Anti-CD20 antibody composition

Abstract

A population of anti-CD20 antibody proteins having a translated modification within a specified range is provided herein. Also provided are methods of using and making such populations of anti-CD20 antibody proteins.

Description

Technical Field

[0001] 1. Claim of Priority This application claims the benefit of U.S. Provisional Application No. 63 / 347,852, filed on June 1, 2022; U.S. Provisional Application No. 63 / 421,078, filed on October 31, 2022; and U.S. Provisional Application No. 63 / 445,082, filed on February 13, 2023. The entire contents of the above are incorporated herein by reference.

[0002] 2. Reference to Electronically Filed Sequence Listing This application includes a sequence listing that was electronically filed as an XML file entitled "50581-0004WO1.XML." The XML file was created on May 22, 2023 and is 39,114 bytes in size. The contents of the XML file are incorporated herein by reference in their entirety.

[0003] 3. Field of the Disclosure This disclosure pertains to the field of recombinant anti-CD20 antibodies, methods of producing such antibodies, and uses of such antibodies.

Background Art

[0004] 4. Background of the Disclosure Therapeutic monoclonal antibodies (mAbs) produced in mammalian cells are heterogeneous as a result of post-translational modifications (PTMs). PTMs can occur during the production, purification, storage, and administration of mAbs. PTMs are product quality attributes (PQAs) of therapeutic mAbs. Controlling PQAs within predetermined acceptance criteria is essential for the biopharmaceutical industry as it thereby 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)).

[0005] It is well recognized that sequence variations in antibodies are critically important. While the sequence diversity in the variable domains of antibodies is essential for specific antigen recognition, the linkage to different constant domains results in unique Fc-mediated effector activities. PTMs of these domains provide additional immune mechanisms by which the binding and activity of antibodies can be regulated. PTMs are diverse, ranging from chain addition, such as N- and O-linked glycosylation, mannosylation, cysteinylation, and sulfation; chain trimming, such as C-terminal lysine clipping; amino acid modifications, such as cyclization (to N-terminal pyroglutamic acid), deamidation, oxidation, isomerization, and carbamylation to disulfide scrambling of the interchain disulfide bonds in the hinge region. Thus, each antibody can give rise to a myriad of unique antibody molecules with greatly differing activities and efficacies. Post-translational modifications of antibodies have been observed and studied for decades, but the full impact of microheterogeneity remains to be further investigated. PTMs can affect the function of antibodies, such as pharmacokinetic and pharmacodynamic properties as well as clinical efficacy.

[0006] Specifying the scope of post-translational modifications can be important for maintaining consistent product quality, clinical safety, and efficacy of the mAb population.

Summary of the Invention

[0007] 5. Summary of the Disclosure Compositions are provided herein that include a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising about 20-40% fucosylated glycans and optionally about 10-20% galactosylated glycans.

[0008] In some embodiments, the N-glycan profile comprises 23% to 36% fucosylated glycan, optionally about 30% fucosylated glycan. In some embodiments, the N-glycan profile comprises 16% to 18% galactosylated glycan, optionally about 17% galactosylated glycan.

[0009] In some embodiments, the relative abundance of fucosylated glycan is the percentage of fucosylated glycan among all glycans in the N-glycan profile. In some embodiments, the relative abundance of galactosylated glycan is the percentage of galactosylated glycan among all glycans in the N-glycan profile.

[0010] In some embodiments, the N-glycan profile comprises 12% to 30% biantennary N-glycan, optionally about 18% biantennary N-glycan. In some embodiments, the biantennary N-glycan comprises one or more of G0B, G0FB, G1FB, G2FBS1, and G2FBS2. In some embodiments, the population of anti-CD20 antibody proteins has an N-glycan profile comprising less than 5% sialylated glycan. In some embodiments, the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1%, or 0.5% sialylated glycan. In some embodiments, the N-glycan profile does not contain a detectable amount of sialylated glucan.

[0011] In some embodiments, the population of anti-CD20 antibody proteins has an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycan. In some embodiments, the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycan. In some embodiments, the N-glycan profile comprises about 0.6% Man5 N-glycan. In some embodiments, Man5 N-glycan is the only high-mannose species in the N-glycan profile.

[0012] In some embodiments, the population of anti-CD20 antibody proteins comprises 0.20 to 0.40 moles of isoaspartate per mole of protein. In some embodiments, the population of anti-CD20 antibody proteins comprises 0.25 to 0.35 moles of isoaspartate per mole of protein.

[0013] In some embodiments, the glutamate at position 1 of the heavy chain is pyroglutamate, and the glutamate at position 1 of the light chain is pyroglutamate.

[0014] In some embodiments, the population of anti-CD20 antibody proteins has an N-glycan profile that includes a G1 N-glycan to G0 N-glycan relative abundance ratio of 0.1 to 0.15. In some embodiments, the population of anti-CD20 antibody proteins has an N-glycan profile that includes a G1F N-glycan to G1 N-glycan relative abundance ratio of 0.5 to 0.9.

[0015] In some embodiments, the population of anti-CD20 antibody proteins further comprises at least two N-glycans within the following relative abundance ranges. (a) G0-GN 0.3% - 2%; (b) G0F-GN 0.1% - 2%; (c) G0 30% - 60%; (d) G1-GN 0.1% - 1%; (e) G0B 5% - 20%; (f) G0F 5% - 30%; (g) Man5 0.1% - 1.5%; (h) G0FB 1% - 15%; (i) G1 1% - 13%; (j) G1’ 0.5% - 10%; (k) G1B 0.5% - 6%; (l) G1F 0.5% - 12%; (m) G1F’ 0.1% - 3%; (n) G1FB 0.1% - 3%; (o) G2 0.1% - 2%; and (p) G2F 0.1% - 2%.

[0016] In some embodiments, the population of anti-CD20 antibody proteins further comprises at least two N-glycans in the following relative abundance ranges. (a) G0-GN 0.8% - 1.1%; (b) G0F-GN 0.5% - 1.1%; (c) G0 42.5% - 48.8%; (d) G1-GN 0.3% - 0.6%; (e) G0B 9.5% - 14.1%; (f) G0F 12.8% - 19.7%; (g) Man5 0.4% - 0.7%; (h) G0FB 5.1% - 7.0%; (i) G1 5.7% - 6.4%; (j) G1’ 2.7% - 3.3%; (k) G1B 1.4% - 2.0%; (l) G1F 2.6% - 4.2%; (m) G1F’ 1.1% - 1.6%; (n) G1FB 1.1% - 1.8%; (o) G2 0.5% - 0.7%; and (p) G2F 0.3% - 0.5%.

