Anti-CD20 antibody compositions

Abstract

Provided herein are populations of anti-CD20 antibody proteins having a specified post-translational modification range. Methods of use and methods of manufacture of such anti-CD20 antibody protein populations are also provided.

Description

[0001] 1. Priority declaration

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 347,852, filed on June 1, 2022, U.S. Provisional Application Serial No. 63 / 421,078, filed on October 31, 2022, and U.S. Provisional Application Serial No. 63 / 445,082, filed on February 13, 2023. The entire contents of the foregoing are incorporated by reference.

[0003] 2. Citation of a sequence listing submitted electronically

[0004] This application contains a sequence listing, which has been submitted electronically as an XML file named "50581-0004WO1.XML". This XML file was created on May 22, 2023 and is 39,114 bytes in size. The data in the XML file is incorporated by reference in its entirety. 3. Technical field

[0005] The present disclosure is in the field of recombinant anti-CD20 antibodies, methods of producing the antibodies, and uses of the antibodies. 4. Background Technology

[0006] The therapeutic monoclonal antibodies (mAbs) produced in mammalian cells have heterogeneity due to post-translational modifications (PTMs). PTMs can occur during mAb production, purification, storage, and administration. PTMs are therapeutic mAb product quality attributes (PQAs). Controlling PQAs within predefined acceptance criteria is critical for the biopharmaceutical industry because it ensures consistent product quality and reduces potential impacts on drug safety and efficacy (Xu, X. et al., Journal of Applied Bioanalysis 3(2):21-5(2017)).

[0007] The critical importance of antibody sequence variation is well recognized. Sequence diversity in antibody variable domains is essential for specific antigen recognition, while association with different constant domains results in different Fc-mediated effector activities. PTMs in these domains provide additional immune mechanisms by which antibody binding and activity can be modulated. PTMs vary from chain additions such as N- and O-linked glycosylation, glycation, cysteineation, and sulfation; chain trimming such as C-terminal lysine clipping; amino acid modifications such as cyclization (to N-terminal pyroglutamate), deamidation, oxidation, isomerization, and carbamylation; and disulfide scrambling of interchain disulfide bonds in the hinge region. As a result, each antibody can generate a large number of different antibody molecules with widely varying activities and potencies. Although post-translational modifications of antibodies have been observed and studied for decades, the full impact of microheterogeneity remains to be further investigated. PTMs can affect antibody functions, such as pharmacokinetic and pharmacodynamic properties, as well as clinical efficacy.

[0008] For a mAb population to have consistent product quality, clinical safety, and efficacy, having a defined range of post-translational modifications can be critical. 5. Summary of the invention

[0009] Provided herein are compositions comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising about 20 to 40% fucosylated glycans and optionally about 10 to 20% galactosylated glycans.

[0010] In some embodiments, the N-glycan profile comprises 23% to 36% fucosylated glycans, optionally about 30% fucosylated glycans. In some embodiments, the N-glycan profile comprises 16% to 18% galactosylated glycans, optionally about 17% galactosylated glycans.

[0011] In some embodiments, the relative abundance of the fucosylated glycans is the percentage of the fucosylated glycans among all glycans in the N-glycan profile. In some embodiments, the relative abundance of the galactosylated glycans is the percentage of the galactosylated glycans among all glycans in the N-glycan profile.

[0012] In some embodiments, the N-glycan profile comprises 12% to 30% bisecting N-glycans, optionally about 18% bisecting N-glycans. In some embodiments, the bisecting N-glycans comprise one or more of G0B, G0FB, G1FB, G2FBS1, and G2FBS2. In some embodiments, the anti-CD20 antibody protein population has an N-glycan profile comprising less than 5% sialylated glycans. In some embodiments, the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1%, or 0.5% sialylated glycans. In some embodiments, the N-glycan profile comprises no detectable amount of sialylated glycans.

[0013] In some embodiments, the anti-CD20 antibody protein population has an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans. In some embodiments, the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans. In some embodiments, the N-glycan profile comprises about 0.6% Man5 N-glycans. In some embodiments, the Man5 N-glycans are the only high mannose species in the N-glycan profile.

[0014] In some embodiments, the anti-CD20 antibody protein population comprises 0.20 to 0.40 mol isoaspartic acid / mol protein. In some embodiments, the anti-CD20 antibody protein population comprises 0.25 to 0.35 mol isoaspartic acid / mol protein.

[0015] In some embodiments, the glutamic acid at position 1 of the heavy chain is pyroglutamic acid and the glutamic acid at position 1 of the light chain is pyroglutamic acid.

[0016] In some embodiments, the anti-CD20 antibody protein population has an N-glycan profile comprising G1 and G0 N-glycans at a relative abundance ratio of 0.1 to 0.15. In some embodiments, the anti-CD20 antibody protein population has an N-glycan profile comprising G1F and G1 N-glycans at a relative abundance ratio of 0.5 to 0.9.

[0017] In some embodiments, the anti-CD20 antibody protein population further comprises at least two N-glycans within the following relative abundance ranges:

[0018] (a) 0.3% to 2% GO-GN;

[0019] (b) 0.1% to 2% GOF-GN;

[0020] (c) 30% to 60% G0;

[0021] (d) 0.1% to 1% G1-GN;

[0022] (e) 5% to 20% GOB;

[0023] (f) 5% to 30% GOF;

[0024] (g) 0.1% to 1.5% Man5;

[0025] (h) 1% to 15% GOFB;

[0026] (i) 1% to 13% G1;

[0027] (j) 0.5% to 10% G1';

[0028] (k) 0.5% to 6% G1B;

[0029] (l) 0.5% to 12% G1F;

[0030] (m) 0.1% to 3% G1F';

[0031] (n) 0.1% to 3% G1FB;

[0032] (o) 0.1% to 2% G2; and

[0033] (p) 0.1% to 2% G2F.

[0034] In some embodiments, the anti-CD20 antibody protein population further comprises at least two N-glycans within the following relative abundance ranges:

[0035] (a) 0.8% to 1.1% GO-GN;

[0036] (b) 0.5% to 1.1% GOF-GN;

[0037] (c) 42.5% to 48.8% G0;

[0038] (d) 0.3% to 0.6% G1-GN;

[0039] (e) 9.5% to 14.1% GOB;

[0040] (f) 12.8% to 19.7% GOF;

[0041] (g) 0.4% to 0.7% Man5;

[0042] (h) 5.1% to 7.0% GOFB;

[0043] (i) 5.7% to 6.4% G1;

[0044] (j) 2.7% to 3.3% G1';

[0045] (k) 1.4% to 2.0% G1B;

[0046] (l) 2.6% to 4.2% G1F;

[0047] (m) 1.1% to 1.6% G1F';

[0048] (n) 1.1% to 1.8% G1FB;

[0049] (o) 0.5% to 0.7% G2; and

[0050] (p) 0.3% to 0.5% G2F.

[0051] In some embodiments, the anti-CD20 antibody protein population further comprises at least two N-glycans within the following relative abundance ranges:

[0052] (a) 0.9% G0-GN;

[0053] (b) 0.8% GOF-GN;

[0054] (c) 46.1% G0;

[0055] (d) 0.5% G1-GN;

[0056] (e) 10.9% G0B;

[0057] (f) 17.0% GOF;

[0058] (g) 0.6% Man5;

[0059] (h) 6.0% GOFB;

[0060] (i) 6.1% G1;

[0061] (j) 2.9% G1′;

[0062] (k) 1.6% G1B;

[0063] (l) 3.2% G1F;

[0064] (m) 1.3% G1F';

[0065] (n)1.3G1FB;

[0066] (o) 0.5% G2; and

[0067] (p)0.3% G2F.

[0068] In some embodiments, the anti-CD20 antibody protein population further comprises at least three, four, or five N-glycans within the following relative abundance ranges:

[0069] (a) 0.3% to 2% GO-GN;

[0070] (b) 0.1% to 2% GOF-GN;

[0071] (c) 30% to 60% G0;

[0072] (d) 0.1% to 1% G1-GN;

[0073] (e) 5% to 20% GOB;

[0074] (f) 5% to 30% GOF;

[0075] (g) 0.1% to 1.5% Man5;

[0076] (h) 1% to 15% GOFB;

[0077] (i) 1% to 13% G1;

[0078] (j) 0.5% to 10% G1';

[0079] (k) 0.5% to 6% G1B;

[0080] (l) 0.5% to 12% G1F;

[0081] (m) 0.1% to 3% G1F';

[0082] (n) 0.1% to 3% G1FB;

[0083] (o) 0.1% to 2% G2; and

[0084] (p) 0.1% to 2% G2F.

[0085] In some embodiments, the N-glycan profile of the anti-CD20 antibody protein population is determined using a method comprising: (a) incubating the anti-CD20 antibody protein population with an enzyme, wherein the enzyme catalyzes the release of N-glycans from anti-CD20 antibodies; (b) measuring the relevant abundance of the released N-glycans using one or more methods selected from chromatography, mass spectrometry, capillary electrophoresis, and combinations thereof. In some embodiments, the method further comprises the following steps after step (a) and before step (b): (c) purifying the N-glycans; and (d) labeling the N-glycans with a fluorescent compound. In some embodiments, the enzyme is PNGase F. In some embodiments, the fluorescent compound is 2-aminobenzamide (2-AB).

[0086] In some embodiments, less than 10% of the anti-CD20 antibody proteins in the population are non-glycosylated. In some embodiments, less than 5% of the anti-CD20 antibody proteins in the population are non-glycosylated. In some embodiments, less than 1% of the anti-CD20 antibody proteins in the population are non-glycosylated.

[0087] In some embodiments, the anti-CD20 antibody protein population comprises two or more secondary structures as determined by circular dichroism at 205 nm to 260 nm as follows:

[0088] (a) 8.0% to 10.0% α-helix;

[0089] (b) 32.0% to 36.0% antiparallel β-sheets;

[0090] (c) 5.0% to 6.0% parallel β-sheets;

[0091] (d) 16.0% to 18.0% β-turn; and

[0092] (e) 35.0% to 36.0% random curl.

[0093] In some embodiments, the anti-CD20 antibody protein population comprises the following secondary structure as determined by circular dichroism at 205 nm to 260 nm:

[0094] (a) 8.0% to 10.0% α-helix;

[0095] (b) 32.0% to 36.0% antiparallel β-sheets;

[0096] (c) 5.0% to 6.0% parallel β-sheets;

[0097] (d) 16.0% to 18.0% β-turn; and

[0098] (e) 35.0% to 36.0% random curl.

[0099] In some embodiments, the anti-CD20 antibody protein population comprises two or more secondary structures as determined by circular dichroism at 205 nm to 260 nm as follows:

[0100] (a) about 9.0% α-helix;

[0101] (b) about 33.0% antiparallel β-sheets;

[0102] (c) about 5.6% parallel β-sheets;

[0103] (d) about 17.5% β-turn; and

[0104] (e) About 35.2% random coil.

[0105] In some embodiments, the anti-CD20 antibody protein population further comprises one or more of the following post-translational modifications at a specified abundance:

[0106]

[0107]

[0108] In some embodiments, one or more of the post-translational modifications are measured by peptide mapping using liquid chromatography-mass spectrometry (LC-MS).

[0109] In some embodiments, the population has a total protein amount of 25.5-25.8 mg / mL as measured by absorbance at 280 nm.

[0110] In some embodiments, the anti-CD20 antibody proteins in the population induce greater cytotoxicity in a cell-based antibody-dependent cellular cytotoxicity (ADCC) assay compared to obinutuzumab, ofatumumab, rituximab, veltuzumab, ibritumomab tiuxetan, and / or ocrelizumab.

[0111] In some embodiments, the population has a relative potency of 90% to 163% in a cell-based ADCC assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of 78% to 116% or 73% to 128% in a cell-based complement dependent cytotoxicity (CDC) assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of 92% to 118% or 82% to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some embodiments, the population has a K of 30 to 70 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance. D In some embodiments, the population has a K of 500 to 1000 nM in an FcγRIIIa 158F binding assay as measured by surface plasmon resonance. D In some embodiments, the population has a significantly higher binding affinity for FcγRIIIa 158V or FcγRIIIa 158F than rituximab. In some embodiments, the population has a relative potency of 88% to 113% or 86% to 116% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In some embodiments, the population has a relative potency of 106% to 126% in a CD16 activity assay compared to a commercial reference standard.

[0112] In some embodiments, the population has 99.2% to 99.9% monomers as detected by size exclusion chromatography (SEC). In some embodiments, the population has 0.1% to 0.8% dimers as detected by SEC. In some embodiments, the population has undetectable levels of aggregates as detected by SEC; and / or the population has undetectable levels of fragments as detected by SEC.

[0113] In some embodiments, the population has 93.6 to 95.9% IgG after purification by non-reducing capillary gel electrophoresis (CGE). In some embodiments, the population has 0.1 to 0.3% high molecular weight species (HMWS) after purification by non-reducing CGE. In some embodiments, the population has 0.7 to 1.2% free light chains (LC) after purification by non-reducing CGE. In some embodiments, the population has 97.7 to 98.0% heavy chain plus light chain species (HC+LC) after purification by reducing CGE.

[0114] In some embodiments, the population has 20% to 25% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF). In some embodiments, the population has 50% to 60% predominant isoforms as detected by iCIEF. In some embodiments, the population has 20% to 30% basic isoforms as detected by iCIEF. In some embodiments, the population has an average molar ratio of free thiols to anti-CD20 antibodies of about 2.0 to 2.2.

[0115] In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of the N-terminal residue. In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of up to 5 N-terminal residues. In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of up to 10 N-terminal residues. In some embodiments, the heavy chain terminal lysine residue of the anti-CD20 antibodies in the population is truncated.

[0116] Also provided herein are pharmaceutical formulations comprising the compositions described herein, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 10 mg / mL to 50 mg / mL. In some embodiments, the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg / mL.

[0117] In some embodiments, the pharmaceutical formulation further comprises one or more of the following: sodium chloride, trisodium citrate dehydrate, polysorbate 80, and hydrochloric acid. In some embodiments, the pharmaceutical formulation comprises about 9.0 mg / mL of sodium chloride, about 7.4 mg / mL of trisodium citrate dehydrate, about 0.7 mg / mL of polysorbate 80, and / or about 0.4 mg / mL of hydrochloric acid.

[0118] In some embodiments, the anti-CD20 antibody is present in a single dosage form.

[0119] Also provided herein is a pharmaceutical formulation comprising:

[0120] (i) a composition as described herein, wherein the composition comprises a single dosage form of the anti-CD20 antibody protein population, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg / mL,

[0121] (ii) about 9.0 mg / mL of sodium chloride,

[0122] (iii) about 7.4 mg / mL of trisodium citrate dehydrate,

[0123] (iv) about 0.7 mg / mL of polysorbate 80, and

[0124] (v) about 0.4 mg / mL of hydrochloric acid.

[0125] Also provided herein is a single batch preparation of a population of anti-CD20 antibody proteins or a pharmaceutical formulation described herein, wherein the single batch comprises at least 100 g, at least 120 g, or at least 150 g of anti-CD20 antibody protein.

[0126] Also provided herein are anti-CD20 antibody protein populations or pharmaceutical preparations described herein, which are produced in a 15,000L or 20,000L bioreactor.

[0127] Also provided herein are methods of treating multiple sclerosis (MS) in a subject in need thereof by administering to the subject in need thereof a therapeutically effective amount of a composition or pharmaceutical formulation described herein.

[0128] In some embodiments, the composition or pharmaceutical formulation is administered as follows: i) a first infusion at a dose of about 150 mg of the anti-CD20 antibody protein, ii) a second infusion two weeks later at a dose of about 450 mg of the anti-CD20 antibody protein, and iii) subsequent infusions every 24 weeks or six months at a dose of about 450 mg of the anti-CD20 antibody protein.

[0129] In some embodiments, administration of the composition or pharmaceutical formulation to a subject results in one or more of the following pharmacokinetic parameters:

[0130] (a) AUC of 2,160 μg / mL to 3,840 μg / mL;

[0131] (b) Cmax of 118,011 ng / mL to 159,989 ng / mL;

[0132] (c) a Cmin of 40 ng / mL to 375 ng / mL; and

[0133] (d) Cavg of 6,437 ng / mL to 11,443 ng / mL.

[0134] In some embodiments, administration of the composition or pharmaceutical formulation to a subject results in one or more of the following pharmacokinetic parameters:

[0135] (a) AUC of approximately 3,000 μg / mL;

[0136] (b) Cmax of approximately 139,000 ng / mL;

[0137] (c) a Cmin of about 139 ng / mL; and

[0138] (d) Cavg of approximately 8,940 ng / mL.

[0139] In some embodiments, the method comprises a treatment period of at least 96 weeks.

[0140] In some embodiments, the subject has been premedicated with a corticosteroid 30 to 60 minutes before administering the composition or pharmaceutical preparation. In some embodiments, the corticosteroid is methylprednisone or dexamethasone. In some embodiments, methylprednisone is administered at a dosage of about 100 mg and / or dexamethasone is administered at a dosage of about 10 to 20 mg.

[0141] In some embodiments, the subject has been premedicated with an antihistamine 30 to 60 minutes prior to administration of the composition or pharmaceutical formulation. In some embodiments, the antihistamine is diphenhydramine HCl. In some embodiments, diphenhydramine HCl is administered at a dose of about 25 to 50 mg.

[0142] In some embodiments, the subject has been premedicated with an antipyretic 30 to 60 minutes prior to administration of the composition or pharmaceutical formulation. In some embodiments, the antipyretic is acetaminophen or an antipyretic bioequivalent thereto.

[0143] In some embodiments, the subject has an Expanded Disability Status Scale (EDSS) score of 0 to 5.5 before treatment.

[0144] Also provided herein are methods of treating multiple sclerosis (MS) in a subject in need thereof, by administering to a subject in need thereof a therapeutically effective amount of a composition or pharmaceutical formulation described herein, wherein administration of the composition or pharmaceutical formulation results in no evidence of disease activity (NEDA) in the subject 24 to 96 weeks after administration.

[0145] In some embodiments, administration of the composition or pharmaceutical formulation results in NEDA in the subject 24 weeks following administration.

[0146] Also provided herein are methods of treating multiple sclerosis (MS) in a subject in need thereof by administering to the subject a therapeutically effective amount of a composition or pharmaceutical formulation described herein, wherein administration of the composition or pharmaceutical formulation results in a temporary decrease in the subject's lymphocyte count.

[0147] In some embodiments, lymphocyte counts are normalized on day 8 of administration.

[0148] In some embodiments, the MS is a relapsing form of MS (RMS).

[0149] Also provided herein is a method of reducing the annualized relapse rate (ARR) of a subject with a relapsing form of multiple sclerosis (MS) by administering to the subject an effective amount of a composition or pharmaceutical formulation described herein, the method comprising: administering an intravenous infusion of the composition or pharmaceutical formulation in a multiple infusion dosing regimen comprising: a) a first infusion comprising 150 mg of anti-CD20 antibody protein on day 1; b) a second infusion comprising 450 mg of anti-CD20 antibody protein about 2 weeks after the first infusion; c) a first subsequent infusion comprising 450 mg of anti-CD20 antibody protein about 24 weeks or about six months from the first infusion; and d) one or more subsequent infusions comprising 450 mg of anti-CD20 antibody protein about 24 weeks or about six months from the previous infusion.

[0150] In some embodiments, the effective amount of the composition or pharmaceutical formulation is sufficient to provide an ARR of 0.091 or an ARR of 0.076.

[0151] In some embodiments, the duration of the second infusion, the first subsequent infusion, and one or more subsequent infusions of the anti-CD20 antibody protein is about one hour.

[0152] Also provided herein is a method of treating a relapsing form of multiple sclerosis (MS) in a subject in need thereof by administering to the subject an effective amount of a composition or pharmaceutical formulation described herein, the method comprising: administering an intravenous infusion of the composition or pharmaceutical formulation in a multiple infusion dosing regimen comprising:

[0153] a) a first infusion on day 1 comprising 150 mg of anti-CD20 antibody protein;

[0154] b) a second infusion containing 450 mg of anti-CD20 antibody protein approximately 2 weeks after the first infusion;

[0155] c) a first subsequent infusion comprising 450 mg of anti-CD20 antibody protein at about 24 weeks or about six months from the first infusion; and

[0156] d) one or more subsequent infusions comprising 450 mg of anti-CD20 antibody protein about 24 weeks or about six months from the previous infusion,

[0157] The duration of the second infusion, the first subsequent infusion, and the one or more subsequent infusions of the anti-CD20 antibody protein is about one hour.

[0158] In some embodiments, the method further includes premedicating the subject with a corticosteroid and an antihistamine 30 to 60 minutes before administering the composition or pharmaceutical preparation. In some embodiments, the corticosteroid is methylprednisone or dexamethasone. In some embodiments, methylprednisone is administered at a dosage of about 100 mg and / or dexamethasone is administered at a dosage of about 10 to 20 mg.

[0159] In some embodiments, the composition or pharmaceutical preparation for intravenous infusion is prepared in 250 mL of 0.9% Sodium Chloride Injection.

[0160] In some embodiments, the first subsequent infusion is about 24 weeks from the first infusion. In some embodiments, one or more subsequent infusions are about 24 weeks from the previous infusion. In some embodiments, the first subsequent infusion is about 6 months from the first infusion. In some embodiments, one or more subsequent infusions are about 6 months from the previous infusion.

[0161] In some embodiments, the duration of the first infusion of anti-CD20 antibody protein is about four hours. In some embodiments, the first infusion of anti-CD20 antibody protein is infused at the following rates: 10 mL / hour for the first 30 minutes; 20 mL / hour for the next 30 minutes; 35 mL / hour for the next hour; and 100 mL / hour for the remaining two hours.

[0162] In some embodiments, the second infusion, the first subsequent infusion, and one or more subsequent infusions of the anti-CD20 antibody protein are infused at a rate of 100 mL / hour for the first 30 minutes and 400 mL / hour for the remaining 30 minutes.

[0163] In some embodiments, a multiple infusion dosing regimen of an anti-CD20 antibody protein reduces or delays progression of MS symptoms.

[0164] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve a reduction in the total number of gadolinium-enhancing T1 lesions per MRI scan compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0165] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve a reduction in the total number of new and enlarging T2 hyperintensive lesions per MRI scan compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0166] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an increase in no evidence of disease activity (NEDA) status compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0167] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an increase in Confirmed Disability Improvement (CDI) compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0168] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an increase in Multiple Sclerosis Functional Composite (MSFC) score compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0169] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an improvement in the Timed 25-Foot Walk (T25FW) score compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0170] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an improvement in 9-Hole Pegtest (9-HPT) scores compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0171] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve a significant reduction in the volume and number of new T1 hypointense lesions per MRI scan compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0172] In some embodiments, the multiple infusion dosing regimen of the anti-CD20 antibody protein results in a geometric mean steady-state AUC of 3000 mcg / mL per day (CV=28%) and a mean maximum concentration of 139 mcg / mL (CV=15%).

[0173] Also provided herein are methods for inactivating viruses or exogenous factors in rat myeloma cells expressing an anti-CD20 antibody protein described in the compositions described herein, wherein the method maintains suitability for antibody production in a 15,000L or 20,000L bioreactor.

[0174] Also provided herein are methods for reducing the immunogenicity of an anti-CD20 antibody protein described in a composition described herein, wherein the method maintains suitability for antibody production in a 15,000L or 20,000L bioreactor.

[0175] Also provided herein is a method for treating multiple sclerosis, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein administration of the pharmaceutical formulation results in no evidence of disease activity (NEDA) in the subject 24 weeks after administration. In some embodiments, administration of the pharmaceutical formulation results in NEDA in the subject 24 to 96 weeks after administration.

[0176] Also disclosed herein is a method for treating multiple sclerosis, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical preparation comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein administration of the pharmaceutical preparation temporarily reduces the lymphocyte count of the subject. In some embodiments, the lymphocyte count is normalized on day 8 of administration.

[0177] In some embodiments of any of the methods described herein, the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans.

[0178] In some embodiments of any of the methods described herein, multiple sclerosis (MS) is a relapsing form of MS (RMS). In certain embodiments, RMS comprises a clinically isolated syndrome ("clinically isolated syndrome, CIS"); relapsing-remitting MS ("relapsing-remitting MS, RRMS"); or active secondary progressive MS ("active secondary progressive MS, SPMS"). In some embodiments of any of the methods described herein, the subject is diagnosed with RMS according to the McDonald Criteria (2010) or by other suitable methods known to those skilled in the art. In some embodiments of any of the methods described herein, the subject is human.

[0179] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10 to 20% galactosylated glycans.

[0180] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 35% fucosylated glycans.

[0181] In some embodiments, the N-glycan profile comprises 28 to 33% fucosylated glycans. In some embodiments, the N-glycan profile comprises about 30% fucosylated glycans.

[0182] In some embodiments, the N-glycan profile comprises 16 to 18% galactosylated glycans. In some embodiments, the N-glycan profile comprises about 17% galactosylated glycans.

[0183] In some embodiments, the relative abundance of fucosylated glycans is the percentage of fucosylated glycans among all glycans of the N-glycan profile. In some embodiments, the relative abundance of galactosylated glycans is the percentage of galactosylated glycans among all glycans of the N-glycan profile.

[0184] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising at least about 10% bisecting N-glycans.

[0185] In some embodiments, the N-glycan profile comprises 12% to 30% bisecting N-glycans. In some embodiments, the N-glycan profile comprises about 18% bisecting N-glycans. In some embodiments, the bisecting N-glycans comprise one or more of G0B, GOFB, G1FB, G2FBS1, and G2FBS2.

[0186] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising less than 5% sialylated glycans.

[0187] In some embodiments, the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1%, or 0.5% sialylated glycans. In some embodiments, the N-glycan profile comprises undetectable amounts of sialylated glycans.

[0188] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising 5 to 15% G0B N-glycans.

[0189] In some embodiments, the N-glycan profile comprises 9 to 11% GOB N-glycans. In some embodiments, the N-glycan profile comprises about 10% GOB N-glycans.

[0190] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans.

[0191] In some embodiments, the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans. In some embodiments, the N-glycan profile comprises about 0.6% Man5 N-glycans. In some embodiments, Man5 N-glycans are the only high mannose species in the N-glycan profile.

[0192] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins comprises 0.20 to 0.40 mol isoaspartic acid / mol protein.

[0193] In some embodiments, the population of anti-CD20 antibody proteins comprises 0.25 to 0.35 mol isoaspartic acid per mol protein.

[0194] Also provided herein are compositions comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the glutamic acid at position 1 of the heavy chain is pyroglutamic acid, and wherein the glutamic acid at position 1 of the light chain is pyroglutamic acid.

[0195] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising G1 to G0 N-glycans in a relative abundance ratio of 0.1 to 0.15.

[0196] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising G1F to G1 N-glycans in a relative abundance ratio of 0.5 to 0.9.

[0197] In some embodiments, the anti-CD20 antibody protein population further comprises at least two N-glycans within the following relative abundance ranges: (a) 0.3% to 2% G0-GN; (b) 0.1% to 2% G0F-GN; (c) 0.1% to 1% G1-GN; (d) 5% to 20% G0B; (e) 5% to 30% G0F; (f) 0.1% to 1.5% Man5; (g) 1% to 15% G0FB; (h) 1% to 13% G1; (i) 0.5% to 10% G1'; (j) 0.5% to 6% G1B; (k) 0.5% to 12% G1F; (l) 0.1% to 3% G1F'; (m) 0.1% to 3% G1FB; (n) 0.1% to 2% G2; and (o) 0.1% to 2% G2F.

[0198] In some embodiments, the anti-CD20 antibody protein population further comprises at least two N-glycans within the following relative abundance ranges: (a) 0.8% to 1.1% G0-GN; (b) 0.5% to 1.1% G0F-GN; (c) 0.3% to 0.6% G1-GN; (d) 9.5% to 14.1% G0B; (e) 12.8% to 19.7% G0F; (f) 0.4% to 0.7% Man5; (g) 5.1% to 7.0% G0FB; (h) 5.7% to 6.4% G1; (i) 2.7% to 3.3% G1'; (j) 1.4% to 2.0% G1B; (k) 2.6% to 4.2% G1F; (l) 1.1% to 1.6% G1F'; (m) 1.1% to 1.8% G1FB; (n) 0.5% to 0.7% G2; and (o) 0.3% to 0.5% G2F.

[0199] In some embodiments, the anti-CD20 antibody protein population further comprises at least two N-glycans at the following relative abundances: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 0.5% G1-GN; (d) 10.9% G0B; (e) 17.0% G0F; (f) 0.6% Man5; (g) 6.0% G0FB; (h) 6.1% G1; (i) 2.9% G1'; (j) 1.6% G1B; (k) 3.2% G1F; (l) 1.3% G1F'; (m) 1.3G1FB; (n) 0.5% G2; and (o) 0.3% G2F.

[0200] In some embodiments, the population of anti-CD20 antibody proteins further comprises at least three, four, or five N-glycans in a relative abundance or within a relative abundance range as described herein.

[0201] In some embodiments, the N-glycan profile of the anti-CD20 antibody protein population is determined using a method comprising: (a) incubating the anti-CD20 antibody protein population with an enzyme, wherein the enzyme catalyzes the release of N-glycans from the anti-CD20 antibodies; (b) measuring the relative abundance of the released N-glycans using one or more methods selected from chromatography, mass spectrometry, capillary electrophoresis, and combinations thereof. In some embodiments, the enzyme is PNGase F.

[0202] In some embodiments, the method further comprises the following steps after step (a) and before step (b): (c) purifying the N-glycans; and (d) labeling the N-glycans with a fluorescent compound. In some embodiments, the fluorescent compound is 2-aminobenzamide (2-AB).

[0203] In some embodiments, less than 10% of the anti-CD20 antibody proteins in the population are non-glycosylated. In some embodiments, less than 5% of the anti-CD20 antibody proteins in the population are non-glycosylated. In some embodiments, less than 1% of the anti-CD20 antibody proteins in the population are non-glycosylated.

[0204] In some embodiments, the N-glycan profile of the anti-CD20 antibody protein population is substantially as follows Figure 2 as shown in .

[0205] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins comprises two or more secondary structures as determined by circular dichroism at 205 nm to 260 nm as follows: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-sheet; (c) 5.0% to 6.0% parallel β-sheet; (d) 16.0% to 18.0% β-turn; and (e) 35.0% to 36.0% random coil.

[0206] In some embodiments, the anti-CD20 antibody protein population comprises a secondary structure as determined by circular dichroism at 205 nm to 260 nm as follows: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-sheet; (c) 5.0% to 6.0% parallel β-sheet; (d) 16.0% to 18.0% β-turn; and (e) 35.0% to 36.0% random coil.

