. Thus, UBC was selected as a weak constitutive promoter (e.g., having a lower rate of transcription as compared to the CMV promoters driving ofatumumab expression) in an effort to decrease afucosylation while minimizing detrimental impact on high mannose prevalence in the antibody composition.

[0321] Transfected cells were selected using puromycin starting on Day 2 after transfection with 3 pg / mL and subsequently increased to 5 pg / mL upon viable cell counts exceeding 70%.

[0322] Each genetic glycoengineering approach (GE-1 and GE-2) was tested in standard culture conditions as described in Tables 8 and 9 for GE-1 (Batches 3 and 4) and GE-2 (Batches 11 and 14), respectively. Table 8: Culture conditions for GE-1 batches:

[0323] Batch No: GE-1 :3 GE-1 :4 pH 6.95 6.95

[0324] Galactose 0 0

[0325] Manganese 1.5 1.5

[0326] GlucosexH2O concentration in feed 160.4 160.4

[0327] Process duration 14 14

[0328] Calcium 1.2 1.2

[0329] Viable cell seeding density [E5 / mL] 0.4 0.4

[0330] Table 9: Culture conditions for GE-2 batches:

[0331] Batch No: GE-2:11 GE-2:14 pH 6.95 6.95

[0332] Galactose 0 0

[0333] Manganese 1.5 1.5

[0334] GlucosexH2O concentration in feed 160.4 160.4

[0335] Process duration 14 14

[0336] Calcium 1.2 1.2

[0337] Viable cell seeding density [E5 / mL] 0.4 0.4

[0338] Glycosylation profiles of the resulting genetically glycoengineered CHO cell-produced ofatumumab antibodies are listed in Tables 10 and 11 for the GE-1 and GE-2 approaches, respectively.

[0339] Table 10: Glycan levels (% total integrated area) observed for GE-1 genetically glycoengineered CHO cell-produced ofatumumab batches.

[0340] Batch No: GE-1 :3 GE-1 :4

[0341] Afucosylation % 1.2 1.2

[0342] High mannose % 6.9 9.3

[0343] Galactosylation % 38.3 39.1

[0344] Terminal sialylation % 2.6 2.4

[0345] Table 11 : Glycan levels (% total integrated area) observed for GE-2 genetically glycoengineered CHO cell-produced ofatumumab batches.

[0346] Batch No: GE-2:11 GE-2:14

[0347] Afucosylation % 3.2 3.4

[0348] High mannose % 9.1 10

[0349] Galactosylation % 24.9 23.6

[0350] Terminal sialylation % 1.1 1.3

[0351] Corresponding ADCC and CDC potencies, calculated based on the observed glycosylation profiles, listed in Tables 12 and 13 for GE-1 and GE-2 approaches, respectively.

[0352] Table 12: Relative potency of GE-1 genetically glycoengineered CHO cell-produced ofatumumab batches, shown as % increase or decrease compared to NS / 0 cell-produced ofatumumab.

[0353] Batch No: GE-1 :3 GE-1 :4

[0354] ADCC -2.25% +0.09%

[0355] CDC -8.22% -6.99%

[0356] Table 13: Relative potency of GE-2 genetically glycoengineered CHO cell-produced ofatumumab batches, shown as % increase or decrease compared to NS / 0 cell-produced ofatumumab.

[0357] Batch No: GE-2:11 GE-2:14

[0358] ADCC +12.7% +14.6%

[0359] CDC -20.6% -22.0%

[0360] These results demonstrate two approaches to genetic glycoengineering of CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2, based on (1) restoring afucosylated N-glycan levels back to NS / 0 cell-produced antibody levels (GE-1) and (2) addition of genetic suppression of high mannose species (GE-2). Each of these antibody batches exhibited reduced levels of afucosylation that more closely resemble NS / 0 cell-produced ofatumumab. Moreover, predictive modelling showed that these genetically glycoengineered CHO cell-produced antibody compositions are projected to exhibit relative ADCC and CDC potencies within 30% of NS / 0 cell-produced ofatumumab.

[0361] Example 5: Combination of Metabolic and Genetic Glycoengineering of CHO cell-produced anti- CD20 antibodies

[0362] This example describes methods in which genetic glycoengineering and metabolic glycoengineering approaches were combined to make CHO cell-produced anti-CD20 antibodies that mirror the glycan structure of NS / 0 cell-produced anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2, thereby mirroring the effector function (e.g., ADCC and CDC) of NS / 0 cell- produced anti-CD20 antibodies having the amino acid sequences of SEQ ID NOs: 1 and 2. Metabolic glycoengineering parameters described below were included in GE-1 and GE-2 pools following methods described in Example 4.

[0363] Each genetic glycoengineering approach (GE-1 and GE-2) was tested in two metabolically engineered culture conditions as described in Tables 14 and 15 for GE-1 (Batches 1 , 2, 5, and 6) and GE- 2 (Batches 7-9), respectively.

[0364] Table 14: Metabolically engineered culture conditions for GE-1 batches:

[0365] Batch No: GE-1 :1 GE-1 :2 GE-1 :5 GE-1 :6 pH 7.05 7.05 7.05 7.05

[0366] Galactose 15 15 15 15

[0367] Manganese 25.8 25.8 25.8 25.8

[0368] GlucosexH2O concentration in feed 160.4 160.4 160.4 160.4

[0369] Process duration 14 14 14 14

[0370] Calcium 9.1 9.1 9.1 9.1

[0371] Viable cell seeding density [E5 / mL] 0.4 0.4 0.7 0.7

[0372] Table 15: Metabolically engineered culture conditions for GE-2 batches:

[0373] Batch No: GE-2:7 GE-2:8 GE-2:9 pH 7.05 7.05 7.05

[0374] Galactose 15 15 15

[0375] Manganese 25.8 25.8 25.8

[0376] GlucosexH2O concentration in feed 160.4 160.4 160.4

[0377] Process duration 14 14 14

[0378] Calcium 9.1 9.1 9.1

[0379] Viable cell seeding density [E5 / mL] 0.4 0.7 0.4

[0380] Glycosylation profiles of the resulting genetically and metabolically glycoengineered CHO cell- produced ofatumumab antibodies are listed in Tables 16 and 17 for the GE-1 and GE-2 approaches, respectively.

[0381] Table 16: Glycan levels (% total integrated area) observed for GE-1 genetically and metabolically glycoengineered CHO cell-produced ofatumumab batches.

[0382] Batch No: GE-1 :1 GE-1 :2 GE-1 :5 GE-1 :6

[0383] Afucosylation % 0.9 1.0 1.2 1.3

[0384] High mannose % 5.8 6.7 6.8 8.7

[0385] Galactosylation % 54.9 52.7 55.0 52.7 Terminal sialylation % 3.1 2.8 3.2 2.9

[0386] Table 17: Glycan levels (% total integrated area) observed for GE-2 genetically and metabolically glycoengineered CHO cell-produced ofatumumab batches.

[0387] Batch No: GE-2:7 GE-2:8 GE-2:9

[0388] Afucosylation % 2.7 2.8 2.9

[0389] High mannose % 6.9 7.2 7.0

[0390] Galactosylation % 38.6 41 .3 40.6

[0391] Terminal sialylation % 1.8 1.8 1.9

[0392] Corresponding ADCC and CDC potencies, calculated based on the observed glycosylation profiles, listed in Tables 18 and 19 for metabolically engineered GE-1 and GE-2 approaches, respectively.

[0393] Table 18: Relative potency of GE-1 genetically glycoengineered CHO cell-produced ofatumumab batches, shown as % increase compared to NS / 0 cell-produced ofatumumab.

[0394] Batch No: GE-1 :1 GE-1 :2 GE-1 :5 GE-1 :6

[0395] ADCC +8.25% +8.13% +12.5% +13.1 %

[0396] CDC +11.7% +9.22% +11.9% +9.74%

[0397] Table 19: Relative potency of GE-2 genetically glycoengineered CHO cell-produced ofatumumab batches, shown as % increase or decrease compared to NS / 0 cell-produced ofatumumab.

[0398] Batch No: GE-2:7 GE-2:8 GE-2:9

[0399] ADCC +17.1 % +21.0% +21.4%

[0400] CDC -5.01 % -1.44% -2.29%

[0401] Each of these genetically and metabolically glycoengineered CHO cell-produced anti-CD20 antibodies having SEQ ID NOs: 1 and 2 exhibited reduced levels of afucosylation that more closely resemble NS / 0 cell-produced ofatumumab, and predictive modelling showed that these genetically glycoengineered CHO cell-produced antibody compositions are projected to exhibit relative ADCC and CDC potencies within 30% of NS / 0 cell-produced ofatumumab.

[0402] Example 6: Structural and functional characterization of glycoengineered CHO cell-produced anti- CD20 antibodies

[0403] This example describes methods for characterizing the glycosylation structures and biological function (CD20 binding, CDC, and ADCC) of anti-CD20 antibodies, such as the glycoengineered CHO cell-produced anti-CD20 antibodies generated using the glycoengineering methods described in Examples 3, 4 and 5. A: Quantification of glycosylation variants in glycoengineered anti-CD20 antibody compositions

[0404] The molecular mass of CHO cell-produced anti-CD20 antibodies having a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 2 is analyzed by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-UV / ESI-MS) to determine the relative quantity of GOF, G1 F, and G2F N-glycans. Based on the amino sequences of SEQ ID NO: 1 and SEQ ID NO: 2, the mass of each type of glycan is shown in Table 20, below.

[0405] Table 20: Deconvoluted masses for reduced heavy chain by glycosylation type

[0406] Heavy Chain Glycosylation Mass (Da)

[0407] SEQ ID NO: 2 + GOF 50922

[0408] SEQ ID NO: 2 + G1 F 51084

[0409] SEQ ID NO: 2 + G2F 51246

[0410] ESI-MA for the CHO cell-produced anti-CD20 antibody is performed using a Waters QTOF Micro mass spectrometer. The electrospray ionization spectra for intact antibody are collected and processed using a Maximum Entropy algorithm to resolve the charge envelopes into the parent mass for each ion series. The determined masses are matched to the masses in the Table above. It is assumed that the glycosylation site at Asn302 is fully occupied with N-linked glycan structures typically observed with an IgG antibody produced from an NS / 0 cell line.

[0411] These results can indicate that 32-49% (total integrated area) of the N-glycans in the composition of glycoengineered, CHO cell-produced anti-CD20 antibodies are GOF; 28-35% (total integrated area) of the N-glycans in the composition are G1 F; and 6-13% (total integrated area) of the N-glycans in the composition are G2F.

[0412] B: Quantification of heavy chain C-terminal lysine in a glycoengineered anti-CD20 antibody composition Column exchange chromatography (CEX) is used to measure the relative quantity of C-terminal lysine containing antibody (e.g., anti-CD20 having the full SEQ ID NO: 3, including its terminal lysine). CEX chromatography allows the separation of proteins primarily based on their surface charge distribution. Chemical modifications of the protein (e.g., deamidation of asparagine) may result in variants that are separated from the unmodified variant by CEX.