[0017] In some embodiments, the population of anti-CD20 antibody proteins further comprises at least two N-glycans in the following relative abundance ranges. (a) G0-GN 0.9%; (b) G0F-GN 0.8%; (c) G0 46.1%; (d) G1-GN 0.5%; (e) G0B 10.9%; (f) G0F 17.0%; (g) Man5 0.6%; (h) G0FB 6.0%; (i) G1 6.1%; (j) G1’ 2.9%; (k) G1B 1.6%; (l) G1F 3.2%; (m) G1F’ 1.3%; (n) G1FB 1.3%; (o) G2 0.5%; and (p) G2F 0.3%.

[0018] In some embodiments, the population of anti-CD20 antibody proteins further comprises at least three, four, or five N-glycans within the following ranges of relative abundances. (a) G0-GN 0.3% - 2%; (b) G0F-GN 0.1% - 2%; (c) G0 30% - 60%; (d) G1-GN 0.1% - 1%; (e) G0B 5% - 20%; (f) G0F 5% - 30%; (g) Man5 0.1% - 1.5%; (h) G0FB 1% - 15%; (i) G1 1% - 13%; (j) G1’ 0.5% - 10%; (k) G1B 0.5% - 6%; (l) G1F 0.5% - 12%; (m) G1F’ 0.1% - 3%; (n) G1FB 0.1% - 3%; (o) G2 0.1% - 2%; and (p) G2F 0.1% - 2%.

[0019] In some embodiments, the N-glycan profile of a population of anti-CD20 antibody proteins is determined using a method comprising: (a) incubating the population of anti-CD20 antibody proteins with an enzyme that catalyzes the release of N-glycans from the anti-CD20 antibody; (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 method further comprises, after step (a) and before step (b), the following steps: (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).

[0020] In some embodiments, less than 10% of the anti-CD20 antibody proteins in the population are unglycosylated. In some embodiments, less than 5% of the anti-CD20 antibody proteins in the population are unglycosylated. In some embodiments, less than 1% of the anti-CD20 antibody proteins in the population are unglycosylated.

[0021] In some embodiments, the population of anti-CD20 antibody proteins comprises two or more secondary structures determined by circular dichroism at 205 nm to 260 nm as follows. (a) α-helix 8.0% - 10.0%; (b) Antiparallel β-sheet 32.0% - 36.0%; (c) Parallel β-sheet 5.0% - 6.0%; (d) β-turn 16.0% - 18.0%; and (e) Random coil 35.0% - 36.0%.

[0022] In some embodiments, the population of anti-CD20 antibody proteins comprises a secondary structure determined by circular dichroism at 205 nm to 260 nm as follows. (a) α-helix 8.0% - 10.0%; (b) Antiparallel β-sheet 32.0% - 36.0%; (c) Parallel β-sheet 5.0% - 6.0%; (d) β-turn 16.0% - 18.0%; and (e) Random coil 35.0% - 36.0%.

[0023] In some embodiments, the population of anti-CD20 antibody proteins comprises two or more secondary structures determined by circular dichroism at 205 nm - 260 nm as follows. (a) α-helix ~9.0%; (b) Antiparallel β-sheet ~33.0%; (c) Parallel β-sheet ~5.6%; (d) β-turn ~17.5%; and (e) Random coil ~35.2%.

[0024] In some embodiments, the population of anti-CD20 antibody proteins further comprises one or more of the following post-translational modifications in a specified abundance.

[0025] [Table 1]

[0026] In some embodiments, one or more of the post-translational modifications are measured by peptide mapping using liquid chromatography - mass spectrometry (LC-MS).

[0027] In some embodiments, the population has a total protein amount of 25.5 - 25.8 mg / mL as measured by absorbance at 280 nm.

[0028] In some embodiments, the anti-CD20 antibody proteins in the population induce greater cytotoxicity in a cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay compared to obinutuzumab, ofatumumab, rituximab, belimumab, ibritumomab tiuxetan, and / or ocrelizumab.

[0029] In some embodiments, the population has a relative potency of 90-163% compared to a commercially available reference standard in a cell-based ADCC assay. In some embodiments, the population has a relative potency of 78%-116% or 73%-128% compared to a commercially available reference standard in a cell-based complement-dependent cytotoxicity (CDC) assay. In some embodiments, the population has a relative potency of 92-118% or 82-138% compared to a commercially available reference standard in a cell-based CD20 binding activity bioassay. In some embodiments, the population has a K D value of 30-70 nM as measured by surface plasmon resonance in an FcγRIIIa-158V binding assay. In some embodiments, the population has a K D value of 500-1000 nM as measured by surface plasmon resonance in an FcγRIIIa-158F binding assay. 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 88-113% or 86-116% compared to a commercially available reference standard as measured by ELISA in a C1q binding assay. In some embodiments, the population has a relative potency of 106-126% compared to a commercially available reference standard in a CD16 activity assay.

[0030] In some embodiments, the population has 99.2-99.9% monomers as detected by size exclusion chromatography (SEC). In some embodiments, the population has 0.1-0.8% dimers as detected by SEC. In some embodiments, the population has undetectable levels of aggregates as detected by SEC and / or undetectable levels of fragments as detected by SEC.

[0031] In some embodiments, the population has 93.6 - 95.9% IgG by non - reducing capillary gel electrophoresis (CGE) after purification. In some embodiments, the population has 0.1 - 0.3% high molecular weight species (HMWS) by non - reducing CGE after purification. In some embodiments, the population has 0.7 - 1.2% free light chain (LC) by non - reducing CGE after purification. In some embodiments, the population has 97.7 - 98.0% heavy chain plus light chain species (HC+LC) by reducing CGE after purification.

[0032] In some embodiments, the population has 20 - 25% acidic isoforms as detected by imaging capillary isoelectric focusing (iCIEF). In some embodiments, the population has 50 - 60% major isoforms as detected by iCIEF. In some embodiments, the population has 20 - 30% basic isoforms as detected by iCIEF. In some embodiments, the population has an average molar ratio of free thiol to anti - CD20 antibody of about 2.0 - 2.2.

[0033] In some embodiments, the amino acid sequence of the anti - CD20 antibody in the population includes a deletion of the N - terminal residue. In some embodiments, the amino acid sequence of the anti - CD20 antibody in the population includes a deletion of up to 5 N - terminal residues. In some embodiments, the amino acid sequence of the anti - CD20 antibody in the population includes a deletion of up to 10 N - terminal residues. In some embodiments, the terminal lysine amino acid residue of the heavy chain of the anti - CD20 antibody in the population is truncated.

[0034] Pharmaceutical formulations comprising the compositions described herein are also provided herein, and 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.

[0035] In some embodiments, the pharmaceutical formulation further comprises one or more of the following: sodium chloride, trisodium citrate anhydrous, 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 anhydrous, about 0.7 mg / mL of polysorbate 80, and / or about 0.4 mg / mL of hydrochloric acid.

[0036] In some embodiments, the anti-CD20 antibody is present in a single-dose form.

[0037] (i) A composition as described herein, comprising a population of anti-CD20 antibody proteins in a single-dose form, 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 anhydrous, (iv) about 0.7 mg / mL of polysorbate 80, and (v) about 0.4 mg / mL of hydrochloric acid A pharmaceutical formulation comprising the above is also provided herein.