[0207] In some embodiments, the anti-CD20 antibody protein population comprises two or more secondary structures as determined by circular dichroism at 205 nm to 260 nm as follows: (a) about 9.0% α-helix; (b) about 33.0% antiparallel β-sheet; (c) about 5.6% parallel β-sheet; (d) about 17.5% β-turn; and (e) about 35.2% random coil.

[0208] Also provided herein are compositions comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain ("HC") comprising the amino acid sequence of SEQ ID NO: 1 and a light chain ("LC") comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins further comprises one or more of the following post-translational modifications at a specified abundance:

[0209]

[0210]

[0211] In some embodiments, the population of anti-CD20 antibody proteins comprises two, three, four, five or more post-translational modifications.

[0212] In some embodiments, the population of anti-CD20 antibody proteins comprises the following post-translational modifications at specified abundance:

[0213] Location Location [bold] Modification Abundance HC(39-63) QTPRQGLEWIGGIYPGNGDTSYNQK(SEQ ID NO:19) Deamidation 4.5

[0214] In some embodiments, the population of anti-CD20 antibody proteins comprises the following post-translational modifications at specified abundance:

[0215] Location Location [bold] Modification Abundance LC(19-38) VTMTCRASSSVSYMHWYQQK(SEQ ID NO:30) Oxidation 1.3

[0216] In some embodiments, one or more of the post-translational modifications are measured by peptide mapping using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, deamidation is measured by isoaspartic acid detection or peptide mapping using LC-MS.

[0217] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins as described herein, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain ("HC") comprising the amino acid sequence of SEQ ID NO: 1 and a light chain ("LC") comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has the following properties:

[0218]

[0219]

[0220] In some embodiments, as measured by absorbance at 280nm, the population has a total protein amount of 25.5 to 25.8mg / mL. In some embodiments, as measured by absorbance at 280nm, the population has a total protein amount of about 25.6mg / mL.

[0221] In some embodiments, compared with obinutuzumab, ofatumumab, rituximab, veltuzumab, ibrutinib and / or ocrelizumab, the group induces greater cytotoxicity in cell-based antibody-dependent cellular cytotoxicity (ADCC) assays. In some embodiments, compared with commercial reference standards, the group has a relative efficacy of 90 to 163% in cell-based ADCC assays. In some embodiments, compared with commercial reference standards, the group has a relative efficacy of about 117% in cell-based ADCC assays. In some embodiments, cell-based ADCC assays use effector cells selected from CD16 effector cells and primary NK cells. In some embodiments, the group performs more than 100% of commercial reference standards in cell-based ADCC using CD16 effector cells.

[0222] In some embodiments, the population exhibits greater B cell depletion activity in a human whole blood B cell depletion assay compared to obinutuzumab, ofatumumab, rituximab, veltuzumab, ibritumomab tiuxetan, and / or ocrelizumab.

[0223] In some embodiments, the population has a relative potency of 78% to 116% or 73% to 128% in a cell-based complement dependent cytotoxicity (CDC) assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 91% in a cell-based CDC assay compared to a commercial reference standard.

[0224] In some embodiments, the population has a relative potency of 92 to 118% or 82 to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 109% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard.

[0225] In some embodiments, the population has a K of 30 to 70 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance. D In some embodiments, the population has a K of about 59 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance. D value.

[0226] In some embodiments, the population has a K of 500 to 1000 nM in an FcγRIIIa158F binding assay as measured by surface plasmon resonance. DIn some embodiments, the population has a K of about 760 nM in an FcγRIIIa 158F binding assay as measured by surface plasmon resonance. D value.

[0227] In some embodiments, the population has a significantly higher binding affinity for FcγRIIIa 158V or FcγRIIIa 158F than rituximab.

[0228] In some embodiments, the population has a relative potency of 88 to 113% or 86 to 116% in a CIq binding assay as measured by ELISA compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 99% in a CIq binding assay as measured by ELISA compared to a commercial reference standard.

[0229] In some embodiments, the population has a relative potency of 106 to 126% in a CD 16 activity assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 115% in a CD 16 activity assay compared to a commercial reference standard.

[0230] In some embodiments, the anti-CD20 antibody protein population has the following purification profile:

[0231]

[0232] In some embodiments, the population has 99.2 to 99.9% monomers as detected by size exclusion chromatography (SEC). In some embodiments, the population has 0.1 to 0.8% dimers as detected by SEC. In some embodiments, the population has undetectable levels of aggregates as detected by SEC. In some embodiments, the population has undetectable levels of fragments as detected by SEC.

[0233] In some embodiments, the population has 93.6 to 95.9% IgG after purification by non-reducing capillary gel electrophoresis (CGE). In some embodiments, the population has 0.1 to 0.3% high molecular weight species (HMWS) after purification by non-reducing CGE. In some embodiments, the population has 0.7 to 1.2% free light chains (LC) after purification by non-reducing CGE.

[0234] In some embodiments, the population has 97.7 to 98.0% heavy chain plus light chain material (HC+LC) after purification by reduced CGE.

[0235] In some embodiments, the anti-CD20 antibody protein population has the following distribution of charged isoforms:

[0236]

[0237] In some embodiments, the population has 20 to 25% acidic isoforms as detected by imaging capillary isoelectric focusing (iCIEF). In some embodiments, the population has 50 to 60% major isoforms as detected by iCIEF. In some embodiments, the population has 20 to 30% basic isoforms as detected by iCIEF.

[0238] In some embodiments, the population has an average molar ratio of free thiol to anti-CD20 antibody of about 2.0 to 2.2.

[0239] In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of the N-terminal residue. In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of up to 5 N-terminal residues. In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of up to 10 N-terminal residues.

[0240] In some embodiments, the terminal lysine residue of the heavy chain of the anti-CD20 antibody in the population is truncated. In some embodiments, administration of the anti-CD20 antibody to a human patient results in one or more of the following pharmacokinetic parameters: (a) AUC of 2,160 μg / mL to 3,840 μg / mL; (b) Cmax of 118,011 ng / mL to 159,989 ng / mL; (c) Cmin of 40 ng / mL to 375 ng / mL; and (d) Cavg between 6,437 ng / mL and 11,443 ng / mL, and wherein the anti-CD20 antibody is administered as follows: i) the first infusion at a dose of about 150 mg, ii) the second infusion two weeks later at a dose of about 450 mg, and iii) subsequent infusions every six months at a dose of about 450 mg.

[0241] In some embodiments, administration of an anti-CD20 antibody to a human patient results in one or more of the following pharmacokinetic parameters: (a) an AUC of about 3,000 μg / mL; (b) a Cmax of about 139,000 ng / mL; (c) a Cmin of about 139 ng / mL; and (d) a Cavg of about 8,940 ng / mL.

[0242] In some embodiments, the antibody proteins of the population are present in a single dosage form.

[0243] Also provided herein are pharmaceutical formulations comprising a composition described herein, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg / mL.

[0244] Also provided herein is a pharmaceutical formulation comprising an anti-CD20 antibody described herein, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the pharmaceutical formulation comprises one or more of the following: sodium chloride, trisodium citrate dehydrate, polysorbate 80, and hydrochloric acid.

[0245] In some embodiments, the pharmaceutical formulation comprises about 9.0 mg / mL of sodium chloride. In some embodiments, the pharmaceutical formulation comprises about 7.4 mg / mL of trisodium citrate dehydrate. In some embodiments, the pharmaceutical formulation comprises about 0.7 mg / mL of polysorbate 80. In some embodiments, the pharmaceutical formulation comprises about 0.4 mg / mL of hydrochloric acid.

[0246] Also provided herein is a single batch preparation of the anti-CD20 antibody protein population described herein, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the single batch comprises at least 100 g of anti-CD20 antibody protein.

[0247] Also provided herein is a single batch preparation of the anti-CD20 antibody protein population described herein, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the single batch comprises at least 120 g of anti-CD20 antibody protein.

[0248] Also provided herein is a single batch preparation of the anti-CD20 antibody protein population described herein, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the single batch comprises at least 150 g of anti-CD20 antibody protein.

[0249] Also provided herein is a population of anti-CD20 antibody proteins described herein, which is produced in a 15,000L or 20,000L bioreactor.

[0250] Also provided herein are methods of treating an autoimmune disease, wherein the method comprises administering to a subject in need thereof a composition as described herein, and wherein the autoimmune disease is selected from the group consisting of psoriasis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, inflammatory muscle disease, allergic rhinitis, vaginitis, interstitial cystitis, scleroderma, osteoporosis, eczema, allogeneic or xenogeneic transplantation, transplant rejection, graft-versus-host disease, lupus erythematosus, inflammatory diseases, type 1 diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis, myasthenia gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, allergic conjunctivitis, atopic dermatitis, chronic obstructive pulmonary disease, glomerulonephritis, neuroinflammatory diseases, and uveitis.

[0251] Also provided herein are methods of treating multiple sclerosis, wherein the method comprises administering to a subject in need thereof a composition described herein.

[0252] In some embodiments, multiple sclerosis (MS) is a relapsing form of MS. In some embodiments, the relapsing form of MS is selected from clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), and active secondary progressive MS (SPMS). In some embodiments, the relapsing form of MS is clinically isolated syndrome (CIS). In some embodiments, the relapsing form of MS is relapsing-remitting multiple sclerosis (RRMS). In some embodiments, the relapsing form of MS is active secondary progressive multiple sclerosis (SPMS).

[0253] Also provided herein are methods of treating a neoplastic disease, wherein the method comprises administering to a subject in need thereof a composition described herein.

[0254] In some embodiments, the neoplastic disease is acute B lymphoblastic leukemia, B cell lymphoma, mature B cell lymphoma, including B-type chronic lymphocytic leukemia (B-CLL), small B cell lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone MALT-type lymphoma, nodal marginal zone lymphoma with or without monocytoid B cells, splenic marginal zone lymphoma (with or without villous lymphocytes), tricholeucocytic leukaemia, diffuse large B cell lymphoma, or Burkitt's lymphoma.

[0255] In some embodiments, the subject is a human.

[0256] Also provided herein are methods for inactivating viruses or exogenous factors in rat myeloma cells expressing an anti-CD20 antibody protein in a composition described herein, wherein the method maintains suitability for antibody production in a 15,000L or 20,000L bioreactor.

[0257] Also provided herein are methods for reducing the immunogenicity of an anti-CD20 antibody protein in a composition described herein, wherein the method maintains suitability for antibody production in a 15,000L or 20,000L bioreactor.

[0258] Also provided herein is a method of analyzing a TG-1101 (TG Therapeutics, Inc.) preparation comprising: (i) providing a separated N-glycan fraction from a TG-1101 (TG Therapeutics, Inc.) preparation;

[0259] (ii) analyzing the N-glycan fraction to determine whether one or more N-glycans are the following N-glycans within the following relative abundance ranges: (a) 0.3% to 2% G0-GN; (b) 0.1% to 2% G0F-GN; (c) 0.1% to 1% G1-GN; (d) 5% to 20% G0B; (e) 5% to 30% G0F; (f) 0.1% to 1.5% Man5; (g) 1% to 15% G0FB; (h) 1% to 13% G1; (i) 0.5% to 10% G1'; (j) 0.5% to 6% G1B; (k) 0.5% to 12% G1F; (l) 0.1% to 3% G1F'; (m) 0.1% to 3% G1FB; (n) 0.1% to 2% G2; and (o) 0.1% to 2% G2F.

[0260] In some embodiments, the method includes analyzing the N-glycan fraction to determine whether one or more N-glycans are the following N-glycans within the following relative abundance ranges: (a) 0.8% to 1.1% G0-GN; (b) 0.5% to 1.1% G0F-GN; (c) 0.3% to 0.6% G1-GN; (d) 9.5% to 14.1% G0B; (e) 12.8% to 19.7% G0F; (f) 0.4% to 0.7% Man5; (g) 5.1% to 7.0% G0FB; (h) 5.7% to 6.4% G1; (i) 2.7% to 3.3% G1'; (j) 1.4% to 2.0% G1B; (k) 2.6% to 4.2% G1F; (l) 1.1% to 1.6% G1F'; (m) 1.1% to 1.8% G1FB; (n) 0.5% to 0.7% G2; and (o) 0.3% to 0.5% G2F.

[0261] In some embodiments, the method includes analyzing the N-glycan fraction to determine whether one or more N-glycans are in the following relative abundances: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 0.5% G1-GN; (d) 10.9% G0B; (e) 17.0% G0F; (f) 0.6% Man5; (g) 6.0% G0FB; (h) 6.1% G1; (i) 2.9% G1'; (j) 1.6% G1B; (k) 3.2% G1F; (l) 1.3% G1F'; (m) 1.3G1FB; (n) 0.5% G2; and (o) 0.3% G2F.

[0262] Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins further comprises at least two N-glycans at the following relative abundances: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 0.5% G1-GN; (d) 10.9% G0B; (e) 17.0% G0F; (f) 0.6% Man5; (g) 6.0% G0FB; (h) 6.1% G1; (i) 2.9% G1'; (j) 1.6% G1B; (k) 3.2% G1F; (l) 1.3% G1F'; (m) 1.3G1FB; (n) 0.5% G2; and (o) 0.3% G2F, and wherein the anti-CD20 antibody protein population is produced in rat hybridoma cells.

[0263] In some embodiments, the rat hybridoma cell is a YB2 / 0 cell. 6. Description of the drawings

[0264] Figure 1 Describe the structures and abbreviations of various N-glycans.

[0265] Figure 2 The glycosylation profiles of the anti-CD20 antibody protein samples provided herein are illustrated.

[0266] Figure 3 Illustrated is the complete MS spectra of the anti-CD20 antibody protein samples provided herein.

[0267] Figure 4 Illustrated are dose response curves for antibody-dependent cellular cytotoxicity (ADCC) activity using Raji cells and KILR CD16a cells.

[0268] Figure 5 Dose response curves for ADCC activity using Raji cells and primary NK cells are illustrated.

[0269] Figure 6 Dose response curves for antibody-dependent cellular phagocytosis (ADCP) activity are illustrated.

[0270] Figure 7 Dose response curves for complement dependent cytotoxicity (CDC) activity are illustrated.

[0271] Figure 8 CD20 binding dose response curve is illustrated.

[0272] Fig. 9 Illustrate CD20 binding dose response curve using FACS.

[0273] Fig.10 C1q binding dose response curve is illustrated.

[0274] Figure 11A-11C Illustration of human whole blood B cell depletion from three donors. B cell depletion was calculated based on data using CD19 as a B cell marker.

[0275] Fig.12 Describe the goodness-of-Fit (GOF) diagnosis of the final model of TG-1101.

[0276] Fig.13 pcVPC of the final PK model for TG-1101 is illustrated by study. Blue dots are observed concentrations corrected for prediction; blue lines are the 50th (solid), 5th (dashed), and 95th (dashed) percentiles of observed concentrations; black lines are the simulated 50th (solid), 5th (dashed), and 95th (dashed) percentiles. Gray bands are the 95% PI of the corresponding black lines based on 500 simulations. Yellow dashes indicate bin intervals. Numbers 201, 301, 302, and 101-304-703 represent study numbers TG1101-RMS201, TG1101-RMS301, and TG1101-RMS302, respectively, and studies in subjects with hematological malignancies in the previous analysis set. Abbreviations: pcVPC-prediction-corrected visual predictive test; PI-prediction interval; pred-corr-prediction-corrected; popPK-population pharmacokinetics.

[0277] Fig.14Forest plot illustrating the covariate effects of TG-1101 drug exposure. The first and second vertical dashed lines correspond to ratios of 0.8 and 1.25, respectively. The vertical solid line corresponds to a ratio of 1 and represents a typical subject. The points and whiskers represent the estimate and 90% confidence interval, respectively. The blue-gray horizontal bars show the range of exposure due to inter-subject variability. The typical subject was defined as a male subject from North America / Western Europe, weighing 73 kg, ADA negative, and having been treated for <416 days. Abbreviations: ADA-anti-drug antibodies; AUCss-area under the serum TG-1101 concentration-time curve at steady state; BSV-between-subject or inter-individual variability; CI-confidence interval; CMAXSS-maximum TG-1101 concentration at steady state; CMINSS-minimum TG-1101 concentration at steady state; N / A-not applicable.

[0278] Fig.15 A map of expression vector HK463-25 is depicted, which contains the immunoglobulin heavy and light chain cDNA sequences of the anti-CD20 antibody TG-1101 described herein.

[0279] Figure 16A-16C The development and maintenance of no evidence of disease activity (NEDA) on ublituximab were described. Fig.16A is a bar graph showing NEDA-3 ratios by treatment for EPOCH (weeks 0-24 vs. weeks 24-96). a Denominator based on participants in the analysis of weeks 24-96. Pooled post hoc analysis. Modified intention-to-treat population. Fig. 16B is a bar graph showing NEDA-3 ratios by treatment for EPOCH (Weeks 24-48 vs. Weeks 48-96). a Denominator based on participants in the analysis of weeks 48-96. Pooled post hoc analysis. Modified intention-to-treat population. Fig. 16C is a bar graph showing components driving EDA in participants treated with utuximab. a Participants may have >1 EDA component. Pooled post hoc analysis. Modified intention-to-treat population. CDP, confirmed disability progression; EDA, evidence of disease activity; Gd+, gadolinium enhancement; n / e, new / expanded; NEDA, no evidence of disease activity; NEDA-3, 3-parameter NEDA; EPOCH, combination of etoposide phosphate, prednisone, vincristine sulfate (Oncovin), cyclophosphamide, and doxorubicin hydrochloride (hydroxydaunorubicin).

[0280] Figure 17A-Figure 17C To describe the characterization of cytopenias with utuximab in the Phase 3 ULTMATE I and II studies in participants with relapsing forms of multiple sclerosis. Fig.17A is a bar graph showing hematology laboratory abnormalities in participants treated with utuximab (all study visits). a (493 / 543). Percentages are based on the number of participants with no missing baseline and at least 1 postbaseline assessment. Pooled Post Hoc Analyses. Safety Population. Fig. 17B is a bar graph showing hematology laboratory abnormalities in participants treated with utuximab (excluding Day 2). a (175 / 543). Percentages are based on the number of participants with no missing baseline and at least 1 postbaseline assessment. Pooled Post Hoc Analyses. Safety Population. Fig. 17C is a bar graph showing the proportion of participants treated with utuximab who had low lymphocyte counts by visit. a (476 / 543). Pooled post hoc analysis. Safety population. Percentages are based on number of participants in population / treatment group. BL, baseline.

[0281] Figure 18A-18B is a bar graph showing the relative reduction in annualized relapse outcome (ARR) in RMS patients treated with utuximab compared to teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.18A ) and ULTIMATE II ( Fig.18B )’s ARR results.

[0282] Figure 19A-19B is a bar graph showing the relative reduction in the total number of gadolinium (Gd)+T1 lesions on MRI in patients with RMS treated with utuximab compared to teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.19A ) and ULTIMATE II ( Fig.19B ) results.

[0283] Figure 20A-20B is a bar graph showing the relative reduction in the number of new or enlarged T2 lesions on MRI in RMS patients treated with utuximab compared to teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig. 20A ) and ULTIMATE II ( Fig. 20B ) results.

[0284] Figure 21A-21Bis a bar graph showing the improvement in the proportion (%) of patients with no evidence of disease activity (NEDA) status in RMS patients treated with utuximab relative to teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.21A ) and ULTIMATE II ( Fig.21B )’s NEDA results.

[0285] Figure 22A-22B is a graph comparing the percentage of RMS patients with confirmed disability progression (CDP) in a pre-specified pooled analysis of patients treated with utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.22A Display 12-week CDP, Fig. 22B Displays 24-week CDP.

[0286] Figure 23A-23B is a graph comparing the percentage of RMS patients who achieved confirmed disability improvement (CDI) in a pre-specified pooled analysis of patients administered utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.23A Shows 12-week CDI, Fig. 23B Shows 24-week CDI.

[0287] Figure 24A-24B is a bar graph showing significant improvement in mean Multiple Sclerosis Functional Composite (MSFC) scores from baseline to week 96 in RMS patients treated with utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.24A ) and ULTIMATE II ( Fig. 24B ) results.

[0288] Figure 25A-25B is a bar graph showing significant improvement from baseline to 96 weeks in mean timed 25-foot walk (T25FW) scores in RMS patients treated with utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.25A ) and ULTIMATE II ( Fig.25B ) results.

[0289] Figure 26A-26B: is a bar graph showing the change from baseline to 96 weeks in the mean Paced Auditory Serial Additional Test (PASAT) scores of RMS patients treated with utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.26A ) and ULTIMATE II ( Fig.26B ). The mean PASAT score changes were similar between treatment groups.

[0290] Figure 27A-27B is a bar graph showing that the mean 9-hole peg test (9-HPT) scores of RMS patients treated with utuximab relative to teriflunomide increased significantly from baseline to week 96 in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.27A ) and ULTIMATE II ( Fig.27B ) results.

[0291] Figure 28A-28B Graph showing percent brain volume change (PBVC) based on MRI from week 24 to week 96 (post hoc analysis) in RMS patients treated with utuximab and teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Fig.28A ) and ULTIMATE II ( Fig.28B ) results. 7. Specific implementation methods

[0292] Anti-CD20 antibody protein groups with specified post-translational modification ranges are provided herein. The primary amino acid sequences of such antibodies are provided in Section 7.3. The types of such post-translational modifications and their corresponding abundance in anti-CD20 antibody groups are described in Section 7.4. Compositions (including single batch compositions) of the anti-CD20 antibody protein groups with specified post-translational modification ranges and purity ranges are described in Section 7.5 (e). The quantitative determination of such post-translational modifications in the anti-CD20 antibody protein groups is described in Section 7.5. The determination of the biological and clinical significance of such post-translational modifications in the anti-CD20 antibody protein groups is described in Section 7.7 (a). The method of using such anti-CD20 antibody protein groups to treat and prevent medical conditions is described in Section 7.8. The pharmacokinetic and pharmacodynamic properties of the compositions provided herein in human patients are described in Section 7.9. Methods for making such anti-CD20 antibody protein populations are described in Section 7.10.

[0293] As used herein, "TG-1101" (TG Therapeutics, Inc.) (also known as Utuximab, UBX, UTX, TG-1101, TGTX-1101, Utuxin TM , LFB-R603, TG20, EMAB603) are the source antibodies of the anti-CD20 antibodies described herein, which have unique glycosylation profiles produced by the disclosed methods.

[0294] Source antibody TG-1101 is a monoclonal antibody targeting epitopes on CD20, such as IRAHT (SEQ ID NO: 37) and EPAN (SEQ ID NO: 38). See Fox, E. et al., Mult. Scler. 27: 420-429 (March 2021); Babiker et al., Expert Opin Investig Drugs 27: 407-412 (2018); Cotchett, KR et al., Multiple Sclerosis and Related Disorders 49: 102787 (2021); Miller et al., Blood 120: Abstract No. 2756 (2012); Deng, C. et al., J. Clin. Oncol. 31: Abstract No. 8575 (2013). TG-1101 is also described in U.S. Patent Nos. 9,234,045 and 9,873,745.

[0295] 7.1 Abbreviations and Conventions

[0296] The following abbreviations are used throughout this application.

[0297]

[0298]

[0299] 7.2 Definitions

[0300] As used herein, the term "anti-CD20 antibody protein group" refers to a composition of anti-CD20 antibody proteins of the abundance of post-translational modifications being tested. The individual anti-CD20 antibody proteins in the group may contain similar or different post-translational modifications. In some embodiments, the anti-CD20 antibody protein group refers to all anti-CD20 antibody proteins present in a single dosage form. In some embodiments, the anti-CD20 antibody protein group refers to all anti-CD20 antibody proteins present in a single batch. In some embodiments, the amount of the anti-CD20 antibody protein group is sufficient to determine whether the anti-CD20 antibody protein of the batch meets or fails to reach a predetermined acceptable comparison value range when compared to a reference standard.

[0301] As used herein, the term "single batch" in the context of an anti-CD20 antibody protein refers to a composition derived from a single production or run from a single bioreactor of a specified volume. For example, an anti-CD20 antibody protein obtained from a single run of a 15,000 L bioreactor can be referred to as a single batch. In certain embodiments, the capacity of the bioreactor is at least 100; 200; 300; 400; 500; 750; 1,000; 2,000; 3,000; 4,000; 5,000; 7,500; 10,000; 15,000; 20,000; or at least 25,000 L.

[0302] As used herein, and unless otherwise indicated, the term "about" or "approximately" means the acceptable error of a particular value determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1 or 2 standard deviations. In certain embodiments, the term "about" or "approximately" means within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0303] 7.3 Primary amino acid sequence of anti-CD20 antibody

[0304] In some aspects, the anti-CD20 antibody proteins provided herein are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2. The sequences are provided in the sequence listing below.

[0305] In some aspects, the anti-CD20 antibody proteins provided herein are expressed by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1, or amino acids at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, or amino acids at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 2.

[0306] In some aspects, the anti-CD20 antibody proteins provided herein comprise the VH CDR1, CDR2, and CDR3 regions of sequences SEQ ID NOs: 3, 4, and 5, and the VL CDR1, CDR2, and CDR3 regions of sequences SEQ ID NOs: 8, 9, and 10.

[0307] In some aspects, the anti-CD20 antibody protein provided herein comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:11.

[0308] In some aspects, the nucleic acid sequence encoding the heavy chain of the anti-CD20 antibody protein provided herein comprises the nucleic acid sequence of SEQ ID NO: 35. In some aspects, the nucleic acid sequence encoding the light chain of the anti-CD20 antibody protein provided herein comprises the nucleic acid sequence of SEQ ID NO: 36.

[0309] In some aspects, the anti-CD20 antibody proteins provided herein bind to the same epitope as TG-1101 (TG Therapeutics, Inc.).

[0310] In some aspects, the anti-CD20 antibody proteins provided herein are chimeric immunoglobulin G1 (IgG1) anti-CD20 monoclonal antibody proteins, each assembled from a tetramer of two light chains (213 amino acids) and two heavy chains (448 amino acids).

[0311] In some aspects, the anti-CD20 antibody proteins provided herein are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, and contain pyroglutamic acid instead of glutamine at position 1 of the light chain and / or heavy chain, thereby obtaining a heavy chain of the amino acid sequence of SEQ ID NO: 13 and / or a light chain of the amino acid sequence of SEQ ID NO: 14.

[0312] In some aspects, the anti-CD20 antibody proteins provided herein are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, and comprise a C-terminal lysine deletion in the heavy chain, thereby generating the amino acid sequence of SEQ ID NO: 15.

[0313] In some embodiments, provided herein are anti-CD20 antibody protein compositions or populations, wherein at least 50%, 60%, 70%, 80%, 90%, 95% or 98% comprise a deletion of (i) pyroglutamic acid (instead of glutamine) at position 1 of the heavy chain, (ii) pyroglutamic acid (instead of glutamine) at position 1 of the light chain, and / or (iii) the C-terminal lysine of the heavy chain.

[0314] In some aspects, the anti-CD20 antibody is expressed by one or more nucleic acid sequences encoding a light chain comprising the amino acid sequence of SEQ ID NO:16.

[0315] 7.4 Anti-CD20 Antibody Compositions

[0316] The anti-CD20 antibody compositions provided herein can be described by various post-translational modifications and / or by their three-dimensional conformation (see Section 7.5(e)). The corresponding levels of various post-translational modifications can be quantified as described in Section 7.5. Without being bound by theory, these structural properties of the anti-CD20 antibody compositions provided herein lead to the biological and clinical properties described in Sections 7.7 and 7.9 below.

[0317] The anti-CD20 antibody compositions produced in vitro have various post-translational modifications. It should be understood that each individual anti-CD20 antibody protein may have its own unique post-translational modification pattern. In order to describe the properties of a group of multiple anti-CD20 antibody proteins, the overall presence of a specific post-translational modification can be quantified. Without being bound by theory, the level of a specific post-translational modification in the anti-CD20 protein group can determine the biological and clinical properties of the composition (such as the dosage of a pharmaceutical preparation). Without being bound by theory, the post-translational modification is achieved by expression in rat hybridoma cells (e.g., YB2 / 0 cells) in cell culture.

[0318] In some embodiments, the type of post-translational modification that can be used to describe the anti-CD20 antibody compositions provided herein is glycosylation. Various glycosylation is known. In one aspect, the glycosylation is N-glycosylation. The N-glycans that may be present may be Figure 1 Any of the N-glycans shown in . The level of N-glycosylation is discussed below in Section 7.4(a) and can be quantified using the assays in Section 7.5.

[0319] In some embodiments, the type of post-translational modification that can be used to describe the anti-CD20 antibody compositions provided herein is deamidation. Deamidation is a chemical reaction in which the amide functional group in the side chain of the amino acid asparagine or glutamine is removed or converted to another functional group. Typically, asparagine is converted to aspartic acid or isoaspartic acid. The level of deamidation at a specific amino acid position in the anti-CD20 antibody compositions provided herein is described in Section 7.4 (b) below and can be measured as described in Section 7.5 herein.

[0320] (a) N-glycosylation

[0321] Various forms of N-glycosylation may be present in the anti-CD20 antibody compositions provided herein. Without being bound by theory, the relative distribution of various N-glycans or individual sugar residues present in those N-glycans in individual anti-CD20 antibody proteins in the anti-CD20 antibody protein population may determine the biological and clinical properties (such as those discussed in Sections 7.7 and 7.9) of the anti-CD20 antibody compositions provided herein. The anti-CD20 antibody compositions provided herein may be described by any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or all of the N-glycans or individual sugar residues described in the following subsections.

[0322] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, or 15 N-glycans within the following relative abundance ranges:

[0323] (a) 0.3% to 2% GO-GN;

[0324] (b) 0.1% to 2% GOF-GN;

[0325] (c) 30% to 60% G0;

[0326] (d) 0.1% to 1% G1-GN;

[0327] (e) 5% to 20% GOB;

[0328] (f) 5% to 30% GOF;

[0329] (g) 0.1% to 1.5% Man5;

[0330] (h) 1% to 15% GOFB;

[0331] (i) 1% to 13% G1;

[0332] (j) 0.5% to 10% G1';

[0333] (k) 0.5% to 6% G1B;

[0334] (l) 0.5% to 12% G1F;

[0335] (m) 0.1% to 3% G1F';

[0336] (n) 0.1% to 3% G1FB;

[0337] (o) 0.1% to 2% G2; and

[0338] (p) 0.1% to 2% G2F.