[0413] CHO cell-produced anti-CD20 antibodies having a light chain of SEQ ID NO: 1 and a heavy chain of SEQ ID NO: 2 are analyzed by CEX to determine the relative abundance of the sum of basic peaks, which represents the relative abundance of C-terminal lysine containing variants, relative to the sum total amount of basic variants, acidic variants, and the main peak.

[0414] Less than 10% of the heavy chains of the CHO cell-produced anti-CD20 antibodies produced in this example contain a C-terminal lysine. C: Monosaccharide composition of a glycoengineered anti-CD20 antibody composition

[0415] Monosaccharide composition analysis is performed by reverse phase HPLC (RP-HPLC) analysis. Fucose, galactose, mannose, and N-acetylglucosamine are detected and molar ratios calculated relative to the trimanosyl core. This analysis can provide a total glycosylation in the composition, as a percent w / w protein.

[0416] D: Biological characterization of a glycoengineered anti-CD20 antibody composition

[0417] Biological activity is characterized by a cellular target binding assay, a functional CDC assay, a functional ADCC assay, an ADCC surrogate assay, a functional ADCP assay, an ADCP surrogate assay, an ELISA quantifying complement C1q binding, and SPR-based methods for affinity to human Fc receptors, FcyRla, human FcyRII, FcyRIII, and FcRn.

[0418] An NS / 0 cell-produced reference antibody is used as a reference standard for biological characterizations.

[0419] (i) CD20 binding assay

[0420] A cell-based target binding assay is used to assess binding of the CHO cell-produced anti-CD20 antibody to its target on CD20 on the surface of Raji cells. The assay utilizes CHO cell-produced anti- CD20 antibody labeled with Alexa Fluor 488 (OMB-A488) in a competitive binding assay format to allow quantification of cell-bound ofatumumab by flow cytometry. In the absence of antibody, a sub-saturating dose of labeled antibody binds CD20 on the surface of Raji cells. Unlabeled antibody present in a test sample competitively inhibits binding of labeled antibody to its antigen, resulting in decreased cell-bound fluorescence. Thus, the amount of cell-bound labeled antibody is inversely proportional to the concentration of unlabeled antibody.

[0421] The binding activity of test antibody is determined by comparison to an NS / 0 cell-produced reference standard, and the samples and standard are normalized on the basis of protein content. Relative potency is calculated using a parallel line assay according to the European Pharmacopoeia chapter 5.3. The result is expressed as relative potency of the test sample compared to the reference standard.

[0422] A successful glycoengineering process would show that the glycoengineered, CHO cell-produced anti-CD20 antibody retains binding affinity to CD20 (i.e., 70-130% relative binding), as compared to an NS / 0 cell-produced reference standard (e.g., NS / 0 cell-produced ofatumumab).

[0423] (ii) CDC effector function assay

[0424] A cell-based functional assay is used to assess the ability of the CHO cell-produced anti-CD20 antibody to mediate CDC using Raji target cells. When NS / O cell-produced ofatumumab binds CD20 in the presence of a complement source, it induces the CDC cascade. In the assay used, Raji B cells endogenously expressing CD20 were incubated with different concentrations of CHO cell-produced anti-CD20 antibody and a fixed concentration of rabbit complement. Concentration-dependent killing of the Raji cells is analyzed after two hours by determination of the ATP concentration in each well, based on the luminescence produced by an ATP-consuming luciferin-luciferase system.

[0425] CDC may also be assessed using Bjab cells, peripheral blood mononuclear cells (PBMC) or Ri 1 cells.

[0426] The functional activity of CHO cell-produced anti-CD20 antibody is determined by comparison to an NS / O-produced reference standard. The samples and standard are normalized on the basis of protein content. Relative potency is then calculated using a parallel line assay according to the European Pharmacopoeia chapter 5.3. The result is expressed as relative potency of a sample (in percent) compared to the reference standard.

[0427] A successful glycoengineering process would show that the glycoengineered, CHO cell-produced anti-CD20 antibody retains CDC potency (i.e. , 70-130% relative CDC potency), as compared to the NS / 0 cell-produced reference standard.

[0428] (Hi) ADCC effector function assay

[0429] A cell-based functional assay is used to assess the ability of the CHO cell-produced anti-CD20 antibody to mediate ADCC.

[0430] The biological ADCC activity of the CHO cell-produced anti-CD20 antibody was measured based on its ability to trigger ADCC of Raji cells by NK effector cells. Fluorescently labeled Raji cells endogenously expressing human CD20 were incubated with different concentrations of the CHO cell- produced anti-CD20 antibody and excess of natural killer cells. Concentration-dependent killing of the Raji target cells were analyzed by measuring the release of the fluorochrome from the lysed Raji cells in each well.

[0431] ADCC may also be assessed using Bjab cells or Jurkat T cells or HEK293 cells engineered to stably express the hFcyR3A reporter on their surface and a reporter gene.

[0432] The ADCC activity of the CHO cell-produced anti-CD20 antibody test samples is determined by comparison to the NS / 0 cell-produced reference standard. The samples and the standard are normalized on the basis of protein content. Relative potency is then calculated using a parallel line assay according to the European Pharmacopoeia, Chapter 5.3. The result is expressed as relative potency of a sample (in percent) compared to the primary reference standard.

[0433] A successful glycoengineering process would show that the glycoengineered, CHO cell-produced anti-CD20 antibody retains ADCC potency (i.e., 70-130% relative ADCC potency), as compared to the NS / 0 cell-produced reference standard. Table 21: Sequences

[0434] VII. Numerated Embodiments

[0435] The following numerated embodiments are provided as part of the invention:

[0436] 1 . An antibody composition comprising CHO cell-produced antibodies, wherein the CHO cell- produced antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell- produced antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated.

[0437] 2. The antibody composition of embodiment 1 , wherein 1-6% of the N-glycans in the composition are afucosylated.

[0438] 3. The antibody composition of embodiment 1 or 2, wherein the antibody composition is devoid of N- glycolyl neuraminic acid (NGNA).

[0439] 4. The antibody composition of any one of embodiments 1-3, wherein:

[0440] (a) 32-49% of the N-glycans in the composition are GOF;

[0441] (b) 28-35% of the N-glycans in the composition are G1 F;

[0442] (c) 6-13% of the N-glycans in the composition are G2F; and / or

[0443] (d) less than 6% of the N-glycans in the composition are mannose-5. 5. An antibody composition comprising CHO cell-produced antibodies, wherein the CHO cell- produced antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell- produced antibodies in the composition comprise an Fc region comprising an N-glycan, wherein:

[0444] (a) 32-49% of the N-glycans in the composition are GOF;

[0445] (b) 28-35% of the N-glycans in the composition are G1 F;

[0446] (c) 6-13% of the N-glycans in the composition are G2F; and / or

[0447] (d) less than 6% of the N-glycans in the composition are mannose-5.

[0448] 6. The antibody composition of embodiment 5, wherein less than 10% of the N-glycans in the composition are afucosylated.

[0449] 7. The antibody composition of embodiment 6, wherein 1-6% of the N-glycans in the composition are afucosylated.

[0450] 8. The antibody composition of any one of embodiments 5-7, wherein the antibody composition is devoid of NGNA.

[0451] 9. An antibody composition comprising CHO cell-produced antibodies, wherein the CHO cell- produced antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell- produced antibodies in the composition comprise an Fc region comprising an N-glycan, wherein:

[0452] (a) 32-49% of the N-glycans in the composition are GOF;

[0453] (b) 28-35% of the N-glycans in the composition are G1 F;

[0454] (c) 6-13% of the N-glycans in the composition are G2F,

[0455] (d) less than 6% of the N-glycans in the composition are mannose-5, and

[0456] (e) less than 10% of the N-glycans in the composition are afucosylated.

[0457] 10. The antibody composition of embodiment 7, wherein 1-6% of the N-glycans in the composition are afucosylated.

[0458] 11 . The antibody composition of embodiment 9 or 10, wherein the antibody composition is devoid of NGNA.

[0459] 12. An antibody composition comprising antibodies, wherein each of the antibodies comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the antibody composition is devoid of NGNA. 13. The antibody composition of embodiment 12, wherein the antibodies are CHO cell-produced antibodies.

[0460] 14. The antibody composition of embodiment 12 or 13, wherein the antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated.

[0461] 15. The antibody composition of any one of embodiments 12-14, wherein 1-6% of the N-glycans in the composition are afucosylated.

[0462] 16. The antibody composition of any one of embodiments 12-15, wherein:

[0463] (a) 32-49% of the N-glycans in the composition are GOF;

[0464] (b) 28-35% of the N-glycans in the composition are G1 F;

[0465] (c) 6-13% of the N-glycans in the composition are G2F; and / or

[0466] (d) less than 6% of the N-glycans in the composition are mannose-5.

[0467] 17. The antibody composition of any one of embodiments 1-16, wherein the composition comprises between 1 .0 and 2.5% N-glycans, as weight / weight of total protein.

[0468] 18. The antibody composition of any one of embodiments 1-17, wherein the composition comprises 40 to 150 mmol sialic acid per mol CHO-produced antibodies.

[0469] 19. The antibody composition of embodiment 18, wherein the sialic acid is NeuGc.

[0470] 20. The antibody composition of any one of embodiments 1-19, wherein at least 25% of the N- glycans in the composition are terminally galactosylated.

[0471] 21 . The antibody composition of any one of embodiments 1-20, wherein at least 2% of the N-glycans in the composition are terminally sialylated.

[0472] 22. The antibody composition of any one of embodiments 1-21 , wherein the CHO-cell produced antibodies have a mean binding affinity (KD) to Fc gamma receptor (FcyR) la from 8 to 11 nM.

[0473] 23. The antibody composition of any one of embodiments 1-22, wherein the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllaHRfrom 15 to 20 pM. 24. The antibody composition of any one of embodiments 1-23, wherein the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllaLRfrom 2 to 7 pM.

[0474] 25. The antibody composition of any one of embodiments 1-24, wherein the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRllb from 26 to 37 pM.

[0475] 26. The antibody composition of any one of embodiments 1-25, wherein the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRlllb from 7 to 12 pM.

[0476] 27. The antibody composition of any one of embodiments 1-26, wherein the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRI I laF158from 2 to 7 pM.

[0477] 28. The antibody composition of any one of embodiments 1-27, wherein the CHO-cell produced antibodies have a mean binding affinity (KD) to FcyRI llaV158from 0.1 to 0.6 pM.

[0478] 29. The antibody composition of any one of embodiments 1-28, wherein the CHO-cell produced antibodies have a mean binding affinity (KD at pH 6.0) to FcRn from 0.3 to 0.8 pM.

[0479] 30. The antibody composition of any one of embodiments 1-29, wherein the CHO cell-produced antibodies have a mean binding affinity (KD) to CD20 within 30% of a reference murine cell-produced antibody.