[0038] Also provided herein is a preparation of a single batch of the population of anti-CD20 antibody proteins or 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.

[0039] Also provided herein is a population of anti-CD20 antibody proteins or pharmaceutical formulation described herein produced in a 15,000 L or 20,000 L bioreactor.

[0040] Also provided herein is a method of treating multiple sclerosis (MS) in a subject in need thereof by administering to the subject a therapeutically effective amount of the composition or pharmaceutical formulation described herein.

[0041] In some embodiments, the composition or pharmaceutical formulation is administered as: i) a first infusion of an anti-CD20 antibody protein at a dose of about 150 mg, ii) a second infusion of an anti-CD20 antibody protein at a dose of about 450 mg two weeks later, and iii) subsequent infusions of an anti-CD20 antibody protein at a dose of about 450 mg every 24 weeks or every six months.

[0042] In some embodiments, administration of the composition or pharmaceutical formulation to a subject results in one or more of the following pharmacokinetic parameters: (a) An AUC of 2,160 μg / mL to 3,840 μg / mL; (b) A Cmax of 118,011 ng / mL to 159,989 ng / mL; (c) A Cmin of 40 ng / mL to 375 ng / mL; and (d) A Cavg of 6,437 ng / mL to 11,443 ng / mL resulting in one or more of the foregoing.

[0043] In some embodiments, administration of the composition or pharmaceutical formulation to a subject 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 resulting in one or more of the foregoing.

[0044] In some embodiments, the method includes a treatment period of at least 96 weeks.

[0045] In some embodiments, the subject receives a corticosteroid pre-medication 30 to 60 minutes prior to administration of the composition or pharmaceutical formulation. In some embodiments, the corticosteroid is methylprednisolone or dexamethasone. In some embodiments, methylprednisolone is administered at a dose of about 100 mg and / or dexamethasone is administered at a dose of about 10 - 20 mg.

[0046] In some embodiments, the subject has received a prophylactic dose of 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.

[0047] In some embodiments, the subject has received a prophylactic dose of an antipyretic 30 to 60 minutes prior to administration of the composition or pharmaceutical formulation. In some embodiments, the antipyretic is acetaminophen or a pyrogenic biological equivalent thereof.

[0048] In some embodiments, the subject has an Expanded Disability Status Scale (EDSS) score of 0 to 5.5 prior to treatment.

[0049] Also provided herein is a method of treating multiple sclerosis (MS) in a subject in need thereof by administering to the subject a therapeutically effective amount of the 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.

[0050] In some embodiments, administration of the composition or pharmaceutical formulation results in NEDA in the subject 24 weeks after administration.

[0051] Also provided herein is a method of treating multiple sclerosis (MS) in a subject in need thereof by administering to the subject a therapeutically effective amount of the composition or pharmaceutical formulation described herein, wherein administration of the composition or pharmaceutical formulation results in a transient decrease in the number of lymphocytes in the subject.

[0052] In some embodiments, the number of lymphocytes normalizes by day 8 of administration.

[0053] In some embodiments, the MS is relapsing MS (RMS).

[0054] Also provided herein is a method of reducing the annualized relapse rate (ARR) in a subject having relapsing multiple sclerosis (MS) by administering to the subject an effective amount of the composition or pharmaceutical formulation described herein. The method includes the step of performing intravenous infusion of the composition or pharmaceutical formulation in a multiple-injection dosing regimen, the dosing regimen including: a) a first infusion comprising 150 mg of an anti-CD20 antibody protein on day 1; b) a second infusion comprising 450 mg of an anti-CD20 antibody protein approximately 2 weeks after the first infusion; c) a first subsequent infusion comprising 450 mg of an anti-CD20 antibody protein at approximately 24 weeks or approximately 6 months from the first infusion, and d) one or more subsequent infusions comprising 450 mg of an anti-CD20 antibody protein at approximately 24 weeks or approximately 6 months from the previous infusion.

[0055] In some embodiments, an effective amount of the composition or pharmaceutical formulation is sufficient to result in an ARR of 0.091 or an ARR of 0.076.

[0056] In some embodiments, 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 1 hour.

[0057] Also provided herein is a method of treating relapsing multiple sclerosis (MS) in a subject in need thereof by administering to the subject an effective amount of the composition or pharmaceutical formulation described herein. The method includes the step of performing intravenous infusion of the composition or pharmaceutical formulation in a multiple-injection dosing regimen, the dosing regimen including a) a first infusion comprising 150 mg of an anti-CD20 antibody protein on day 1; b) a second infusion comprising 450 mg of an anti-CD20 antibody protein approximately 2 weeks after the first infusion; c) a first subsequent infusion comprising 450 mg of an anti-CD20 antibody protein at approximately 24 weeks or approximately 6 months from the first infusion, and d) one or more subsequent infusions comprising 450 mg of an anti-CD20 antibody protein at approximately 24 weeks or approximately 6 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 1 hour.

[0058] In some embodiments, the method further comprises the step of pre-dosing the subject with corticosteroids and antihistamines 30 to 60 minutes prior to administration of the composition or pharmaceutical formulation. In some embodiments, the corticosteroid is methylprednisolone or dexamethasone. In some embodiments, methylprednisolone is administered at a dose of about 100 mg and / or dexamethasone is administered at a dose of about 10 - 20 mg.

[0059] In some embodiments, the intravenous infusion of the composition or pharmaceutical formulation is prepared in 250 mL of 0.9% sodium chloride for injection.

[0060] In some embodiments, the first subsequent infusion is at about 24 weeks from the first infusion. In some embodiments, the one or more subsequent infusions are at about 24 weeks from the previous infusion. In some embodiments, the first subsequent infusion is at about 6 months from the first infusion. In some embodiments, the one or more subsequent infusions are at about 6 months from the previous infusion.

[0061] In some embodiments, the duration of the first infusion of the anti-CD20 antibody protein is about 4 hours. In some embodiments, the first infusion of the anti-CD20 antibody protein is infused at a rate of 10 mL per hour for the first 30 minutes, 20 mL per hour for the next 30 minutes, 35 mL per hour for the next 1 hour, and 100 mL per hour for the remaining 2 hours.

[0062] In some embodiments, the second infusion, the first subsequent infusion, and the one or more subsequent infusions of the anti-CD20 antibody protein are infused at a rate of 100 mL per hour for the first 30 minutes and 400 mL per hour for the remaining 30 minutes.

[0063] In some embodiments, multiple infusion dosing regimens of the anti-CD20 antibody protein reduce or delay the progression of MS symptoms.

[0064] 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-enhanced T1 lesions by MRI scan compared to subjects receiving daily oral administration of 14 mg of teriflunomide during the same treatment period.

[0065] 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 hyperintense lesions by MRI scan compared to subjects receiving daily oral administration of 14 mg of teriflunomide during the same treatment period.