[0339] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, or 15 N-glycans within the following relative abundance ranges:

[0340] (a) 0.8% to 1.1% GO-GN;

[0341] (b) 0.5% to 1.1% GOF-GN;

[0342] (c) 42.5% to 48.8% G0;

[0343] (d) 0.3% to 0.6% G1-GN;

[0344] (e) 9.5% to 14.1% GOB;

[0345] (f) 12.8% to 19.7% GOF;

[0346] (g) 0.4% to 0.7% Man5;

[0347] (h) 5.1% to 7.0% GOFB;

[0348] (i) 5.7% to 6.4% G1;

[0349] (j) 2.7% to 3.3% G1';

[0350] (k) 1.4% to 2.0% G1B;

[0351] (l) 2.6% to 4.2% G1F;

[0352] (m) 1.1% to 1.6% G1F';

[0353] (n) 1.1% to 1.8% G1FB;

[0354] (o) 0.5% to 0.7% G2; and

[0355] (p) 0.3% to 0.5% G2F.

[0356] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, or 15 N-glycans within the following relative abundance ranges:

[0357] (a) 0.9% G0-GN;

[0358] (b) 0.8% GOF-GN;

[0359] (c) 46.1% G0;

[0360] (d) 0.5% G1-GN;

[0361] (e) 10.9% G0B;

[0362] (f) 17.0% GOF;

[0363] (g) 0.6% Man5;

[0364] (h) 6.0% GOFB;

[0365] (i) 6.1% G1;

[0366] (j) 2.9% G1′;

[0367] (k) 1.6% G1B;

[0368] (l) 3.2% G1F;

[0369] (m) 1.3% G1F';

[0370] (n)1.3G1FB;

[0371] (o) 0.5% G2; and

[0372] (p)0.3% G2F.

[0373] In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.3% to about 2% GO-GN, about 0.8% to about 1.1% GO-GN, or about 0.9% GO-GN. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 2% GOF-GN, about 0.5% to about 1.1% GOF-GN, or about 0.8% GOF-GN. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 30% to 60% GO, about 42.5% to 48.8% GO, or about 46.1% GO. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 1% G1-GN, about 0.3% to about 0.6% G1-GN, or about 0.5% G1-GN. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 5% to about 20% G0B, about 5% to about 15% G0B, about 9.5% to about 14.1% G0B, about 10.9% G0B, or about 10% G0B. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 5% to about 30% G0F, about 12.8% to about 19.7% G0F, or about 17.0% G0F. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 1.5% Man5, about 0.4% to about 0.7% Man5, or about 0.6% Man5. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 1% to about 15% GOFB, about 5.1% to about 7.0% GOFB, or about 6.0% GOFB. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 1% to about 13% G1, about 5.7% to about 6.4% G1, or about 6.1% G1. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.5% to about 10% G1', about 2.7% to about 3.3% G1', or about 2.9% G1'. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.5% to about 6% G1B, about 1.4% to about 2.0% G1B, or about 1.6% G1B.In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.5% to about 12% G1F, about 2.6% to about 4.2% G1F, or about 3.2% G1F. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 3% G1F', about 1.1% to about 1.6% G1F', or about 1.3% G1F'. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 3% G1FB, about 1.1% to about 1.8% G1FB, or about 1.3% G1FB. In certain embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 2% G2, about 0.5% to about 0.7% G2, or about 0.5% G2. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 2% G2F, about 0.3% to about 0.5% G2F, or about 0.3% G2F.

[0374] In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.3% to about 2% G0-GN, about 0.1% to about 2% G0F-GN, about 30% to 60% G0, about 0.1% to about 1% G1-GN, about 5% to about 20% G0B, about 5% to about 30% G0F, about 0.1% to about 1.5% Man5, about 1% to about 15% G0FB, about 1% to about 13% G1, about 0.5% to about 10% G1', about 0.5% to about 6% G1B, about 0.5% to about 12% G1F, about 0.1% to about 3% G1F', about 0.1% to about 3% G1FB, about 0.1% to about 2% G2, and about 0.1% to about 2% G2F. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile.

[0375] In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising about 0.8% to about 1.1% G0-GN, about 0.5% to about 1.1% G0F-GN, about 42.5% to 48.8% G0, about 0.3% to about 0.6% G1-GN, about 9.5% to about 14.1% G0B, about 12.8% to about 19.7% G0F, about 0.4% to about 0.7% Man5, about 5.1% to about 7.0% G0FB, about 5.7% to about 6.4% G1, about 2.7% to about 3.3% G1', about 1.4% to about 2.0% G1B, about 2.6% to about 4.2% G1F, about 1.1% to about 1.6% G1F', about 1.1% to about 1.8% G1FB, about 0.5% to about 0.7% The relative abundance of G2 and about 0.3% to about 0.5% G2F. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile.

[0376] In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising a relative abundance of about 0.9% G0-GN, about 0.8% G0F-GN, about 46.1% G0, about 0.5% G1-GN, about 10.9% G0B, about 17.0% G0F, about 0.6% Man5, about 6.0% G0FB, about 6.1% G1, about 2.9% G1', about 1.6% G1B, about 3.2% G1F, about 1.3% G1F', about 1.3% G1FB, about 0.5% G2, and about 0.3% G2F. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile.

[0377] In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising G1 and G0 N-glycans at a relative abundance ratio of about 0.1 to about 0.15. In some embodiments, the anti-CD20 antibody protein population comprises an N-glycan profile comprising G1F and G1 N-glycans at a relative abundance ratio of about 0.5 to about 0.9.

[0378] In one embodiment, the anti-CD20 antibody composition has Figure 2 N-glycan profile shown in .

[0379] Specific ranges and values ​​for various N-glycans are provided below.Anti-CD20 antibody protein compositions or populations provided herein can be described by any of these N-glycan prevalences or by any population or by all of these N-glycans.

[0380] (i) Galactosylation

[0381] In some embodiments, the anti-CD20 antibody compositions provided herein comprise 10 to 20% galactosylated glycans. Figure 1 Those with galactose residues (such as Figure 1 The N-glycans of the core (shown as the hollow circles in ). Assays for determining the percentage of galactosylation (or galactosylated N-glycans) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody proteins is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of galactosylated N-glycans is the percentage of galactosylated N-glycans in the N-glycans cleaved using enzymatic digestion.

[0382] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17% or at least 18% and at most 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or at most 20% galactosylated glycans. In some embodiments, the anti-CD20 antibody compositions provided herein comprise 11% to 19%, 12% to 18%, 13% to 17%, or 14% to 16% galactosylated glycans. For example, the anti-CD20 antibody compositions provided herein may comprise about 17% galactosylated glycans (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%).

[0383] (ii) Fucosylation

[0384] In some embodiments, the anti-CD20 antibody compositions provided herein comprise 20% to 40% fucosylated glycans; 23% to 36% fucosylated glycans; 20% to 35% fucosylated glycans; 28% to 33% fucosylated glycans; or about 33% fucosylated glycans (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). Fucosylated glycans are Figure 1 Those N-glycans with fucose residues shown in Figure 1 ). Assays for determining the percentage of fucosylation (or fucosylated N-glycans) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody proteins is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of fucosylated N-glycans is the percentage of fucosylated N-glycans in the N-glycans cleaved using enzymatic digestion.

[0385] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, or at least 33% and at most 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 39%, 40%, 41%, or at most 42% fucosylated glycans.

[0386] (iii) Galactosylation to fucosylation ratio

[0387] In some embodiments, provided anti-CD20 antibody compositions are characterized by a particular ratio of total fucosylated glycans to total galactosylated glycans (or "fucose / galactose ratio"). The fucose / galactose ratio may be between 1.5 and 2.1; 1.5 and 2; 1.5 and 1.9; 1.5 and 1.8; 1.6 and 2.1; 1.7 and 2.1; 1.8 and 2.8; 1.6 and 2.0; 1.7 and 1.9; 1.6 and 1.8; or the fucose / galactose ratio may be about 1.75 (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). Fucosylated glycans are Figure 1 Those with fucose residues (such as Figure 1 The N-glycans of galactosylation are shown in the figure. Figure 1 Those with galactose residues (such as Figure 1 The N-glycans of the core are shown in the open circles in the figure. Assays for determining the percentage of fucosylation (or fucosylated N-glycans) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody proteins is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of fucosylated N-glycans is the percentage of fucosylated N-glycans in the N-glycans cleaved using enzymatic digestion.

[0388] (iv) Bisecting N-glycans

[0389] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least 10%, 15%, 20%, 25%, or at least 30% bisecting N-glycans; 10% to 30%, 12% to 30%, 12% to 25%, 12% to 20%, 15% to 30%, 15% to 25%, 15% to 20%, 18% to 30%, or 18% to 25% bisecting N-glycans. Figure 1 The third GlcNAc shown in FIG is attached to the mannose residue closest to the protein backbone (eg Figure 1The percentage of fucosylated N-glycans is the percentage of bisecting N-glycans in the N-glycans cleaved from the core. The percentage of fucosylated N-glycans is the percentage of bisecting N-glycans in the N-glycans cleaved using enzymatic digestion.

[0390] (v)sialylation

[0391] In some embodiments, the anti-CD20 antibody compositions provided herein contain less than 10%, 8%, 5%, 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycans. In some embodiments, the anti-CD20 antibody compositions provided herein contain 10% to 0.5% sialylated glycans; 10% to 5% sialylated glycans; 5% to 0.5% sialylated glycans; 4% to 0.5% sialylated glycans; 2% to 0.5% sialylated glycans; or undetectable amounts of sialylated glycans. Assays for determining the percentage of sialylation (or sialylated N-glycans) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody proteins is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of sialylated N-glycans is the percentage of sialylated N-glycans among N-glycans cleaved using enzymatic digestion.

[0392] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least undetectable amounts, 0.5%, 1%, 2%, 3%, 4%, or at least 5%, and at most 0.5%, 1%, 2%, 3%, 4%, 5%, or at most 10% sialylated glycans. In some embodiments, the anti-CD20 antibody compositions provided herein comprise undetectable amounts of sialylated glycans.

[0393] (vi) G0B N-glycan

[0394] In some embodiments, the anti-CD20 antibody compositions provided herein comprise 5% to 15% GOB N-glycans; 9% to 11% GOB N-glycans; or about 10% GOB N-glycans (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). GOB N-glycans such as Figure 1As shown in . The assay for determining the percentage of G0B N-glycans is described in Section 7.5. In brief, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated so that all N-glycans are cut from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of G0B N-glycans is the percentage of G0B N-glycans in the N-glycans cut using enzymatic digestion.

[0395] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least 5%, 6%, 7%, 8%, 9%, 10%, or at least 11% and at most 7%, 8%, 9%, 10%, 11%, 12%, or at most 13% GOB N-glycans.

[0396] (vii) Man5 N-glycan

[0397] In some embodiments, the anti-CD20 antibody compositions provided herein comprise 0.1% to 1.5% Man5 N-glycans; 0.4% to 0.7% G0B N-glycans; or about 0.6% Man5 N-glycans (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). Man5 N-glycans such as Figure 1 As shown in . The assay for determining the percentage of Man5 N-glycans is described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of Man5 N-glycans is the percentage of Man5 N-glycans in the N-glycans cleaved using enzymatic digestion.

[0398] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or at least 0.7% and at most 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or at most 0.9% Man5 N-glycans.

[0399] In some embodiments, the Man5 N-glycan is the only high mannose species in the N-glycan profile.

[0400] (viii) G0 N-glycans

[0401] In some embodiments, the anti-CD20 antibody compositions provided herein comprise about 42% to about 52.8% G0 N-glycans (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). G0 N-glycans such as Figure 1As shown in . The assay for determining the percentage of G0 N-glycans is described in Section 7.5. In brief, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated so that all N-glycans are cut from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of G0 N-glycans is the percentage of G0 N-glycans in the N-glycans cut using enzymatic digestion.

[0402] In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, or at least 48% and at most 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 52.8%, 53%, 54%, 55%, 56%, 57%, or at most 58% GON-glycans.

[0403] (ix) G1 to G0 N-glycan ratio

[0404] In some embodiments, provided anti-CD20 antibody compositions are characterized by a specified abundance ratio of G1 to G0 N-glycans. The abundance ratio of G1 to G0 N-glycans may be 0.02 to 0.3; 0.05 to 0.25; 0.08 to 0.22; 0.09 to 0.2; 0.1 to 0.19; 0.1 to 0.18; 0.1 to 0.17; 0.1 to 0.16; or the abundance ratio of G1 to G0 N-glycans may be 0.1 to 0.15. G1 and G0 N-glycans may be 0.02 to 0.3; 0.05 to 0.25; 0.08 to 0.22; 0.09 to 0.2; 0.1 to 0.19; 0.1 to 0.18; 0.1 to 0.17; 0.1 to 0.16; or the abundance ratio of G1 to G0 N-glycans may be 0.1 to 0.15. Figure 1 As shown in . Assays for determining the percentage of G1 or G0 glycans are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of G1 or G0 glycans is the percentage of G1 or G0 glycans in the N-glycans cleaved using enzymatic digestion, respectively.

[0405] (x) G1F to G1 N-glycan ratio

[0406] In some embodiments, provided anti-CD20 antibody compositions are characterized by a specified abundance ratio of G1F to G1 N-glycans. The abundance ratio of G1F to G1 N-glycans may be 0.1 to 1.2; 0.2 to 1.1; 0.3 to 1; 0.4 to 1; 0.5 to 1; or the abundance ratio of G1 to G0 N-glycans may be 0.5 to 0.9. G1F and G1 N-glycans may be 0.1 to 1.2; 0.2 to 1.1; 0.3 to 1; 0.4 to 1; 0.5 to 1; or the abundance ratio of G1 to G0 N-glycans may be 0.5 to 0.9. Figure 1. The analytical method for determining the percentage of G1F or G1 glycans is described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of G1F or G1 glycans is the percentage of G1F or G1 glycans in the N-glycans cleaved using enzymatic digestion.

[0407] (b) Deamidation

[0408] In some embodiments, the anti-CD20 antibody protein population as described herein comprises asparagine deamidation at one or more asparagine residues present in the heavy chain. In some embodiments, the one or more deamidated asparagine residues present in the heavy chain are selected from Asn-33 (as shown in SEQ ID NO: 18), Asn-55 (as shown in SEQ ID NO: 19), Asn-61 (as shown in SEQ ID NO: 19), Asn-160 (as shown in SEQ ID NO: 22), Asn-202 (as shown in SEQ ID NO: 22), Asn-204 (as shown in SEQ ID NO: 22), Asn-277 (as shown in SEQ ID NO: 25), Asn-287 (as shown in SEQ ID NO: 25), Asn-362 (as shown in SEQ ID NO: 27) or Asn-385 (as shown in SEQ ID NO: 28). In some embodiments, the anti-CD20 antibody protein population as described herein comprises asparagine deamidation at one or more asparagine residues present in the light chain. In some embodiments, the one or more deamidated asparagine residues present in the light chain are selected from Asn-136 (as shown in SEQ ID NO:31), Asn-137 (as shown in SEQ ID NO:31), Asn-151 (as shown in SEQ ID NO:32), or Asn-157 (as shown in SEQ ID NO:32).

[0409] (c) Oxidation

[0410] In some embodiments, the anti-CD20 antibody protein population comprises methionine oxidation at one or more methionine residues present in the heavy chain. In some embodiments, the one or more methionine residues present in the heavy chain are selected from Met-20, Met-34, Met-81, Met-253 or Met-428 as shown in SEQ ID NO: 17, 18, 21, 24 or 29, respectively. In some embodiments, the anti-CD20 antibody protein population comprises methionine oxidation at one or more methionine residues present in the light chain. In some embodiments, the one or more methionine residues present in the light chain are selected from Met-21 or Met-32 as shown in SEQ ID NO: 30.

[0411] (d) Pyroglutamate

[0412] In some embodiments, the anti-CD20 antibody protein population comprises pyroglutamination at the N-terminal glutamine residue in the heavy chain or the light chain. In some embodiments, the pyroglutamination at the N-terminal glutamine residue is present in the heavy chain, e.g., pGlu-1 as shown in SEQ ID NO: 13. In some embodiments, the pyroglutamination at the N-terminal glutamine residue is present in the light chain, e.g., pGlu-1 as shown in SEQ ID NO: 14. In some embodiments, the glutamic acid at position 1 of the heavy chain is pyroglutamic acid, and the glutamic acid at position 1 of the light chain is pyroglutamic acid.

[0413] (e) Lysine truncation

[0414] In some embodiments, the anti-CD20 antibody protein group comprises a deletion of a C-terminal lysine amino acid residue present in a heavy chain or a light chain. In some embodiments, the heavy chain C-terminal lysine amino acid residue of the anti-CD20 antibodies in the group is truncated. In some embodiments, the anti-CD20 antibody protein group comprises a deletion of a C-terminal lysine at the heavy chain. In some embodiments, the heavy chain of the anti-CD20 antibody protein comprises the amino acid sequence of SEQ ID NO: 15.

[0415] (f) Conformation

[0416] The three-dimensional conformation or protein folding can be determined using the assays described in Section 7.5(e).

[0417] In some embodiments, the anti-CD20 antibody protein provided herein (e.g., expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2) composition or population has a specified three-dimensional folding pattern or conformation. In some embodiments, the anti-CD20 antibody protein population comprises two or more of the following secondary structures as determined by circular dichroism (CD) spectroscopy at 205 nm to 260 nm: α-helices in the range of 3.0% to 15.0%; antiparallel β-sheets in the range of 25.0% to 40.0%; parallel β-sheets in the range of 0.5% to 12.0%; β-turns in the range of 10.0% to 25.0%; and random coils in the range of 30.0% to 42.0%.

[0418] In some embodiments, the anti-CD20 antibody protein population comprises two or more of the following secondary structures as determined by circular dichroism (CD) spectroscopy at 205 nm to 260 nm: α-helices in the range of 8.0% to 10.0%; antiparallel β-sheets in the range of 32.0% to 36.0%; parallel β-sheets in the range of 5.0% to 6.0%; β-turns in the range of 16.0% to 18.0%; and random coils in the range of 35.0% to 36.0%.

[0419] In some embodiments, the anti-CD20 antibody protein population comprises two or more of the following secondary structures as determined by circular dichroism (CD) spectroscopy at 205 nm to 260 nm: about 9.0% α-helices; about 33.0% antiparallel β-sheets; about 5.6% parallel β-sheets; about 17.5% β-turns; and about 35.2% random coils, wherein the term "about" means ±5%.

[0420] 7.5 Assays for Quantifying Post-Translational Modifications

[0421] In some embodiments, the present invention provides a method for determining the amount of post-translational modification in an anti-CD20 antibody protein population. In some embodiments, the post-translational modification is selected from asparagine deamidation, methionine oxidation, glycosylation, pyroglutamate formation, and lysine truncation.

[0422] (a) General digestion

[0423] In some embodiments, the method for determining the amount of post-translational modification in an anti-CD20 antibody protein population includes the step of digesting the anti-CD20 antibody protein population with an endoprotease. In some embodiments, the anti-CD20 antibody protein population is reduced prior to digestion. In some embodiments, the anti-CD20 antibody protein population is alkylated prior to digestion. In some embodiments, the endoprotease is selected from Asp-N, Lys-C or trypsin. In some embodiments, the digestion step is performed at 37°C for at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, overnight, and / or less than 24 hours. In some embodiments, the anti-CD20 antibody protein population is digested overnight at 37°C using Asp-N or Lys-C. In some embodiments, the anti-CD20 antibody protein population is digested overnight at 37°C using trypsin at a ratio of 50:1 (w:w). In some embodiments, the digested anti-CD20 antibody protein population is purified from the digestion reaction components and / or undigested anti-CD20 antibody proteins.

[0424] (b) Method for determining N-glycosylation

[0425] Exemplary assays and their results are discussed in the Examples in Sections 8.1 and 8.2 below.

[0426] In some embodiments, the method includes a step of deglycosylation of the anti-CD20 antibody protein population, thereby producing released N-glycans for labeling. In some embodiments, deglycosylation includes cleaving glycosidic bonds between one or more or all N-glycans of the anti-CD20 antibody protein population. In some embodiments, deglycosylation includes releasing some or most or substantially all N-glycans from the anti-CD20 antibody protein population. In some embodiments, deglycosylation releases greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 97%, greater than 98%, greater than 99% or 100% of the N-glycans present on the anti-CD20 antibody protein population.

[0427] In some embodiments, deglycosylation comprises contacting the anti-CD20 antibody protein population with one or more deglycosylation agents that cleave N-glycans or N-linked oligosaccharides. In some embodiments, the deglycosylation agent is a deglycosylation enzyme or chemical agent. In some embodiments, the deglycosylation enzyme is PNGase F.

[0428] In some embodiments, deglycosylation comprises contacting the anti-CD20 antibody protein population with one or more deglycosylation enzymes or chemicals at a deglycosylation temperature of about 25°C to about 50°C, about 37°C to about 50°C, about 25°C, about 37°C, about 42°C, or about 50°C. In some embodiments, the deglycosylation temperature is 37°C. The deglycosylation rate can be increased by increasing the deglycosylation temperature. In some embodiments, deglycosylation comprises contacting the anti-CD20 antibody protein population with one or more deglycosylation enzymes or chemicals for a period of time sufficient to release some or most or substantially all N-glycans from the anti-CD20 antibody protein population. In some embodiments, deglycosylation comprises contacting the anti-CD20 antibody protein population with the one or more deglycosylation enzymes or chemicals for at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, and / or less than 48 hours. In some embodiments, deglycosylation comprises contacting the anti-CD20 antibody protein population with the one or more deglycosylation enzymes or chemicals for a period of about 30 minutes to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 8 hours, about 8 hours to about 12 hours, about 12 hours to about 20 hours, about 20 to about 30 hours, greater than about 30 hours, and / or less than 48 hours. In some embodiments, deglycosylation comprises contacting the anti-CD20 antibody protein population with one or more deglycosylation enzymes or chemicals for about 12 to about 20 hours. In some embodiments, deglycosylation comprises contacting the anti-CD20 antibody protein population with PNGase F at about 37°C for about 12 to about 20 hours. In some embodiments, deglycosylation comprises contacting the anti-CD20 antibody with PNGase F at 37°C for a period of 12 to 20 hours. In some embodiments, deglycosylation using PNGase F occurs in the presence of a non-ionic detergent (i.e., NP-40).

[0429] In some embodiments, deglycosylation includes denaturing the CD20 antibody protein population before contacting the anti-CD20 antibody protein population with the one or more deglycosylation enzymes or chemicals. In some embodiments, denaturation includes heat denaturation, chemical denaturation, or a combination of the two. In some embodiments, heat denaturation includes incubating the anti-CD20 antibody protein population at a denaturation temperature and denaturation time sufficient to defold some or most or all of the immunoglobulin folding domains of the anti-CD20 antibody protein population. In some embodiments, the denaturation temperature is at least 50°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C and / or less than about 100°C. In some embodiments, the denaturation temperature is about 50°C to about 60°C, about 60°C to about 70°C, about 70°C to about 80°C, about 80°C to about 90°C, or 90°C to about 100°C. In some embodiments, the denaturation temperature is about 50°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, or about 100°C. In some embodiments, the denaturation temperature is 70°C. In some embodiments, chemical denaturation comprises incubating the anti-CD20 antibody protein group at a denaturation temperature or high temperature (i.e., thermal denaturation temperature) of at least 25°C, at least 30°C, at least 37°C. In some embodiments, the denaturation time is at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 10 hours, or at least 24 hours. In some embodiments, the denaturation time is about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 10 hours, or about 24 hours. In some embodiments, the denaturation time is 10 minutes. In some embodiments, chemical denaturation includes contacting the anti-CD20 antibody protein group with one or more chemical denaturants. In some embodiments, one or more chemical denaturants are selected from ionic detergents, non-ionic detergents, zwitterionic detergents, chaotropic detergents or reducing agents. In some embodiments, the chemical denaturant is selected from sodium dodecyl sulfate (SDS), urea or dithiothreitol (DDT). The effect of certain chemical denaturants may interfere with deglycosylation (i.e., enzymatic deglycosylation) and can be offset in solution. In some embodiments, the chemical denaturant is offset by adding a non-ionic detergent to the solution. In some embodiments, the chemical denaturant is SDS and the additional non-ionic detergent is NP-40. In some embodiments, denaturing the anti-CD20 antibody protein group includes heat denaturation at 70°C for 10 minutes.

[0430] In some embodiments, the method includes purifying the N-glycans released by the anti-CD20 antibody protein population from the deglycosylation reaction mixture. In some embodiments, the released N-glycans are substantially free of deglycosylation reagents and deglycosylated or unreacted anti-CD20 antibodies. In some embodiments, the released N-glycans are substantially free of salt and / or detergent. In some embodiments, the released N-glycans are purified by hydrophilic interactions. In some embodiments, the released N-glycans are purified by chromatography comprising a hydrophilic stationary phase and a reverse eluent. In some embodiments, the released N-glycans are purified by hydrophilic interaction liquid chromatography (HILIC). In some embodiments, the released N-glycans are purified via a Waters HILIC MassPrep μElution plate according to the manufacturer's protocol. In some embodiments, the released N-glycans are purified to a purity level comparable to the purity level obtained when purified via a Waters HILIC MassPrep μElution plate according to the manufacturer's protocol.

[0431] In some embodiments, the method includes a step of labeling the released N-glycans of the anti-CD20 antibody protein group, thereby producing labeled N-glycans for detection. In some embodiments, labeling the released N-glycans includes chemical derivatization, such as providing a detectable charge, ultraviolet activity or fluorescent properties to the released N-glycans. In some embodiments, labeling the released N-glycans includes reductive amination, hydrazide labeling, methylation, Michael addition or permethylation. In some embodiments, labeling the released N-glycans includes contacting the released N-glycans with a label selected from 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), 2-aminopyridine (PA), 2-aminonaphthalene trisulfonic acid (ANTS) or 1-aminopyrene-3,6,8-trisulfonic acid (APTS). In some embodiments, the label is 2-AB. In some embodiments, labeling the released N-glycans by reductive amination includes using a reducing agent. In some embodiments, the reducing agent is selected from sodium cyanoborohydride or 2-methylpyridine borane. In some embodiments, the reducing agent is cyanoborohydride. In some embodiments, the N-glycans released by reductive amination labeling include contacting the released N-glycans with a label and a reducing agent suitable for reductive amination at a reaction temperature and reaction time sufficient for labeling. In some embodiments, the reaction temperature is about 25°C to about 40°C, about 40°C to about 50°C, about 50°C to about 60°C, about 60°C to about 70°C, or about 70°C to about 80°C. In certain embodiments, the reaction temperature is about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, or about 75°C. In some embodiments, the reaction temperature is 65°C. In some embodiments, the reaction time is at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 6 hours. In some embodiments, the reaction time is about 30 minutes to about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, or about 4 hours to about 6 hours. In some embodiments, the reaction time is about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 6 hours. In some embodiments, the reaction time is 3 hours. In some embodiments, labeling the N-glycans released from the anti-CD20 antibody protein population includes reductive amination using 2-AB and cyanoborohydride at 65° C. for 3 hours to produce labeled N-glycans.

[0432] In some embodiments, the method includes purifying the labeled N-glycans of the anti-CD20 antibody protein group from the labeling reaction mixture. In certain embodiments, the labeled N-glycans are substantially free of unreacted labels. In certain embodiments, the labeled N-glycans are substantially free of unreacted released N-glycans. In certain embodiments, the labeled N-glycans are substantially free of reducing agents (i.e., cyanoborohydrides). In certain embodiments, the labeled N-glycans are purified by hydrophilic interactions. In certain embodiments, the labeled N-glycans are purified by chromatography comprising a hydrophilic stationary phase and a reverse eluent. In certain embodiments, the labeled N-glycans are purified by hydrophilic interaction liquid chromatography (HILIC). In some embodiments, the labeled N-glycans are purified via Waters HILIC MassPrep μElution plates according to the manufacturer's protocol. In some embodiments, the labeled N-glycans are purified to a level of purity comparable to that obtained when purified via a Waters HILIC MassPrep μElution plate according to the manufacturer's protocol.

[0433] In some embodiments, the method includes the steps of separating labeled N-glycans of an anti-CD20 antibody protein population and determining the N-glycan profile of the population. In some embodiments, separating labeled N-glycans includes separating based on hydrophilicity. In some embodiments, separating labeled N-glycans includes chromatography comprising a hydrophilic stationary phase and a reverse eluent. In specific embodiments, the chromatography is hydrophilic interaction liquid chromatography (HILIC). In some embodiments of the method, separating labeled N-glycans includes chromatography comprising an amide stationary phase. In some embodiments, the amide stationary phase is a polysaccharide BEH amide column. In certain embodiments, the reverse eluent comprises one or more mobile phases, wherein the mobile phase comprises an acidic ammonium buffer and / or acetonitrile.

[0434] In some embodiments, chromatography is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate labeled N-glycans. In some embodiments, chromatography is performed using a chromatography system (i.e., HPLC or UPLC system) that controls these parameters. In some embodiments, the chromatography system is coupled to a detector for detecting labeled N-glycans of an anti-CD20 antibody protein population. In some embodiments, the detector is a fluorescence detector that detects fluorescence from a label (e.g., a label that contacts the released N-glycans of an anti-CD20 antibody protein population in a labeling step). In some embodiments, the label is 2-AB. In some embodiments, detecting fluorescence includes excitation with 360nm wavelength light and observing fluorescence emission at 428nm wavelength light. In some embodiments, the chromatography system is a HILIC-UPLC system. In some embodiments, separation and detection of labeled N-glycans is performed using a hydrophilic interaction stationary phase, a reverse eluent, and a Waters UPLC equipped with a fluorescence detector. In some embodiments, the hydrophilic interaction stationary phase comprises a polysaccharide BEH amide column ( 1.7 μm, 2.1 mm X 150 mm). In some embodiments, the stationary phase (i.e., column) temperature is 50°C. In some embodiments, the flow rate is 0.50 mL / min. In some embodiments, the reverse eluent comprises one or more mobile phases, the mobile phase comprising a first mobile phase and a second mobile phase, the first mobile phase comprising about 250 mM ammonium formate, pH about 4.4, and the second mobile phase comprising acetonitrile. In some embodiments, the mobile phase gradient comprises the first mobile phase increasing from 22.0% to 44.1% in 38.5 minutes.