[0480] 31 . The antibody composition of any one of embodiments 1-30, wherein the CHO-cell produced antibodies have a relative antibody dependent cytotoxicity (ADCC) potency within 30% of a reference murine cell-produced antibody.

[0481] 32. The antibody composition of any one of embodiments 1-31 , wherein the CHO-cell produced antibodies have a relative complement dependent cytotoxicity (CDC) potency within 30% of a reference murine cell-produced antibody.

[0482] 33. The antibody composition of any one of embodiments 1-32, wherein the CHO-cell produced antibodies have a relative antibody dependent cellular phagocytosis (ADCP) potency within 30% of a reference murine cell-produced antibody.

[0483] 34. The antibody composition of any one of embodiments 1-33, wherein the CHO-cell produced antibodies have a relative C1q binding potency within 10% of a reference murine cell-produced antibody. 35. The antibody composition of any one of embodiments 30-34, wherein the reference murine cell- produced antibody is NS / 0 cell-produced ofatumumab.

[0484] 36. The antibody composition of any one of embodiments 1-35, wherein the CHO-cell produced antibodies are glycoengineered antibodies.

[0485] 37. A CHO cell-produced antibody, wherein the CHO cell-produced antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell-produced antibody comprises:

[0486] (a) a binding affinity (KD) to FcyRla from 8 to 11 nM;

[0487] (b) a binding affinity (KD) to FcyRllaHRfrom 15 to 20 pM;

[0488] (c) a binding affinity (KD) to FcyRllaLRfrom 2 to 7 pM;

[0489] (d) a binding affinity (KD) to FcyRllb from 26 to 37 pM;

[0490] (e) a binding affinity (KD) to FcyRlllb from 7 to 12 pM;

[0491] (f) a binding affinity (KD) to FcyRI I laF158from 2 to 7 pM;

[0492] (g) a binding affinity (KD) to FcyRI llaV158from 0.1 to 0.6 pM;

[0493] (h) a binding affinity (KD) to FcRn from 0.3 to 0.8 pM;

[0494] (i) binding affinity (KD) to CD20 within 30% of a reference murine cell-produced antibody

[0495] (j) a relative CDC potency within 30% of a reference murine cell-produced antibody;

[0496] (k) a relative ADCP potency within 30% of a reference murine cell-produced antibody; and / or

[0497] (l) a relative C1q binding potency within 10% of a reference murine cell-produced antibody.

[0498] 38. The CHO cell-produced antibody of embodiment 37, wherein the CHO cell-produced antibody is a glycoengineered antibody.

[0499] 39. The CHO cell-produced antibody of embodiment 37 or 38, wherein the CHO cell-produced antibody is devoid of NGNA.

[0500] 40. A CHO cell expressing an anti-CD20 antibody, wherein the anti-CD20 antibody is the CHO cell- produced antibody of any one of embodiments 37-39.

[0501] 41 . The CHO cell of embodiment 40, wherein the CHO cell comprises one or more transgenes genes encoding Fut8, Man2a1 , or Mgat2.

[0502] 42. A culture comprising CHO cells that express anti-CD20 antibodies, wherein the anti-CD20 antibodies are the CHO cell-produced antibodies of any one of embodiments 37-39. 43. A culture comprising the CHO cell-produced antibodies of any one of embodiments 37-39.

[0503] 44. The culture embodiment 43, wherein the glycoengineered CHO cells comprise one or more transgenes genes encoding Fut8, Man2a1 , or Mgat2.

[0504] 45. The culture of any one of embodiments 42-44, wherein the culture comprises putrescine, L- fucose, dexamethasone, insulin, and / or galactose.

[0505] 46. The culture of any one of embodiments 42-45, wherein the culture comprises putrescine in an amount of about 1 g / L or less.

[0506] 47. The culture of embodiment 46, wherein the culture comprises putrescene in an amount of about 1 g / L.

[0507] 48. The culture of any one of embodiments 42-47, wherein the culture comprises L-fucose in an amount of about 5 g / L or less.

[0508] 49. The culture of embodiment 48, wherein the culture comprises L-fucose in an amount of about 5 g / L.

[0509] 50. The culture of any one of embodiments 42-49, wherein the culture comprises dexamethasone in an amount of about 20 pM or less.

[0510] 51. The culture of embodiment 50, wherein the culture comprises dexamethasone in an amount of about 20 pM.

[0511] 52. The culture of any one of embodiments 42-51 , wherein the culture comprises insulin in an amount of about 2 mg / mL or less.

[0512] 53. The culture of embodiment 52, wherein the culture comprises insulin in an amount of about 2 mg / mL.

[0513] 54. The culture of any one of embodiments 42-53, wherein the culture comprises galactose in an amount of about 15 g / L or less.

[0514] 55. The culture of embodiment 54, wherein the culture comprises galactose in an amount of about 15 g / L. 56. The culture of any one of embodiments 42-55, wherein the culture comprises manganese in an amount of about 50 pM or less.

[0515] 57. The culture of embodiment 56, wherein the culture comprises manganese in an amount from about 1 .5 pM to about 50 pM.

[0516] 58. The culture of embodiment 57, wherein the culture comprises manganese in an amount of about 25.75 pM.

[0517] 59. The culture of any one of embodiments 42-58, wherein the culture comprises glucose in an amount of about 175 g / L or less.

[0518] 60. The culture of embodiment 59, wherein the culture comprises glucose in an amount from about 100 g / L to about 175 g / L.

[0519] 61 . The culture of embodiment 60, wherein the culture comprises glucose in an amount of about 137.5 g / mL.

[0520] 62. The culture of any one of embodiments 42-61 , wherein the culture comprises calcium in an amount greater than 1 .2 mM.

[0521] 63. The culture of embodiment 62, wherein the culture comprises 2-3 mM calcium.

[0522] 64. The culture of embodiment 63, wherein the culture comprises about 2.4 mM calcium.

[0523] 65. An antibody composition produced by a process comprising:

[0524] (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise:

[0525] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and

[0526] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0527] (b) culturing the CHO cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L-fucose, dexamethasone, and / or insulin, and

[0528] (c) isolating the antibodies to obtain the antibody composition.

[0529] 66. An antibody composition produced by a process comprising: (a) providing CHO cells comprising:

[0530] (i) one or more transgenes encoding Fut8, Man2a1 , and / or Mgat2, and

[0531] (ii) nucleic acid encoding antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0532] (b) culturing the CHO cells under conditions which permit expression of the antibodies; and

[0533] (c) isolating the antibodies to obtain the antibody composition.

[0534] 67. The antibody composition of any one of embodiments 1-36, produced by a process comprising:

[0535] (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise:

[0536] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and

[0537] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0538] (b) culturing the CHO cells under conditions which permit expression of the antibodies, and

[0539] (c) isolating the antibodies to obtain the antibody composition.

[0540] 68. The antibody composition of embodiment 65 or 67, wherein the CHO cells express Fut8, Man2a1 , or Mgat2.

[0541] 69. The antibody composition of any one of embodiments 66-68, wherein the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, insulin, and / or galactose.

[0542] 70. The antibody composition of any one of embodiments 65-69, wherein the CHO cells are cultured in the presence of putrescine in an amount of about 1 g / L or less.

[0543] 71. The antibody composition of embodiment 70, wherein the CHO cells are cultured in the presence of putrescene in an amount of about 1 g / L.

[0544] 72. The antibody composition of any one of embodiments 65-71 , wherein the CHO cells are cultured in the presence of L-fucose in an amount of about 5 g / L or less.

[0545] 73. The antibody composition of embodiment 72, wherein the CHO cells are cultured in the presence of L-fucose in an amount of about 5 g / L.

[0546] 74. The antibody composition of any one of embodiments 65-73, wherein the CHO cells are cultured in the presence of dexamethasone in an amount of about 20 pM or less. 75. The antibody composition of embodiment 74, wherein the CHO cells are cultured in the presence of dexamethasone in an amount of about 20 pM.

[0547] 76. The antibody composition of any one of embodiments 65-75, wherein the CHO cells are cultured in the presence of insulin in an amount of about 2 mg / mL or less.

[0548] 77. The antibody composition of embodiment 76, wherein the CHO cells are cultured in the presence of insulin in an amount of about 2 mg / mL.

[0549] 78. The antibody composition of any one of embodiments 65-77, wherein the CHO cells are cultured in the presence of galactose in an amount of about 15 g / L or less.

[0550] 79. The antibody composition of embodiment 78, wherein the CHO cells are cultured in the presence of galactose in an amount of about 15 g / L.

[0551] 80. The antibody composition of any one of embodiments 65-79, wherein the CHO cells are cultured in the presence of manganese in an amount of about 50 pM or less.

[0552] 81. The antibody composition of embodiment 80, wherein the CHO cells are cultured in the presence of manganese in an amount from about 1 .5 pM to about 50 pM.

[0553] 82. The antibody composition of embodiment 81 , wherein the CHO cells are cultured in the presence of manganese in an amount of about 25.75 pM.

[0554] 83. The antibody composition of any one of embodiments 65-82, wherein the CHO cells are cultured in the presence of glucose in an amount of about 175 g / L or less.

[0555] 84. The antibody composition of embodiment 83, wherein the CHO cells are cultured in the presence of glucose in an amount from about 100 g / L to about 175 g / L.

[0556] 85. The antibody composition of embodiment 84, wherein the CHO cells are cultured in the presence of glucose in an amount of about 137.5 g / mL.

[0557] 86. The antibody composition of any one of embodiments 65-85, wherein the CHO cells are cultured in the presence of calcium in an amount greater than 1 .2 mM. 87. The antibody composition of embodiment 86, wherein the CHO cells are cultured in the presence of 2-3 mM calcium.

[0558] 88. The antibody composition of embodiment 87, wherein the CHO cells are cultured in the presence of about 2.4 mM calcium.

[0559] 89. A method of producing an antibody composition, the method comprising:

[0560] (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise:

[0561] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and

[0562] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0563] (b) culturing the CHO cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L-fucose, dexamethasone, and / or insulin; and

[0564] (c) isolating the antibodies to obtain the antibody composition.

[0565] 90. A method of producing an antibody composition, the method comprising:

[0566] (a) providing CHO cells comprising:

[0567] (i) Fut8, Man2a1 , or Mgat2, and

[0568] (ii) nucleic acid encoding antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0569] (b) culturing the CHO cells under conditions which permit expression of the antibodies; and

[0570] (c) isolating the antibodies to obtain the antibody composition.

[0571] 91 . A method of producing the antibody composition of any one of embodiments 1-27, the method comprising:

[0572] (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise:

[0573] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and

[0574] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0575] (b) culturing the CHO cells under conditions which permit expression of the antibodies, and

[0576] (c) isolating the antibodies to obtain the antibody composition.

[0577] 92. The method of embodiment 89 or 91 , wherein the CHO cells express Fut8, Man2a1 , or Mgat2. 93. The method of any one of embodiments 90-92, wherein the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, and / or insulin.

[0578] 94. The method of any one of embodiments 89-93, wherein the CHO cells are cultured in the presence of putrescine in an amount of about 1 g / L or less.