[0066] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an increase in the no evidence of disease activity (NEDA) state compared to subjects receiving daily oral administration of 14 mg of teriflunomide during the same treatment period.

[0067] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an increase in the confirmed disability improvement (CDI) compared to subjects receiving daily oral administration of 14 mg of teriflunomide during the same treatment period.

[0068] In some embodiments, subjects administered a multiple infusion dosing regimen of the anti-CD20 antibody protein achieve an increase in the multiple sclerosis functional composite (MSFC) score compared to subjects receiving daily oral administration of 14 mg of teriflunomide during the same treatment period.

[0069] 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 of teriflunomide during the same treatment period.

[0070] In some embodiments, subjects administered multiple infusions of the anti-CD20 antibody protein achieve an improvement in the 9-Hole Peg Test (9-HPT) score compared to subjects who received daily oral administration of 14 mg of teriflunomide during the same treatment period.

[0071] In some embodiments, subjects administered multiple infusions of the anti-CD20 antibody protein achieve a significant reduction in the volume and number of new T1 hypointense lesions by MRI scan compared to subjects who received daily oral administration of 14 mg of teriflunomide during the same treatment period.

[0072] 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 (CV = 28%) per day and a mean maximum concentration of 139 mcg / mL (CV = 15%).

[0073] Also provided herein is a method for inactivating viruses or exogenous contaminating organisms in rat myeloma cells expressing the anti-CD20 antibody protein cited in the compositions described herein, which method maintains suitability for antibody production in a 15,000 L or 20,000 L bioreactor.

[0074] Also provided herein is a method for reducing the immunogenicity of the anti-CD20 antibody protein cited in the compositions described herein, which method maintains suitability for antibody production in a 15,000 L or 20,000 L bioreactor.

[0075] Methods for treating multiple sclerosis are also provided herein, the methods comprising 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 from 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 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.

[0076] Methods for treating multiple sclerosis are also disclosed herein, the methods comprising 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 from 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 wherein administration of the pharmaceutical formulation results in a transient decrease in the number of lymphocytes in the subject. In some embodiments, the number of lymphocytes normalizes by day 8 of administration.

[0077] In some embodiments of any of the methods described herein, the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan.

[0078] In some embodiments of any of the methods described herein, the multiple sclerosis (MS) is relapsing MS (RMS). In certain embodiments, the RMS includes clinically isolated syndrome ("CIS"), relapsing-remitting MS ("RRMS"), or active secondary progressive MS ("SPMS"). In some embodiments of any of the methods described herein, the subject is diagnosed as having RMS according to the McDonald criteria (2010) or another suitable method known to those of skill in the art. In some embodiments of any of the methods described herein, the subject is human.

[0079] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan.

[0080] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-35% fucosylated glycan.

[0081] In some embodiments, the N-glycan profile comprises 28-33% fucosylated glycan. In some embodiments, the N-glycan profile comprises about 30% fucosylated glycan.

[0082] In some embodiments, the N-glycan profile comprises 16-18% galactosylated glycan. In some embodiments, the N-glycan profile comprises about 17% galactosylated glycan.

[0083] In some embodiments, the relative abundance of fucosylated glycan is the percentage of fucosylated glycan among all glycans in the N-glycan profile. In some embodiments, the relative abundance of galactosylated glycan is the percentage of galactosylated glycan among all glycans in the N-glycan profile.

[0084] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising at least about 10% of biantennary N-glycans.

[0085] In some embodiments, the N-glycan profile comprises 12% to 30% biantennary N-glycans. In some embodiments, the N-glycan profile comprises about 18% biantennary N-glycans. In some embodiments, the biantennary N-glycans comprise one or more of G0B, G0FB, G1FB, G2FBS1, and G2FBS2.

[0086] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising less than 5% sialylated glycans.

[0087] 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 does not contain a detectable amount of sialylated glycans.

[0088] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 5 to 15% G0B N-glycans.

[0089] In some embodiments, the N-glycan profile comprises 9-11% G0B N-glycan. In some embodiments, the N-glycan profile comprises about 10% G0B N-glycan.

[0090] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 0.1%-1.5% Man5 N-glycan.

[0091] In some embodiments, the N-glycan profile comprises 0.4%-0.7% Man5 N-glycan. In some embodiments, the N-glycan profile comprises about 0.6% Man5 N-glycan. In some embodiments, Man5 N-glycan is the only high-mannose species in the N-glycan profile.

[0092] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins comprises 0.20-0.40 moles of isoaspartate per mole of protein.

[0093] In some embodiments, the population of anti-CD20 antibody proteins comprises 0.25-0.35 moles of isoaspartate per mole of protein.

[0094] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the glutamate at position 1 of the heavy chain is pyroglutamate and the glutamate at position 1 of the light chain is pyroglutamate.

[0095] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile consisting of G1 N-glycan to G0 N-glycan at a relative abundance ratio of 0.1 to 0.15.

[0096] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile consisting of G1F N-glycan to G1 N-glycan at a relative abundance ratio of 0.5 to 0.9.

[0097] In some embodiments, the population of anti-CD20 antibody proteins further comprises at least two N-glycans within the following relative abundance ranges: (a) G0-GN 0.3% to 2%; (b) G0F-GN 0.1% to 2%; (c) G1-GN 0.1% to 1%; (d) G0B 5% to 20%; (e) G0F 5% to 30%; (f) Man5 0.1% to 1.5%; (g) G0FB 1% to 15%; (h) G1 1% to 13%; (i) G1’ 0.5% to 10%; (j) G1B 0.5% to 6%; (k) G1F 0.5% to 12%; (l) G1F’ 0.1% to 3%; (m) G1FB 0.1% to 3%; (n) G2 0.1% to 2%; and (o) G2F 0.1% to 2%.

[0098] In some embodiments, the population of anti-CD20 antibody proteins further comprises at least two N-glycans within the following ranges of relative abundances: (a) G0-GN 0.8% to 1.1%; (b) G0F-GN 0.5% to 1.1%; (c) G1-GN 0.3% to 0.6%; (d) G0B 9.5% to 14.1%; (e) G0F 12.8% to 19.7%; (f) Man5 0.4% to 0.7%; (g) G0FB 5.1% to 7.0%; (h) G1 5.7% to 6.4%; (i) G1’ 2.7% to 3.3%; (j) G1B 1.4% to 2.0%; (k) G1F 2.6% to 4.2%; (l) G1F’ 1.1% to 1.6%; (m) G1FB 1.1% to 1.8%; (n) G2 0.5% to 0.7%; and (o) G2F 0.3% to 0.5%.

[0099] In some embodiments, the population of anti-CD20 antibody proteins further comprises at least two N-glycans at the following relative abundances: (a) G0-GN 0.9%; (b) G0F-GN 0.8%; (c) G1-GN 0.5%; (d) G0B 10.9%; (e) G0F 17.0%; (f) Man5 0.6%; (g) G0FB 6.0%; (h) G1 6.1%; (i) G1’ 2.9%; (j) G1B 1.6%; (k) G1F 3.2%; (l) G1F’ 1.3%; (m) G1FB 1.3%; (n) G2 0.5%; and (o) G2F 0.3%.