[0435] In some embodiments, detecting fluorescence comprises generating a chromatogram, wherein the dependent variable is selected from the mobile phase volume, the volume of eluent passed through the chromatographic column, or time, and fluorescence is observable. In certain embodiments, determining the N-glycan profile of an anti-CD20 antibody protein population comprises quantifying the relative amounts of labeled G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1', G1B, G1F, G1F', G1FB, G2, and G2F N-glycans of the population. In certain embodiments, chromatography (i.e., HILIC-UPLC) is performed using flow rate, column temperature, mobile phase gradient, and time period parameters sufficient to separate G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1', G1B, G1F, G1F', G1FB, G2, and G2F N-glycans for quantification. In certain embodiments, the amount of N-glycans is quantified by calculating the area under the curve comprising labeled G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1', G1B, G1F, G1F', G1FB, G2, and G2F N-glycans in a chromatogram separating the N-glycans. In certain embodiments, the relative abundance of an N-glycan selected from G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1', G1B, G1F, G1F', G1FB, G2 or G2F is quantified by calculating the peak area percentage of the N-glycan relative to the total peak area of ​​G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1', G1B, G1F, G1F', G1FB, G2 and G2F N-glycans in a chromatogram separating the N-glycans. In certain embodiments, the peak area of ​​the N-glycans in the chromatogram is greater than or equal to 0.25%. In certain embodiments, the peak area of ​​the N-glycans in the chromatogram has a signal-to-noise ratio greater than or equal to 3.0.

[0436] The N-glycan profile of an anti-CD20 antibody protein population can be determined by liquid chromatography coupled to mass spectrometry (LC-MS) peptide mapping, for example, by summing the site-specific glycosylation of the digested anti-CD20 antibody protein population to determine the relative glycosylation amount of the population.

[0437] In some embodiments, LC-MS peptide mapping includes determining the molecular weight of peptides derived from the anti-CD20 antibody protein group digested by Lys-C to determine the presence and amount of glycosylated residues (i.e., G0, G0F, G0B, G0FB, G1 or G1F). In some embodiments, the anti-CD20 antibody protein group is reduced and alkylated before digestion. In certain embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate the peptides of the Lys-C digest. In certain embodiments, the relative abundance of the peptides of the Lys-C digest is calculated by integrating the area under the curve compared to the total area.

[0438] In some embodiments, the observed molecular weight is compared to a theoretical mass of a glycosylated peptide selected from Table 1, Column 2. In certain embodiments, the observed molecular weight is compared to a theoretical mass selected from Table 1, Column 4.

[0439] Table 1: Peptides used to determine glycosylation after Lys-C digestion

[0440]

[0441] In some embodiments, the anti-CD20 antibody protein population comprises glycosylation at residue Asn-298 of SEQ ID NO: 33 present in the heavy chain.

[0442] (c) Method for Determining Deamidation

[0443] (i) General deamidation

[0444] Also provided herein is a method for determining the amount of asparagine deamidation in an anti-CD20 antibody protein population. In some embodiments, the amount of asparagine deamidation in an anti-CD20 antibody protein population is the amount of isoaspartic acid residues in the population. In some embodiments, the method includes detecting the amount of isoaspartic acid residues in the digested anti-CD20 antibody protein population.

[0445] (ii) Deamidation by isoquant

[0446] The amount of asparagine deamidation or isoaspartate in the anti-CD20 antibody protein population or the digested anti-CD20 antibody protein population can be determined via an enzymatic method, for example, by adding a methyltransferase (i.e., protein isoaspartate methyltransferase (PIMT)), which catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to isoaspartate. The amount of S-adenosylhomocysteine ​​(SAH) produced by this PIMT-catalyzed reaction is proportional to the amount of isoaspartate present in the sample (~1:1 stoichiometry). SAH can be directly measured by isolating SAH (i.e., via ultra-high performance liquid chromatography (UPLC) or reverse-phase high performance liquid chromatography (RP-HPLC)), detecting at 260 nm, and by comparing to a SAH standard curve (i.e., Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu peptide) to quantify the amount of SAH.

[0447] In some embodiments, the method includes a derivatization step, which includes contacting the anti-CD20 antibody protein group with PIMT and SAM. In some embodiments, the anti-CD20 antibody protein group is contacted with PIMT and SAM at a temperature sufficient to derivatize (i.e., methylate) substantially all aspartic acid residues present in the group and a time period. In some embodiments, the method optionally includes a step of quenching the derivatization reaction. In certain embodiments, the method includes a step of separating SAH by hydrophobicity to quantify isoaspartic acid. In some embodiments, separating SAH includes a chromatography comprising a hydrophobic interaction stationary phase and a reverse eluent. In some embodiments, separating SAH includes a chromatography using a C18 stationary phase. In some embodiments, the C18 stationary phase is a Hydro-RP column. In certain embodiments, the reverse eluent comprises one or more mobile phases (i.e., weakly acidic phosphate buffer and / or methanol). In certain embodiments, chromatography is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate SAH from the derivatization reaction component. In some embodiments, chromatography is performed using a chromatography system (i.e., an HPLC or UPLC system) that controls these parameters. In certain embodiments, the chromatography system is coupled to a detector for detecting SAH after derivatization (i.e., absorbance at 260nm). In some embodiments, a hydrophilic interaction stationary phase, a reverse eluent, and an RP-HPLC or UPLC system equipped with a detector for detecting the absorbance of SAH at about 260nm are used to separate and detect SAM. In some embodiments, the hydrophobic interaction stationary phase comprises Synergi Hydro-RP (4.6mm X 150mm). In some embodiments, the stationary phase (i.e., column) temperature is about 25°C or room temperature. In a specific embodiment, the flow rate is 1mL / min. In some embodiments, the reverse eluent comprises one or more mobile phases, the mobile phase comprising a first mobile phase and a second mobile phase, the first mobile phase comprising about 50mM potassium phosphate, a pH of about 6.2, and the second mobile phase comprising methanol. In some embodiments, the mobile phase gradient comprises the second mobile phase, the second mobile phase is 10.0% at 0 minutes, 40% at 7.5 minutes, 80% at 10.5 minutes, 80% at 12.5 minutes, 10% at 13.5 minutes, 10% at 20 minutes, and 10% at 25 minutes. In some embodiments, the amount of SAH is quantified by comparing its integrated curve area with a SAH standard curve. In some embodiments, the SAH standard curve is prepared using Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu.In some embodiments, the amount of asparagine deamidation or isoaspartate in a population of anti-CD20 antibody proteins is determined using a Promega isoquant kit and an Isoasp-DSIP standard according to the manufacturer's protocol.

[0448] (iii) Deamidation by LC-MS

[0449] The amount of asparagine deamidation or isoaspartic acid in a population of anti-CD20 antibody proteins or a population of digested anti-CD20 antibody proteins can be determined via a liquid chromatography coupled to mass spectrometry (LC-MS) peptide mapping method, for example, by summing the site-specific deamidation results of a population of digested anti-CD20 antibody proteins.

[0450] In some embodiments, LC-MS peptide mapping includes determining the molecular weight and / or relative abundance of peptides derived from anti-CD20 antibody protein groups digested with Lys-C to determine the presence and amount of deamidated or isoaspartic acid residues. In some embodiments, the anti-CD20 antibody protein group is reduced and alkylated before digestion. In certain embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate the peptides of Lys-C digests. In certain embodiments, the relative abundance of the peptides of Lys-C digests is calculated by integrating the area under the curve compared to the total area.

[0451] In some embodiments, the observed molecular weight is compared to a theoretical mass for a deamidated peptide selected from Table 2, Column 2. In certain embodiments, the observed molecular weight is compared to a theoretical mass selected from Table 2, Column 3.

[0452] Table 2: Peptides used to determine deamidation after Lys-C digestion

[0453]

[0454]

[0455] In some embodiments, LC-MS peptide mapping includes determining the molecular weight and / or relative abundance of peptides derived from anti-CD20 antibody protein groups digested by Asp-N to determine the presence and amount of deamidated or isoaspartic acid residues. In some embodiments, the anti-CD20 antibody protein group is reduced and alkylated before digestion. In certain embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate the peptides of Asp-N digests. In certain embodiments, the relative abundance of the peptides of Asp-N digests is calculated by integrating the area under its curve compared to the total area.

[0456] In certain embodiments, the observed molecular weight is compared to a theoretical mass for a deamidated peptide selected from Table 3, Column 2. In certain embodiments, the observed molecular weight is compared to a theoretical mass selected from Table 3, Column 3.

[0457] Table 3: Peptides used to determine deamidation after Asn-N digestion

[0458]

[0459] (d) Method for determining oxidation

[0460] (i) General oxidation

[0461] Also provided herein is a method for determining the level of methionine oxidation in an anti-CD20 antibody protein population described herein. In some embodiments, the amount of methionine oxidation in an anti-CD20 antibody protein population is the amount of Met sulfoxide (MetO) residues in the population. In some embodiments, the method includes detecting the amount of Met sulfoxide (MetO) residues in the population.

[0462] (ii) Oxidation by LC-MS

[0463] The amount of methionine oxidation or Met sulfoxide (MetO) residues in a population of anti-CD20 antibody proteins or a population of digested anti-CD20 antibody proteins can be determined via a liquid chromatography coupled to mass spectrometry (LC-MS) peptide mapping method, for example, by summing the site-specific oxidation results of a population of digested CD20 antibody proteins.

[0464] In some embodiments, LC-MS peptide mapping includes determining the molecular weight of peptides derived from anti-CD20 antibody protein groups digested by Lys-C to determine the presence and amount of oxidation or MetO residues. In some embodiments, the anti-CD20 antibody protein group is reduced and alkylated before digestion. In some embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate the peptides of Lys-C digests. In some embodiments, the relative abundance of the peptides of Lys-C digests is calculated by integrating the area under the curve compared to the total area.

[0465] In some embodiments, the observed molecular weight is compared to a theoretical mass of an oxidized peptide selected from Table 4, Column 2. In some embodiments, the observed molecular weight is compared to a theoretical mass selected from Table 4, Column 3.

[0466] Table 4: Peptides used to determine oxidation after Lys-C digestion

[0467]

[0468] In some embodiments, the anti-CD20 antibody protein group comprises methionine oxidation at one or more methionine residues present in the heavy chain. In some embodiments, the one or more methionine residues present in the heavy chain are selected from Met-20, Met-34, Met-81, Met-253, or Met-428 as shown in SEQ ID NO: 17, 18, 21, 24 or 29, respectively. In certain embodiments, the anti-CD20 antibody protein group comprises methionine oxidation at one or more methionine residues present in the light chain. In some embodiments, the one or more methionine residues present in the light chain are selected from Met-21 or Met-32 as shown in SEQ ID NO: 30.

[0469] (e) Determination of protein conformation - circular dichroism

[0470] In some embodiments, circular dichroism (CD) spectroscopy is used to analyze the secondary structure of the anti-CD20 antibody protein by measuring the difference in absorption between left-circularly polarized light and right-circularly polarized light due to structural asymmetry. CD spectroscopy uses a far ultraviolet spectrum with a wavelength of approximately 170 to 260 nm. At these wavelengths, different secondary structures commonly found in proteins can be analyzed, because alpha helices, parallel and antiparallel beta sheets, beta turns, and random coil conformations each produce a characteristic spectrum, and the spectrum of a given protein can be used to estimate its percentage content in secondary structure.

[0471] 7.6 Single batch composition

[0472] Provided herein are anti-CD20 antibody protein compositions or groups described in the scaled-up quantities in Section 7.4. In certain embodiments, these scaled-up quantities are present in a single batch, i.e., a composition derived from a single run of a single bioreactor of a specified volume. For example, an anti-CD20 antibody protein obtained from a single run of a 15,000L bioreactor can be referred to as a single batch. In certain embodiments, the capacity of the bioreactor is at least 100; 200; 300; 400; 500; 750; 1,000; 2,000; 3,000; 4,000; 5,000; 7,500; 10,000; 15,000; 20,000; or at least 25,000L. In certain embodiments, the anti-CD20 antibody protein is present in such a single batch at a concentration of at least 10 mg / mL; 15 mg / mL; 20 mg / mL; 25 mg / mL; or at least 30 mg / mL, as determined using the assay described in Section 7.6(a). In certain embodiments, the anti-CD20 antibody protein is present in such a single batch at a concentration of 10 to 35 mg / mL; 10 to 30 mg / mL; 10 to 25 mg / mL; 10 to 20 mg / mL; 10 to 15 mg / mL; 15 to 35 mg / mL; 15 to 30 mg / mL; 15 to 25 mg / mL; 15 to 20 mg / mL; 20 to 35 mg / mL; 20 to 30 mg / mL; 20 to 25 mg / mL; 25 to 35 mg / mL; or 25 to 30 mg / mL, as determined using the assay described in Section 7.6(a). In certain embodiments, the anti-CD20 antibody protein is present in such a single batch at a concentration of about 15 mg / mL; about 20 mg / mL; about 25 mg / mL; about 30 mg / mL; or about 35 mg / mL, as determined using the assay described in Section 7.6(a) (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%).

[0473] In some embodiments, the anti-CD20 antibody proteins disclosed herein are produced on a commercial scale. In certain embodiments, the commercial scale is 10,000 to 25,000 L.

[0474] (a) Total protein

[0475] In some embodiments, the amount of total protein is measured spectrophotometrically. In certain embodiments, the amount of total protein is measured by absorbance at 280 nm.

[0476] In some embodiments, the following determination procedure can be used to measure the amount of total protein. The method uses the ultraviolet absorbance measurement at 280nm and the light scattering correction at 320nm to determine the concentration of the anti-CD20 antibody protein. The calculation of concentration is based on the Beer-Lambert law. As determined by the amino acid composition, the extinction coefficient of the anti-CD20 antibody protein at 280nm is 1.61mL / mg / cm. The extinction coefficient can be used. The method is applicable to sample testing, release testing and stability testing in the process after protein A purification. The sample is diluted to a target protein concentration of 0.4mg / mL in triplicate using 0.9% sodium chloride by weight analysis. Saline is used as a blank for the instrument. Bovine serum albumin is used as a system suitability control and is tested before and after sample measurement. The sample is loaded into a quartz cuvette with a 1cm optical path and the absorbance is measured at 280 and 320nm. The sample is tested in triplicate. In certain embodiments, the amount of total protein can be measured using the key materials and equipment listed in Table 5. In certain embodiments, the amount of total protein can be measured using the protein concentration system suitability criteria listed in Table 6.

[0477] Table 5: Key materials and equipment used in protein concentration analysis

[0478] project use BSA Ensuring spectrophotometer performance UV / Vis Spectrophotometer Protein concentration readings

[0479] UV = ultraviolet light; Vis = visible light

[0480] Table 6: Protein Concentration System Suitability Criteria

[0481] System suitability type Suitability criteria Blank absorbance 0.000±0.004AU BSA concentration BSA concentration must be within 0.08 mg / mL of the COA value Sample absorbance 0.2-0.9AU CV of triplicate protein concentration measurements CV≤2.0%

[0482] AU = absorbance unit; CV = coefficient of variation

[0483] In some embodiments, the anti-CD20 antibody protein population provided herein has an amount of total protein of 25.5-25.8 mg / mL. In certain embodiments, the population has an amount of total protein of about 25.6 mg / mL.

[0484] 7.7 Biological properties

[0485] In some embodiments, the anti-CD20 antibody protein populations provided herein have the biological properties detailed in this section. In certain embodiments, the biological properties can be measured using the assays described in Section 7.7(a). The biological properties of the anti-CD20 antibody protein compositions provided herein are described in Section 7.7(b).

[0486] (a) Determination

[0487] (i) Cell-based antibody-dependent cellular cytotoxicity (ADCC)

[0488] In some embodiments, cell-based ADCC assays use Raji cells as target cells. In certain embodiments, Raji cells express CD20. In certain embodiments, cell-based ADCC assays use effector cells selected from CD16 effector cells and primary NK cells. In certain embodiments, cell-based ADCC assays use Raji cells as target cells and use KILR CD16a effector cells as effector cells. In certain embodiments, cell-based ADCC assays use Raji cells as target cells and use primary NK cells as effector cells. In certain embodiments, KILR CD16a effector cells are human CD8+T lymphocytes of a single donor origin engineered to express CD16 / FcγRIII on their plasma membrane surface.

[0489] In some embodiments, the cell-based ADCC assay uses target cell lysis as a readout. In some embodiments, target cell lysis is measured using CytoTox Glo TM (Promega) for quantification. In some embodiments, the cell-based ADCC assay shows the relative potency of the group relative to the commercial reference standard. In some embodiments, the cell-based ADCC assay produces a dose response curve and EC50 value. In some embodiments, the composition or group of anti-CD20 antibody proteins provided herein performs at more than 100% of the commercial reference standard RS-117808 in cell-based ADCC using CD16 effector cells.

[0490] In some embodiments, ADCC activity can be measured using CD16 activity assay. In some embodiments, CD16 activity assay uses alternative readings to assess ADCC activity. In some embodiments, CD16 activity assay uses WIL2-S as a target cell. In certain embodiments, the effector cell used is a stably transformed Jurkat cell line expressing a chimeric molecule, which comprises an FcγRIIIa extracellular domain connected to the transmembrane domain and intracellular domain of the γ chain of the mast cell / basophil Fc receptor for IgE. In certain embodiments, cells are merged and treated with serially diluted anti-CD20 antibodies in the presence of PMA (phorbol 12-myristate 13-acetate). In certain embodiments, the activation of effector cells induces the release of IL-2, which is measured by a commercial ELISA kit. In certain embodiments, efficacy is described as a percentage relative to a reference standard.

[0491] In some embodiments, the population has a relative potency of 106% to 126% in a CD16 activity assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 115% in a CD16 activity assay compared to a commercial reference standard. In some embodiments, the commercial reference standard is RS-117808.

[0492] In some embodiments, the cell-based ADCC assay uses the following assay procedure. Reference standards, QC reference controls and test materials (250, 50, 16.7, 5.6, 1.9, 0.6, 0.2, 0.04 pg / mL) with eight-point serial dilutions of a concentration range of 250.00 pg / mL to 0.04 pg / mL are used. Two independent material preparations are prepared and assayed on duplicate plates. Assay controls are prepared in triplicate and include: single target cell controls, target cell death controls, single effector cell controls, and effector cell and target cell controls. KILR cells are obtained from Eurofins. They come from a master cell bank made from a unique human donor. The master cell bank and working cell bank system and the identification protocol allow suppliers to create thousands of vials in a reproducible manner, and TG maintains a safe supply of this key reagent. Procedures such as the feed supplement identification process ensure that highly reproducible cells are used in each assay. KILR cells were thawed and cultured at 1x10^6 cells / mL in medium supplemented with IL-2 and allowed to stand for at least 6 days before use and should not be used more than 14 days after thawing. For the KILR ADCC assay, Raji cells, which were also managed through the master cell bank and working cell bank system, were cultured at 1x10^6 cells / mL. 5 The KILR effector cells were seeded at 5x10 cells / mL and the reference standard, internal assay control, and sample dilutions were added to the Raji cells. 5 Cells / mL were seeded to achieve a final effect:target (E:T) ratio of 5:1. Cells were cultured at 36±1°C, 5±1% CO2 for 18 to 22 hours. At the end of the incubation, CytoTox GLo TM Preparations were made and the plates were incubated for 30 ± 10 minutes. The plates were read using a SpectraMax plate reader. Data were analyzed using SoftMax Pro using a 4-parameter logistic fit with the aid of weighted nonlinear regression. The efficacy of the resulting data was evaluated relative to a reference standard using SoftMax Pro software. Representative reference standards and sample dose response curves are shown. Results are described in percent efficacy relative to the primary reference standard or its derivatives.

[0493] In some embodiments, cell-based ADCC determinations are used as potency determinations for batch release. In some embodiments, cell-based ADCC determinations are used as potency determinations for stability testing. In some embodiments, cell-based ADCC determinations are used as potency determinations for manufacturing quality control and process. In some embodiments, cell-based ADCC determinations are used for comparative studies (e.g., in research and development or clinical studies).

[0494] In some embodiments, the higher potency of the population in a cell-based ADCC assay is associated with a lower level of fucose content in the N-glycan profile of the population. In some embodiments, the higher potency of the population in a cell-based ADCC assay is due to a lower level of fucose content in the N-glycan profile of the population.

[0495] (ii) Antibody-dependent cellular phagocytosis (ADCP)

[0496] In some embodiments, Daudi cells expressing CD20 can be used as target cells (labeled by PKH26) to measure antibody-dependent cellular phagocytosis (ADCP) activity. In some embodiments, human monocytes are separated from PBMC and differentiated in vitro using GM-CSF to produce macrophages. In some embodiments, macrophages are co-cultured with target cells labeled with PKH26 (previously incubated with serially diluted anti-CD20 antibody samples). In some embodiments, target cell phagocytosis is assessed by flow cytometry. In some embodiments, the dose-response curve of the sample and the calculated IC50 can be displayed.

[0497] (iii) Complement-dependent cytotoxicity (CDC)

[0498] In some embodiments, cell-based CDC assays use Jeko-1 cells (as target cells) and rabbit complement system. In certain embodiments, cell-based CDC assays use Raji cells (as target cells) and human complement system. In some embodiments, cell-based CDC assays use target cell lysis as readout.

[0499] In some embodiments, the cell-based CDC assay uses the following assay procedure. A nine-point serial dilution of reference standard, QC reference control, and test material (10,000.00, 3333.33, 1666.67, 833.33, 416.67, 208.33, 104.17, 52.08, 10.42 ng / mL) is used at a concentration range of 10,000 ng / mL to 10.42 ng / mL. Two independent material preparations are prepared and assayed on duplicate plates. Assay negative controls are prepared in triplicate and include target cell and complement controls as well as target cell controls alone. In this assay, 3x10 5 Jeko-1 cells (obtained from ATCC and maintained by the master library system) were inoculated with 10 cells / mL and incubated for 60 to 90 minutes. Reference standards, internal assay controls and samples diluted in duplicate in cell culture medium were added. Complement was then added to the wells, and the plate was incubated at 36°C ± 1°C for approximately 2 hours, followed by incubation at room temperature for 25 ± 5 minutes. Controls included target cells plus complement controls and target cell controls only to provide a basal level of target cell viability during the assay. Cell Titer-Glo reagent was then added and incubated at room temperature for another 30 ± 10 minutes. At the end of the assay, the plate was read using a SpectraMax M5 plate reader. Using SoftMax Pro, the data were analyzed using a 4-parameter logistic fit by means of weighted nonlinear regression. The efficacy of the obtained data was evaluated using SoftmaxPro software relative to the reference standard. The results are described in % efficacy relative to the reference standard. Representative reference standards and sample dose response curves are shown.

[0500] (iv) CD20 binding activity

[0501] In some embodiments, the cell-based CD20 binding assay uses Jeko-1 cells and an MSD assay. In some embodiments, the cell-based CD20 binding assay generates a dose-dependent binding curve and an EC50 value.

[0502] In some embodiments, the cell-based CD20 binding assay uses the following assay procedure. An eight-point serial dilution of reference standard, QC reference control, and test material is prepared at a concentration range of 40,000.00 ug / mL to 0.2 ng / mL (40,000.00, 4,000.00, 1,000.00, 333.30, 111.10, 37.00, 4.60, 0.23 ng / mL). Two independent test material preparations are prepared for each 2-plate test material evaluation. Assay controls include a no cell control (reference standard / test material dilution + detection reagent, cells omitted) and a cell-only control (cells + detection reagent, reference standard / test material omitted). Jeko-1 cells are plated at 3x10 5 Cells / mL were seeded onto MSD high binding plates in PBS in a final volume of 100 μL per well and incubated at 35 to 37°C for 2 hours ± 10 minutes. Unbound cells were removed by washing with PBS and the plates were blocked with 45% FBS. 50 μL of reference standard, QC reference control or test material dilution was added and the plates were incubated with shaking at room temperature for 1 hour ± 10 minutes. After incubation and three PBS washes, 50 μL of anti-human Fc detection antibody conjugated with STREP-SULFOTAG was added and incubated with shaking at room temperature for 1 hour ± 5 minutes. The plates were washed again with PBS and 150 μL of MesoScale reading buffer containing tripropylamine (TPA) was added as a co-reactant to generate light for electrochemiluminescence readings. The plates were read immediately on an MSD plate reader using Workbench 4.0. The resulting data were evaluated using PLA software and analyzed using a restricted 4-parameter logistic model to generate relative binding, 95% confidence intervals, and results relative to the reference standard. Binding activity results are reported as a percentage of potency relative to a reference standard. Relative potency test results are calculated from representative reference standards and test sample dose response curves.

[0503] (v) FcγRIIIa 158V and FcγRIIIa 158F binding activity

[0504] In some embodiments, the FcγRIIIa binding assay uses surface plasmon resonance (SPR). In certain embodiments, the FcγRIIIa binding assay produces a sensorgram showing dose-dependent binding, saturation and dissociation. In certain embodiments, the FcγRIIIa binding assay is calculated by association and dissociation rates and steady-state kinetics to calculate the dissociation constant. In certain embodiments, the binding affinity of the group to FcγRIIIa 158V is about one order of magnitude higher than the binding affinity of the group to FcγRIIIa 158F. In certain embodiments, the binding affinity of the group to FcγRIIIa 158V is significantly higher than Rituxan. In certain embodiments, the binding affinity of the group to FcγRIIIa 158F is significantly higher than Rituxan. In certain embodiments, the binding affinity of the group to both FcγRIIIa 158V and FcγRIIIa 158F is significantly higher than Rituxan.

[0505] In some embodiments, the FcγRIIIa binding assay uses the following assay procedure. The FcγRIIIa 158V receptor (1.2 μg / mL) is immobilized on the chip surface using covalent amine coupling chemistry. Eight-point serial dilutions of reference standards, QC reference controls, and test materials are prepared at a concentration range of 2000nM to 15.6nM, with a dilution factor of 2. Independent replicates of sample dilutions are injected onto the chip, and the surface is regenerated between each cycle. Binding is measured in response units (RU). The kinetics of the binding reaction are determined by measuring the SPR change caused by the increase in mass close to the surface of the biosensor chip. The change in mass of the complex over time is visualized as a sensorgram. The equilibrium dissociation constant (K) of each sample for each receptor relative to the reference standard is determined. D ) and relative affinities. The rate of change of the SPR signal was analyzed for the FcγRIIIa 158V variant using a 1:1 Langmuir model to generate apparent rate constants for the association and dissociation phases of the reaction as well as the equilibrium dissociation constant. K D The steady state affinity of the FcγRIIIa 158F variant was determined. The binding signal was exported to PLA to determine the relative binding response. The results are reported as % potency for each FcγRIIIa variant (158V and 158F) relative to the reference standard.

[0506] (vi) C1q binding activity

[0507] In some embodiments, C1q binding activity can be measured using ELISA. In certain embodiments, C1q binding activity can be measured using the following assay procedure. A seven-point serial dilution of reference standards, QC reference controls, and test materials is prepared at a concentration range of 15.00 μg / mL to 0.12 μg / mL. The reference standards, QC reference controls, and test material dilutions are coated on an ELISA plate, and the plate is incubated at room temperature for 1 hour ± 30 minutes (shaking 150 to 200 rpm). After coating, the plate is washed (3 times with PBS / 0.05% Tween), blocked (with 1% BSA and incubated at room temperature for 1 hour ± 10 minutes and shaking at 150 to 200 rpm), and washed (3 times with PBS / 0.05% Tween). C1q conjugated with peroxidase is then added, and the plate is incubated at room temperature for 1.5 hours ± 30 minutes (shaking at 150 to 200 rpm). After incubation and washing, tetramethylbenzidine (TMB) substrate solution was added and the plates were incubated at room temperature for 7 minutes (-1 minute / +30 seconds). This produced a colorimetric reaction proportional to the level of C1q binding. The reaction was terminated by adding 1M sulfuric acid and the color was measured at 450nm using a Molecular Devices SpectraMax microplate reader. The data were analyzed using SoftMax Pro with the aid of weighted nonlinear regression using a 4-parameter logistic fit. The data were evaluated for potency relative to a reference standard using SoftMax Pro software. C1q binding activity results are reported as a percentage of potency relative to a reference standard. A representative dose response curve for one assay can be shown.

[0508] (vii) B cell depletion activity

[0509] In some embodiments, B cell depletion activity can be measured in human whole blood B cell depletion assay. In certain embodiments, B cell depletion activity can be measured in autologous normal human whole blood B cell depletion assay. In certain embodiments, B cell depletion can be measured by displaying cells in CD45 positive lymphocyte gate and counting CD3 positive T cells, CD19 positive B cells and CD20 positive B cells. In certain embodiments, B cell depletion percentage (100-([100 / B- / T- cell ratio in sample without antibody] x [B- / T- cell ratio in sample containing antibody])) can be calculated and plotted relative to sample concentration.

[0510] In some embodiments, B cell depletion activity can be measured using blood from three healthy donors. In certain embodiments, B cell depletion can be measured by displaying cells in a CD45 positive lymphocyte gate and counting CD3 positive T cells, CD19 positive B cells, and CD20 positive B cells.

[0511] (b) Biological properties

[0512] In some embodiments, the biological properties of the anti-CD20 antibody protein compositions or populations provided herein can be measured and described in the assays described in Section 7.7(a) and using comparisons to reference standards. In certain embodiments, the reference standard is a commercial reference standard.

[0513] In some embodiments, the reference standard is an anti-CD20 antibody. In certain embodiments, the reference standard is GAZYVA (Obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab).

[0514] In some embodiments, the commercial reference standard is RS-117808. In certain embodiments, the anti-CD20 antibody protein population provided herein has the biological properties as shown in Table 7. RS-117808 ("antibody Utuximab (TG-1101)") was deposited at the American Type Culture Collection (ATCC) under the terms of the Budapest Treaty, located at 10801 University Boulevard, Manassas, VA 20110, and was received by ATCC on April 15, 2022, and assigned informal patent deposit number PTA-127294.

[0515] Table 7: Biological properties compared with reference standards

[0516]

[0517] (i) Cell-based antibody-dependent cellular cytotoxicity (ADCC)

[0518] In some embodiments, the compositions or populations of anti-CD20 antibody proteins provided herein exhibit a relative potency in cell-based ADCC using a CD16 effector cell assay (see Section 7.7(a)(i)) of at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% of the commercial reference standard RS-117808. In certain embodiments, the compositions or populations of anti-CD20 antibody proteins provided herein exhibit a relative potency in cell-based ADCC using a CD16 effector cell assay (see Section 7.7(a)(i)) of the commercially available (Genentech / Biogen).

[0519] In some embodiments, the population induces cytotoxicity in a cell-based antibody-dependent cellular cytotoxicity (ADCC) assay. In certain embodiments, the population induces greater cytotoxicity in an ADCC assay compared to a commercial reference standard.