[0579] 95. The method of embodiment 94, wherein the CHO cells are cultured in the presence of putrescene in an amount of about 1 g / L.

[0580] 96. The method of any one of embodiments 89-95, wherein the CHO cells are cultured in the presence of L-fucose in an amount of about 5 g / L or less.

[0581] 97. The method of embodiment 96, wherein the CHO cells are cultured in the presence of L-fucose in an amount of about 5 g / L.

[0582] 98. The method of any one of embodiments 89-97, wherein the CHO cells are cultured in the presence of dexamethasone in an amount of about 20 pM or less.

[0583] 99. The method of embodiment 98, wherein the CHO cells are cultured in the presence of dexamethasone in an amount of about 20 pM.

[0584] 100. The method of any one of embodiments 89-99, wherein the CHO cells are cultured in the presence of insulin in an amount of about 2 mg / mL or less.

[0585] 101. The method of embodiment 100, wherein the CHO cells are cultured in the presence of insulin in an amount of about 2 mg / mL.

[0586] 102. The method of any one of embodiments 89-101 , wherein the CHO cells are cultured in the presence of galactose in an amount of about 15 g / L or less.

[0587] 103. The method of embodiment 102, wherein the CHO cells are cultured in the presence of galactose in an amount of about 15 g / L.

[0588] 104. The method of any one of embodiments 89-103, wherein the CHO cells are cultured in the presence of manganese in an amount of about 50 pM or less. 105. The method of embodiment 104, wherein the CHO cells are cultured in the presence of manganese in an amount from about 1 .5 pM to about 50 pM.

[0589] 106. The method of embodiment 105, wherein the CHO cells are cultured in the presence of manganese in an amount of about 25.75 pM.

[0590] 107. The method of any one of embodiments 89-106, wherein the CHO cells are cultured in the presence of glucose in an amount of about 175 g / L or less.

[0591] 108. The method of embodiment 107, wherein the CHO cells are cultured in the presence of glucose in an amount from about 100 g / L to about 175 g / L.

[0592] 109. The method of embodiment 108, wherein the CHO cells are cultured in the presence of glucose in an amount of about 137.5 g / mL.

[0593] 110. The method of any one of embodiments 89-109, wherein the CHO cells are cultured in the presence of calcium in an amount greater than 1 .2 mM.

[0594] 111. The method of embodiment 110, wherein the CHO cells are cultured in the presence of 2-3 mM calcium.

[0595] 112. The method of embodiment 111 , wherein the CHO cells are cultured in the presence of about 2.4 mM calcium.

[0596] 113. A method of treating multiple sclerosis, the method comprising administering an effective amount of a CHO cell-produced antibody composition to a patient in need thereof, wherein the CHO cell- produced antibody composition comprises anti-CD20 antibodies comprising:

[0597] (a) a light chain comprising the amino acid sequence of SEQ ID NO: 1 ; and

[0598] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.

[0599] 114. The method of embodiment 113, wherein the anti-CD20 antibodies comprise:

[0600] (a) the antibody composition of any one of embodiments 1-36 or 65-88; or

[0601] (b) the CHO cell-produced antibody of any one of embodiments 37-39.

[0602] 115. The method of embodiment 113 or 114, wherein the CHO cell-produced antibody composition is administered to the patient once per month, once every two months, once every three months, once every four months, once every six months, once every eight months, or once yearly. 116. The method of embodiment 115, wherein the CHO cell-produced antibody composition is administered to the patient once per month.

[0603] 117. The method of embodiment 116, wherein the CHO cell-produced antibody composition administered to the patient once per month comprises about 20 mg of the anti-CD20 antibodies.

[0604] 118. The method of embodiment 115, wherein the CHO cell-produced antibody composition is administered to the patient once every two months.

[0605] 119. The method of embodiment 118, wherein the CHO cell-produced antibody composition administered to the patient once every two months comprises about 135 mg of the anti-CD20 antibodies.

[0606] 120. The method of any one of embodiments 113-119, wherein the CHO cell-produced antibody composition is administered by subcutaneous injection.

[0607] 121. The method of any one of embodiments 113-120, wherein the multiple sclerosis is relapsing multiple sclerosis (RMS).

[0608] 122. The method of any one of embodiments 113-121 , wherein the multiple sclerosis is relapsingremitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS), or clinically isolated syndrome (CIS).

[0609] VIII. Further Numerated Embodiments

[0610] The following further numerated embodiments are provided as part of the invention:

[0611] 1 . An antibody composition comprising CHO cell-produced antibodies, wherein the CHO cell- produced antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell- produced antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated.

[0612] 2. The antibody composition of embodiment 1 , wherein 6% or fewer of the N-glycans in the composition are afucosylated.

[0613] 3. The antibody composition of embodiment 2, wherein 1-6% of the N-glycans in the composition are afucosylated.

[0614] 4. The antibody composition of any one of embodiments 1-3, wherein 0.9% to 4.5% of the N-glycans in the composition are afucosylated, 1 .8% to 10.3% of the N-glycans in the composition are high mannose glycans and / or 23% to 55% of the N-glycans in the composition are galactosylated.

[0615] 5. The antibody composition of embodiment 4, wherein 0.9% to 3.4% of the N-glycans in the composition are afucosylated, 5.8% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 55% of the N-glycans in the composition are galactosylated.

[0616] 6. The antibody composition of embodiment 5, wherein 0.9% to 1 .3% of the N-glycans in the composition are afucosylated, 5.8% to 9.3% of the N-glycans in the composition are high mannose glycans, and / or 38% to 55% of the N-glycans in the composition are galactosylated.

[0617] 7. The antibody composition of embodiment 5, wherein 2.7% to 3.4% of the N-glycans in the composition are afucosylated, 6.9% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 41 % of the N-glycans in the composition are galactosylated.

[0618] 8. The antibody composition of embodiment 4, wherein 3.4% and 4.5% of the N-glycans in the composition are afucosylated, 1 .8% to 5% of the N-glycans in the composition are high mannose glycans, and / or 29% to 45% of the N-glycans in the composition are galactosylated.

[0619] 9. The antibody composition of any one of embodiments 1-8, wherein: (a) 32-49% of the N-glycans in the composition are GOF;

[0620] (b) 28-35% of the N-glycans in the composition are G1 F;

[0621] (c) 6-13% of the N-glycans in the composition are G2F; and / or

[0622] (d) less than 6% of the N-glycans in the composition are mannose-5.

[0623] 10. The antibody composition of any one of embodiments 1-9, wherein the antibody composition is devoid of N-glycolyl neuraminic acid (NGNA).

[0624] 11. The antibody composition of any one of embodiments 1-10, wherein less than 15% of the heavy chains comprise a C-terminal lysine.

[0625] 12. The antibody composition of any one of embodiments 1-11 , wherein at least 0.01 % of the heavy chains comprises C-terminal amidation.

[0626] 13. An antibody composition, wherein the antibody composition comprises antibodies comprising a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein less than 15% of the heavy chains comprise C-terminal lysine, optionally wherein the antibodies comprise one or more native fucose residues.

[0627] 14. The antibody composition of embodiment 13, wherein the antibody composition is devoid of N- glycolyl neuraminic acid (NGNA).

[0628] 15. The antibody composition of embodiment 13 or 14, wherein at least 0.01% of the heavy chains comprises C-terminal amidation.

[0629] 16. An antibody composition, wherein the antibody composition comprises antibodies comprising a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein at least 0.01 % of the heavy chains comprises C-terminal amidation.

[0630] 17. The antibody composition of embodiment 16, wherein the antibody composition is devoid of NGNA.

[0631] 18. The antibody composition of embodiment 16 or 17, wherein less than 15% of the heavy chains comprise C-terminal lysine. 19. An antibody composition, wherein the antibody composition comprises antibodies comprising a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the antibody composition is devoid of NGNA, optionally wherein the antibodies comprise one or more native fucose residues.

[0632] 20. The antibody composition of embodiment 19, wherein at least 0.01% of the heavy chains comprises C-terminal amidation.

[0633] 21 . The antibody composition of embodiment 19 or 20, wherein less than 15% of the heavy chains comprise C-terminal lysine.

[0634] 22. The antibody composition of any one of embodiments 13-21 , wherein less than 10% of the N- glycans in the composition are afucosylated.

[0635] 23. The antibody composition of embodiment 22, wherein 6% or fewer of the N-glycans in the composition are afucosylated.

[0636] 24. The antibody composition of embodiment 23, wherein 1-6% of the N-glycans in the composition are afucosylated.

[0637] 25. The antibody composition of any one of embodiments 13-24, wherein 0.9% to 4.5% of the N- glycans in the composition are afucosylated, 1.8% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 55% of the N-glycans in the composition are galactosylated.

[0638] 26. The antibody composition of embodiment 25, wherein 0.9% to 3.4% of the N-glycans in the composition are afucosylated, 5.8% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 55% of the N-glycans in the composition are galactosylated.

[0639] 27. The antibody composition of embodiment 26, wherein 0.9% to 1 .3% of the N-glycans in the composition are afucosylated, 5.8% to 9.3% of the N-glycans in the composition are high mannose glycans, and / or 38% to 55% of the N-glycans in the composition are galactosylated.

[0640] 28. The antibody composition of embodiment 26, wherein 2.7% to 3.4% of the N-glycans in the composition are afucosylated, 6.9% to 10.3% of the N-glycans in the composition are high mannose glycans, and / or 23% to 41 % of the N-glycans in the composition are galactosylated. 29. The antibody composition of embodiment 25, wherein 3.4% and 4.5% of the N-glycans in the composition are afucosylated, 1 .8% to 5% of the N-glycans in the composition are high mannose glycans, and / or 29% to 45% of the N-glycans in the composition are galactosylated.

[0641] 30. The antibody composition of any one of embodiments 13-29, wherein the antibody composition is a CHO cell-produced antibody composition.

[0642] 31 . The antibody composition of any one of embodiments 13-30, wherein:

[0643] (a) 32-49% of the N-glycans in the composition are GOF;

[0644] (b) 28-35% of the N-glycans in the composition are G1 F;

[0645] (c) 6-13% of the N-glycans in the composition are G2F; and / or

[0646] (d) less than 6% of the N-glycans in the composition are mannose-5.

[0647] 32. An antibody composition comprising CHO cell-produced antibodies, wherein the CHO cell- produced antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell- produced antibodies in the composition comprise an Fc region comprising an N-glycan, wherein:

[0648] (a) 32-49% of the N-glycans in the composition are GOF;

[0649] (b) 28-35% of the N-glycans in the composition are G1 F;

[0650] (c) 6-13% of the N-glycans in the composition are G2F; and / or

[0651] (d) less than 6% of the N-glycans in the composition are mannose-5.

[0652] 33. The antibody composition of embodiment 32, wherein less than 10% of the N-glycans in the composition are afucosylated.

[0653] 34. The antibody composition of embodiment 33, wherein 1-6% of the N-glycans in the composition are afucosylated.