[0100] In some embodiments, the population of anti-CD20 antibody proteins further comprises at least three, four, or five N-glycans within the relative abundances or ranges of relative abundances described herein.

[0101] In some embodiments, the N-glycan profile of a population of anti-CD20 antibody proteins is determined using a method comprising: (a) incubating the population of anti-CD20 antibody proteins with an enzyme that catalyzes the release of N-glycans from the anti-CD20 antibody; and (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.

[0102] In some embodiments, the method further comprises, after step (a) and before step (b), the following steps: (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).

[0103] In some embodiments, less than 10% of the anti-CD20 antibody proteins in the population are unglycosylated. In some embodiments, less than 5% of the anti-CD20 antibody proteins in the population are unglycosylated. In some embodiments, less than 1% of the anti-CD20 antibody proteins in the population are unglycosylated.

[0104] In some embodiments, the N-glycan profile of the population of anti-CD20 antibody proteins is substantially as shown in Figure 2.

[0105] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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 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) α-helix 8.0% to 10.0%; (b) anti-parallel β-sheet 32.0% to 36.0%; (c) parallel β-sheet 5.0% to 6.0%; (d) β-turn 16.0% to 18.0%; and (e) random coil 35.0% to 36.0%.

[0106] In some embodiments, the population of anti-CD20 antibody proteins comprises a secondary structure as determined by circular dichroism at 205 nm to 260 nm as follows: (a) α-helix 8.0% to 10.0%; (b) anti-parallel β-sheet 32.0% to 36.0%; (c) parallel β-sheet 5.0% to 6.0%; (d) β-turn 16.0% to 18.0%; and (e) random coil 35.0% to 36.0%.

[0107] In some embodiments, 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) α-helix approximately 9.0%; (b) anti-parallel β-sheet approximately 33.0%; (c) parallel β-sheet approximately 5.6%; (d) β-turn approximately 17.5%; and (e) random coil approximately 35.2%.

[0108] Compositions comprising a population of anti-CD20 antibody proteins are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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, and the population of anti-CD20 antibody proteins further comprises one or more of the following post-translational modifications in a specified abundance.

[0109] [Table 2]

[0110] In some embodiments, the population of anti-CD20 antibody proteins comprises two, three, four, five, or more post-translational modifications.

[0111] In some embodiments, the population of anti-CD20 antibody proteins is present in amounts that specify the following post-translational modifications.

[0112] [Table 3]

[0113] In some embodiments, the population of anti-CD20 antibody proteins is present in amounts that specify the following post-translational modifications.

[0114] [Table 4]

[0115] 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 detection of isoaspartate or by peptide mapping using LC-MS.

[0116] Compositions comprising the population of anti-CD20 antibody proteins described herein are also provided herein, wherein the anti-CD20 antibodies in the population are expressed from 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, and the population of anti-CD20 antibody proteins has the following characteristics.

[0117] [Table 5]

[0118] In some embodiments, the population has a total protein mass of 25.5 - 25.8 mg / mL as measured by absorbance at 280 nm. In some embodiments, the population has a total protein mass of about 25.6 mg / mL as measured by absorbance at 280 nm.

[0119] In some embodiments, the population induces greater cytotoxicity in a cell-based antibody-dependent cell cytotoxicity (ADCC) assay compared to obinutuzumab, ofatumumab, rituximab, belimumab, ibritumomab tiuxetan, and / or ocrelizumab. In some embodiments, the population has a relative potency of 90 - 163% compared to a commercially available reference standard in a cell-based ADCC assay. In some embodiments, the population has a relative potency of about 117% compared to a commercially available reference standard in a cell-based ADCC assay. In some embodiments, the cell-based ADCC assay uses effector cells selected from CD16 effector cells and primary NK cells. In some embodiments, the population functions above 100% of a commercially available reference standard in cell-based ADCC using CD16 effector cells.

[0120] 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, belimumab, ibritumomab tiuxetan, and / or ocrelizumab.

[0121] In some embodiments, the population has a relative potency of 78% - 116% or 73% - 128% compared to a commercially available reference standard in a cell-based complement-dependent cytotoxicity (CDC) assay. In some embodiments, the population has a relative potency of about 91% compared to a commercially available reference standard in a cell-based CDC assay.

[0122] In some embodiments, the population has a relative potency of 92 - 118% or 82 - 138% compared to a commercially available reference standard in a cell - based CD20 - binding activity bioassay. In some embodiments, the population has a relative potency of about 109% compared to a commercially available reference standard in a cell - based CD20 - binding activity bioassay.

[0123] In some embodiments, the population has a K D value of 30 - 70 nM as measured by surface plasmon resonance in an FcγRIIIa - 158V - binding assay. In some embodiments, the population has a K D value of about 59 nM as measured by surface plasmon resonance in an FcγRIIIa - 158V - binding assay.

[0124] In some embodiments, the population has a K D value of 500 - 1000 nM as measured by surface plasmon resonance in an FcγRIIIa - 158F - binding assay. In some embodiments, the population has a K D value of 760 nM as measured by surface plasmon resonance in an FcγRIIIa - 158F - binding assay.

[0125] In some embodiments, the population has a higher binding affinity for FcγRIIIa 158V or FcγRIIIa 158F than rituximab.

[0126] In some embodiments, the population has a relative potency of 88 - 113% or 86 - 116% compared to a commercially available reference standard as measured by ELISA in a C1q - binding assay. In some embodiments, the population has a relative potency of about 99% compared to a commercially available reference standard as measured by ELISA in a C1q - binding assay.

[0127] In some embodiments, the population has a relative potency of 106 - 126% compared to a commercially available reference standard in a CD16 - activity assay. In some embodiments, the population has a relative potency of about 115% compared to a commercially available reference standard in a CD16 - activity assay.

[0128] In some embodiments, the population of anti-CD20 antibody proteins has the following purification profile.

[0129] [Table 6]

[0130] In some embodiments, the population has 99.2 - 99.9% monomers as detected by size exclusion chromatography (SEC). In some embodiments, the population has 0.1 - 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.

[0131] In some embodiments, the population has 93.6 - 95.9% IgG as detected by non-reducing capillary gel electrophoresis (CGE) after purification. In some embodiments, the population has 0.1 - 0.3% high molecular weight species (HMWS) as detected by non-reducing CGE after purification. In some embodiments, the population has 0.7 - 1.2% free light chain (LC) as detected by non-reducing CGE after purification.

[0132] In some embodiments, the population has 97.7 - 98.0% heavy chain plus light chain species (HC+LC) as detected by reducing CGE after purification.

[0133] In some embodiments, the population of anti-CD20 antibody proteins has the following distribution of charged isoforms.