[0520] In some embodiments, compared with anti-CD20 antibodies, the group induces greater cytotoxicity in ADCC assays. In certain embodiments, compared with GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and / or OCREVUS (ocrezumab), the group induces greater cytotoxicity in ADCC assays. In certain embodiments, compared with GAZYVA (obinutuzumab), the group induces greater cytotoxicity in ADCC assays. In certain embodiments, compared with ARZERRA (ofatumumab), the group induces greater cytotoxicity in ADCC assays. In certain embodiments, compared with RITUXAN (rituximab), the group induces greater cytotoxicity in ADCC assays. In certain embodiments, compared with veltuzumab (IMMU-106), the group induces greater cytotoxicity in ADCC assays. In certain embodiments, the population induces greater cytotoxicity in an ADCC assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population induces greater cytotoxicity in an ADCC assay compared to OCREVUS (ocrelizumab).

[0521] In some embodiments, the group has at least 1000%, 750%, 500%, 250%, 100%, 75%, 50% or at least 25% relative efficacy in cell-based ADCC assays compared to commercial reference standards. In certain embodiments, the group has at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5 times, 10 times, 15 times, 20 times, 30 times, 50 times, 75 times, 100 times, 125 times, 150 times or at least 200 times relative efficacy in cell-based ADCC assays compared to commercial reference standards. In certain embodiments, the group has at least more than 100% relative efficacy in cell-based ADCC assays compared to commercial reference standards. In certain embodiments, the population has a relative potency of greater than 100% in a cell-based ADCC assay compared to a commercial reference standard.

[0522] In some embodiments, the population has a relative potency in a cell-based ADCC assay of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in a cell-based ADCC assay of 5% to 50%, 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold compared to a commercial reference standard.

[0523] In some embodiments, the commercial reference standard is an anti-CD20 antibody. In certain embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and / or OCREVUS (ocrezumab). In certain embodiments, compared with GAZYVA (obinutuzumab), the group has a relative efficacy of about 38% to about 3 times in a cell-based ADCC assay. In certain embodiments, compared with ARZERRA (ofatumumab), the group has a relative efficacy of about 10 times to about 31 times in a cell-based ADCC assay. In certain embodiments, compared with RITUXAN (rituximab), the group has a relative efficacy of about 28 times to about 2250 times in a cell-based ADCC assay. In certain embodiments, the population has about 25-fold greater relative potency in a cell-based ADCC assay compared to OCREVUS (ocrelizumab).

[0524] In some embodiments, the group has a relative efficacy of 60 to 200%, 70 to 190%, 80% to 180%, 85 to 170%, or 90 to 163% in a cell-based ADCC assay compared to a commercial reference standard. In certain embodiments, the group has a relative efficacy of 90 to 163% in a cell-based ADCC assay compared to a commercial reference standard. In certain embodiments, the group has a relative efficacy of about 117% in a cell-based ADCC assay compared to a commercial reference standard. In certain embodiments, the commercial reference standard is RS-117808.

[0525] In some embodiments, the EC50 value of the ADCC effectiveness measured in the ADCC assay based on cell is between 2 and 6pg / mL.In certain embodiments, the EC50 value of the ADCC effectiveness measured in the ADCC assay based on cell is between 0.2 to 20pg / mL, 0.3 to 18pg / mL, 0.4 to 15pg / mL, 0.5 to 12pg / mL, 0.6 to 10pg / mL, 0.7 to 9pg / mL, 0.8 to 8pg / mL, 0.9 to 7pg / mL or 1 to 6pg / mL.In certain embodiments, the EC50 value of ADCC effectiveness is the average EC50 value calculated from the EC50 value obtained in the ADCC assay based on cell for twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, thirty times, forty times, fifty times, sixty times or more times of repetition. In certain embodiments, the EC50 value of the ADCC potency of the population is about 5.45 pg / mL as measured in a cell-based ADCC assay (e.g., using Eurofins / DiscoverX KILR CD16a effector cells). In certain embodiments, the EC50 value of the ADCC potency of the population is about 2.42 pg / mL as measured in a cell-based ADCC assay (e.g., using Eurofins / DiscoverX KILR CD16a effector cells).

[0526] (ii) Antibody-dependent cellular phagocytosis (ADCP)

[0527] In some embodiments, the group induces antibody-dependent cellular phagocytosis (ADCP). In certain embodiments, the group induces greater phagocytosis in an ADCP assay compared to a commercial reference standard. In certain embodiments, the group induces greater phagocytosis in an ADCP assay compared to an anti-CD20 antibody.

[0528] In some embodiments, the group induces greater phagocytosis in the ADCP assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and / or OCREVUS (ocrelizumab). In certain embodiments, the group induces phagocytosis in the ADCP assay compared to GAZYVA (obinutuzumab). In certain embodiments, the group induces greater cytotoxicity in the CDC assay compared to ARZERRA (ofatumumab). In certain embodiments, the group induces greater phagocytosis in the ADCP assay compared to RITUXAN (rituximab). In certain embodiments, the group induces greater cytotoxicity in the CDC assay compared to veltuzumab (IMMU-106). In certain embodiments, the group induces greater phagocytosis in the ADCP assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population induces greater phagocytosis in an ADCP assay compared to OCREVUS (ocrelizumab).

[0529] In some embodiments, compared with commercial reference standards, the group has at least 1000%, 750%, 500%, 250%, 100%, 75%, 50% or at least 25% relative efficacy in ADCP determination. In certain embodiments, compared with commercial reference standards, the group has at least 150%, 160%, 170%, 180%, 190%, 200%, 5 times, 10 times, 15 times, 20 times, 30 times, 50 times, 75 times, 100 times, 125 times, 150 times or at least 200 times relative efficacy in ADCP determination. In certain embodiments, compared with commercial reference standards, the group has at least more than 100% relative efficacy in ADCP determination. In certain embodiments, compared with commercial reference standards, the group has more than 100% relative efficacy in ADCP determination.

[0530] In some embodiments, the population has a relative potency in an ADCP assay that is at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in an ADCP assay of 5% to 50%, 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold compared to a commercial reference standard.

[0531] In some embodiments, the commercial reference standard is an anti-CD20 antibody. In certain embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrelizumab). In certain embodiments, compared with ARZERRA (ofatumumab), the group has about 7 times the relative efficacy in the ADCP assay. In certain embodiments, compared with RITUXAN (rituximab), the group has about 15 times the relative efficacy in the ADCP assay. In certain embodiments, compared with GAZYVA (obinutuzumab), the group has about 23 times the relative efficacy in the ADCP assay.

[0532] In some embodiments, the EC50 value of the ADCP efficacy measured in the ADCP assay of the group is 0.1 to 1 ng / mL. In certain embodiments, the EC50 value of the ADCP efficacy measured in the ADCP assay of the group is 1 to 10, 2 to 9, 3 to 8, 4 to 7 or 5 to 6 ng / mL. In certain embodiments, the EC50 value of the ADCP efficacy measured in the ADCP assay of the group is 0.05 to 20 ng / mL, 0.1 to 19 ng / mL, 0.15 to 18 ng / mL, 0.2 to 18 ng / mL, 0.25 to 15 ng / mL, 0.3 to 12 ng / mL, 0.3 to 10 ng / mL, 0.3 to 9 ng / mL, 0.3 to 8 ng / mL, 0.3 to 7 ng / mL, or 0.3 to 6 ng / mL. In certain embodiments, the EC50 value of ADCP potency is the average EC50 value calculated from the EC50 values ​​obtained in two, three, four, five, six, seven, eight, nine, ten or more repeated ADCP assays. In certain embodiments, the EC50 value of ADCP potency as measured in the ADCP assay of the population is about 5.50 ng / mL.

[0533] (iii) Complement-dependent cytotoxicity (CDC)

[0534] In some embodiments, the group induces complement dependent cytotoxicity (CDC). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to a commercial reference standard. In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to an anti-CD20 antibody.

[0535] In some embodiments, the group induces greater cytotoxicity in a CDC assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and / or OCREVUS (ocrelizumab). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to GAZYVA (obinutuzumab). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to ARZERRA (ofatumumab). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to RITUXAN (rituximab). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to veltuzumab (IMMU-106). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population induces greater cytotoxicity in a CDC assay compared to OCREVUS (ocrelizumab).

[0536] In some embodiments, the group has at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, 25%, 12% or at least 5% relative efficacy in cell-based CDC assays compared to commercial reference standards. In certain embodiments, the group has at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5 times, 10 times, 15 times, 20 times, 30 times, 50 times, 75 times, 100 times, 125 times, 150 times or at least 200 times relative efficacy in cell-based CDC assays compared to commercial reference standards. In certain embodiments, the group has at least more than 100% relative efficacy in cell-based CDC assays compared to commercial reference standards. In certain embodiments, the population has a relative potency of greater than 100% in a cell-based CDC assay compared to a commercial reference standard.

[0537] In some embodiments, the population has a relative potency in a cell-based CDC assay of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in a cell-based CDC assay of 5% to 50%, 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold compared to a commercial reference standard.

[0538] In some embodiments, the commercial reference standard is an anti-CD20 antibody. In certain embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrezumab). In certain embodiments, compared with ARZERRA (ofatumumab), the group has a relative efficacy of about 50% in a cell-based CDC assay. In certain embodiments, compared with RITUXAN (rituximab), the group has a relative efficacy of about 37% in a cell-based CDC assay. In certain embodiments, compared with OCREVU (ocrezumab), the group has a relative efficacy of about 1.8 times in a cell-based CDC assay.

[0539] In some embodiments, the group has a relative efficacy of 50 to 150%, 60 to 140%, 70 to 130%, 75 to 120%, or 78 to 116% in a cell-based CDC assay compared to a commercial reference standard. In certain embodiments, the group has a relative efficacy of 78 to 116% in a cell-based CDC assay compared to a commercial reference standard. In certain embodiments, the group has a relative efficacy of 73 to 128% or 74 to 127% in a cell-based CDC assay compared to a commercial reference standard. In certain embodiments, the group has a relative efficacy of about 91% in a cell-based CDC assay compared to a commercial reference standard. In certain embodiments, the commercial reference standard is RS-117808.

[0540] In some embodiments, the EC50 value of the CDC effectiveness measured in the CDC assay based on cell is 0.4 to 0.7 μ g / mL or in 0.4 to 0.6 μ g / mL. In certain embodiments, the EC50 value of the CDC effectiveness measured in the CDC assay based on cell is 0.05 to 5 μ g / mL, 0.1 to 4 μ g / mL, 0.15 to 3 μ g / mL, 0.2 to 2 μ g / mL, 0.25 to 1 μ g / mL, 0.3 to 0.9 μ g / mL, 0.3 to 0.8 μ g / mL or 0.3 to 0.7 μ g / mL. In certain embodiments, the EC50 value of CDC effectiveness is the average EC50 value calculated from the EC50 value obtained in the CDC assay based on cell repeated twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. In certain embodiments, the population has an EC50 value for CDC potency of about 0.5 μg / mL as measured in a cell-based CDC assay.

[0541] (iv) CD20 binding activity

[0542] In some embodiments, the population has CD20 binding activity in a cell-based CD20 binding assay. In certain embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay compared to a commercial reference standard.

[0543] In some embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay compared to an anti-CD20 antibody. In certain embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and / or OCREVUS (ocrelizumab). In certain embodiments, the population has greater CD20 binding activity compared to GAZYVA (obinutuzumab). In certain embodiments, the population has greater CD20 binding activity compared to ARZERRA (ofatumumab). In certain embodiments, the population has greater CD20 binding activity compared to RITUXAN (rituximab). In certain embodiments, the population has greater CD20 binding activity compared to veltuzumab (IMMU-106). In certain embodiments, the population has greater CD20 binding activity compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population has greater CD20 binding activity compared to OCREVUS (ocrelizumab).

[0544] In some embodiments, the population has a relative potency of at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, 25%, 12%, or at least 5% in a CD20 binding assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least greater than 100% in CD20 binding compared to a commercial reference standard. In certain embodiments, the population has a relative potency of greater than 100% in CD20 binding compared to a commercial reference standard.

[0545] In some embodiments, the population has a relative potency in a CD20 binding assay that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in a CD20 binding assay of 5% to 50%, 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold compared to a commercial reference standard.

[0546] In some embodiments, the commercial reference standard is an anti-CD20 antibody. In certain embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrelizumab). In certain embodiments, the population has a relative potency of about 1.6 times to about 5.8 times in a CD20 binding assay compared to GAZYVA (obinutuzumab). In certain embodiments, the population has a relative potency of about 81% to about 4.3 times in a CD20 binding assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has a relative potency of about 1.6 times to about 4.1 times in a CD20 binding assay compared to RITUXAN (rituximab).

[0547] In some embodiments, the population has a relative potency of 50 to 150%, 60 to 140%, 70 to 130%, 80 to 120%, 90 to 120%, or 92 to 118% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 92 to 118% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 82 to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of about 109% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In certain embodiments, the commercial reference standard is RS-117808.

[0548] In some embodiments, the EC50 value of the CD20 binding potency of the population as measured in a cell-based CD20 binding activity bioassay is 0.05 to 0.1 μg / mL. In certain embodiments, the EC50 value of the CD20 binding potency of the population as measured in a cell-based CD20 binding activity bioassay is 0.01 to 0.5 μg / mL, 0.02 to 0.4 μg / mL, 0.03 to 0.3 μg / mL, 0.04 to 0.2 μg / mL, or 0.05 to 0.1 μg / mL. In certain embodiments, the EC50 value of the CD20 binding potency is the average EC50 value calculated from the EC50 values ​​obtained in two, three, four, five, six, seven, eight, nine, ten or more repeated cell-based CD20 binding activity bioassays. In certain embodiments, the EC50 value of the CD20 binding potency of the population as measured in a cell-based CD20 binding activity bioassay is about 0.093 μg / mL. In certain embodiments, the population has an EC50 value of about 0.063 μg / mL for CD20 binding potency as measured in a cell-based CD20 binding activity bioassay.

[0549] (v) FcγRIIIa 158V and FcγRIIIa 158F binding activity

[0550] In some embodiments, the population has FcγRIIIa 158V binding activity in an FcγRIIIa binding assay. In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to a commercial reference standard.

[0551] In some embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to an anti-CD20 antibody. In certain embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and / or OCREVUS (ocrelizumab). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to GAZYVA (obinutuzumab). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to veltuzumab (IMMU-106). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to OCREVUS (ocrelizumab).

[0552] In some embodiments, the population has at least 1000%, 750%, 500%, 250%, 100%, 75%, or at least 50% relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has at least greater than 100% relative FcγRIIIa 158V binding activity in a cell-based ADCC assay compared to a commercial reference standard. In certain embodiments, the population has greater than 100% relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to a commercial reference standard.

[0553] In some embodiments, the population has at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has at least 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold greater relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard.

[0554] In some embodiments, the commercial reference standard is an anti-CD20 antibody. In certain embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrelizumab). In certain embodiments, compared with GAZYVA (obinutuzumab), the group has about 3.8 times the relative FcγRIIIa158V binding activity in the FcγRIIIa binding assay. In certain embodiments, compared with ARZERRA (ofatumumab), the group has about 25.6 times the relative FcγRIIIa 158V binding activity in the FcγRIIIa binding assay. In certain embodiments, the population has about 18.7-fold greater relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has about 16-fold greater relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to OCREVUS (ocrelizumab).

[0555] In some embodiments, the population has FcγRIIIa 158F binding activity in an FcγRIIIa binding assay. In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to a commercial reference standard. In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to an anti-CD20 antibody. In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to an anti-CD20 antibody. In certain embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) and / or OCREVUS (ocrelizumab). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to GAZYVA (obinutuzumab). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to veltuzumab (IMMU-106). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to ZEVALIN (ibritumumab tiuxetan). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to OCREVUS (ocrelizumab).

[0556] In some embodiments, the population has at least 1000%, 750%, 500%, 250%, 100%, 75%, or at least 50% relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has at least greater than 100% relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to a commercial reference standard. In certain embodiments, the population has greater than 100% relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to a commercial reference standard.

[0557] In some embodiments, the population has at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has at least 5% to 50%, 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold greater relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard.

[0558] In certain embodiments, the commercial reference standard is an anti-CD20 antibody. In certain embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrelizumab). In certain embodiments, compared with GAZYVA (obinutuzumab), the group has about 2.6 times the relative FcγRIIIa158F binding activity in the FcγRIIIa binding assay. In certain embodiments, compared with ARZERRA (ofatumumab), the group has about 21.8 times the relative FcγRIIIa 158F binding activity in the FcγRIIIa binding assay. In certain embodiments, the population has about 10.2 times the relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has about 9.9 times the relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to OCREVUS (ocrelizumab). In certain embodiments, the population has a significantly higher binding affinity for FcγRIIIa 158V or FcγRIIIa158F than rituximab.

[0559] In some embodiments, the population has a relative potency of 20 to 300%, 30 to 250%, 40 to 220%, or 50 to 200% in a cell-based FcγRIIIa binding assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 50 to 200% in a cell-based FcγRIIIa binding assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 76 to 130% or 82 to 130% in a cell-based FcγRIIIa 158V binding assay compared to a commercial reference standard. In certain embodiments, the commercial reference standard is RS-117808.

[0560] In some embodiments, the population has a K of 55 to 70 nM as measured in an FcγRIIIa-158V binding assay by surface plasmon resonance. D In certain embodiments, the population has a K of 10 to 100 nM, 15 to 90 nM, 10 to 80 nM, or 30 to 70 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance. D In certain embodiments, the population has a K of 30 to 70 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance. DIn certain embodiments, K D The K values ​​are obtained from FcγRIIIa-158V binding assays repeated two, three, four, five, six, seven, eight, nine, ten or more times. D The average K value is calculated D In certain embodiments, the population has a K of about 59 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance. D In certain embodiments, the population has a K of about 64.1 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance. D value.

[0561] In some embodiments, the population has a K of 600 to 800 nM in an FcγRIIIa158F binding assay as measured by surface plasmon resonance. D In certain embodiments, the population has a K of 100 to 2000 nM, 200 to 1800 nM, 300 to 1700 nM, 400 to 1600 nM, 500 to 1500 nM, 500 to 1200 nM, 600 to 1000 nM, or 600 to 800 nM in an FcγRIIIa 158F binding assay as measured by surface plasmon resonance. D In certain embodiments, the population has a K of 500 to 1000 nM in an FcγRIIIa 158F binding assay as measured by surface plasmon resonance. D In certain embodiments, K D The K values ​​are obtained from FcγRIIIa-158V binding assays repeated two, three, four, five, six, seven, eight, nine, ten or more times. D The average K value is calculated D In certain embodiments, the population has a K of 760 nM in an FcγRIIIa 158F binding assay as measured by surface plasmon resonance. D In certain embodiments, the population has a K of 680.3 nM in an FcγRIIIa 158F binding assay as measured by surface plasmon resonance. D value.

[0562] (vi) C1q binding activity

[0563] In some embodiments, the population has CIq binding activity as measured by ELISA.In certain embodiments, the population has greater CIq binding activity as measured by ELISA compared to a commercial reference standard.

[0564] In some embodiments, the population has greater C1q binding activity as measured by ELISA compared to an anti-CD20 antibody. In certain embodiments, the population has greater C1q binding activity as measured by ELISA compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and / or OCREVUS (ocrelizumab). In certain embodiments, compared to GAZYVA (obinutuzumab), there is greater C1q binding activity as measured by ELISA. In certain embodiments, compared to ARZERRA (ofatumumab), there is greater C1q binding activity as measured by ELISA. In certain embodiments, compared to RITUXAN (rituximab), there is greater C1q binding activity as measured by ELISA. In certain embodiments, the population has greater C1q binding activity as measured by ELISA compared to veltuzumab (IMMU-106). In certain embodiments, the population has greater C1q binding activity as measured by ELISA compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population has greater C1q binding activity as measured by ELISA compared to OCREVUS (ocrelizumab).

[0565] In some embodiments, the population has a relative potency of at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, or at least 25% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least greater than 100% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of greater than 100% in a CIq binding assay as measured by ELISA compared to a commercial reference standard.

[0566] In some embodiments, the population has a relative potency in a CIq binding assay as measured by ELISA of at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in a C1q binding assay as measured by ELISA of 5% to 50%, 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold as compared to a commercial reference standard.

[0567] In some embodiments, the commercial reference standard is an anti-CD20 antibody. In certain embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrelizumab). In certain embodiments, the population has a relative potency of about 142% as measured by ELISA in a C1q binding assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has a relative potency of about 123% as measured by ELISA in a C1q binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has a relative potency of about 112% as measured by ELISA in a C1q binding assay compared to OCREVUS (ocrelizumab).

[0568] In some embodiments, the population has a relative potency of 30 to 180%, 40 to 170%, 50 to 160%, 60 to 150%, 70 to 140%, 80 to 130%, 85 to 120%, or 88 to 113% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 88% to 113% or 86% to 117% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 86 to 116% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of about 99% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the commercial reference standard is RS-117808.

[0569] In some embodiments, the EC50 value of the C1q binding activity of the population as measured by ELISA is 1.5 to 3 μg / mL. In certain embodiments, the EC50 value of the C1q binding activity of the population as measured by ELISA is 0.2 to 9 μg / mL, 0.3 to 8 μg / mL, 0.4 to 7 μg / mL, 0.5 to 6 μg / mL, 0.6 to 5 μg / mL, 0.7 to 4 μg / mL, 0.8 to 3 μg / mL, 0.9 to 2.9 μg / mL, or 1 to 2.8 μg / mL. In certain embodiments, the EC50 value of the C1q binding activity is the average EC50 value calculated from the EC50 values ​​obtained in two, three, four, five, six, seven, eight, nine, ten or more repeated ELISA experiments. In certain embodiments, the EC50 value of the C1q binding activity of the population as measured by ELISA is about 1.92 μg / mL. In certain embodiments, the EC50 value for C1q binding activity of the population as measured by ELISA is about 2.6 μg / mL.

[0570] (vii) B cell depletion activity

[0571] In some embodiments, the population has B cell depletion activity as measured in a human whole blood B cell depletion assay. In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to a commercial reference standard.

[0572] In some embodiments, compared with anti-CD20 antibodies, the group has greater B cell depletion activity as measured in a human whole blood B cell depletion assay. In certain embodiments, compared with GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and / or OCREVUS (ocrelizumab), the group has greater B cell depletion activity as measured in a human whole blood B cell depletion assay. In certain embodiments, compared with GAZYVA (obinutuzumab), the group has greater B cell depletion activity as measured in a human whole blood B cell depletion assay. In certain embodiments, compared with ARZERRA (ofatumumab), the group has greater B cell depletion activity as measured in a human whole blood B cell depletion assay. In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to RITUXAN (rituximab). In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to veltuzumab (IMMU-106). In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to OCREVUS (ocrelizumab).

[0573] In some embodiments, the population has at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, 25%, 12% or at least 5% relative potency in a human whole blood B cell depletion assay compared to a commercial reference standard. In certain embodiments, the population has at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold or at least 200-fold relative potency in a human whole blood B cell depletion assay compared to a commercial reference standard. In certain embodiments, the population has at least more than 100% relative potency in a human whole blood B cell depletion assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of greater than 100% in a human whole blood B cell depletion assay compared to a commercial reference standard.

[0574] In some embodiments, the population has a relative potency in a human whole blood B cell depletion assay of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in a human whole blood B cell depletion assay of 5% to 50%, 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold greater than a commercial reference standard.

[0575] In certain embodiments, the commercial reference standard is an anti-CD20 antibody. In certain embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan), or OCREVUS (ocrelizumab).

[0576] 7.8 Methods of treating and preventing medical conditions

[0577] Treatments for multiple sclerosis

[0578] Provided herein is a method for treating a relapsing form of multiple sclerosis (RMS) in a subject in need thereof by administering to the subject an effective amount of an anti-CD20 antibody composition as described herein. In some embodiments, the relapsing form of multiple sclerosis (RMS) is selected from clinically isolated syndrome ("CIS"); relapsing-remitting type ("RRMS"); or active secondary progressive disease ("SPMS"). In some embodiments, RMS is CIS. In some embodiments, RMS is RRMS. In some embodiments, RMS is SPMS. In some embodiments, the subject is diagnosed with RMS according to McDonald Criteria (2010) or by other suitable methods known to those skilled in the art.

[0579] In some embodiments, the method includes administering an anti-CD20 antibody composition as described herein to a subject with a multiple dosing regimen. In certain embodiments, the method includes administering an anti-CD20 antibody composition as described herein to a subject by intravenous infusion. For example, an anti-CD20 antibody composition as described herein can be administered to a subject by intravenous infusion with a multiple infusion dosing regimen. An anti-CD20 antibody composition as described herein can be administered to a subject by intravenous infusion with a multiple infusion dosing regimen for 48 weeks. Additionally or as an alternative, an anti-CD20 antibody composition as described herein can be administered to a subject by intravenous infusion with a multiple infusion dosing regimen for 96 weeks. In some embodiments, a multiple infusion dosing regimen comprises the first, second, and subsequent intravenous infusions of Utuximab. In some embodiments, the "subsequent infusion" of Utuximab is an infusion of any number of times after the second infusion.

[0580] In some embodiments, the intravenous infusion comprises a multiple dosing regimen (e.g., a multiple infusion dosing regimen) comprising: a) a first infusion on day 1 comprising about 100 to about 200 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 2 weeks after the first infusion comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans). NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); c) a first subsequent infusion at about 24 weeks or about six months after the first infusion, comprising about 400 to about 500 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibodies in the population are composed of a heavy chain encoding an amino acid sequence comprising SEQ ID NO:1 and a light chain encoding an amino acid sequence comprising SEQ ID NO:2; NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions about 24 weeks or about six months after the previous infusion, comprising about 400 to about 500 mg of an anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans).

[0581] In some embodiments, the intravenous infusion comprises a multiple dosing regimen (e.g., a multiple infusion dosing regimen) comprising: a) a first infusion on day 1 comprising 150 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 2 weeks after the first infusion comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); c) a first subsequent infusion about 24 weeks or about six months after the first infusion, comprising 450 mg of an anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions about 24 weeks or about six months after the previous infusion, comprising 450 mg of an anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); In some embodiments, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; and subsequent infusions (e.g., the first subsequent infusion and / or one or more subsequent infusions) are administered within 1 hour.

[0582] In some embodiments, the method comprises administering to the subject a first anti-CD20 antibody composition (e.g., GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab)) by intravenous infusion, followed by administering to the subject a second anti-CD20 antibody as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans).In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising a prescribed dosage of an anti-CD20 antibody composition (e.g., GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan), or OCREVUS (ocrelizumab)); b) a second infusion about 2 weeks after the first infusion comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibody in the population consists of a heavy chain encoding an amino acid sequence comprising SEQ ID NO: 1 and a heavy chain encoding an amino acid sequence comprising SEQ ID NO: 2). NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); c) a first subsequent infusion at about 24 weeks or about six months after the first infusion, comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are composed of a heavy chain encoding an amino acid sequence comprising SEQ ID NO:1 and a light chain encoding an amino acid sequence comprising SEQ ID NO:2; NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions about 24 weeks or about six months after the previous infusion, comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans).

[0583] In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising a prescribed dose of an anti-CD20 antibody composition (e.g., GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan), or OCREVUS (ocrelizumab)); b) a second infusion about 2 weeks after the first infusion comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibody in the population consists of a heavy chain encoding an amino acid sequence comprising SEQ ID NO: 1 and a heavy chain encoding an amino acid sequence comprising SEQ ID NO: 2). NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); c) a first subsequent infusion about 24 weeks or about six months after the first infusion, comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions about 24 weeks or about six months after the previous infusion, comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); In some embodiments, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; and subsequent infusions (e.g., the first subsequent infusion and / or one or more subsequent infusions) are administered within 1 hour.

[0584] In some embodiments, the method comprises a treatment period of at least 48 weeks. In some embodiments, the method comprises a treatment period of at least 96 weeks.

[0585] In some embodiments, the subject is human. In some embodiments, the human subject is an adult. In some embodiments, the subject has experienced at least one relapse in the previous year before treatment or has experienced two relapses in the previous two years before treatment. In some embodiments, the subject has or had T1 gadolinium (Gd) enhanced lesions in the previous year before treatment with an anti-CD20 antibody composition as described herein. In some embodiments, before treatment with an anti-CD20 antibody composition as described herein, the subject has an Expanded Disability Status Scale (EDSS) score of 0 to 5.5.

[0586] In some embodiments of the method, the subject has not been treated with nonsteroidal therapy for MS in the past five years prior to treatment with an anti-CD20 antibody composition as described herein. In some embodiments, the subject has not been treated for MS. In certain embodiments, the subject is negative for hepatitis B virus (HBV). In some embodiments, the subject is negative for hepatitis B virus surface antigen (HBsAg). In some embodiments, the subject is negative for anti-hepatitis B virus core antibody. In certain embodiments, prior to treatment with an anti-CD20 antibody composition as described herein, the subject has not been immunized with a vaccine for at least 2 weeks or at least 4 weeks.

[0587] In some embodiments of the method, about 30 to about 60 minutes before the administration of an anti-CD20 antibody composition as described herein, the subject is premedicated with a certain amount of a corticosteroid. In some embodiments, the corticosteroid is methylprednisone or a bioequivalent corticosteroid thereof. In some embodiments, the amount of the corticosteroid is about 100 mg of methylprednisone. In some embodiments, the corticosteroid is dexamethasone or a bioequivalent corticosteroid thereof. In some embodiments, the amount of the corticosteroid is about 10 to 20 mg of dexamethasone. In certain embodiments, about 30 to about 60 minutes before the administration of an anti-CD20 antibody composition as described herein, the subject is premedicated with a certain amount of an antipyretic. In some embodiments, the antipyretic is acetaminophen or a bioequivalent antipyretic thereof. In some embodiments, about 30 to about 60 minutes before the administration of an anti-CD20 antibody composition as described herein, the subject is premedicated with a certain amount of an antihistamine. In some embodiments, the antihistamine is diphenhydramine HCl or a bioequivalent antihistamine thereof. In some embodiments, the amount of antihistamine is about 25 to 50 mg diphenhydramine HCl. In some embodiments, the subject is premedicated with an amount of a corticosteroid (as described above) and an amount of an antihistamine (as described above) about 30 to about 60 minutes prior to administration of an anti-CD20 antibody composition as described herein. In some embodiments, the corticosteroid and / or antihistamine is administered orally to the subject.