[0654] 35. The antibody composition of any one of embodiments 32-34, wherein the antibody composition is devoid of NGNA.

[0655] 36. An antibody composition comprising CHO cell-produced antibodies, wherein the CHO cell- produced antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell- produced antibodies in the composition comprise an Fc region comprising an N-glycan, wherein:

[0656] (a) 32-49% of the N-glycans in the composition are GOF;

[0657] (b) 28-35% of the N-glycans in the composition are G1 F; (c) 6-13% of the N-glycans in the composition are G2F,

[0658] (d) less than 6% of the N-glycans in the composition are mannose-5, and

[0659] (e) less than 10% of the N-glycans in the composition are afucosylated.

[0660] 37. The antibody composition of embodiment 36, wherein 1-6% of the N-glycans in the composition are afucosylated.

[0661] 38. The antibody composition of embodiment 36 or 37, wherein the antibody composition is devoid of NGNA.

[0662] 39. An antibody composition comprising antibodies, wherein each of the antibodies comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the antibody composition is devoid of NGNA.

[0663] 40. The antibody composition of embodiment 39, wherein the antibodies are CHO cell-produced antibodies.

[0664] 41 . The antibody composition of embodiment 39 or 40, wherein the antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated.

[0665] 42. The antibody composition of any one of embodiments 39-41 , wherein 1-6% of the N-glycans in the composition are afucosylated.

[0666] 43. The antibody composition of any one of embodiments 39-42, wherein:

[0667] (a) 32-49% of the N-glycans in the composition are GOF;

[0668] (b) 28-35% of the N-glycans in the composition are G1 F;

[0669] (c) 6-13% of the N-glycans in the composition are G2F; and / or

[0670] (d) less than 6% of the N-glycans in the composition are mannose-5.

[0671] 44. The antibody composition of any one of embodiments 1-43, wherein the antibodies comprise between 1 .0 and 2.5% N-glycans, as weight / weight of total protein.

[0672] 45. The antibody composition of any one of embodiments 1-44, wherein the composition comprises 40 to 150 mmol sialic acid per mol antibodies.

[0673] 46. The antibody composition of embodiment 45, wherein the sialic acid is NeuGc. 47. The antibody composition of any one of embodiments 1-46, wherein at least 25% of the N- glycans in the composition are terminally galactosylated.

[0674] 48. The antibody composition of any one of embodiments 1-47, wherein at least 2% of the N-glycans in the composition are terminally sialylated.

[0675] 49. The antibody composition of any one of embodiments 1-47, wherein the antibodies have a mean binding affinity (KD) to Fc gamma receptor (FcyR) la from 8 to 11 nM.

[0676] 50. The antibody composition of any one of embodiments 1-49, wherein the antibodies have a mean binding affinity (KD) to FcyRllaHRfrom 15 to 20 pM.

[0677] 51 . The antibody composition of any one of embodiments 1-50, wherein the antibodies have a mean binding affinity (KD) to FcyRllaLRfrom 2 to 7 pM.

[0678] 52. The antibody composition of any one of embodiments 1-51 , wherein the antibodies have a mean binding affinity (KD) to FcyRllb from 26 to 37 pM.

[0679] 53. The antibody composition of any one of embodiments 1-52, wherein the antibodies have a mean binding affinity (KD) to FcyRlllb from 7 to 12 pM.

[0680] 54. The antibody composition of any one of embodiments 1-53, wherein the antibodies have a mean binding affinity (KD) to FcyRI I laF158from 2 to 7 pM.

[0681] 55. The antibody composition of any one of embodiments 1-54, wherein the antibodies have a mean binding affinity (KD) to FcyRII laV158from 0.1 to 0.6 pM.

[0682] 56. The antibody composition of any one of embodiments 1-55, wherein the antibodies have a mean binding affinity (KD at pH 6.0) to FcRn from 0.3 to 0.8 pM.

[0683] 57. The antibody composition of any one of embodiments 1-56, wherein the antibodies have a mean binding affinity (KD) to CD20 within 30% of a reference murine cell-produced antibody.

[0684] 58. The antibody composition of any one of embodiments 1-57, wherein the antibodies have a relative antibody dependent cytotoxicity (ADCC) potency within 30% of a reference murine cell-produced antibody. 59. The antibody composition of any one of embodiments 1-58, wherein the antibodies have a relative complement dependent cytotoxicity (CDC) potency within 30% of a reference murine cell- produced antibody.

[0685] 60. The antibody composition of any one of embodiments 1-59, wherein the antibodies have a relative antibody dependent cellular phagocytosis (ADCP) potency within 30% of a reference murine cell- produced antibody.

[0686] 61 . The antibody composition of any one of embodiments 1-60, wherein the antibodies have a relative C1q binding potency within 10% of a reference murine cell-produced antibody.

[0687] 62. The antibody composition of any one of embodiments 57-61 , wherein the reference murine cell- produced antibody is NS / 0 cell-produced ofatumumab.

[0688] 63. The antibody composition of any one of embodiments 1-62, wherein the antibodies are glycoengineered antibodies.

[0689] 64. A pharmaceutical formulation comprising the antibody composition of any one of embodiments 1- 63 and a pharmaceutically acceptable carrier.

[0690] 65. The pharmaceutical formulation of embodiment 64, further comprising an antioxidant.

[0691] 66. The pharmaceutical formulation of embodiment 65, wherein the antioxidant is methionine.

[0692] 67. The pharmaceutical formulation of embodiment 65 or 66, wherein the antioxidant is at a concentration from 0.1 to 20 mM.

[0693] 68. The pharmaceutical formulation of any one of embodiments 64-67, wherein the pharmaceutical formulation is at a pH from 5.2-5.8.

[0694] 69. The pharmaceutical formulation of any one of embodiments 64-68, wherein the pharmaceutical formulation comprises arginine, sodium acetate, sodium chloride, EDTA, and / or polysorbate.

[0695] 70. The pharmaceutical formulation of embodiment 69, wherein the pharmaceutical formulation comprises 1 % (w / v) arginine, 50 mM sodium acetate, 51 mM sodium chloride, 0.05 mM EDTA, and 0.02% (w / v) polysorbate 80. 71 . The pharmaceutical formulation of any one of embodiments 64-70, wherein the antibodies are present in an amount of about 50 mg / mL or about 90 mg / mL.

[0696] 72. A prefilled syringe comprising the pharmaceutical formulation of any one of embodiments 64-71.

[0697] 73. An autoinjector comprising the pharmaceutical formulation of any one of embodiments 64-71 or the prefilled syringe of embodiment 72.

[0698] 74. A CHO cell-produced antibody, wherein the CHO cell-produced antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell-produced antibody comprises:

[0699] (a) a binding affinity (KD) to FcyRla from 8 to 11 nM;

[0700] (b) a binding affinity (KD) to FcyRllaHRfrom 15 to 20 pM;

[0701] (c) a binding affinity (KD) to FcyRllaLRfrom 2 to 7 pM;

[0702] (d) a binding affinity (KD) to FcyRllb from 26 to 37 pM;

[0703] (e) a binding affinity (KD) to FcyRlllb from 7 to 12 pM;

[0704] (f) a binding affinity (KD) to FcyRI I laF158from 2 to 7 pM;

[0705] (g) a binding affinity (KD) to FcyRI llaV158from 0.1 to 0.6 pM;

[0706] (h) a binding affinity (KD) to FcRn from 0.3 to 0.8 pM;

[0707] (i) binding affinity (KD) to CD20 within 30% of a reference murine cell-produced antibody

[0708] (j) a relative ADCC potency within 30% of a reference murine cell-produced antibody;

[0709] (k) a relative CDC potency within 30% of a reference murine cell-produced antibody;

[0710] (l) a relative ADCP potency within 30% of a reference murine cell-produced antibody; and / or

[0711] (m) a relative C1q binding potency within 10% of a reference murine cell-produced antibody.

[0712] 75. The CHO cell-produced antibody of embodiment 74, wherein the CHO cell-produced antibody is a glycoengineered antibody.

[0713] 76. The CHO cell-produced antibody of embodiment 74 or 75, wherein the CHO cell-produced antibody is devoid of NGNA.

[0714] 77. A CHO cell expressing an anti-CD20 antibody, wherein the anti-CD20 antibody is the antibody of the composition of any one of embodiments 1-63 or the CHO cell-produced antibody of any one of embodiments 74-76. 78. The CHO cell of embodiment 77, wherein the CHO cell comprises one or more transgenes genes encoding Fut8, Man2a1 , or Mgat2.

[0715] 79. The CHO cell of embodiment 77, wherein the CHO cell comprises a transgene encoding a fucosyltransferase.

[0716] 80. The CHO cell of embodiment 79, wherein the CHO cell comprises a first transgene encoding the anti-CD20 antibody and a second transgene encoding the fucosyltransferase, wherein expression of the anti-CD20 antibody is driven by a first promoter, and the fucosyltransferase is driven by a second promoter, wherein the first promoter has a higher rate of transcription in the host cell than the second promoter.

[0717] 81. A recombinant host cell expressing an anti-CD20 antibody and a fucosyltransferase, wherein the anti-CD20 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the CHO cell comprises a first transgene encoding the anti-CD20 antibody and a second transgene encoding the fucosyltransferase, wherein expression of the anti-CD20 antibody is driven by a first promoter, and the fucosyltransferase is driven by a second promoter, wherein the first promoter has a higher rate of transcription in the host cell than the second promoter.

[0718] 81A. A recombinant host cell expressing an anti-CD20 antibody and a fucosyltransferase, wherein the anti-CD20 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the recombinant host cell comprises a first transgene encoding the anti-CD20 antibody and a second transgene encoding the fucosyltransferase, wherein expression of the anti-CD20 antibody is driven by a first promoter, and the fucosyltransferase is driven by a second promoter, wherein the first promoter has a higher rate of transcription in the host cell than the second promoter.

[0719] 82. The recombinant host cell of embodiment 81 or 81 A, which is a mammalian host cell.

[0720] 83. The recombinant host cell of embodiment 81 , 81 A or 82, which is not an NS / 0 cell.

[0721] 84. The recombinant host cell of embodiment 83, which is a CHO cell.

[0722] 85. The recombinant host cell of any one of embodiments 81-84, wherein the second promoter is a ubiquitin C (UBC) promoter. 86. The recombinant host cell of any one of embodiments 81-85, wherein the anti-CD20 antibody is the antibody of the composition of any one of embodiments 1-63.

[0723] 87. A culture comprising recombinant host cells that express recombinant anti-CD20 antibodies and recombinant fucosyltransferase, wherein the recombinant anti-CD20 antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, and wherein mRNA transcripts for the recombinant anti-CD20 antibodies exceed the number of mRNA transcripts for the recombinant fucosylatransferase.

[0724] 88. The culture of embodiment 87, wherein the mRNA transcripts for the recombinant anti-CD20 antibodies exceed the number of the mRNA transcripts for the recombinant fucosylatransferase by a factor of at least two.