[0134] [Table 7]

[0135] In some embodiments, the population has acidic isoforms detected by imaging capillary isoelectric focusing (iCIEF) and has 20-25%. In some embodiments, the population has major isoforms detected by iCIEF and has 50-60%. In some embodiments, the population has basic isoforms detected by iCIEF and has 20-30%.

[0136] In some embodiments, the population has an average molar ratio of free thiol to anti-CD20 antibody of about 2.0-2.2.

[0137] In some embodiments, the amino acid sequence of the anti-CD20 antibody in the population includes a deletion of the N-terminal residue. In some embodiments, the amino acid sequence of the anti-CD20 antibody in the population includes a deletion of up to 5 N-terminal residues. In some embodiments, the amino acid sequence of the anti-CD20 antibody in the population includes a deletion of up to 10 N-terminal residues.

[0138] In some embodiments, the terminal lysine amino acid residue of the heavy chain of the anti-CD20 antibody in the population is truncated.

[0139] In some embodiments, the administration of the anti-CD20 antibody to a human patient results in one or more of the following pharmacokinetic parameters: (a) an AUC of 2,160 μg / mL - 3,840 μg / mL; (b) a Cmax of 118,011 ng / mL - 159,989 ng / mL; (c) a Cmin of 40 ng / mL - 375 ng / mL; and (d) a Cavg of 6,437 ng / mL - 11,443 ng / mL, and the anti-CD20 antibody is administered as i) an infusion of a dose of about 150 mg at the first time, ii) a second infusion of a dose of about 450 mg two weeks later, and iii) subsequent infusions of a dose of about 450 mg every six months.

[0140] In some embodiments, the administration of the 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.

[0141] In some embodiments, the population of antibody proteins is present in a single-dose form.

[0142] Pharmaceutical formulations comprising the compositions described herein are also provided herein, and the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg / mL.

[0143] Pharmaceutical formulations comprising the anti-CD20 antibody described herein are also provided herein, the anti-CD20 antibody comprising 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 the pharmaceutical formulation comprising one or more of the following: sodium chloride, trisodium citrate anhydrous, polysorbate 80, and hydrochloric acid.

[0144] 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 anhydrous. 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.

[0145] Preparations of a single batch of the population of anti-CD20 antibody proteins described herein are also provided herein, the anti-CD20 antibody comprising 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 the single batch comprising at least 100 g of anti-CD20 antibody protein.

[0146] Preparations of a single batch of the population of anti-CD20 antibody proteins described herein are also provided herein, the anti-CD20 antibody comprising 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 the single batch comprising at least 120 g of anti-CD20 antibody protein.

[0147] Also provided herein is a preparation of a single batch of a population of anti-CD20 antibody proteins 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, and the single batch comprises at least 150 g of anti-CD20 antibody protein.

[0148] Also provided herein is a population of anti-CD20 antibody proteins described herein produced in a 15,000 L or 20,000 L bioreactor.

[0149] Also provided herein is a method of treating an autoimmune disease, the method comprising administering a composition described herein to a subject in need thereof, 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, graft rejection, graft-versus-host disease, systemic lupus erythematosus, inflammatory disease, type 1 diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis, myasthenia gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic recurrent hepatitis, primary biliary cirrhosis, allergic conjunctivitis, atopic dermatitis, chronic obstructive pulmonary disease, glomerulonephritis, neuroinflammatory disease, and uveitis.

[0150] Also provided herein is a method of treating multiple sclerosis, the method comprising administering a composition described herein to a subject in need thereof.

[0151] In some embodiments, the multiple sclerosis (MS) is relapsing MS. In some embodiments, the relapsing MS is selected from clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), or active secondary progressive MS (SPMS). In some embodiments, the relapsing MS is clinically isolated syndrome (CIS). In some embodiments, the relapsing MS is relapsing-remitting multiple sclerosis (RRMS). In some embodiments, the relapsing MS is active secondary progressive multiple sclerosis (SPMS).

[0152] Methods for treating neoplastic diseases are also provided herein, which methods include administering a composition described herein to a subject in need thereof.

[0153] In some embodiments, the neoplastic disease is acute lymphoblastic leukemia, B-cell lymphoma, mature B-cell lymphoma including B-cell chronic lymphocytic leukemia (B-CLL), small cell type 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), hairy cell leukemia, diffuse large B-cell lymphoma, or Burkitt lymphoma.

[0154] In some embodiments, the subject is human.

[0155] Methods for inactivating viruses or adventitious contaminants in rat myeloma cells expressing an anti-CD20 antibody protein in a composition described herein are also provided herein, which methods maintain suitability for antibody production in a 15,000 L or 20,000 L bioreactor.

[0156] Methods for reducing the immunogenicity of an anti-CD20 antibody protein in a composition described herein are also provided herein, which methods maintain suitability for antibody production in a 15,000 L or 20,000 L bioreactor.

[0157] A method for analyzing a TG-1101 (TG Therapeutics, Inc.) preparation, comprising: (i) preparing an N-glycan fraction isolated from a TG-1101 (TG Therapeutics, Inc.) preparation; (ii) analyzing the N-glycan fraction to determine that one or more N-glycans are within the following relative abundance ranges (a) G0 - GN 0.3% - 2%; (b) G0F - GN 0.1% - 2%; (c) G1 - GN 0.1% - 1%; (d) G0B 5% - 20%; (e) G0F 5% - 30%; (f) Man5 0.1% - 1.5%; (g) G0FB 1% - 15%; (h) G1 1% - 13%; (i) G1’ 0.5% - 10%; (j) G1B 0.5% - 6%; (k) G1F 0.5% - 12%; (l) G1F’ 0.1% - 3%; (m) G1FB 0.1% - 3%; (n) G2 0.1% - 2%; and (o) G2F 0.1% - 2% a step of determining whether it is any of the following N - glycans within A method including this is also provided in this specification.

[0158] In some embodiments, the method analyzes an N - glycan fraction to determine whether one or more N - glycans are within the following relative abundance ranges (a) G0 - GN 0.8% - 1.1%; (b) G0F - GN 0.5% - 1.1%; (c) G1 - GN 0.3% - 0.6%; (d) G0B 9.5% - 14.1%; (e) G0F 12.8% - 19.7%; (f) Man5 0.4% - 0.7%; (g) G0FB 5.1% - 7.0%; (h) G1 5.7% - 6.4%; (i) G1’ 2.7% - 3.3%; (j) G1B 1.4% - 2.0%; (k) G1F 2.6% - 4.2%; (l) G1F’ 1.1% - 1.6%; (m) G1FB 1.1% - 1.8%; (n) G2 0.5% - 0.7%; and (o) G2F 0.3% - 0.5% including a step of determining whether it is any of the following N - glycans within