[0588] In some embodiments, the method alleviates or delays the progress of one or more MS symptoms of the subject. In certain embodiments, the method reduces the annualized relapse rate (ARR) of the subject after administering an anti-CD20 antibody (e.g., Utuximab) composition as described herein. In some embodiments, ARR is the total number of relapses of the subject divided by the sum of the duration of treatment (i.e., it is the sum of the RMS relapse counts of the subject divided by the sum of the duration of treatment of the subject (in years)). In some embodiments, ARR is the number of times the subject confirms relapse by an independent relapse adjudication panel (IRAP) each year. In some embodiments, the reduction of ARR is assessed at about 96 weeks after the first infusion. In some embodiments, compared with subjects who received oral administration of 14 mg teriflunomide daily during the same treatment period, subjects who were administered multiple infusion dosing regimens of anti-CD20 antibody compositions described herein achieved significantly reduced ARR. For example, compared with the administration of teriflunomide, the administration of anti-CD20 antibody compositions described herein can significantly reduce ARR, and the relapse rate is relatively reduced by 59%. In addition or as an alternative, administration of the anti-CD20 antibody compositions described herein can significantly reduce the ARR compared to administration of teriflunomide, and the relapse rate is relatively reduced by 49%. In some embodiments, treatment with the anti-CD20 antibody compositions described herein results in an ARR of about 0.100 to about 0.050; such as about 0.100 to about 0.090, about 0.090 to about 0.080, about 0.080 to about 0.070, about 0.070 to about 0.060, or about 0.060 to about 0.050 (e.g., about 0.099, 0.098, 0.097, 0.096, 0.095, 0.094, 0.093, 0.092, 0.091, 0.090, 0.089, 0.088, 0.087, 0.086, 0.085 0.054, 0.053, 0.052, 0.051, or 0.050).

[0589] In some embodiments, the method reduces the total number of T1 gadolinium (Gd) enhanced lesions in a subject after administering an anti-CD20 antibody composition as described herein. In some embodiments, the reduction of Gd enhanced T1 lesions is assessed by MRI scanning. In some embodiments, the reduction of Gd enhanced T1 lesions is assessed at about 96 weeks after the first infusion. In some embodiments, subjects who are administered a multiple infusion dosing regimen of anti-CD20 antibodies achieve a reduction in the total number of Gd enhanced T1 lesions per MRI scan compared to subjects who receive 14 mg of teriflunomide orally daily during the same treatment period. In some embodiments, the administration of an anti-CD20 antibody (e.g., Utuximab) significantly reduces the total number of Gd enhanced T1 lesions compared to the administration of teriflunomide, with a relative reduction of about 97%.

[0590] In some embodiments, the method reduces the number of newly added or expanded T2 high-intensity lesions in the subject after administering an anti-CD20 antibody composition as described herein. In some embodiments, the reduction in the number of newly added or expanded T2 high-intensity lesions is assessed by MRI scanning. In some embodiments, the reduction in the number of newly added or expanded T2 high-intensity lesions is assessed at about 96 weeks after the first infusion. In some embodiments, compared with subjects who receive 14 mg of teriflunomide orally daily during the same treatment period, subjects who are administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., utuximab) achieve a reduction in the total number of newly added and expanded T2 high-intensity lesions per MRI scan. In some embodiments, compared with the administration of teriflunomide, the administration of an anti-CD20 antibody (e.g., utuximab) reduces the total number of newly added or expanded T2 high-intensity lesions, a relative reduction of about 90 to 92%.

[0591] In some embodiments, the method achieves confirmed disability progression in the subject after administration of an anti-CD20 antibody composition as described herein. In some embodiments, confirmed disability progression comprises an increase of greater than or equal to 1.0 points relative to the subject's baseline EDSS score due to MS, wherein the baseline EDSS score is 5.5 or less. In some embodiments, confirmed disability progression comprises an increase of greater than or equal to 0.5 points relative to the subject's baseline EDSS score due to MS, wherein the baseline EDSS score is greater than 5.5.

[0592] In some embodiments, the method makes the subject have no evidence of disease activity (NEDA). In some cases, NEDA includes one or more of the following: confirming no recurrence, no gadolinium-enhanced (Gd+) T1 lesions, no newly added and / or enlarged T2 lesions, and no 12-week confirmed disability progression. In some embodiments, the method of the present disclosure causes NEDA in the subject about 24 weeks after administering a pharmaceutical formulation as described herein (e.g., an anti-CD20 antibody composition). For example, at about 24-96 weeks (e.g., 24-48 weeks, 24-72 weeks, 48-72 weeks, 72-96 weeks, or 48-96 weeks (e.g., 24 weeks, 36 weeks, 48 ​​weeks, 60 weeks, 72 weeks, 84 weeks, 96 weeks, or any range therebetween)) after administration, the administration of a pharmaceutical formulation as described herein can cause NEDA in the subject. In some embodiments, subjects who are administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., Utuximab) achieve an increase in NEDA status compared to subjects who receive 14 mg of teriflunomide orally daily during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., Utuximab) increases NEDA status by 197% compared to administration of teriflunomide. In some embodiments, administration of an anti-CD20 antibody (e.g., Utuximab) increases NEDA status by 277% compared to administration of teriflunomide.

[0593] In some embodiments, subjects who were administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., Utuximab) achieved an increase in confirmed disability improvement (CDI) compared to subjects who received 14 mg of teriflunomide orally daily during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., Utuximab) increased CDI by 116% at 12 weeks compared to teriflunomide. In some embodiments, administration of an anti-CD20 antibody (e.g., Utuximab) increased CDI by 103% at 24 weeks compared to administration of teriflunomide.

[0594] In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., Utuximab) achieve an increase in the Multiple Sclerosis Functional Composite (MSFC) score compared to subjects receiving oral administration of 14 mg teriflunomide daily during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., Utuximab) increases the MSFC score by about 76% to 90% compared to administration of teriflunomide.

[0595] In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., Utuximab) achieve an improvement in the Timed 25-Foot Walk (T25FW) score compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period. In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., Utuximab) achieve an improvement in the 9-Hole Peg Test (9-HPT) score compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0596] In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., utuximab) achieve a significant reduction in both the volume and number of new T1 hypointense lesions per MRI scan compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

[0597] In some embodiments, as disclosed herein, the treatment for MS (eg, RMS) further comprises administering an anti-CD20 antibody in combination with one or more additional therapeutic agents. For example, the anti-CD20 antibody in the disclosed method for treating RMS can be administered in combination with other compounds, drugs, and / or agents for treating RMS. Such compounds, drugs, and / or agents may include, for example, small molecule drugs, monoclonal antibodies, or other B cell depleting agents. In some embodiments, the methods described herein are used in combination with existing MS treatment care standards. In some cases, anti-CD20 antibodies are used in combination with Bruton's tyrosine kinase (BTK) inhibitors for the treatment of RMS, and BTK inhibitors include, for example, fenbutinib (fenebrutinib), ivobutinib (evobrutinib), tolebrutinib (tolebrutinib), orelabrutinib (orelabrutinib) BIIB091, AC0058, PRN473 (Dolgin, Nature Biotechnology 39: 3-12 (2021), and those compounds described in U.S. Pat. No. 9,951,077). In some cases, anti-CD20 antibodies are used in combination regimens that exploit a non-overlapping mechanism of utuximab to deplete B cells.

[0598] Anti-CD20 antibodies (e.g., Utuximab) can be combined with injectable, oral, or infused medications. Injectable medications for combined treatment with anti-CD20 antibodies can include interferons, including but not limited to AVONEX (interferon beta-1a), BETASERON (interferon beta-1b), EXTAYIA (interferon beta-1b), PLEGRIDY (pegylated interferon beta-1a), and REBIF (interferon beta-1a). Other injectable medications may include COPAXONE (glatiram acetate). Oral medications combined with Utuximab may include AUBAGIO (teriflunomide), fumarate-based compositions (including BAFIERTAM), dimethyl fumarate, TECFIDERA, and VUMERITY. Additionally or alternatively, anti-CD20 antibodies (e.g., Utuximab) can be used in combination with the monoclonal antibody Tysabri (natalizumab). Orally administered agents that can be used in combination with anti-CD20 antibodies include GILENYI (fingolimod), MAYZENT (siponimod), ZEPOSIA (ozanimod), and PONVORY (ponesimod). Additional orally administered agents include MAVENCLAD (cladribine).

[0599] Anti-CD20 antibodies (eg, utuximab) can also be used in combination with infusional medications, including LEMTRADE (alemtuzumab) and NOVANTRONE (mitoxantrone).

[0600] In some embodiments, an anti-CD20 antibody of the present disclosure (e.g., utoximab or an anti-CD20 antibody that binds to the same epitope as utoximab) can be administered in a subject (e.g., a patient) with known resistance to one or more anti-CD20 antibodies because utoximab targets a unique epitope region on the CD20 antigen that is not targeted by other anti-CD20 monoclonal antibodies.

[0601] In some embodiments, the additional therapeutic agent may comprise a B cell depleting agent that is not an anti-CD20 antibody. In some cases, the B cell depleting agent is a PI3K inhibitor.

[0602] In some embodiments, the additional therapeutic agent comprises a B cell depleting agent, which is an anti-CD20 antibody like the anti-CD20 antibodies described herein (e.g., utuximab). For example, utuximab can be used in combination with an additional anti-CD20 antibody composition, such as (Orelizumab), (ofatumumab) and Rituxan (rituximab).

[0603] The additional therapeutic agent may be administered orally, parenterally, intravenously, or subcutaneously.

[0604] In some embodiments, the methods described herein may result in adverse events (TEAEs) occurring during treatment. In some cases, TEAEs include cytopenias or a decrease in blood cell counts in a subject. Blood cell counts can be evaluated by blood tests such as complete blood counts (CBC). Blood cell counts can be obtained by cell counting methods known in the art, including but not limited to manual methods (e.g., by using a blood cell counter) and automated methods (e.g., by using an automatic cell counter). In some cases, cytopenias include a decrease or reduction in the blood cell count of a subject by about 20 to 100% (e.g., about 20 to 30%, about 20 to 40%, about 30 to 40%, about 30 to 50%, about 40 to 50%, about 40 to 60%, about 50 to 60%, about 50 to 70%, about 60 to 70%, about 60 to 80%, about 70 to 80%, about 70 to 90%) compared to a control or normal blood cell count. , about 80 to 90%, about 80 to 100%, about 90 to 100%, or any range therebetween (e.g., about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 100%, or any range therebetween)). A control or normal blood cell count may comprise a blood cell count of a healthy subject to whom the pharmaceutical formulation of the present disclosure has not yet been administered. Additionally or alternatively, a blood cell count of a subject before treatment by the present method may serve as a control or normal blood cell count. Cytopenia or reduced blood cell count may comprise one or more of lymphocytopenia, neutropenia, leukopenia, and anemia.

[0605] In some cases, administration of a pharmaceutical formulation as described herein (e.g., an anti-CD20 antibody composition) results in a subject having cytopenias about 1 to 3 days (e.g., about 1 day, about 2 days, or about 3 days) after administration. For example, administration of a pharmaceutical formulation may result in lymphopenias in a subject about 2 days after administration. In some cases, cytopenias (e.g., lymphopenias, neutropenias, leukopenias, and / or anemias) are temporary, such that the subject's blood cell counts normalize (e.g., become the same or similar to a control or normal blood cell count) about 15 days (e.g., 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, or 4 days) after administration of the pharmaceutical formulation. For example, a subject's lymphocyte count may normalize 8 days after administration of the pharmaceutical formulation.

[0606] Methods for treating cancer

[0607] Also provided herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an anti-CD20 antibody composition as described herein. In some embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, gynecological cancer (i.e., cervical cancer, ovarian cancer, uterine cancer, vaginal cancer, or vulvar cancer), head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, mesothelioma, myeloma, prostate cancer, skin cancer, or thyroid cancer.

[0608] Also provided herein is a method for treating a disease or condition associated with excessive B cell proliferation in a subject in need thereof, comprising administering to the subject an effective amount of an anti-CD20 antibody composition as described herein. In some embodiments, the disease or condition associated with excessive B cell proliferation is a blood cancer. In some embodiments, the blood cancer is a lymphoma, leukemia or myeloma. In some embodiments, the blood cancer is selected from B cell lymphoma, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom's macroglobulinemia (WM), diffuse large B cell lymphoma (DLBCL), marginal zone lymphoma (MZL), hairy cell leukemia (HCL), Burkitt's lymphoma (BL), Richter's transformation or primary central nervous system lymphoma (PCNSL). In various aspects of the methods, the anti-CD20 antibody composition as described herein comprises an anti-CD20 antibody that is an IgG1 antibody comprising heavy chains each comprising the amino acid sequence of SEQ ID NO:1 and light chains each comprising the amino acid sequence of SEQ ID NO:2.

[0609] In some embodiments, the blood cancer is a B-cell lymphoma. In some embodiments, the B-cell lymphoma is relapsed or refractory. In certain embodiments, the blood cancer is non-Hodgkin's lymphoma (NHL). In some embodiments, NHL is relapsed or refractory. In certain embodiments, the blood cancer is Waldenstrom's macroglobulinemia (WM). In some embodiments, WM is relapsed or refractory. In certain embodiments, the blood cancer is marginal zone lymphoma (MZL). In some embodiments, MZL is relapsed or refractory. In certain embodiments, the blood cancer is chronic lymphocytic leukemia (CLL). In some embodiments, CLL is relapsed or refractory. In certain embodiments, the blood cancer is small lymphocytic lymphoma (SLL). In some embodiments, SLL is relapsed or refractory. In certain embodiments, the blood cancer is primary central nervous system lymphoma (PCNSL). In some embodiments, PCNSL is relapsed or refractory.

[0610] In some embodiments, the method of treating a blood cancer comprises administering an anti-CD20 antibody composition as described herein to a subject by intravenous infusion. In some embodiments, the intravenous infusion comprises: a) on days 1 and 2 of cycle 1 (each cycle is 28 days) (divided into a 150 mg dose on day 1 and a 750 mg dose on day 2), day 8 and day 15; day 1 of cycles 2 to 6; and day 1 of every 3 cycles after cycle 6 (e.g., cycle 9, 12, 15, etc.), an infusion of about 900 mg of an anti-CD20 antibody composition as described herein. In some embodiments, the blood cancer is CLL.

[0611] In some embodiments, the methods described herein may result in adverse events (TEAEs) occurring during treatment. In some cases, TEAEs include cytopenias or a decrease in blood cell counts in a subject. Blood cell counts can be assessed by blood tests, such as complete blood counts (CBCs). Blood cell counts can be obtained by cell counting methods known in the art, including but not limited to manual methods (e.g., by using a blood cell counter) and automated methods (e.g., by using an automated cell counter). In some cases, cytopenias include a decrease or reduction in the blood cell count of a subject by about 20 to 100% (e.g., about 20 to 30%, about 20 to 40%, about 30 to 40%, about 30 to 50%, about 40 to 50%, about 40 to 60%, about 50 to 60%, about 50 to 70%, about 60 to 70%, about 60 to 80%, about 70 to 80%, about 70 to 90%) compared to a control or normal blood cell count. , about 80 to 90%, about 80 to 100%, about 90 to 100%, or any range therebetween (e.g., about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 100%, or any range therebetween)). A control or normal blood cell count may comprise a blood cell count of a healthy subject to whom the pharmaceutical formulation of the present disclosure has not yet been administered. Additionally or alternatively, a blood cell count of a subject before treatment by the present method may serve as a control or normal blood cell count. Cytopenia or reduced blood cell count may comprise one or more of lymphocytopenia, neutropenia, leukopenia, and anemia.

[0612] Multiple infusion doses

[0613] In any of the embodiments described herein, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion comprising about 500 to about 700 mg, about 800 to about 1000 mg, or about 1100 to about 1300 mg of the anti-CD20 antibody composition; c) a third infusion about 2 weeks after the first infusion comprising about 500 to about 700 mg, about 800 to about 1000 mg, or about 1100 to about 1300 mg of an anti-CD20 antibody composition; d) a fourth infusion about 3 weeks after the first infusion, comprising about 500 to about 700 mg, about 800 to about 1000 mg, or about 1100 to about 1300 mg of an anti-CD20 antibody composition; and e) one or more subsequent infusions about one month after the previous infusion, comprising about 400 to about 500 mg, about 500 to about 700 mg, about 800 to about 1000 mg, or about 1100 to about 1300 mg of an anti-CD20 antibody composition. In some cases, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; the fourth infusion is administered within 1 hour; and one or more subsequent infusions are administered within 1 hour.

[0614] In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibody in the population consists of a heavy chain encoding an amino acid sequence comprising SEQ ID NO: 1 and a heavy chain encoding an amino acid sequence comprising SEQ ID NO: 2). NO:2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion, comprising 600, 900 or 1200 mg of the anti-CD20 antibody composition; c) a third infusion about 2 weeks after the first infusion, comprising 600, 900 or 1200 mg of the anti-CD20 antibody composition; d) a fourth infusion about 3 weeks after the first infusion, comprising 600, 900 or 1200 mg of the anti-CD20 antibody composition; and e) one or more subsequent infusions about one month after the previous infusion, comprising 450, 600, 900 or 1200 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; the fourth infusion is administered within 1 hour; and one or more subsequent infusions are administered within 1 hour.

[0615] In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion comprising about 500 to about 700 mg of the anti-CD20 antibody composition; c) a third infusion about 2 weeks after the first infusion comprising about 800 to about 1000 mg of the anti-CD20 antibody composition; and d) one or more subsequent infusions about one month after the previous infusion comprising about 400 to about 1000 mg of the anti-CD20 antibody composition. In certain instances, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; and one or more subsequent infusions are administered within 1 hour.

[0616] In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion comprising 600 mg of the anti-CD20 antibody composition; c) a third infusion about 2 weeks after the first infusion comprising 900 mg of the anti-CD20 antibody composition; and d) one or more subsequent infusions about one month after the previous infusion comprising 450, 600 or 900 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; and one or more subsequent infusions are administered within 1 hour.

[0617] In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising about 5 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and b) one or more subsequent infusions about one week after the previous infusion comprising about 5 to about 450 mg of the anti-CD20 antibody composition, wherein the dose of each subsequent infusion is higher than the previous infusion. In some cases, the first infusion is administered within 4 hours; and one or more subsequent infusions are administered within 1 hour.

[0618] In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising about 100 to about 200 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and b) one or more subsequent infusions about one week after the previous infusion comprising about 400 to about 500 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered within 4 hours; and one or more subsequent infusions are administered within 1 hour.

[0619] In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising 150 mg of the anti-CD20 antibody composition; and b) one or more subsequent infusions about one week after the previous infusion comprising 450 mg of the anti-CD20 antibody composition. In some embodiments, the method comprises seven or more subsequent infusions. In some cases, the first infusion is administered within 4 hours; one or more subsequent infusions are administered within 1 hour.

[0620] In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising about 500 to about 1000 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion comprising about 500 to about 1000 mg of the anti-CD20 antibody; c) a third infusion about 2 weeks after the first infusion comprising about 500 to about 1000 mg of the anti-CD20 antibody composition; and d) one or more subsequent infusions about one month after the previous infusion comprising about 500 to about 1000 mg of the anti-CD20 antibody composition. In certain embodiments, one or more subsequent infusions each contain 600 mg of an anti-CD20 antibody composition as described herein. In certain embodiments, the first infusion, the second infusion, the third infusion, and one or more subsequent infusions each contain 600 mg of an anti-CD20 antibody composition as described herein. In some embodiments, the first infusion, the second infusion, the third infusion, and one or more subsequent infusions each contain 900 mg of an anti-CD20 antibody composition as described herein. In some cases, the first infusion is administered within 4 hours; the second is administered within 1 hour; the third infusion is administered within 1 hour; and one or more subsequent infusions are administered within 1 hour.

[0621] In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising about 100 to about 200 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion comprising about 700 to about 800 mg of the anti-CD20 antibody composition; c) a third infusion about 2 weeks after the first infusion comprising about 850 to about 950 mg of the anti-CD20 antibody composition; and d) one or more subsequent infusions about one month after the previous infusion comprising about 850 to about 950 mg of the anti-CD20 antibody composition. In some embodiments, the first infusion comprises 150 mg of the anti-CD20 antibody composition, the second infusion comprises 750 mg of the anti-CD20 antibody composition, and the third and one or more subsequent infusions comprise 900 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; and one or more subsequent infusions are administered within 1 hour.

[0622] Subjects

[0623] In some embodiments, the subject is human. In some embodiments, the human subject is an adult. In some embodiments, the subject suffers from relapsed or refractory B-cell lymphoma. In some embodiments, the subject has previously been treated with at least one previous course of rituximab or a rituximab-based therapy. In some embodiments, the subject has previously been treated with at least one previous course of fludarabine or a fludarabine-based therapy. In some embodiments, the subject has not received treatment for B-cell lymphoma. In some embodiments, the subject is eligible for high-dose or combination chemotherapy and / or stem cell transplantation. In some embodiments, before treatment with an anti-CD20 antibody composition as described herein, the subject has an Eastern Coast Collaborative Cancer Clinical Research Organization (ECOG) score of 0 to 2. In some embodiments, before treatment with an anti-CD20 antibody composition as described herein, the subject has a peripheral blood lymphocyte count greater than 5,000 / μL.

[0624] In some embodiments, the anti-CD20 antibody compositions provided herein can be used to treat and / or prevent chronic inflammatory demyelinating polyneuropathy (CIDP); myositis; lupus nephritis; other forms of MS-PPMS, SPMS; myasthenia gravis (MG); antiphospholipid syndrome; thrombotic thrombocytopenic purpura (TTP); ulcerative colitis; minimal change nephrotic syndrome (MCNS); aplastic anemia; autoimmune glomerulopathy; rheumatoid arthritis (RA); interstitial lung disease; myasthenia gravis (MG); ); subepidermal autoimmune blistering disease; infectious pulmonary disease; acquired hemophilia; refractory mixed cryoglobulinemia; essential immune thrombocytopenia; graft-versus-host disease (GVHD); autoimmune blistering disease; anti-myelin-associated glycoprotein (MAG) polyneuropathy; granulomatosis with polyangiitis (GPA); neuromyelitis optica; systemic lupus erythematosus; pemphigus; post-transplantation lymphoproliferative disease; autoimmune hemolytic anemia; cerebral vasculitis; microscopic polyangiitis (MPA); or idiopathic nephrotic syndrome.

[0625] 7.9 Pharmacokinetic properties

[0626] Also provided herein are methods of treating a human patient having a disease (eg, an autoimmune disease) comprising administering to the patient an anti-CD20 antibody provided herein.

[0627] In some embodiments, the anti-CD20 antibody is administered as follows: i) a first infusion at a dose of about 150 mg, ii) a second infusion two weeks later at a dose of about 450 mg, and iii) subsequent infusions every six months at a dose of about 450 mg.

[0628] In some embodiments, administration of an anti-CD20 antibody produces an area under the curve (AUC) of about 2,160 μg / mL to about 3,840 μg / mL. In certain embodiments, the AUC is about 3,000 μg / mL. In certain embodiments, the AUC is a steady-state AUC.

[0629] In some embodiments, administration of an anti-CD20 antibody produces a Cmax of about 118,011 ng / mL to about 159,989 ng / mL. In certain embodiments, the Cmax is about 139,000 ng / mL. In certain embodiments, the Cmax is a steady-state Cmax.

[0630] In some embodiments, administration of an anti-CD20 antibody produces a Cmin of about 0 ng / mL to about 375 ng / mL. In certain embodiments, the Cmin is about 139 ng / mL. In certain embodiments, the Cmin is a steady-state Cmin.

[0631] In some embodiments, administration of an anti-CD20 antibody produces a Cavg of about 6,437 ng / mL to about 11,443 ng / mL. In certain embodiments, the Cavg is about 8,940 ng / mL. In certain embodiments, the Cavg is a steady-state Cavg.

[0632] In some embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, psoriasis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, inflammatory muscle disease, allergic rhinitis, vaginitis, interstitial cystitis, scleroderma, osteoporosis, eczema, allogeneic or xenogeneic transplantation (organ, bone marrow, stem cells and other cells and tissues), transplant rejection, graft-versus-host disease, lupus erythematosus, inflammatory diseases, type 1 diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g., Hashimoto's thyroiditis and autoimmune thyroiditis), myasthenia gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, allergic conjunctivitis, atopic dermatitis, chronic obstructive pulmonary disease, glomerulonephritis, neuroinflammatory diseases and uveitis.

[0633] In some embodiments, the autoimmune disease is multiple sclerosis. In certain embodiments, multiple sclerosis is a relapsing form of multiple sclerosis. In certain embodiments, the relapsing form of multiple sclerosis is clinically isolated syndrome (CIS); relapsing-remitting MS (RRMS); active secondary progressive MS (SPMS); or primary progressive MS (PPMS). In certain embodiments, the relapsing form of MS is clinically isolated syndrome (CIS). In certain embodiments, the relapsing form of MS is relapsing-remitting multiple sclerosis (RRMS). In certain embodiments, the relapsing form of MS is active secondary progressive multiple sclerosis (SPMS). In certain embodiments, the relapsing form of MS is primary progressive MS (PPMS).

[0634] In certain embodiments, the anti-CD20 antibody is administered intravenously.

[0635] 7.10 Manufacturing method

[0636] Also provided herein are methods for making the anti-CD20 antibody protein populations provided herein with a specified range of post-translational modifications. Example 15 provides an exemplary method for making the anti-CD20 antibody protein populations provided herein.

[0637] In some embodiments, the method of making the anti-CD20 antibody protein population having a specified range of post-translational modifications as described above comprises: i) culturing rat hybridoma cells at a first culture pH of about 7.0 to about 7.55 for 0 to 3 days, ii) culturing rat hybridoma cells at a second culture pH of about 6.5 to about 6.99 on day 3, iii) maintaining the culture pH at a second culture pH of about 6.5 to about 6.99 from culture day 3 to day 14 of the cell culture, and iv) controlling the culture pCO2 content to less than about 200 mmHg during the entire culture period. In some embodiments, the second culture pH is about 6.60 to about 6.96 (e.g., the second culture pH is 6.8).

[0638] In some embodiments, the second culture pH results in a higher integrated viable cell density (IVCD) and higher titer at harvest.

[0639] In some embodiments, the second culture pH results in a lower percentage of fucosylation.

[0640] In some embodiments, rat hybridoma cells expressing the recombinant protein are cultured in a basal medium, ie, a chemically defined animal component-free (ADCF) medium.

[0641] In some embodiments, the basal medium is supplemented with a feed medium.

[0642] In some embodiments, the method further comprises an initial temperature set point of about 37°C, wherein the initial temperature set point is set between culture day 0 and culture day 1.

[0643] In some aspects, the method further comprises a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3.

[0644] In some embodiments, the method further comprises a third temperature set point of about 32°C to about 33°C, wherein the third temperature set point is set on culture day 3 and maintained until harvest. The term "harvest" refers to the point in the mammalian cell culture process when cells containing the recombinant protein are separated and removed from the cell culture medium and subjected to additional processing (such as, for example, centrifugation, filtration or purification).

[0645] In some embodiments, harvesting the cells occurs on day 12, 13, or 14 of the cell culture process, or when cell viability drops below 20%, whichever occurs first.

[0646] In some embodiments, the method further comprises harvesting the recombinant protein produced by the rat hybridoma cells.

[0647] In some embodiments, the method further comprises purifying the recombinant protein by affinity chromatography and / or ion exchange chromatography. In some embodiments, the affinity chromatography comprises Protein A purification.

[0648] In some embodiments, the method increases the yield of the recombinant protein. For example, the recombinant protein is increased by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150% relative to the recombinant protein produced by the culture method that does not adopt the culture conditions cited above.

[0649] Sequence Listing

[0650]

[0651]

[0652]

[0653]

[0654] 7.11 Preparation of Utuximab for Intravenous Infusion in the Disclosed Treatment Methods

[0655] In some embodiments, the Utuximab infusion is prepared in 250 mL of 0.9% Sodium Chloride Injection. For example, the Utuximab infusion can be prepared in an infusion bag containing 0.9% Sodium Chloride Injection.

[0656] In some embodiments, the first infusion of 150 mg of Utuximab is prepared by: (1) inspecting a vial of 150 mg / 6 mL Utuximab solution for any particulate matter or discoloration and not using the solution if it contains discrete foreign particulate matter; (2) if the vial is free of particulate matter, using a vial of 150 mg / 6 mL Utuximab solution to prepare a 250 mL infusion bag for the first infusion; (3) withdrawing 6 mL of 0.9% Sodium Injection from the infusion bag and discarding it; (4) withdrawing 6 mL of Utuximab from the vial; (5) diluting 6 mL (150 mg) of Utuximab into the 250 mL infusion bag containing 0.9% Sodium Injection for immediate administration; and (6) mixing the infusion bag by gently inverting without shaking.

[0657] In some embodiments, a second or subsequent infusion of 450 mg of Utuximab is prepared by: (1) inspecting three vials of 150 mg / 6 mL Utuximab solution for any particulate matter or discoloration and not using the solution if it contains discrete foreign particulate matter; (2) if the vials are free of particulate matter, using the three vials of 150 mg / 6 mL Utuximab solution to prepare a 250 mL infusion bag for the second or subsequent infusion; (3) withdrawing 18 mL of 0.9% Sodium Injection from the infusion bag and discarding it; (4) removing 18 mL of Utuximab from the vial; (5) diluting 18 mL (450 mg) of Utuximab into the 250 mL infusion bag containing 0.9% Sodium Injection for immediate administration; and (6) mixing the infusion bag by gently inverting without shaking.

[0658] In some embodiments, the contents of the infusion bag are at room temperature prior to starting the intravenous infusion of Utuximab.

[0659] 7.12 Administration of Utuximab in the Treatment Methods Disclosed herein

[0660] In some embodiments, before starting Utuximab administration, the subject is pre-screened for hepatitis B virus (HBV). If the subject's hepatitis B surface antigen (HbsAg) test is positive, Utuximab should not be administered. In some embodiments, the subject administered with Utuximab infusion is negative for hepatitis B virus surface antigen (HBsAg).

[0661] In some embodiments, the subject is premedicated with a corticosteroid and an antihistamine 30 to 60 minutes before administration of Utuximab or an anti-CD20 antibody that binds to the same epitope as Utuximab, both of which can be administered orally or intravenously. In some embodiments, the corticosteroid and / or antihistamine is administered orally to the subject. In some cases, the pre-treatment dose of a corticosteroid is about 100 mg of methylprednisone, 10 to 20 mg of dexamethasone, or an equivalent corticosteroid.

[0662] In some embodiments, Utuximab is administered in a multiple dosing schedule. In some cases, Utuximab is administered by intravenous infusion. For example, Utuximab can be administered by intravenous infusion in a multiple infusion dosing schedule.

[0663] In some embodiments, Utuximab is administered to patients with RMS by intravenous infusion for 48 weeks in a multiple infusion dosing schedule. In some embodiments, Utuximab is administered to patients with RMS by intravenous infusion for 96 weeks in a multiple infusion dosing schedule.