[0725] 89. The culture of embodiment 88, wherein the mRNA transcripts for the recombinant anti-CD20 antibodies exceed the number of the mRNA transcripts for the recombinant fucosylatransferase by a factor of at least four.

[0726] 90. A culture comprising CHO cells that express anti-CD20 antibodies, wherein the anti-CD20 antibodies are the CHO cell-produced antibodies of any one of embodiments 74-76.

[0727] 91 . A culture comprising the CHO cell-produced antibodies of any one of embodiments 74-76.

[0728] 92. The culture of embodiment 91 , wherein the culture has a pH of about 6.9 or higher.

[0729] 93. The culture of embodiment 92, wherein the culture has a pH of about 6.9 to about 7.1.

[0730] 94. The culture of embodiment 93, wherein the pH is about 6.9, about 7.0, or about 7.1 .

[0731] 95. The culture any one of embodiments 91-94, wherein the glycoengineered CHO cells comprise one or more transgenes encoding Fut8, Man2a1 , or Mgat2.

[0732] 96. The culture of embodiment 95, wherein the glycoengineered CHO cells comprise a transgene encoding Fut8.

[0733] 97. The culture of any one of embodiments 91-96, wherein the culture comprises putrescine, L- fucose, dexamethasone, insulin, and / or galactose. 98. The culture of any one of embodiments 91-97, wherein the culture comprises putrescine in an amount of about 1 g / L or less.

[0734] 99. The culture of embodiment 98, wherein the culture comprises putrescene in an amount of about 0.5 g / L to about 1 g / L.

[0735] 100. The culture of any one of embodiments 91-99, wherein the culture comprises L-fucose in an amount of about 5 g / L or less.

[0736] 101. The culture of embodiment 100, wherein the culture comprises L-fucose in an amount of about 5 g / L.

[0737] 102. The culture of any one of embodiments 91-101 , wherein the culture comprises dexamethasone in an amount of about 20 pM or less.

[0738] 103. The culture of embodiment 102, wherein the culture comprises dexamethasone in an amount of about 20 pM.

[0739] 104. The culture of any one of embodiments 91-103, wherein the culture comprises insulin in an amount of about 2 mg / mL or less.

[0740] 105. The culture of embodiment 104, wherein the culture comprises insulin in an amount of about 2 mg / mL.

[0741] 106. The culture of any one of embodiments 91-105, wherein the culture comprises galactose in an amount of about 15 g / L or less.

[0742] 107. The culture of embodiment 106, wherein the culture comprises galactose in an amount of about 15 g / L.

[0743] 108. The culture of any one of embodiments 91-107, wherein the culture comprises manganese in an amount of about 50 pM or less.

[0744] 109. The culture of embodiment 108, wherein the culture comprises manganese in an amount from about 1 .5 pM to about 50 pM. 109A. The culture of embodiment 109, wherein the culture comprises manganese is an amount of 25.8 pM.

[0745] 110. The culture of embodiment 109, wherein the culture comprises manganese in an amount of about 25.75 pM.

[0746] 111. The culture of any one of embodiments 91-110, wherein the culture comprises glucose in an amount of about 175 g / L or less.

[0747] 112. The culture of embodiment 111 , wherein the culture comprises glucose in an amount from about 100 g / L to about 175 g / L.

[0748] 112A. The culture of embodiment 112, wherein the culture comprises glucose in an amount of about

[0749] 160.4 g / L.

[0750] 113. The culture of embodiment 112, wherein the culture comprises glucose in an amount of about

[0751] 137.5 g / mL.

[0752] 114. The culture of any one of embodiments 91-113, wherein the culture comprises calcium in an amount greater than 1 .2 mM.

[0753] 114A. The culture of embodiment 114, wherein the culture comprises 9.1 mM calcium.

[0754] 115. The culture of embodiment 114, wherein the culture comprises 2-3 mM calcium.

[0755] 116. The culture of embodiment 115, wherein the culture comprises about 2.4 mM calcium.

[0756] 117. An antibody composition produced by a process comprising:

[0757] (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise:

[0758] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and

[0759] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0760] (b) culturing the CHO cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L-fucose, dexamethasone, and / or insulin, and

[0761] (c) isolating the antibodies to obtain the antibody composition.

[0762] 118. An antibody composition produced by a process comprising: (a) providing CHO cells comprising:

[0763] (i) one or more transgenes encoding Fut8, Man2a1 , and / or Mgat2, and

[0764] (ii) nucleic acid encoding antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0765] (b) culturing the CHO cells under conditions which permit expression of the antibodies; and

[0766] (c) isolating the antibodies to obtain the antibody composition.

[0767] 119. The antibody composition of any one of embodiments 1-63, produced by a process comprising:

[0768] (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise:

[0769] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and

[0770] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0771] (b) culturing the CHO cells under conditions which permit expression of the antibodies, and

[0772] (c) isolating the antibodies to obtain the antibody composition.

[0773] 120. The antibody composition of embodiment 117 or 119, wherein the CHO cells express Fut8, Man2a1 , or Mgat2.

[0774] 121. The antibody composition of any one of embodiments 118-120, wherein the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L- fucose, dexamethasone, insulin, and / or galactose.

[0775] 122. The antibody composition of any one of embodiments 117-121 , wherein the CHO cells are cultured in the presence of putrescine in an amount of about 1 g / L or less.

[0776] 123. The antibody composition of embodiment 122, wherein the CHO cells are cultured in the presence of putrescene in an amount of about 1 g / L.

[0777] 124. The antibody composition of any one of embodiments 117-123, wherein the CHO cells are cultured in the presence of L-fucose in an amount of about 5 g / L or less.

[0778] 125. The antibody composition of embodiment 124, wherein the CHO cells are cultured in the presence of L-fucose in an amount of about 5 g / L.

[0779] 126. The antibody composition of any one of embodiments 117-125, wherein the CHO cells are cultured in the presence of dexamethasone in an amount of about 20 pM or less. 127. The antibody composition of embodiment 126, wherein the CHO cells are cultured in the presence of dexamethasone in an amount of about 20 pM.

[0780] 128. The antibody composition of any one of embodiments 117-127, wherein the CHO cells are cultured in the presence of insulin in an amount of about 2 mg / mL or less.

[0781] 129. The antibody composition of embodiment 128, wherein the CHO cells are cultured in the presence of insulin in an amount of about 2 mg / mL.

[0782] 130. The antibody composition of any one of embodiments 117-129, wherein the CHO cells are cultured in the presence of galactose in an amount of about 15 g / L or less.

[0783] 131. The antibody composition of embodiment 130, wherein the CHO cells are cultured in the presence of galactose in an amount of about 15 g / L.

[0784] 132. The antibody composition of any one of embodiments 117-131 , wherein the CHO cells are cultured in the presence of manganese in an amount of about 50 pM or less.

[0785] 133. The antibody composition of embodiment 132, wherein the CHO cells are cultured in the presence of manganese in an amount from about 1 .5 pM to about 50 pM.

[0786] 133A. The antibody composition of embodiment 133, wherein the CHO cells are cultured in the presence of manganese in an amount of 25.8 pM.

[0787] 134. The antibody composition of embodiment 133, wherein the CHO cells are cultured in the presence of manganese in an amount of about 25.75 pM.

[0788] 135. The antibody composition of any one of embodiments 117-134, wherein the CHO cells are cultured in the presence of glucose in an amount of about 175 g / L or less.

[0789] 136. The antibody composition of embodiment 135, wherein the CHO cells are cultured in the presence of glucose in an amount from about 100 g / L to about 175 g / L.

[0790] 136A. The antibody composition of embodiment 136, wherein the CHO cells are cultured in the presence of glucose in an amount of about 160.4 g / L. 137. The antibody composition of embodiment 136, wherein the CHO cells are cultured in the presence of glucose in an amount of about 137.5 g / mL.

[0791] 138. The antibody composition of any one of embodiments 117-137, wherein the CHO cells are cultured in the presence of calcium in an amount greater than 1 .2 mM.

[0792] 138A. The antibody composition of embodiment 138, wherein the CHO cells are cultured in the presence of 9.1 mM calcium.

[0793] 139. The antibody composition of embodiment 138, wherein the CHO cells are cultured in the presence of 2-3 mM calcium.

[0794] 140. The antibody composition of embodiment 139, wherein the CHO cells are cultured in the presence of about 2.4 mM calcium.

[0795] 141. A method of producing an antibody composition, the method comprising:

[0796] (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise:

[0797] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and

[0798] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0799] (b) culturing the CHO cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L-fucose, dexamethasone, and / or insulin; and

[0800] (c) isolating the antibodies to obtain the antibody composition.

[0801] 142. The method of embodiment 141 , wherein less than 5% of N-glycans in the antibody composition are afucosylated.

[0802] 143. The method of embodiment 141 or 142, wherein the conditions comprise culture at pH 6.95 or higher.

[0803] 143A. The method of any one of embodiments 141-143, wherein the conditions comprise culture at pH 7.1 or lower.

[0804] 144. The method of embodiment 143, wherein the conditions comprise culture at pH 7.1 or higher. 145. The method of any one of embodiments 141-144, wherein the conditions comprise culture in the presence of putrescine.

[0805] 146. The method of embodiment 145, wherein the putrescine is in an amount from about 0.5 to about 1 .0 mg / mL.

[0806] 147. The method of any one of embodiments 141-146, wherein the conditions comprise culture in the presence of fucose.

[0807] 148. The method of embodiment 147, wherein the fucose is in an amount from about 2.5 mg / mL to about 5.0 mg / mL.

[0808] 149. The method of embodiment 148, wherein the fucose is in an amount of about 5.0 mg / mL.

[0809] 150. The method of any one of embodiments 141-149, wherein the conditions comprise culture in the presence of insulin.

[0810] 151 . The method of embodiment 150, wherein the insulin is in an amount from about 1 ug / mL to about 2 ug / mL.

[0811] 152. The method of any one of embodiments 141-151 , wherein the conditions comprise culture in the presence of dexamethasone.

[0812] 153. The method of embodiment 152, wherein the dexamethasone is in an amount from about 10 uM to about 20 uM.

[0813] 154. A method of producing an antibody composition, the method comprising:

[0814] (a) providing CHO cells comprising:

[0815] (i) Fut8, Man2a1 , or Mgat2, and

[0816] (ii) nucleic acid encoding antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0817] (b) culturing the CHO cells under conditions which permit expression of the antibodies; and

[0818] (c) isolating the antibodies to obtain the antibody composition.

[0819] 155. A method of producing the antibody composition of any one of embodiments 1-63, the method comprising: (a) providing CHO cells comprising nucleic acid encoding antibodies, wherein the antibodies comprise:

[0820] (i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and

[0821] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;

[0822] (b) culturing the CHO cells under conditions which permit expression of the antibodies, and

[0823] (c) isolating the antibodies to obtain the antibody composition.

[0824] 156. The method of embodiment 154 or 155, wherein the CHO cells express Fut8, Man2a1 , or Mgat2.