[0159] In some embodiments, the method analyzes an N - glycan fraction to determine whether one or more N - glycans are the following relative abundances (a) G0-GN 0.9%; (b) G0F-GN 0.8%; (c) G1-GN 0.5%; (d) G0B 10.9%; (e) G0F 17.0%; (f) Man5 0.6%; (g) G0FB 6.0%; (h) G1 6.1%; (i) G1’ 2.9%; (j) G1B 1.6%; (k) G1F 3.2%; (l) G1F’ 1.3%; (m) G1FB 1.3%; (n) G2 0.5%; and (o) G2F 0.3% including the step of determining whether it is

[0160] Compositions are also provided herein that include a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed from 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 the population of anti-CD20 antibody proteins has the following relative abundances: (a) G0-GN 0.9%; (b) G0F-GN 0.8%; (c) G1-GN 0.5%; (d) G0B 10.9%; (e) G0F 17.0%; (f) Man5 0.6%; (g) G0FB 6.0%; (h) G1 6.1%; (i) G1’ 2.9%; (j) G1B 1.6%; (k) G1F 3.2%; (l) G1F’ 1.3%; (m) G1FB 1.3%; (n) G2 0.5%; and (o) G2 0.3%, and further includes at least two N-glycans, and the population of anti-CD20 antibody proteins is produced in rat hybridoma cells.

[0161] In some embodiments, the rat hybridoma cells are YB2 / 0 cells.

Brief Description of the Drawings

[0162] 6. Brief Description of the Drawings

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Figure 11-3

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Mode for Carrying Out the Invention

[0163] 7. Detailed Description of the Disclosure A population of anti-CD20 antibody proteins having a specified range of post-translational modifications is 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 respective abundances in the anti-CD20 antibody population are described in Section 7.4. Compositions comprising such a single batch composition of a population of anti-CD20 antibody proteins having a specified range of post-translational modifications and a range of purity are described in Section 7.5(e). Assays for quantifying such post-translational modifications in a population of anti-CD20 antibody proteins are described in Section 7.5. Assays for analyzing a population of anti-CD20 antibody proteins are described in Section 7.5. Assays for demonstrating the biological and clinical significance of such post-translational modifications in a population of anti-CD20 antibody proteins are described in Section 7.7(a). Methods of using such a population of anti-CD20 antibody proteins for the treatment and prevention of medical conditions are 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 of making such a population of anti-CD20 antibody proteins are described in Section 7.10.

[0164] As used herein, “TG-1101” (TG Therapeutics, Inc.) (also known as ofatumumab, UBX, UTX, TG-1101, TGTX-1101, Utuxin™, LFB-R603, TG20, EMAB603) is the starting antibody for the anti-CD20 antibodies described herein having a unique glycosylation profile produced by the disclosed method.

[0165] The starting antibody TG-1101 is a monoclonal antibody that targets CD20 epitopes 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.

[0166] 7.1 Abbreviations and Conventions The following abbreviations are used throughout this application.

[0167]

Table 8-1

[0168]

Table 8-2

[0169] 7.2 Definitions As used herein, the term "population of anti-CD20 antibody proteins" refers to a composition of anti-CD20 antibody proteins that has been tested for the abundance of post-translational modifications. Individual anti-CD20 antibody proteins in the population can include the same or different post-translational modifications. In some embodiments, the population of anti-CD20 antibody proteins refers to all of the anti-CD20 antibody proteins present within a single dosage form. In some embodiments, the population of anti-CD20 antibody proteins refers to all of the anti-CD20 antibody proteins present within a single batch. In some embodiments, the population of anti-CD20 antibody proteins is an amount sufficient to determine whether a batch of anti-CD20 antibody proteins meets a predetermined tolerance of a comparative value when compared to a reference standard.

[0170] As used herein, the term "single batch" with respect to an anti-CD20 antibody protein refers to a composition derived from a single production or run in a single bioreactor of a specified volume. For example, anti-CD20 antibody proteins obtained from a single run of a 15,000 L bioreactor may be referred to as a single batch. In certain embodiments, the bioreactor has a volume of 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.

[0171] As used herein, unless otherwise specified, the terms "about" or "substantially" mean an acceptable error for a particular value as 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 terms "about" or "substantially" mean within 1 or 2 standard deviations. In certain embodiments, the terms "about" or "substantially" mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0172] 7.3 Primary Amino Acid Sequence of Anti-CD20 Antibody In some embodiments, the anti-CD20 antibody protein provided herein is expressed from 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 below in the Sequence Listing.

[0173] In some embodiments, the anti-CD20 antibody protein provided herein is expressed from one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is 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 the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 2.

[0174] In some embodiments, the anti-CD20 antibody protein provided herein comprises the VH CDR1, CDR2, and CDR3 regions of the sequences of SEQ ID NOs: 3, 4, and 5, and the VL CDR1, CDR2, and CDR3 regions of the sequences of SEQ ID NOs: 8, 9, and 10.

[0175] In some embodiments, the anti-CD20 antibody protein provided herein comprises VH of SEQ ID NO: 6 and VL of SEQ ID NO: 11.

[0176] In some embodiments, 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 embodiments, 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.

[0177] In some embodiments, the anti-CD20 antibody protein provided herein binds to the same epitope as TG-1101 (TG Therapeutics, Inc.).

[0178] In some embodiments, the anti-CD20 antibody proteins provided herein are chimeric immunoglobulin G1 (IgG1) anti-CD20 monoclonal antibody proteins each consisting of a tetrameric assembly derived from two light chains (213 amino acids) and two heavy chains (448 amino acids).

[0179] In some embodiments, the anti-CD20 antibody proteins provided herein are expressed from 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 pyroglutamate instead of glutamine at position 1 of the light chain and / or heavy chain, thereby resulting in the amino acid sequence of SEQ ID NO: 13 for the heavy chain and / or the amino acid sequence of SEQ ID NO: 14 for the light chain.

[0180] In some embodiments, the anti-CD20 antibody proteins provided herein are expressed from 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 a deletion of the C-terminal lysine of the heavy chain, thereby resulting in the amino acid sequence of SEQ ID NO: 15.

[0181] In some embodiments, compositions or populations of anti-CD20 antibody proteins are provided herein, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, or 98% contain (i) pyroglutamate at position 1 of the heavy chain (instead of glutamine), (ii) pyroglutamate at position 1 of the light chain (instead of glutamine), and / or (iii) a deletion of the C-terminal lysine of the heavy chain.

[0182] In some embodiments, the anti-CD20 antibody is expressed from one or more nucleic acid sequences encoding a light chain comprising the amino acid sequence of SEQ ID NO: 16.

[0183] 7.4 Anti-CD20 Antibody Compositions The anti-CD20 antibody compositions provided herein can be described by various post-translational modifications and / or their three-dimensional conformations (see Section 7.5(e)). The level of each of the 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 result in the biological and clinical properties described in Sections 7.7 and 7.9 below.