[0664] The multiple infusion dosing regimen may comprise a first, second, and subsequent intravenous infusions of Utuximab. In some embodiments, a "subsequent infusion" of Utuximab may be any number of infusions after the first and second infusions.

[0665] In some embodiments, the multiple infusion dosing regimen comprises a first infusion and a second infusion, 150 mg of Utuximab intravenous infusion (first infusion), followed by 450 mg of Utuximab intravenous infusion two weeks later (second infusion). In some embodiments, the multiple infusion dosing regimen further comprises subsequent infusions of 450 mg of Utuximab intravenous infusion every six months.

[0666] In some embodiments, the duration of the first infusion of Utuximab or an anti-CD20 antibody that binds to the same epitope as Utuximab is about four hours. For example, the infusion rate of the first infusion of Utuximab or an anti-CD20 antibody that binds to the same epitope as Utuximab may be 10 mL / hour for the first 30 minutes; 20 mL / hour for the next 30 minutes; 35 mL / hour for the next hour; and 100 mL / hour for the remaining two hours. In some embodiments, the duration of the second infusion and subsequent infusions of Utuximab is about one hour. For example, the infusion rate of the second infusion and / or subsequent infusions of Utuximab may be 100 mL / hour for the first 30 minutes, and 400 mL / hour for the remaining 30 minutes. If the infusion is interrupted or slowed, the infusion duration may be longer.

[0667] In some embodiments, the subject is monitored for at least one hour after the first two infusions of Utuximab are completed. Subsequent infusions do not require post-infusion monitoring unless an infusion-related reaction (IRR) and / or allergic reaction is observed.

[0668] In some embodiments, if the subject has symptoms of life-threatening infusion-related reactions, the intravenous infusion of Utuximab is stopped and permanently suspended. In some embodiments, if the subject has symptoms of severe infusion-related reactions, the intravenous infusion of Utuximab is discontinued, and once the symptoms of the infusion-related reactions of the subject have subsided, it is restarted. In some embodiments, the intravenous infusion of Utuximab is restarted at half the infusion rate at the beginning of the infusion-related reaction. If the subject tolerates half the infusion speed, the infusion rate of Utuximab can be increased to the original infusion rate. In some embodiments, if the subject has symptoms of mild to moderate infusion-related reactions, the infusion rate of Utuximab is reduced by half, wherein half the infusion rate is maintained for at least 30 minutes. If the subject tolerates half the infusion speed, the infusion rate of Utuximab can be increased to the original infusion rate.

[0669] In some embodiments, Utuximab is administered by intravenous infusion such that the geometric mean steady-state AUC is 3000 mcg / mL per day (CV=28%) and the mean maximum concentration is 139 mcg / mL (CV=15%).

[0670] In some embodiments, administration of Utuximab may be via a route of administration other than intravenous infusion (eg, subcutaneous injection, intramuscular injection, oral, epidermal, spinal, or inhalation).

[0671] 7.13 Pharmaceutical Compositions

[0672] "Pharmaceutical composition" may refer to a composition that is acceptable for pharmaceutical administration, e.g., to humans. Such compositions may include impurity materials at levels no greater than acceptable levels for pharmaceutical administration (such levels including the absence of such impurities), and may include, in addition to any active agent, pharmaceutically acceptable excipients, vehicles, carriers, stabilizers, and other inactive ingredients, for example, to prepare such compositions for administration.

[0673] The present disclosure provides pharmaceutical compositions comprising Utuximab or an antibody that binds to the same epitope as Utuximab for use in treating a subject having a relapsing form of multiple sclerosis according to any of the methods disclosed herein.

[0674] In some embodiments, the pharmaceutical composition comprises Utuximab. In some embodiments, the pharmaceutical composition comprising Utuximab is formulated with a pharmaceutical carrier. Suitable pharmaceutical carriers are those known to those skilled in the art (Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (1990)).

[0675] The pharmaceutical composition may include any number of excipients. Available excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents and combinations thereof. The selection and use of suitable excipients are compiled in Gennaro, Remington: The Science and Practice of Pharmacy, the 20th edition (Lippincott Williams & Wilkins 2003) and are taught, the disclosure of which is incorporated herein by reference.

[0676] The pharmaceutical compositions described herein may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect it from the effects of acid and other natural conditions that may inactivate it. As used herein, "parenteral administration" may refer to modes of administration other than enteral and topical administration (usually by injection), and may include, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Additionally or as an alternative, the pharmaceutical compositions described herein may be administered via non-parenteral routes, such as topical, epidermal or mucosal administration routes (e.g., intranasal, oral, vaginal, rectal, sublingual or topical).

[0677] In some embodiments, the pharmaceutical composition comprising Utuximab is for administration to a subject via intravenous infusion.

[0678] The following examples are offered by way of illustration and not limitation.

[0679] 8. Examples

[0680] 8.1 Example 1 - Glycosylation profile

[0681] The glycosylation profile of the anti-CD20 antibody protein sample provided herein is determined by measuring fluorescently labeled N-glycans (the fluorescent label is 2-aminobenzamide), which are cleaved from the anti-CD20 antibody protease using PNGase F. The labeled glycans are resolved using a hydrophilic interaction column equipped. After separation, the glycans are passed through a fluorescence detector. Peak identification from the sample chromatogram is based on retention time and relative to peaks in a glycan standard that has been confirmed by mass spectrometry. The relative percentage of each N-glycan is calculated based on the N-glycan peak area divided by the total peak area of ​​all N-glycans. The glycosylation profile is shown in Figure 2 middle.

[0682] 8.2 Example - Intact Mass Method

[0683] The glycosylation profile of the anti-CD20 antibody protein provided herein was evaluated under non-reducing conditions by intact mass analysis (LC-MS). During the chromatography step using SEC and a mobile phase containing TFA, acetonitrile and water, the anti-CD20 antibody protein sample provided herein was first exchanged into an appropriate buffer for MS. The sample was then introduced into ESI-QTOF for intact mass analysis. The mass spectrum was deconvoluted and peaks were assigned based on mass. The relative abundance of each anti-CD20 antibody protein containing N-glycans provided herein was calculated by obtaining the abundance of N-glycans and dividing by the total abundance of all identified peaks. The results are provided in the following table and Figure 3 middle.

[0684] Table 8: Intact molecular weight of anti-CD20 antibody protein samples by LC-MS

[0685]

[0686]

[0687] Abbreviation: ND = not detected.

[0688] 8.3 Example 3 - Cell-based ADCC assay

[0689] (a) Materials and methods

[0690] Antibody-dependent cellular cytotoxicity (ADCC) is mediated by the Fc part of TG-1101 (TG Therapeutics, Inc.) bound to the FcγIIIA receptor on the effector cell. The assay used for this analysis uses the "KILR CD16a effector cells" of Eurofins-DiscoverX, which are human CD8+T lymphocytes from a single donor source that are engineered to express CD16 (FcγRIII) on their plasma membrane surface. Compared with PMBC preparations separated from fresh blood, these cytotoxic T cells can provide reduced background killing, increased accuracy and reproducibility. Raji cells are used as target cells, and ADCC activity is measured by the dissolution of target cells.

[0691] KILR cells were obtained from Eurofins, and Raji cells were obtained from ATCC. The master cell bank and working cell bank system were used to ensure the quality of the reagents. 5 Raji cells were inoculated at 5x10 cells / mL. 5 KILR effector cells were seeded at 10 cells / mL with a final effector:target (E:T) ratio of 5:1. Eight-point serial dilutions of the sample (250, 50, 16.7, 5.6, 1.9, 0.6, 0.2, 0.04 pg / mL) were used at a concentration range of 250.00 pg / mL to 0.04 pg / mL. The cell mixture and the sample were incubated at 36±1°C, 5±1% CO2 for 18 to 22 hours. At the end of the incubation, CytoTox GLo TMPreparation, and the plate was incubated for 30 ± 10 minutes. The plate was read using a SpectraMax plate reader. Two independent material preparations were prepared and measured on duplicate plates. The assay control was prepared in triplicate and included: a single target cell control, a target cell death control, a single effector cell control, and an effector cell and target cell control. Using SoftMax Pro, the data were analyzed using a 4-parameter logistic fit by means of weighted nonlinear regression. The results are described as a percentage of ADCC activity compared to the Utuximab reference standard. The EC50 of each sample was generated based on a 4-parameter logistic fit curve to obtain more information. The test method is a validated assay CTSOP482, which is used for TG-1101 (TG Therapeutics, Inc.) drug substance and drug release and stability testing.

[0692] (b) Results

[0693] The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. The commercial reference standard of TG-1101 (TG Therapeutics, Inc.) RS-117808 (117808) was used as a control, as Figure 4 As shown in, all anti-CD20 samples exhibited dose-dependent ADCC activity, and TG-1101 (TG Therapeutics, Inc.) had the highest ADCC activity compared to other anti-CD20s. Compared to the TG-1101 (TG Therapeutics, Inc.) reference standard, the ADCC activity expressed as a percentage of the TG-1101 (TG Therapeutics, Inc.) reference standard is shown in Table 9, Gazava has a relatively similar ADCC activity to TG-1101 (TG Therapeutics, Inc.), while Rituxan, Ocrevus and Arzerra have significantly lower ADCC activity. The ED50 of each anti-CD20 is also shown in Table 9, and overall TG-1101 (TG Therapeutics, Inc.) and Gazyva have lower EC50s than Arzerra, Rituxan and Ocrevus. The EC50 of TG-1101 (TG Therapeutics, Inc.) is approximately 25-fold lower than that of Ocrevus.

[0694] Table 9: ADCC activity and EC50

[0695]

[0696] 8.4 Example 4 - Cell-based ADCC assay using primary NK cells

[0697] (a) Materials and methods

[0698] The ADCC assay was performed using Raji cells expressing CD20 as target cells, primary NK cells as effector cells, and LDH as a readout for target cell lysis. Raji cells (ATCC, Cat# CCL-86TM) were cultured at 1×10 5 Cells / well were seeded on the plate. Primary NK cells were isolated from human donor PBMCs using the Miltenyibiotec kit (Cat#130-092-657). NK92 / CD16a cells and primary NK cells with an E / T ratio of 5:1 were used in this assay. Eight-point serial dilutions of the sample were used in triplicates ranging from 0.01 μg / mL to 0 ug / ml, with a dilution factor of 10. The target cells were incubated with the sample dilutions in a 37°C incubator for 30 minutes. Effector cells were added to the target cell culture, followed by incubation for 6 hours, after which the supernatant was collected. OD492nm data minus the background (OD650nm) was used to calculate LDH release. The percentage of cell lysis was calculated according to the following formula: Cell lysis % = 100*(OD sample data-OD tumor cells plus effector cells) / (OD maximum release-OD minimum release).

[0699] (b) Results

[0700] The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, and Arzerra. Figure 5 As shown in Table 10, all anti-CD20 samples exhibited dose-dependent ADCC activity. The calculated EC50 values ​​are summarized in Table 10. TG-1101 (TG Therapeutics, Inc.) and Gazyva exhibited higher ADCC activity and lower EC50 than Rituxan and Arzerra.

[0701] Table 10: EC50 of ADCC assay using primary NK cells

[0702]

[0703] 8.5 Example 5 - Cell-based ADCP assay

[0704] (a) Materials and methods

[0705] Antibody-dependent cellular phagocytosis (ADCP) is another potential mechanism of action (MOA) of anti-CD20. ADCP activity was assessed using an assay using CD20-expressing Daudi cells as target cells (ATCC, Cat#CCL-213, labeled by PKH26). Human monocytes were isolated from PBMCs of 20 human donors (using the Human Pan Monocyte Isolation Kit, Miltenyi Biotec, Cat#130-096-537) and differentiated in vitro using GM-CSF to generate macrophages. An E / T ratio of 5:1 was used; and ADCP was assessed by flow cytometry in this assay. Samples with eight-point serial dilutions ranging from 100 μg / mL to 0 ug / ml (dilution factor of 10) were incubated in triplicate with target cells labeled by PKH26. Macrophages were then co-cultured with target cells labeled by PKH26 for 22 hours. Target cell phagocytosis was assessed by flow cytometry. Controls in the assay included a target cell control of Daudi cells stained only with PKH26; an effector cell control of MDMs stained only with PKH67.

[0706] Effector cell and target cell controls and non-specific IgG1 antibody; Effector cell and target cell controls (background control). ADCP was determined by FACS as the percentage of PKH26 / PKH67 double positive cell counts / PKH26.

[0707] (b) Results

[0708] The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, and Arzerra. Figure 6 As shown in Table 11, all anti-CD20 samples exhibited dose-dependent ADCP activity. The calculated EC50 values ​​are summarized in Table 11. EC50 was in the ng / ml range, and the ADCP activity of all samples was considered similar considering the nature of the assay method.

[0709] Table 11: EC50 of ADCP assay

[0710]

[0711] Blind code: 50K068:70T:2003 = Gazyva; 52W243:70T:2003 = Arzerra;

[0712] 54A157:70T:2003=Rituxan

[0713] 8.6 Example 6 - Cell-based CDC assay

[0714] (a) Materials and methods

[0715] Complement-dependent cytotoxicity (CDC) is mediated by binding of the Fc portion of TG-1101 (TG Therapeutics, Inc.) to the C1q receptor in the complement system. The CDC activity assay used in this analysis is a cell-based assay that uses the CD20-expressing human mantle cell lymphoma cell line Jeko-1 and rabbit serum as a complement source. CDC-mediated cell lysis was performed by Cell Titer-Glo TM The assay was performed using the ELISA kit (Promega). Nine-point serial dilutions of the samples were used in a concentration range of 10,000 ng / mL to 10.42 ng / mL (10,000.00, 3333.33, 1666.67, 833.33, 416.67, 208.33, 104.17, 52.08, 10.42 ng / mL). Two independent preparations were made for each sample and assayed on duplicate plates. Negative controls were prepared in triplicate and included target cell and complement controls as well as target cell alone controls.

[0716] 3x10 5 Jeko-1 cells obtained from ATCC and maintained by the master library system were inoculated with 10 cells / mL and incubated for 60 to 90 minutes. Add sample diluent, then add complement, and incubate the plate at 37 ° C for about 2 hours and at room temperature for 25 minutes. The control of only target cells plus complement and the control of only target cells provide the basal level of target cell viability during the assay. Then add Cell Titer-Glo reagent and incubate for another 30 minutes at room temperature. At the end of the assay, the plate readings were read using a SpectraMax M5 plate reader. Using SoftMax Pro, the data were analyzed using a 4-parameter logistic fit by means of weighted nonlinear regression. PLA software was used to evaluate the parallelism and efficacy of the obtained data relative to the reference standard. The results are described in % efficacy relative to the reference standard. The EC50 of each sample is generated based on a 4-parameter logistic fit curve to obtain additional information. The test method is a validated assay CTSOP463 for TG-1101 (TG Therapeutics, Inc.) drug substance and drug release and stability testing.

[0717] (b) Results

[0718] The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus and Arzerra. TG-1101 (TG Therapeutics, Inc.) commercial reference standard RS-117808 (117808) was used as a control, such as Figure 7 As shown in, except Gazyva (known to have reduced CDC activity), all samples exhibited dose-dependent CDC activity. Rituxan and Arzerra have comparable CDC activity; Ubli and Ocrevus have comparable CDC activity. The CDC activity expressed as a percentage of the TG-1101 (TG Therapeutics, Inc.) reference standard is shown in Table 12. Compared with TG-1101 (TG Therapeutics, Inc.), Gazava has a relatively similar ADCC activity to TG-1101 (TG Therapeutics, Inc.), while the ADCC activity of Rituxan, Ocrevus and Arzerra is significantly lower. The ED50 of each anti-CD20 is also shown in Table 12, which shows a similar comparison result to the CDC activity level.

[0719] Table 12: CDC activity and EC50

[0720]

[0721] 8.7 Example 7 - Cell-based CD20 binding assay

[0722] (a) Materials and methods

[0723] The CD20 binding used for this analysis is a cell-based binding assay using the CD20-expressing human mantle cell lymphoma cell line Jeko-1 and the MSD (MesoScale Discovery) assay format. Jeko-1 target cells are seeded onto MSD plates, the samples are incubated and allowed to bind to the Jeko-1 cells, and the CD20 binding assay is performed using a streptavidin-conjugated SULFOTAG TMThe electrochemiluminescent signal was emitted by adding an anti-human Fc detection antibody. An eight-point serial dilution of the test specimen was used with a concentration range of 40,000.00 μg / ml to 0.23 ng / mL (40,000.00, 4,000.00, 1,000.00, 333.30, 111.10, 37.00, 4.60, 0.23 ng / mL). Two independent test material preparations were prepared for each 2 plates evaluated. Assay controls included a cell-free control (reference standard / test material diluent + detection reagent, cells omitted) and a cell-only control (cells + detection reagent, reference standard / test material omitted).

[0724] 3x10 5 Jeko-1 cells obtained from ATCC and managed by the master library system were inoculated into MSD high binding plates containing PBS (final volume of 100 μL per well) and incubated at 35 to 37°C for 2 hours ± 10 minutes. Unbound cells were removed by washing with PBS, the plates were closed and then washed. 50 μL of sample diluent was added, and the plates were incubated with shaking at room temperature for 1 hour ± 10 minutes. After incubation and 3 washes, 50 μL of anti-human Fc detection antibody conjugated with STREP-SULFOTAG was added and incubated with shaking at room temperature for 1 hour ± 10 minutes. The plate was washed again, and 150 μL of MesoScale reading buffer containing tripropylamine (TPA) was added as a co-reactant to generate light for electrochemiluminescence readings. The plate was read immediately on the MSD Reader using Workbench 4.0. The data obtained were evaluated using PLA software and analyzed using a restricted 4-parameter logistic model. The binding activity results are described as a percentage of the efficacy relative to the reference standard. EC50 for each sample was generated based on a 4-parameter logistic fit curve to obtain additional information. This test method is a validated assay CTSOP466, used for TG-1101 (TG Therapeutics, Inc.) drug substance and drug product release and stability testing.

[0725] (b) Results

[0726] The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. The commercial reference standard of TG-1101 (TG Therapeutics, Inc.), RS-117808 (117808), was used as a control. Figure 8As shown in Table 13, all samples exhibited dose-dependent CD20 binding. In addition to Gazyva (a type II anti-CD20 known to have a target occupancy of approximately 50%), Utuximab, Rituxan, Ocrevus, and Arzerra also had similar maximum binding. CD20 binding activity expressed as a percentage of the TG-1101 (TG Therapeutics, Inc.) reference standard and binding EC50 are shown in Table 13. The CD20 binding affinities of the four anti-CD20s were similar.

[0727] Table 13: CD20 binding activity and EC50

[0728]

[0729] The EC50 values ​​listed are negative, and the logarithmic transformation from the PLA software converts the concentrations to base 2. The actual ED50 (in μg / ml) is 2 n , where n = the number listed.

[0730] 8.8 Example 8 - Cell Surface CD20 Binding by FACS

[0731] (a) Materials and methods

[0732] Binding of TG-1101 (TG Therapeutics, Inc.) to CD20 on the surface of Raji and Daudi cells was characterized by FACS analysis at LakePharma. Six-point serial dilutions of samples were used in duplicates with a concentration range of 40 μg / mL to 0 ug / ml, with a dilution factor of 5. Cells were incubated with sample dilutions; binding was detected using a PE-conjugated anti-human secondary antibody.

[0733] (b) Results

[0734] The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Arzerra, and Rituxan. Fig. 9 As shown in Table 14, all anti-CD20 samples exhibited dose-dependent CD20 binding to both Daudi cells and Raji cells. The binding characteristics as evaluated by FACS assay were similar to those evaluated by MSD assay. With the exception of Gazyva (a type II anti-CD20 known to have approximately 50% target occupancy), Utuximab, Rituxan, and Arzerra also had similar maximal binding. The calculated EC50 values ​​are summarized in Table 14. The CD20 binding affinities of the four anti-CD20 antibodies were similar.

[0735] Table 14: EC50 of cell surface CD20 binding by FACS

[0736]

[0737] 8.9 Example 9 - FcγRIIIA Binding Assay

[0738] (a) Materials and methods

[0739] The assay used in this analysis is based on a surface plasmon resonance (SPR) method that measures binding to both FcγRIIIa158V and FcγRIIIa 158F receptors. The method follows a direct binding assay in which the FcγRIIIa receptor is directly fixed to a flow cell on the surface of a sensor chip, and the sample is injected onto the chip to assess binding. FcγRIIIa 158V receptor (3 μg / mL) or FcγRIIIa 158F receptor (6 μg / mL) is fixed to the chip surface using covalent amine coupling chemistry. Eight-point serial dilutions were prepared in a concentration range of 1000 nM to 15.6 nM, with a dilution factor of 2. Independent duplicates of the sample dilutions were injected onto the chip, and then the surface was regenerated between each cycle. Binding is measured in response units (RU). The kinetics of the binding reaction is determined by measuring the SPR changes caused by the increase in mass near the surface of the biosensor chip. The changes in the mass of the complex are visualized as a sensorgram as a function of time.

[0740] The equilibrium dissociation constant (K) of each sample was determined for each receptor. D The rate of change of the SPR signal was analyzed for the FcγRIIIa 158V variant using a 1:1 Langmuir model to generate apparent rate constants for the association and dissociation phases of the reaction, as well as the equilibrium dissociation constant. Steady-state affinity was used to determine K for the FcγRIIIa 158F variant. D The binding signal was exported to PLA to determine the relative binding response, relative affinity and relative binding of the sample and was reported relative to the TG-1101 (TG Therapeutics, Inc.) reference standard. This test method is a validated assay CTSOP477 for TG-1101 (TG Therapeutics, Inc.) drug substance release testing.

[0741] (b) Results

[0742] The samples tested include TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus and Arzerra. As shown in Table 15, among the anti-CD20 antibodies tested, TG-1101 (TG Therapeutics, Inc.) has the highest binding affinity for both FcγRIIIa 158V and FcγRIIIa 158F receptors. Gazyva ranks second in binding affinity. For the high-affinity receptor FcγRIIIa158V, the affinity of TG-1101 (TG Therapeutics, Inc.) is about 15 times higher than that of Ocrevus; for the low-affinity receptor FcγRIIIa158F, the affinity of TG-1101 (TG Therapeutics, Inc.) is about 10 times higher than that of Ocrevus. The relative affinity and relative binding values ​​using TG-1101 (TG Therapeutics, Inc.) reference standard as a reference are shown in Table 16. The results demonstrate that TG-1101 (TG Therapeutics, Inc.) has higher relative binding and relative affinity than all other anti-CD20s.

[0743] Table 15: KD for FcγRIIIa 158V and FcγRIIIa 158V by SPR binding

[0744]

[0745] *: Average of 4 values

[0746] Table 16: Summary of relative affinity and relative binding results

[0747]

[0748] 8.10 Example 10 - Fc receptor binding by Octet

[0749] (a) Materials and methods

[0750] The analysis was performed by LakePharma. Binding characterization was performed on an Octet HTX instrument at 25°C. The human Fc receptor panel was loaded onto the Anti-PentaHis (H1S1K) biosensor. The loaded sensor was immersed in a serial dilution of the sample (starting at 300nM, 1:3 dilution, 7 points). The kinetic constants were calculated using a monovalent (1:1) binding model.

[0751] (b) Results

[0752] The samples tested include TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus and Arzerra. As shown in Table 17, among the anti-CD20 antibodies tested, TG-1101 (TG Therapeutics, Inc.) has the highest binding affinity to both FcγRIIIa 158V and FcγRIIIa 158F receptors, confirming the SPR data. In all anti-CD20s, the binding affinity to FCRN is similar, which can affect PK. Compared with Ocrevus, TG-1101 (TG Therapeutics, Inc.) also has higher affinity to FcγRIIA and FcγRIIIB.

[0753] Table 17: K for Fc receptors D Results Summary

[0754]

[0755] 8.11 Example 11-C1q Binding Assay

[0756] (a) Materials and methods

[0757] The C1q binding assay used for this analysis is an ELISA assay. The sample is coated on an ELISA plate and HRP-conjugated human C1q is incubated with the sample on the plate. In the presence of the substrate TMB, the bound HRP will produce a colorimetric signal. A 7-point serial dilution of the test material is prepared at a concentration range of 15.00ug / ml to 0.12ug / ml, with a dilution factor of 2. The sample dilutions are coated on the ELISA plate and the plate is incubated at room temperature for 1 hour ± 30 minutes. After coating, the plate is washed, blocked and washed again. Peroxidase-conjugated C1q is added and the plate is incubated at room temperature for 1.5 hours ± 30 minutes. After incubation and washing, tetramethylbenzidine (TMB) substrate solution is added and the plate is incubated at room temperature for 7 minutes (-1 minute / +30 seconds). This will produce a colorimetric reaction proportional to the level of C1q binding. The reaction is stopped by adding 1M sulfuric acid and the color is measured at 450nm using a Molecular Devices SpectraMax microplate reader. Data were analyzed using SoftMax Pro with the aid of weighted nonlinear regression using a 4-parameter logistic fit. Binding activity results were reported as a percentage of potency relative to the TG-1101 (TG Therapeutics, Inc.) reference standard. EC50s were generated for each sample based on a 4-parameter logistic fit curve to obtain additional information. The test method is a validated assay, CTSOP455, for TG-1101 (TG Therapeutics, Inc.) drug substance and drug product release and stability testing.

[0758] (b) Results

[0759] The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus and Arzerra. TG-1101 (TG Therapeutics, Inc.) commercial reference standard RS-117808 (117808) was used as a control, such as Fig.10 As shown in Table 18, Gazyva had minimal C1q binding (as expected), while the other four anti-CD20s exhibited dose-dependent C1q binding. The maximum binding of TG-1101 (TG Therapeutics, Inc.) was slightly higher. The C1q binding activity expressed as a percentage of the TG-1101 (TG Therapeutics, Inc.) reference standard is shown in Table 18. The ED50 of each anti-CD20 is also shown in Table 18, and the EC50 of TG-1101 (TG Therapeutics, Inc.) is slightly lower than all anti-CD20s, indicating that TG-1101 (TG Therapeutics, Inc.) has a higher affinity for C1q.

[0760] Table 18: C1q binding activity and EC50

[0761]

[0762] 8.12 Example 12 - Human Whole Blood B Cell Depletion Assay

[0763] (a) Materials and methods

[0764] TG-1101 (TG Therapeutics, Inc.) was characterized in an autologous normal human whole blood B cell depletion assay and compared with Gazyva, Rituxan, Ocrevus and Arzerra. Whole blood from three human donors was used, and donors with FCGR3A_SNP target (rs396991) 158V / 158V genotype were selected. Sample diluents with a concentration range of 0.000001 to 100 μg / ml were added to whole blood and incubated at 37 ° C for 24 hours in a humidified cell culture incubator. Markers of aliquots of blood were stained, including CD45 (lymphocyte population), CD3 (T cells), CD19 (B cells) and CD20 (B cells). B cell depletion was evaluated by displaying cells in the CD45 positive lymphocyte gate and counting CD3 positive T cells, CD19 positive B cells and CD20 positive B cells. The percentage of B cell depletion was calculated (100-([100 / B- / T-cell ratio in samples without antibody] x [B- / T-cell ratio in samples with antibody])) and plotted against sample concentration.

[0765] (b) Results

[0766] The samples tested included TG-1101 (TG Therapeutics, Inc.), commercial reference standard RS-117808 (117808) (API manufactured by commercial process (C2) at Samsung Biologics (PPQ1)), Gazyva, Rituxan, Ocrevus, and Arzerra. FIG. 11A to FIG. 11CAs shown in , all samples showed dose-dependent B cell depletion activity, although there were some minor differences between donors. Overall, TG-1101 (TG Therapeutics, Inc.) and Gazyva have higher B cell depletion activity than Arzerra, Rituxan and Ocrevus. Three anti-CD20 antibodies and an anti-CD19 antibody were used in this experiment. The B cell depletion ED50 calculated using various antibodies for B cell markers is summarized in Table 19. Overall, the EC50 of TG-1101 (TG Therapeutics, Inc.) and Gazyva is also lower than that of Arzerra, Rituxan and Ocrevus. On average, the EC50 of TG-1101 (TG Therapeutics, Inc.) is more than ten times lower than that of Ocrevus.

[0767] Table 19: Summary of ED50s for B cell depletion of multiple B cell markers

[0768]

[0769] 8.13 Example 13 - Calculation of Pharmacokinetic (PK) Values

[0770] Relevant steady-state PK parameters are calculated according to methods known in the art. Steady state is achieved when the amount of drug eliminated per unit time is equal to the amount of drug reaching the systemic circulation per unit time. Therefore, the half-life represents the time required for the plasma concentration of the drug to decrease by 50% to reach steady state. Where; t = time, Vd = volume of distribution, Cl = clearance. The half-life is calculated using the following formula:

[0771]

[0772] AUC represents the total dose of drug exposure over time. AUC is used as a metric when determining the equivalent dose of a drug product and the resulting tissue or plasma exposure. AUC is equivalent to the average concentration over a time interval. Where t = time, and Cpt = the last measured drug concentration relative to time. AUC is calculated using the following formula:

[0773]

[0774] Cmax is obtained by measuring the highest point of the drug concentration during the observation period, after the absorption phase ends and the elimination phase begins. Cmin is obtained by measuring the highest point of the drug concentration during the observation period, after the absorption phase ends and the elimination phase begins.

[0775] 8.14 Example 14 - Method for Determining Population Pharmacokinetic (PPK) Values ​​of TG-1101 for Treating Autoimmune Diseases

[0776] Pop PK analysis of TG-1101 was performed by pooling TG-1101 serum concentration-time, dose, demographic, and covariate data from one Phase 2 study (TG1101-RMS201) and two Phase 3 studies (TG1101-RMS301 and TG1101-RMS302) in RMS subjects. The datasets were combined with previous TG-1101 datasets in subjects with hematological malignancies.

[0777] All subjects who were administered at least one dose of TG-1101 in studies TG1101-RMS201, TG1101-RMS301, and TG1101-RMS302 were included in the data set for PK analysis. Subjects who did not have at least one quantifiable post-dose TG-1101 concentration were included in the data set and marked. The exposure of these subjects was determined based on typical population PK parameters.