[0825] 157. The method of any one of embodiments 141-156, wherein the conditions which permit expression of the antibodies comprise culturing the CHO cells in the presence of putrescine, L-fucose, dexamethasone, and / or insulin.

[0826] 158. The method of any one of embodiments 141-157, wherein the CHO cells are cultured in the presence of putrescine in an amount of about 1 g / L or less.

[0827] 159. The method of embodiment 158, wherein the CHO cells are cultured in the presence of putrescene in an amount of about 1 g / L.

[0828] 160. The method of any one of embodiments 141-159, wherein the CHO cells are cultured in the presence of L-fucose in an amount of about 5 g / L or less.

[0829] 161 . The method of embodiment 160, wherein the CHO cells are cultured in the presence of L-fucose in an amount of about 5 g / L.

[0830] 162. The method of any one of embodiments 141-161 , wherein the CHO cells are cultured in the presence of dexamethasone in an amount of about 20 pM or less.

[0831] 163. The method of embodiment 162, wherein the CHO cells are cultured in the presence of dexamethasone in an amount of about 20 pM.

[0832] 164. The method of any one of embodiments 141-163, wherein the CHO cells are cultured in the presence of insulin in an amount of about 2 mg / mL or less.

[0833] 165. The method of embodiment 164, wherein the CHO cells are cultured in the presence of insulin in an amount of about 2 mg / mL. 166. The method of any one of embodiments 141-165, wherein the CHO cells are cultured in the presence of galactose in an amount of about 15 g / L or less.

[0834] 167. The method of embodiment 166, wherein the CHO cells are cultured in the presence of galactose in an amount of about 15 g / L.

[0835] 168. The method of any one of embodiments 141-167, wherein the CHO cells are cultured in the presence of manganese in an amount of about 50 pM or less.

[0836] 169. The method of embodiment 168, wherein the CHO cells are cultured in the presence of manganese in an amount from about 1 .5 pM to about 50 pM.

[0837] 169A. The method of embodiment 169, wherein the CHO cells are cultured in the presence of manganese in an amount of 25.8 pM.

[0838] 170. The method of embodiment 169, wherein the CHO cells are cultured in the presence of manganese in an amount of about 25.75 pM.

[0839] 171. The method of any one of embodiments 141-170, wherein the CHO cells are cultured in the presence of glucose in an amount of about 175 g / L or less.

[0840] 172. The method of embodiment 171 , wherein the CHO cells are cultured in the presence of glucose in an amount from about 100 g / L to about 175 g / L.

[0841] 172A. The method of embodiment 172, wherein the CHO cells are cultured in the presence of glucose in an amount of about 160.4 g / L.

[0842] 173. The method of embodiment 172, wherein the CHO cells are cultured in the presence of glucose in an amount of about 137.5 g / mL.

[0843] 174. The method of any one of embodiments 141-173, wherein the CHO cells are cultured in the presence of calcium in an amount greater than 1 .2 mM.

[0844] 174A. The method of embodiment 174, wherein the CHO cells are cultured in the presence of calcium in an amount of 9.1 mM. 175. The method of embodiment 174, wherein the CHO cells are cultured in the presence of 2-3 mM calcium.

[0845] 176. The method of embodiment 175, wherein the CHO cells are cultured in the presence of about 2.4 mM calcium.

[0846] 177. The method of any one of embodiments 141-176, further comprising preparing a pharmaceutical formulation by adding a pharmaceutically acceptable carrier.

[0847] 178. The method of embodiment 177, wherein the pharmaceutical formulation comprises arginine, sodium acetate, sodium chloride, EDTA, and / or polysorbate.

[0848] 179. The method of embodiment 178, wherein the pharmaceutical formulation comprises 1% (w / v) arginine, 50 mM sodium acetate, 51 mM sodium chloride, 0.05 mM EDTA, and 0.02% (w / v) polysorbate 80.

[0849] 180. The method of any one of embodiments 177-179, wherein the antibodies are present in the pharmaceutical formulation in an amount of about 50 mg / mL or about 90 mg / mL.

[0850] 181. The method of any one of embodiments 177-180, wherein the pharmaceutical formulation is at a pH from 5.2-5.8.

[0851] 182. The method of any one of embodiments 177-181 , further comprising preparing a pharmaceutical product comprising a container by dispensing the pharmaceutical formulation into the container.

[0852] 183. The method of embodiment 182, wherein the container is a prefilled syringe or an autoinjector.

[0853] 184. A method of treating multiple sclerosis, the method comprising administering an effective amount of an antibody composition to a patient in need thereof, wherein the antibody composition comprises anti-CD20 antibodies comprising:

[0854] (a) a light chain comprising the amino acid sequence of SEQ ID NO: 1 ; and

[0855] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.

[0856] 184A. A pharmaceutical composition for use in a method of treating multiple sclerosis, comprising an antibody composition, wherein the method comprises administering an effective amount of antibody composition to a patient in need thereof, wherein the antibody composition comprises anti-CD20 antibodies comprising:

[0857] (a) a light chain comprising the amino acid sequence of SEQ ID NO: 1 ; and

[0858] (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.

[0859] 185. The method of embodiments 184 or the pharmaceutical composition of embodiment 184A, wherein the antibody composition:

[0860] (a) is the antibody composition of any one of embodiments 1-63 or 117-140;

[0861] (b) is the antibody composition made by the method of any one of embodiments 141-184;

[0862] (c) comprises the CHO cell-produced antibody of any one of embodiments 74-76; or

[0863] (d) is the pharmaceutical formulation of any one of 64-71.

[0864] 186. The method or pharmaceutical composition of any one of embodiments 184-185, wherein the antibody composition is administered to the patient once per month, once every two months, once every three months, once every four months, once every six months, once every eight months, or once yearly.

[0865] 187. The method or pharmaceutical composition of embodiment 186, wherein the antibody composition is administered to the patient once per month.

[0866] 188. The method or pharmaceutical composition of embodiment 187, wherein the antibody composition administered to the patient once per month comprises about 20 mg of the anti-CD20 antibodies.

[0867] 189. The method or pharmaceutical composition of embodiment 193, wherein the antibody composition is administered to the patient once every two months.

[0868] 190. The method or pharmaceutical composition of embodiment 196, wherein the CHO cell-produced antibody composition administered to the patient once every two months comprises about 135 mg of the anti-CD20 antibodies.

[0869] 191. The method or pharmaceutical composition of any one of embodiments 184-190, wherein the CHO cell-produced antibody composition is administered by subcutaneous injection.

[0870] 192. The method or pharmaceutical composition of any one of embodiments 184-191 , wherein the multiple sclerosis is relapsing multiple sclerosis (RMS). 193. The method or pharmaceutical composition of any one of embodiments 184-192, wherein the multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS), or clinically isolated syndrome (CIS).

Claims

CLAIMS1 . A composition comprising antibodies, wherein the antibodies each comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the antibodies in the composition comprise an Fc region comprising an N-glycan, wherein less than 10% of the N-glycans in the composition are afucosylated, and wherein:(a) less than 1 % of the N-glycans in the composition are N-glycolyl neuraminic acid (NGNA) glycans;(b) at least 0.01 % of the heavy chains comprise C-terminal amidation; and / or(c) less than 15% of the heavy chains comprise a C-terminal lysine.

2. The composition of claim 1 , wherein the antibodies in the composition are devoid of N-glycolyl neuraminic acid (NGNA).

3. The composition of claim 1 or claim 2, wherein the antibodies have:(a) a relative antibody dependent cytotoxicity (ADCC) potency within 30% of a reference murine cell-produced antibody;(b) a relative complement dependent cytotoxicity (CDC) potency within 30% of a reference murine cell-produced antibody;(c) a relative antibody dependent cellular phagocytosis (ADCP) potency within 30% of a reference murine cell-produced antibody; and / or(d) a relative C1q binding potency within 10% of a reference murine cell-produced antibody.

4. The composition of claim 3, wherein the reference murine cell-produced antibody is an NsO cell- produced antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2.

5. The composition of any one of the preceding claims, wherein 6% or fewer of the N-glycans in the composition are afucosylated.

6. The composition of any one of the preceding claims, wherein 1-6% of the N-glycans in the composition are afucosylated.

7. The composition of any one of the preceding claims, wherein no more than 4.5% of the N-glycans in the composition are afucosylated.

8. The composition of claim 5, wherein:(a) 0.9% to 4.5% of the N-glycans in the composition are afucosylated;(b) 0.9% to 3.4% of the N-glycans in the composition are afucosylated;(c) 0.9% to 1 .3% of the N-glycans in the composition are afucosylated;(d) 2.7% to 3.4% of the N-glycans in the composition are afucosylated; and / or(e) 3.4% to 4.5% of the N-glycans in the composition are afucosylated.

9. The composition of any one of the preceding claims, wherein no more than 10.3% of the N-glycans in the composition are high mannose glycans.

10. The composition of any one of the preceding claims, wherein:(a) 1 .8% to 10.3% of the N-glycans in the composition are high mannose glycans;(b) 5.8% to 10.3% of the N-glycans in the composition are high mannose glycans;(c) 5.8% to 9.3% of the N-glycans in the composition are high mannose glycans; and / or(d) 6.9% to 10.3% of the N-glycans in the composition are high mannose glycans.11 . The composition of any one of the preceding claims, wherein no more than 55% of the N-glycans in the composition are galactosylated.

12. The composition of any one of the preceding claims, wherein:(a) 23% to 55% of the N-glycans in the composition are galactosylated;(b) 38% to 55% of the N-glycans in the composition are galactosylated;(c) 23% to 41 % of the N-glycans in the composition are galactosylated; and / or(d) 29% to 45% of the N-glycans in the composition are galactosylated.

13. The composition of any one of the preceding claims, wherein at least 1 % of the N-glycans in the composition are terminally sialylated.

14. The composition of any one of the preceding claims, wherein:(a) less than about 57% of the N-glycans in the composition are G0F;(b) more than about 18% of the N-glycans in the composition are G1 F; and / or(c) more than about 1 .7% of the N-glycans in the composition are G2F.

15. The composition of any one of the preceding claims, wherein:(a) 32-49% of the N-glycans in the composition are G0F;(b) 28-35% of the N-glycans in the composition are G1 F;(c) 6-13% of the N-glycans in the composition are G2F; and / or(d) less than 6% of the N-glycans in the composition are mannose-5.

16. A composition comprising antibodies produced by a process comprising:(a) providing non-murine cells comprising nucleic acids encoding the antibodies, wherein the antibodies comprise:(i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 ; and(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;(b) culturing the non-murine cells under conditions which permit expression of the antibodies, wherein the conditions comprise culturing the non-murine cells in the presence of putrescine, L-fucose, dexamethasone, insulin, glucose, galactose, manganese and / or calcium; and(c) isolating the antibodies to obtain the composition.

17. A composition comprising antibodies produced by a process comprising:(a) providing non-murine cells comprising(i) one or more transgenes encoding a fucosyltransferase, a glycoside hydrolase, and / or a GIcNAc transferase; and(ii) nucleic acids encoding the antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;(b) culturing the non-murine cells under conditions which permit expression of the antibodies; and(c) isolating the antibodies to obtain the composition.

18. The composition of claim 17, wherein the non-murine cells comprise a transgene encoding a fucosyltransferase, and wherein the expression of the fucosyltransferase is under the control of a weak promoter, optionally wherein the weak promoter is a ubiquitin C (UBC) promoter.

19. The composition of claim 17, wherein the non-murine cells comprise transgenes encoding a fucosyltransferase, a glycoside hydrolase and a GIcNAc transferase.

20. The composition of any one of claims 17-19, wherein:(a) the fucosyltransferase is Fut8;(b) the glycoside hydrolase is Man2a1 ; and / or(c) the GIcNAc transferase is Mgat2.

21. The composition of any one of claims 17-20, wherein the conditions which permit expression of the antibodies comprise culture in the presence of putrescine, L-fucose, dexamethasone, insulin, glucose, galactose, manganese and / or calcium.

22. The composition of any one of claims 16-21 , wherein the conditions which permit expression of the antibodies comprise culture in the presence of putrescine, L-fucose, dexamethasone, and / or insulin.

23. The composition of any one of claims 16-21 , wherein the conditions which permit the expression of the antibodies comprise culture in the presence of:(a) putrescine, L-fucose, dexamethasone, insulin, galactose, manganese and / or glucose; or(b) galactose, manganese, glucose and / or calcium.

24. The composition of any one of claims 16-23, wherein the conditions comprise culture at a pH of about6.9 or higher, optionally a pH of about 6.9 to about 7.1 , further optionally:(a) a pH of 6.95; or(b) a pH of 7.05.

25. The composition of any one of claims 16-24, wherein the conditions comprise culture in the presence of:(a) putrescine in an amount of about 1 g / L or less, optionally in an amount of about 1 g / L;(b) L-fucose in an amount of about 5 g / L or less, optionally in an amount of about 5 g / L;(c) dexamethasone in an amount of about 20 pM or less, optionally in an amount of about 20 pM;(d) insulin in an amount of about 2 mg / L or less, optionally in an amount of about 2 mg / L;(e) galactose in an amount of about 15 g / L or less, optionally in an amount of about 15 g / L;(f) manganese in an amount of about 50 pM or less, optionally in an amount from about 1 .5 pM to about 50 pM, further optionally in amount of about 25.75 pM;(g) glucose in an amount of about 175 g / L or less, optionally in an amount from about 100 g / L to about 175 g / L, further optionally in an amount of about 137.5 g / L; and / or(h) calcium in an amount greater than 1.2 mM, optionally in an amount of 2-3 mM calcium, further optionally in an amount of about 2.4 mM.

26. The composition of any one of claims 16-25, wherein the non-murine cells are immortalized cells derived from the epithelial cells of the ovary of the Chinese hamster.

27. The composition of any one of claims 16-25, wherein the non-murine cells are CHO cells, optionally recombinant CHO cells.

28. The composition of any one of claims 1-27, wherein the antibodies:(a) have a mean binding affinity (KD) to Fc gamma receptor (FcyR) la from 8 to 11 nM;(b) have a mean KD to FcyRllaHRfrom 15 to 20 pM;(c) have a mean KD to FcyRllaLRfrom 2 to 7 pM;(d) have a mean KD to FcyRllb from 26 to 37 pM;(e) have a mean Ko to FcyRlllb from 7 to 12 pM;(f) have a mean KD to FcyRI I laF158from 2 to 7 pM;(g) have a mean Ko to FcyRlllaV158from 0.1 to 0.6 pM; and / or(h) have a mean KD at pH 6.0 to FcRn from 0.3 to 0.8 pM.

29. A non-murine cell expressing the antibodies of the composition of any one of claims 1-28.

30. The non-murine cell of claim 29, wherein the non-murine cell comprises one or more transgenes encoding a fucosyltransferase, a glycoside hydrolase, and / or a GIcNAc transferase.

31. The non-murine cell of claim 30, wherein the non-murine cell comprises a transgene encoding a fucosyltransferase, wherein the expression of the fucosyltransferase is under the control of a weak promoter, optionally wherein the weak promoter is a ubiquitin C (UBC) promoter.

32. The non-murine cell of claim 31 , wherein the mRNA transcripts for the antibodies exceed the number of mRNA transcripts for the fucosyltransferase, optionally by a factor of at least two or by a factor of at least four.

33. The non-murine cell of claim 30, wherein the non-murine cell comprises transgenes encoding a fucosyltransferase, a glycoside hydrolase, and a GIcNAc transferase.

34. The non-murine cell of any one of claims 30-33, wherein:(a) the fucosyltransferase is Fut8;(b) the glycoside hydrolase is Man2a1 ; and / or(c) the GIcNAc transferase is Mgat2.

35. The non-murine cell of any one of claims 29-34, wherein the non-murine cells are immortalized cells derived from the epithelial cells of the ovary of the Chinese hamster.

36. The non-murine cell of any one of claims 29-34, wherein the non-murine cells are CHO cells, optionally recombinant CHO cells.

37. A cell culture comprising the non-murine cell of any one of claims 29-36.

38. The cell culture of claim 37, wherein the cell culture further comprises putrescine, L-fucose, dexamethasone, insulin, galactose, manganese, glucose, and / or calcium.

39. The cell culture of claim 38, wherein the cell culture further comprises putrescine, L-fucose, dexamethasone, and / or insulin.

40. The cell culture of claim 38, wherein the cell culture further comprises:(a) putrescine, L-fucose, dexamethasone, insulin, galactose, manganese and / or glucose; or(b) galactose, manganese, glucose and / or calcium.

41. The cell culture of any one of claims 37-40, wherein the cell culture has a pH of about 6.9 or higher, optionally wherein the cell culture has a pH of about 6.9 to about 7.1 , further optionally wherein:(a) the cell culture has a pH of 6.95; or(b) the cell culture has a pH of 7.05.

42. The cell culture of any one of claims 37-41 , wherein the cell culture comprises:(a) putrescine in an amount of about 1 g / L or less, optionally in an amount of about 1 g / L;(b) L-fucose in an amount of about 5 g / L or less, optionally in an amount of about 5 g / L;(c) dexamethasone in an amount of about 20 pM or less, optionally in an amount of about 20 pM;(d) insulin in an amount of about 2 mg / L or less, optionally in an amount of about 2 mg / L;(e) galactose in an amount of about 15 g / L or less, optionally in an amount of about 15 g / L;(f) manganese in an amount of about 50 pM or less, optionally in an amount from about 1 .5 pM to about 50 pM, further optionally in amount of about 25.75 pM;(g) glucose in an amount of about 175 g / L or less, optionally in an amount from about 100 g / L to about 175 g / L, further optionally in an amount of about 137.5 g / L; and / or(h) calcium in an amount greater than 1.2 mM, optionally in an amount of 2-3 mM calcium, further optionally in an amount of about 2.4 mM.

43. A method of producing a composition comprising antibodies, the method comprising:(a) providing non-murine cells comprising nucleic acids encoding the antibodies, wherein the antibodies comprise:(i) a light chain comprising the amino acid sequence of SEQ ID NO: 1 , and(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;(b) culturing the non-murine cells under conditions which permit expression of the antibodies, wherein the conditions comprise culture in the presence of putrescine, L-fucose, dexamethasone, insulin, glucose, galactose, manganese and / or calcium, and(c) isolating the antibodies to obtain the composition.

44. A method of producing a composition comprising antibodies, comprising,(a) providing non-murine cells comprising:(i) one or more transgenes encoding a fucosyltransferase, a glycoside hydrolase, and / or a GIcNAc transferase; and(ii) nucleic acids encoding the antibodies, wherein the antibodies comprise a light chain comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2;(b) culturing the non-murine cells under conditions which permit expression of the antibodies; and(c) isolating the antibodies to obtain the composition.

45. The method of claim 44, wherein the non-murine cells comprise a transgene encoding a fucosyltransferase, wherein the expression of the fucosyltransferase is under the control of a weak promoter, optionally wherein the weak promoter is a ubiquitin C (UBC) promoter.

46. The method of claim 44, wherein the non-murine cells comprise transgenes encoding a fucosyltransferase, a glycoside hydrolase and a GIcNAc transferase.

47. The method of any one of claims 44-46, wherein:(a) the fucosyltransferase is Fut8;(b) the glycoside hydrolase is Man2a1 ; and / or(c) the GIcNAc transferase is Mgat2.

48. The method of any one of claims 44-47, wherein the conditions which permit expression of the antibodies comprise culture in the presence of putrescine, L-fucose, dexamethasone, insulin, glucose, galactose, manganese and / or calcium.

49. The method of any one of claims 43-48, wherein the conditions which permit expression of the antibodies comprise culture in the presence of putrescine, L-fucose, dexamethasone, and / or insulin.

50. The method of any one of claims 43-48, wherein the conditions which permit expression of the antibodies comprise culture in the presence of:(a) putrescine, L-fucose, dexamethasone, insulin, galactose, manganese and / or glucose; or(b) galactose, manganese, glucose and / or calcium.

51. The method of any one of claims 43-50, wherein the conditions which permit expression of the antibodies comprise culture at a pH of about 6.9 or higher, optionally a pH of about 6.9 to about 7.1 , further optionally wherein:(a) a pH of 6.95; or(b) a pH of 7.05.

52. The method of any one of claims 43-51 , wherein the conditions comprise culture in the presence of:(a) putrescine in an amount of about 1 g / L or less, optionally in an amount of about 1 g / L;(b) L-fucose in an amount of about 5 g / L or less, optionally in an amount of about 5 g / L;(c) dexamethasone in an amount of about 20 pM or less, optionally in an amount of about 20 pM;(d) insulin in an amount of about 2 mg / L or less, optionally in an amount of about 2 mg / L;(e) galactose in an amount of about 15 g / L or less, optionally in an amount of about 15 g / L;(f) manganese in an amount of about 50 pM or less, optionally in an amount from about 1 .5 pM to about 50 pM, further optionally in amount of about 25.75 pM;(g) glucose in an amount of about 175 g / L or less, optionally in an amount from about 100 g / L to about 175 g / L, further optionally in an amount of about 137.5 g / L; and / or(h) calcium in an amount greater than 1.2 mM, optionally in an amount of 2-3 mM calcium, further optionally in an amount of about 2.4 mM.

53. A pre-filled syringe or autoinjector comprising the composition of any one of claims 1-28, antibodies expressed by the non-murine cell of any one of claims 29-36, antibodies expressed in the cell culture of any one of claims 37-42, or a composition produced by the method of any one of claims 43-52.

54. A method of treating multiple sclerosis in a subject in need thereof, comprising administering to the subject the composition of any one of claims 1-28, antibodies expressed by the non-murine cell of any one of claims 29-36, antibodies expressed in the cell culture of any one of claims 37-42, or a composition produced by the method of any one of claims 43-52.

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