[0778] In the Phase 2a clinical trial TG1101-RMS201, TG-1101 was administered as a single agent and compared to placebo to investigate the level of B cell depletion by TG-1101 and to determine the optimal dose and infusion schedule of TG-1101 in RMS subjects. Based on the results of this study, a dose of 150 mg was given on Day 1 of Week 1 (infused over 4 hours), followed by a dose of 450 mg on Day 15 of Week 3 (infused over one hour), resulting in a median of >99% B cell depletion at Week 4 that was sustained through Week 24. The dosing regimen was well tolerated by subjects, with infusion-related reactions (Grades 1 and 2) being the most commonly reported adverse events. Two Phase 3 studies have been completed in RMS subjects. The two Phase 3 studies, TG1101-RMS301 (also known as ULTIMATE I) and TG1101-RMS302 (ULTIMATE II), were randomized, double-blind, double-dummy, active-controlled studies comparing TG-1101 with oral teriflunomide to evaluate the ARR, safety, and tolerability in RMS subjects.

[0779] Overall, the dataset for PopPK analysis included a total of 8672 PK samples collected from 931 subjects. Pre-dose samples accounted for 10.02% of the PK samples, while records with missing information or outliers (>10 standard deviations from the mean TG-1101 concentration at the nominal dosing time) accounted for 0.20% and were excluded. Post-dose samples of BLQ accounted for 3.47% of the data. The exclusion resulted in 36 subjects having no quantifiable post-dose PK samples. Therefore, the PK analysis dataset included 7485 PK samples from 895 subjects, of which 5624 PK samples were from 591 RMS subjects.

[0780] The final PopPK parameter estimates are presented in Table 20. The PK parameter estimates for a typical subject (defined as a male subject from North America or Western Europe, weighing 73 kg, and ADA negative) are as follows: CL is estimated to be 11.6 mL / h, IIV is 38.1%; Ve is estimated to be 3.18 L (IIV = 15.0%); Vp is estimated to be 3.60 L (IIV = 21.3%); Q is estimated to be 11.6 mL / h.

[0781] Weight and ADA were found to be statistically significant predictors of TG-1101CL. ADA-positive subjects had a slight increase of 14% in TG-1101CL compared to subjects without quantifiable ADA. For the RMS subpopulation with a wide range of weights (45.1 to 154 kg), CL ranged from a low of 22% to a high of 48% compared to a typical subject weighing 73 kg. In addition, in the later period (417 days after the start of treatment), the median CL reduction was 12.5%.

[0782] Weight, sex, and region were found to be statistically significant predictors of Vc. Subjects from Eastern Europe had slightly higher Vc than Western Europeans and North Americans (10%), and females had slightly lower Vc than males (7%). For the RMS subpopulation with a wide range of weights (45.1 to 154 kg), Vc ranged from a low of 19% to a high of 38% for a typical subject weighing 73 kg.

[0783] After incorporating body weight into the model, age, hemoglobin concentration, platelet count, white blood cell count, renal impairment, or hepatic impairment had no effect on TG-1101 PK.

[0784] Table 20: Parameter estimates for the final TG-1101 PK model

[0785]

[0786] Abbreviations: CL-clearance; CV%-coefficient of variation percentage; ETA-individual-specific random effect; IIV-interindividual variability; Q-intercompartmental clearance; RSE-relative standard error; SD-standard deviation; SE-standard error; U2-TG-1101+umbralisib; Vc-volume of the central compartment; Vp-volume of the peripheral compartment.

[0787] a The RSE for parameter estimates was calculated as 100x(SE / typical value); the RSE for IIV magnitude was calculated as 100x(SE / estimate of variation).

[0788] b Estimates of random effects and IIV are presented as CV% and are calculated as √variance x 100 based on the estimates.

[0789] c Shrinkage (%) was calculated as 100x(1-SD[ETA] / √[variance]).

[0790] d The correlation coefficient between CL and Vc was estimated to be 0.434.

[0791] In evaluating the final TG-1101 PopPK model, standard errors were obtained and presented with the parameter estimates in Table 20. Model parameters were accurately estimated with RSE < 20% for structural and covariate model parameters and RSE < 30% for random effect estimates. The shrinkage for CL and Vc was acceptable (5.32% and 32.6%, respectively), while the shrinkage for Vp was larger (40%).

[0792] Key goodness-of-fit (GOF) diagnostics for the final PopPK model for TG-1101 showed satisfactory fit over time with minimal residual bias between predicted concentration values, and good agreement between predicted and observed concentrations. ETA-covariate relationships were resolved relative to the base model, and no further trends between ETA (for CL and Vc) and covariates were evident in the RMS subpopulation, indicating that the model adequately captured significant covariate relationships. Overall, the prediction-corrected visual prediction test (pcVPC) plots indicated that the model adequately predicted the central tendency of the observed TG-1101 concentrations and adequately captured the range of the data.

[0793] The relative importance of the effects of the covariates included in the final PoPK model was evaluated using forest plots to assess the relative changes in exposure (Cmax,ss, Cmin,ss, and AUCss) when the covariates were changed one at a time (i.e., univariate analysis). The effects of these covariates (including weight, sex, region ADA, and late CL fraction change) on TG-1101 exposure fell within the range of 0.8 to 1.25 compared with the reference exposure (defined as the exposure of male subjects from North America / Western Europe, weighing 73 kg, ADA-negative, and treated for <416 days). Therefore, none of the covariates was considered clinically relevant. In addition, the covariates did not significantly affect the magnitude of IIV for CL or Vc. The combined effect of weight and ADA reduced IIV by only 2.8%, from 39.2% in the base model to 38.1% in the final model (Table 20). Similarly, weight, sex, and region reduced IIV in Vc by 18.5%, from 18.4% in the base model to 15.0% in the final model.

[0794] Fig.12 Goodness of fit (GOF) diagnostics for the final model for TG-1101 are shown. Fig.13 The pcVPC of the final PK model of TG-1101 derived from the study is shown. Fig.14 Forest plot showing the covariate effects of TG-1101 drug exposure.

[0795] The final PopPK model was re-estimated using the data set including the exclusion of outliers. All structural parameters were precisely estimated with no relevant changes in those estimates, whereas the IIV term was inflated by the inclusion of outliers. Therefore, it is expected that excluding outliers during model development would eliminate the effects of these outliers and minimize spurious results in the covariate analysis. The final PopPK model including only the RMS subpopulation was also re-estimated. The fit of the model to the RMS data yielded similar structural parameters and random effects, except for a slightly lower IIV on CL; the precision of the parameter estimates was also similar.

[0796] Conclusion: TG-1101PPK Analysis

[0797] The final model for TG-1101 was used to obtain individual post hoc estimates of PK parameters. For each subject enrolled in studies TG1101-RMS201, TG1101-RMS301, and TG1101-RMS302, the PK parameters (AUCss, Cavg,ss, Cmax,ss, and Cmin,ss) at week 48 were estimated based on the post hoc PK parameters.

[0798] Geometric mean 1 1 / 2 (90% confidence interval [CI]) was calculated to be 21.8 days (21.4, 22.1 days). The median time to steady state was determined to be 15.5 weeks. Therefore, there was no accumulation in subjects who received 150 mg TG-1101 on day 1, followed by 450 mg TG-1101 on days 15, 24, and 48 of the per-protocol regimen. The ratio of the median Cmax at week 24 to the Cmax at day 1 was 3.04 (range 3.00 to 3.42), which is consistent with a 3-fold increase in the number of doses and indicates no accumulation. Similarly, the Cmax ratio at week 48 to week 24 was 1, indicating no accumulation.

[0799] The geometric mean AUCss, Cavgss, Cmaxss and Cminss predicted by the model were 3000ug·d / mL (±28%), 8940ng / mL (±28%), 139000 (±15.1%) and 139 (±170%), respectively.

[0800] 8.15 Example 15 - Commercial Scale Manufacturing Method for Preparing TG-1101 in YB2 / 0 Rat Hybridoma Cells

[0801] In this example, a method for producing TG-1101 expressed in YB2 / 0 rat hybridoma cells at 15,000 L is described. The flow chart in Table 21 illustrates an overview of the production process of TG-1101.

[0802] Table 21: TG-1101 manufacturing process flow chart

[0803]

[0804] In summary, the production of each batch of TG-1101 starts from thawing working cell bank (WCB) vials, as further described below. Cultures are amplified by a series of shake flasks and seed bioreactors to meet the inoculum requirements of 15,000L production bioreactors, which are operated in fed-batch mode. The bioreactor is harvested and clarified by centrifugation and then deep filtration. The clarified harvest is purified by three chromatographic steps, which include protein A, cation exchange and anion exchange, which are designed to purify TG-1101 and reduce process impurities (such as host cell proteins and residual DNA). The purification process (as shown in Example 4) contains steps to ensure virus safety, including virus inactivation (solvent / detergent) and virus filtration steps. Final UFDF and formulation steps are used to concentrate TG-1101 and buffer exchange into formulation buffer and reach required product concentration. The ready-to-fill drug substance was formulated to obtain a concentration of 25.0 mg / mL of TG-1101 in 25 mM sodium citrate, 154 mM sodium chloride, 0.07% polysorbate 80, pH 6.5. After filling, the TG-1101 drug substance was frozen at ≤-60°C and then stored frozen at ≤-35°C.

[0805] Additional description of upstream and downstream process operations is provided below.

[0806] (a) Expression vectors, production cell lines and cell banks

[0807] The host cell line used to generate the TG-1101 production cell line was the rat cell line YB2 / 0. The production cell line R603-12D11 was developed by transfecting the expression vector HK463-25 (containing the immunoglobulin heavy and light chain cDNA sequences of TG-1101) into the YB2 / 0 host cell line. Fig.15 The expression vector map of HK463-25 is described to produce TG-1101 in a 15,000 L bioreactor.

[0808] Expression vector

[0809] The expression vector HK463-25 includes various elements optimized for stable expression in YB2 / 0 host cell lines. The Rous sarcoma virus long terminal repeat (RSV LTR) promoter is used for the constitutive expression of heavy chain and light cha...

Claims

1. A composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising about 20 to 40% fucosylated glycans and optionally about 10 to 20% galactosylated glycans.

2. The composition according to claim 1, wherein: The N-glycan profile comprises 23% to 36% fucosylated glycans, optionally about 30% fucosylated glycans; and / or The N-glycan profile comprises 16% to 18% galactosylated glycans, optionally about 17% galactosylated glycans.

3. The composition according to claim 1 or 2, wherein: The relative abundance of the fucosylated glycans is the percentage of the fucosylated glycans among all glycans in the N-glycan profile; and / or The relative abundance of the galactosylated glycans is the percentage of the galactosylated glycans among all the glycans in the N-glycan profile.

4. The composition of any one of claims 1 to 3, wherein the N-glycan profile comprises 12% to 30% bisecting N-glycans, optionally about 18% bisecting N-glycans.

5. The composition of claim 4, wherein the bisecting N-glycan comprises one or more of G0B, GOFB, G1FB, G2FBS1 and G2FBS2.

6. The composition of any one of claims 1 to 5, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising less than 5% sialylated glycans, Optionally wherein the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycans, Optionally wherein the N-glycan profile comprises undetectable amounts of sialylated glycans.

7. The composition of any one of claims 1 to 6, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans, Optionally wherein the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans, Optionally wherein the N-glycan profile comprises about 0.6% Man5 N-glycans, Optionally wherein the Man5 N-glycan is the only high mannose species in the N-glycan profile.

8. The composition according to any one of claims 1 to 7, wherein the anti-CD20 antibody protein population comprises 0.20 to 0.40 mol isoaspartic acid / mol protein, optionally 0.25 to 0.35 mol isoaspartic acid / mol protein.

9. The composition according to any one of claims 1 to 8, wherein the glutamic acid at position 1 of the heavy chain is pyroglutamic acid and the glutamic acid at position 1 of the light chain is pyroglutamic acid.

10. The composition of any one of claims 1 to 9, wherein the anti-CD20 antibody protein population has an N-glycan profile comprising G1 to G0 N-glycans at a relative abundance ratio of 0.1 to 0.15 and / or G1F to G1 N-glycans at a relative abundance ratio of 0.5 to 0.

9.

11. The composition of any one of claims 1 to 10, wherein the anti-CD20 antibody protein population further comprises at least two N-glycans within the following relative abundance ranges: (a) 0.3% to 2% GO-GN; (b) 0.1% to 2% G0F-GN; (c) 30% to 60% G0; (d) 0.1% to 1% G1-GN; (e) 5% to 20% GOB; (f) 5% to 30% GOF; (g) 0.1% to 1.5% Man5; (h) 1% to 15% GOFB; (i) 1% to 13% G1; (j) 0.5% to 10% G1'; (k) 0.5% to 6% G1B; (l) 0.5% to 12% G1F; (m) 0.1% to 3% G1F'; (n) 0.1% to 3% G1FB; (o) 0.1% to 2% G2; and (p) 0.1% to 2% G2F, Optionally wherein the anti-CD20 antibody protein population further comprises at least two N-glycans within the following relative abundance ranges: (a) 0.8% to 1.1% GO-GN; (b) 0.5% to 1.1% G0F-GN; (c) 42.5% to 48.8% G0; (d) 0.3% to 0.6% G1-GN; (e) 9.5% to 14.1% GOB; (f) 12.8% to 19.7% GOF; (g) 0.4% to 0.7% Man5; (h) 5.1% to 7.0% GOFB; (i) 5.7% to 6.4% G1; (j) 2.7% to 3.3% G1'; (k) 1.4% to 2.0% G1B; (l) 2.6% to 4.2% G1F; (m) 1.1% to 1.6% G1F'; (n) 1.1% to 1.8% G1FB; (o) 0.5% to 0.7% G2; and (p) 0.3% to 0.5% G2F, Optionally wherein the anti-CD20 antibody protein population further comprises at least two N-glycans within the following relative abundance ranges: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 46.1% G0; (d) 0.5% G1-GN; (e) 10.9% G0B; (f) 17.0% GOF; (g) 0.6% Man5; (h) 6.0% GOFB; (i) 6.1% G1; (j) 2.9% G1'; (k) 1.6% G1B; (l) 3.2% G1F; (m) 1.3% G1F'; (n)1.3G1FB; (o) 0.5% G2; and (p)0.3%G2F.

12. The composition of claim 11, wherein the anti-CD20 antibody protein population further comprises at least three, four or five N-glycans within the following relative abundance ranges: (a) 0.3% to 2% GO-GN; (b) 0.1% to 2% G0F-GN; (c) 30% to 60% G0; (d) 0.1% to 1% G1-GN; (e) 5% to 20% GOB; (f) 5% to 30% GOF; (g) 0.1% to 1.5% Man5; (h) 1% to 15% GOFB; (i) 1% to 13% G1; (j) 0.5% to 10% G1'; (k) 0.5% to 6% G1B; (l) 0.5% to 12% G1F; (m) 0.1% to 3% G1F'; (n) 0.1% to 3% G1FB; (o) 0.1% to 2% G2; and (p) 0.1% to 2% G2F.

13. The composition of any one of claims 1 to 12, wherein the N-glycan profile of the anti-CD20 antibody protein population is determined using a method comprising: (a) incubating the anti-CD20 antibody protein population with an enzyme, wherein the enzyme catalyzes the release of N-glycans from the anti-CD20 antibodies; (b) measuring the relative abundance of the released N-glycans using one or more methods selected from chromatography, mass spectrometry, capillary electrophoresis, and combinations thereof, Optionally, the method further comprises the following steps after step (a) and before step (b): (c) purifying the N-glycans; and (d) labeling the N-glycan with a fluorescent compound, Optionally wherein the enzyme is PNGase F and / or the fluorescent compound is 2-aminobenzamide (2-AB).

14. The composition of any one of claims 1 to 13, wherein less than 10% of the anti-CD20 antibody proteins in the population are non-glycosylated, Optionally wherein less than 5% of the anti-CD20 antibody proteins in the population are aglycosylated, Optionally wherein less than 1% of the anti-CD20 antibody proteins in the population are aglycosylated.

15. The composition according to any one of claims 1 to 14, wherein the anti-CD20 antibody protein population comprises two or more secondary structures as determined by circular dichroism at 205 nm to 260 nm as follows: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-sheets; (c) 5.0% to 6.0% parallel β-sheets; (d) 16.0% to 18.0% β-turn; and (e) 35.0% to 36.0% random curl, Optionally, the anti-CD20 antibody protein population comprises the following secondary structure as determined by circular dichroism at 205 nm to 260 nm: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-sheets; (c) 5.0% to 6.0% parallel β-sheets; (d) 16.0% to 18.0% β-turn; and (e) 35.0% to 36.0% random curl, Optionally, the anti-CD20 antibody protein population comprises two or more secondary structures as determined by circular dichroism at 205 nm to 260 nm as follows: (a) about 9.0% α-helix; (b) about 33.0% antiparallel β-sheets; (c) about 5.6% parallel β-sheets; (d) about 17.5% β-turn; and (e) About 35.2% random coil.

16. The composition of any one of claims 1 to 15, wherein the anti-CD20 antibody protein population further comprises one or more of the following post-translational modifications at a specified abundance: Optionally wherein one or more of the post-translational modifications are measured by peptide mapping using liquid chromatography-mass spectrometry (LC-MS).

17. The composition of any one of claims 1 to 16, wherein the population has a total protein amount of 25.5-25.8 mg / mL as measured by absorbance at 280 nm.

18. The composition of any one of claims 1 to 17, wherein the anti-CD20 antibody proteins in the population induce greater cytotoxicity in a cell-based antibody-dependent cellular cytotoxicity (ADCC) assay compared to obinutuzumab, ofatumumab, rituximab, veltuzumab, ibrutinib and / or ocrelizumab.

19. A composition according to any one of claims 1 to 18, wherein: The population had a relative potency ranging from 90% to 163% in a cell-based ADCC assay compared to a commercial reference standard; The population has a relative potency of 78% to 116% or 73% to 128% in a cell-based complement-dependent cytotoxicity (CDC) assay compared to a commercial reference standard; The population has a relative potency of 92% to 118% or 82% to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard; The population had a K of 30 to 70 nM in the FcγRIIIa-158V binding assay as measured by surface plasmon resonance. D value; The population had a K of 500 to 1000 nM in the FcγRIIIa 158F binding assay as measured by surface plasmon resonance. D value; The population has a significantly higher binding affinity to FcγRIIIa 158V or FcγRIIIa 158F than rituximab; The population has a relative potency of 88% to 113% or 86% to 116% in a CIq binding assay as measured by ELISA compared to a commercial reference standard; and / or The population had a relative potency of 106% to 126% in the CD16 activity assay compared to the commercial reference standard.

20. The composition according to any one of claims 1 to 19, wherein The population has 99.2% to 99.9% monomers as detected by size exclusion chromatography (SEC); the population has 0.1% to 0.8% dimers as detected by SEC; the population has undetectable levels of aggregates as detected by SEC; and / or the population has undetectable levels of fragments as detected by SEC.

21. A composition according to any one of claims 1 to 20, wherein the population has 93.6 to 95.9% IgG after purification by non-reducing capillary gel electrophoresis (CGE); the population has 0.1 to 0.3% high molecular weight species (HMWS) after purification by non-reducing CGE; the population has 0.7 to 1.2% free light chains (LC) after purification by non-reducing CGE; the population has 97.7 to 98.0% heavy chain plus light chain species (HC+LC) after purification by reducing CGE.

22. The composition according to any one of claims 1 to 21, wherein The population has 20% to 25% acidic isoform as detected by imaged capillary isoelectric focusing (iCIEF); the population has 50% to 60% major isoform as detected by iCIEF; the population has 20% to 30% basic isoform as detected by iCIEF; and / or the population has an average molar ratio of free thiol to anti-CD20 antibody of about 2.0 to 2.

2.

23. A composition according to any one of claims 1 to 22, wherein: The amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of N-terminal residues, optionally a deletion of up to 5 N-terminal residues, optionally a deletion of up to 10 N-terminal residues; and / or The anti-CD20 antibodies in the population are truncated at the terminal lysine residue of the heavy chain.

24. A pharmaceutical formulation comprising the composition of any one of claims 1 to 23, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 10 mg / mL to 50 mg / mL, optionally wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg / mL.

25. The pharmaceutical formulation of claim 24, further comprising one or more of the following: sodium chloride, trisodium citrate dehydrate, polysorbate 80, and hydrochloric acid.

26. The pharmaceutical formulation of claim 25, comprising about 9.0 mg / mL sodium chloride, about 7.4 mg / mL trisodium citrate dehydrate, about 0.7 mg / mL polysorbate 80 and / or about 0.4 mg / mL hydrochloric acid.

27. The pharmaceutical formulation of any one of claims 24 to 26, wherein the anti-CD20 antibody is present in a single dosage form.

28. A pharmaceutical preparation comprising: (i) the composition according to any one of claims 1 to 23, wherein the composition comprises a single dosage form of the anti-CD20 antibody protein population, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg / mL, (ii) about 9.0 mg / mL of sodium chloride, (iii) about 7.4 mg / mL of trisodium citrate dehydrate, (iv) about 0.7 mg / mL of polysorbate 80, and (v) about 0.4 mg / mL of hydrochloric acid.

29. A single batch preparation of a population of anti-CD20 antibody proteins according to any one of claims 1 to 23 or a pharmaceutical formulation according to any one of claims 24 to 28, wherein the single batch comprises at least 100 g, at least 120 g or at least 150 g of anti-CD20 antibody protein.

30. The anti-CD20 antibody protein of any one of claims 1 to 23 or the pharmaceutical formulation of any one of claims 24 to 28, which is produced in a 15,000L or 20,000L bioreactor.

31. A method of treating multiple sclerosis (MS) in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a composition according to any one of claims 1 to 23 or a pharmaceutical formulation according to any one of claims 24 to 28.

32. The method of claim 31, wherein the MS is a relapsing form of MS (RMS), Optionally wherein RMS is selected from clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS) and active secondary progressive MS (SPMS).

33. The method of claim 31 or 32, wherein the composition or the pharmaceutical formulation is administered as follows: i) a first infusion at a dose of about 150 mg of anti-CD20 antibody protein, ii) a second infusion two weeks later at a dose of about 450 mg of anti-CD20 antibody protein, and iii) subsequent infusions every 24 weeks or six months at a dose of about 450 mg of anti-CD20 antibody protein.

34. The method of any one of claims 31 to 33, wherein administration of the composition or the pharmaceutical formulation to the subject results in one or more of the following pharmacokinetic parameters: (a) AUC of 2,160 μg / mL to 3,840 μg / mL; (b) Cmax of 118,011 ng / mL to 159,989 ng / mL; (c) a Cmin of 40 ng / mL to 375 ng / mL; and (d) Cavg of 6,437 ng / mL to 11,443 ng / mL, Optionally wherein administration of said composition or said pharmaceutical formulation to said subject results in one or more of the following pharmacokinetic parameters: (a) AUC of approximately 3,000 μg / mL; (b) Cmax of approximately 139,000 ng / mL; (c) a Cmin of about 139 ng / mL; and (d) Cavg of approximately 8,940 ng / mL.

35. The method of any one of claims 31 to 34, wherein the method comprises a treatment period of at least 96 weeks.

36. The method of any one of claims 31 to 35, wherein the subject has been premedicated with a corticosteroid 30 to 60 minutes prior to administration of the composition or the pharmaceutical formulation, optionally wherein the corticosteroid is methylprednisone or dexamethasone, optionally wherein the methylprednisone is administered at a dose of about 100 mg and / or the dexamethasone is administered at a dose of about 10 to 20 mg.

37. The method of any one of claims 31 to 36, wherein the subject has been premedicated with an antihistamine 30 to 60 minutes prior to administration of the composition or the pharmaceutical formulation, optionally wherein the antihistamine is diphenhydramine HCl, optionally wherein the diphenhydramine HCl is administered at a dose of about 25 to 50 mg.

38. The method of any one of claims 31 to 37, wherein the subject has been premedicated with an antipyretic 30 to 60 minutes prior to administration of the composition or the pharmaceutical formulation, optionally wherein the antipyretic is acetaminophen or an antipyretic bioequivalent thereto.

39. The method of any one of claims 31 to 38, wherein the subject has an Expanded Disability Status Scale (EDSS) score of 0 to 5.5 before treatment.

40. The method of any one of claims 31 to 39, wherein the subject is diagnosed with RMS according to the McDonald Criteria (2010).

41. A method of treating multiple sclerosis (MS) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 1 to 23 or a pharmaceutical formulation according to any one of claims 24 to 28, wherein administration of the composition or the pharmaceutical formulation results in no evidence of disease activity (NEDA) in the subject 24 to 96 weeks after administration.

42. The method of claim 41, wherein administration of the composition or the pharmaceutical formulation results in NEDA in the subject 24 weeks after administration.

43. A method of treating multiple sclerosis (MS) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 1 to 23 or a pharmaceutical formulation according to any one of claims 24 to 28, wherein administration of the composition or the pharmaceutical formulation results in a temporary decrease in lymphocyte count in the subject.

44. The method of claim 43, wherein the lymphocyte count is normalized on day 8 of administration.

45. The method according to any one of claims 41 to 44, wherein MS is a relapsing form of MS (RMS).

46. ​​A method of reducing the annualized relapse rate (ARR) in a subject with a relapsing form of multiple sclerosis (MS), comprising administering to the subject an effective amount of a composition according to any one of claims 1 to 23 or a pharmaceutical formulation according to any one of claims 24 to 28, the method comprising: The composition or pharmaceutical preparation for intravenous infusion is administered in a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising 150 mg of anti-CD20 antibody protein; b) a second infusion containing 450 mg of anti-CD20 antibody protein approximately 2 weeks after the first infusion; c) a first subsequent infusion comprising 450 mg of anti-CD20 antibody protein at about 24 weeks or about six months from the first infusion; and d) one or more subsequent infusions comprising 450 mg of anti-CD20 antibody protein at about 24 weeks or about six months from the previous infusion.

47. The method of claim 46, wherein the effective amount of the composition or the pharmaceutical formulation is sufficient to achieve an ARR of 0.091 or an ARR of 0.

076.

48. The method of claim 46 or 47, wherein the duration of the second infusion, the first subsequent infusion, and the one or more subsequent infusions of anti-CD20 antibody protein is about one hour.

49. A method of treating a relapsing form of multiple sclerosis (MS) in a subject in need thereof, comprising administering to the subject an effective amount of a composition according to any one of claims 1 to 23 or a pharmaceutical formulation according to any one of claims 24 to 28, the method comprising: The composition or the pharmaceutical preparation is administered as an intravenous infusion in a multiple infusion dosing regimen comprising: e) a first infusion comprising 150 mg of anti-CD20 antibody protein on day 1; f) a second infusion containing 450 mg of anti-CD20 antibody protein approximately 2 weeks after the first infusion; g) a first subsequent infusion comprising 450 mg of anti-CD20 antibody protein at about 24 weeks or about six months from the first infusion; and h) one or more subsequent infusions comprising 450 mg of anti-CD20 antibody protein at about 24 weeks or about six months from the previous infusion, The duration of the second infusion, the first subsequent infusion, and the one or more subsequent infusions of the anti-CD20 antibody protein is about one hour.

50. The method of any one of claims 41 to 49, further comprising pre-medicating the subject with a corticosteroid and an antihistamine 30 to 60 minutes prior to administering the composition or the pharmaceutical formulation.

51. The method of claim 50, wherein the corticosteroid is methylprednisone or dexamethasone, optionally wherein the methylprednisone is administered at a dose of about 100 mg and / or the dexamethasone is administered at a dose of about 10 to 20 mg.

52. The method of any one of claims 46 to 51, wherein the composition or the pharmaceutical preparation for intravenous infusion is prepared in 250 mL of 0.9% Sodium Chloride Injection.

53. The method of any one of claims 46 to 52, wherein the first subsequent infusion is about 24 weeks from the first infusion.

54. The method of any one of claims 46 to 53, wherein one or more subsequent infusions are at about 24 weeks from a previous infusion.

55. The method of any one of claims 46 to 54, wherein the first subsequent infusion is about 6 months from the first infusion.

56. The method of any one of claims 46 to 55, wherein one or more subsequent infusions are at about 6 months from a previous infusion.

57. The method of any one of claims 46 to 56, wherein the duration of the first infusion of anti-CD20 antibody protein is about four hours, Optionally, the first infusion of anti-CD20 antibody protein is infused at the following rate: The first 30 minutes are 10 mL / hour; the next 30 minutes are 20 mL / hour; the next hour is 35 mL / hour; and the remaining two hours are 100 mL / hour.

58. The method of any one of claims 48 to 57, wherein the second infusion, the first subsequent infusion, and one or more subsequent infusions of anti-CD20 antibody protein are infused at a rate of 100 mL / hour for the first 30 minutes and 400 mL / hour for the remaining 30 minutes.

59. The method of any one of claims 46 to 58, wherein the multiple infusion dosing regimen of the anti-CD20 antibody protein reduces or delays progression of MS symptoms.

60. The method of any one of claims 46 to 59, wherein subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve a reduction in the total number of gadolinium-enhancing T1 lesions per MRI scan compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

61. The method of any one of claims 46 to 60, wherein subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve a reduction in the total number of new and enlarging T2 hyperintensive lesions per MRI scan compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

62. The method of any one of claims 46 to 61, wherein subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an increase in No Evidence of Disease Activity (NEDA) status compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

63. The method of any one of claims 46 to 62, wherein subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an increase in confirmed disability improvement (CDI) compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

64. The method of any one of claims 46 to 63, wherein subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an increase in Multiple Sclerosis Functional Composite (MSFC) score compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

65. The method of any one of claims 46 to 64, wherein subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an improvement in the Timed 25-Foot Walk (T25FW) score compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

66. The method of any one of claims 46 to 65, wherein subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an improvement in 9-hole peg test (9-HPT) score compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

67. The method of any one of claims 46 to 66, wherein subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve a significant reduction in the volume and number of new T1 hypointense lesions per MRI scan compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

68. The method of any one of claims 46 to 67, wherein the multiple infusion dosing regimen of the anti-CD20 antibody protein results in a geometric mean steady-state AUC of 3000 mcg / mL per day (CV=28%) and a mean maximum concentration of 139 mcg / mL (CV=15%).

69. The method of any one of claims 45 to 68, wherein RMS comprises clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), or active secondary progressive MS (SPMS).

70. The method of any one of claims 45 to 69, wherein the subject is diagnosed with RMS according to the McDonald Criteria (2010).

71. The method of any one of claims 31 to 70, wherein the subject is a human.

72. A method for inactivating viruses or exogenous factors in rat myeloma cells expressing the anti-CD20 antibody protein in the composition of any one of claims 1 to 23, wherein the method maintains the suitability of antibody production in a 15,000L or 20,000L bioreactor.

73. A method for reducing the immunogenicity of an anti-CD20 antibody protein in a composition according to any one of claims 1 to 23, wherein the method maintains the suitability of antibody production in a 15,000L or 20,000L bioreactor.

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