[0184] Anti-CD20 antibody compositions produced in vitro have various post-translational modifications. It is understood that each individual anti-CD20 antibody protein can have its own specific pattern of post-translational modifications. To describe the properties of a population of multiple anti-CD20 antibody proteins, the overall presence of specific post-translational modifications can be quantified. Without being bound by theory, the level of a specific post-translational modification in a population of anti-CD20 proteins can determine the biological and clinical properties of the composition (e.g., the dosage of a pharmaceutical formulation). Without being bound by theory, post-translational modifications are conferred by expression in rat hybridoma cells (e.g., YB2 / 0 cells) in cell culture.

[0185] 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 glycosylations are known. In one aspect, the glycosylation is N-glycosylation. The N-glycans that can be present can be any one of the N-glycans shown in FIG. 1. The level of N-glycosylation is discussed in Section 7.4(a) below and can be quantified using the assays of Section 7.5.

[0186] 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 acids 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 specific amino acid positions in the anti-CD20 antibody compositions provided herein is described in Section 7.4(b) below and can be determined as described in Section 7.5 of this specification.

[0187] (a) N-glycosylation Various forms of N-glycosylation can be present in the anti-CD20 antibody compositions provided herein. Without being bound by theory, the relative distribution of the various N-glycans or the individual sugar residues present within these N-glycans can determine the biological and clinical properties (e.g., the biological and clinical properties discussed in Sections 7.7 and 7.9) of the individual CD20 antibody proteins within the population of anti-CD20 antibody proteins provided herein. The anti-CD20 antibody compositions provided herein can be described by any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or all of the N-glycans, or by the individual sugar residues described in the following subsections.

[0188] In some embodiments, the anti-CD20 antibody compositions provided herein contain at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen N-glycans within the following ranges of relative abundance. (a) G0-GN 0.3% - 2%; (b) G0F-GN 0.1% - 2%; (c) G0 30% - 60%; (d) G1-GN 0.1% - 1%; (e) G0B 5% - 20%; (f) G0F 5% - 30%; (g) Man5 0.1% - 1.5%; (h) G0FB 1% - 15%; (i) G1 1% - 13%; (j) G1’ 0.5% - 10%; (k) G1B 0.5% - 6%; (l) G1F 0.5% - 12%; (m) G1F’ 0.1% - 3%; (n) G1FB 0.1% - 3%; (o) G2 0.1% - 2%; and (p) G2F 0.1% - 2%.

[0189] In some embodiments, the anti - CD20 antibody composition provided herein comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen N - glycans within the following ranges of relative abundance. (a) G0 - GN 0.8% - 1.1%; (b) G0F - GN 0.5% - 1.1%; (c) G0 42.5% - 48.8%; (d) G1 - GN 0.3% - 0.6%; (e) G0B 9.5% - 14.1%; (f) G0F 12.8% - 19.7%; (g) Man5 0.4% - 0.7%; (h) G0FB 5.1% - 7.0%; (i) G1 5.7% - 6.4%; (j) G1’ 2.7% - 3.3%; (k) G1B 1.4% - 2.0%; (l) G1F 2.6% - 4.2%; (m) G1F’ 1.1% - 1.6%; (n) G1FB 1.1% - 1.8%; (o) G2 0.5% - 0.7%; and (p) G2F 0.3% - 0.5%.

[0190] In some embodiments, the anti-CD20 antibody composition provided herein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 N-glycans within the following ranges of relative abundance. (a) G0-GN 0.9%; (b) G0F-GN 0.8%; (c) G0 46.1%; (d) G1-GN 0.5%; (e) G0B 10.9%; (f) G0F 17.0%; (g) Man5 0.6%; (h) G0FB 6.0%; (i) G1 6.1%; (j) G1’ 2.9%; (k) G1B 1.6%; (l) G1F 3.2%; (m) G1F’ 1.3%; (n) G1FB 1.3%; (o) G2 0.5%; and (p) G2F 0.3%.

[0191] In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile with a relative abundance of about 0.3% to about 2% G0-GN, about 0.8% to about 1.1% G0-GN, or about 0.9% G0-GN. In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile with a relative abundance of about 0.1% to about 2% G0F-GN, about 0.5% to about 1.1% G0F-GN, or about 0.8% G0F-GN. In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile with a relative abundance of about 30% to 60% G0, about 42.5% to 48.8% G0, or about 46.1% G0. In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile with 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 population of anti-CD20 antibody proteins comprises an N-glycan profile with 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 population of anti-CD20 antibody proteins comprises an N-glycan profile with 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 population of anti-CD20 antibody proteins comprises an N-glycan profile with 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 population of anti-CD20 antibody proteins comprises an N-glycan profile with a relative abundance of 1% to about 15% G0FB, about 5.1% to about 7.0% G0FB, or about 6.0% G0FB. In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile with 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 population of anti-CD20 antibody proteins comprises an N-glycan profile that includes 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 population of anti-CD20 antibody proteins comprises an N-glycan profile that includes 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 population of anti-CD20 antibody proteins comprises an N-glycan profile that includes 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 population of anti-CD20 antibody proteins comprises an N-glycan profile that includes 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 population of anti-CD20 antibody proteins comprises an N-glycan profile that includes 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 population of anti-CD20 antibody proteins comprises an N-glycan profile that includes 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 population of anti-CD20 antibody proteins comprises an N-glycan profile that includes a relative abundance of about 0.1% to about 2% G2F, about 0.3% to about 0.5% G2F, or about 0.3% G2F.

[0192] In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile with the relative abundances 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.

[0193] In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile with the relative abundances of 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% 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.

[0194] In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile comprising the relative abundances 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.

[0195] In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile comprising a G1 N-glycan to G0 N-glycan relative abundance ratio of about 0.1 to about 0.15. In some embodiments, the population of anti-CD20 antibody proteins comprises an N-glycan profile comprising a G1F N-glycan to G1 N-glycan relative abundance ratio of about 0.5 to about 0.9.

[0196] In one embodiment, the anti-CD20 antibody composition has the N-glycan profile shown in FIG. 2.

[0197] Specific ranges and values for various N-glycans are provided below. The compositions or populations of anti-CD20 antibody proteins provided herein can be described by any one of the occurrence frequencies of any of these N-glycans or any group or all of these N-glycans.

[0198] (i) Galactosylation In some embodiments, the anti-CD20 antibody compositions provided herein comprise 10-20% galactosylated glycan. The galactosylated glycan is the N-glycan shown in Figure 1 that bears galactose residues (shown as white circles in Figure 1). Assays for determining the percentage of galactosylation (or galactosylated N-glycan) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is subjected to enzymatic deglycosylation, whereby all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percent of galactosylated N-glycan is the percent of galactosylated N-glycan among the N-glycans cleaved using enzymatic digestion.

[0199] In some embodiments, the anti-CD20 antibody compositions provided herein comprise between 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 glycan. In some embodiments, the anti-CD20 antibody compositions provided herein comprise 11%-19%, 12%-18%, 13%-17%, or 14%-16% galactosylated glycan. For example, the anti-CD20 antibody compositions provided herein can comprise about 17% galactosylated glycan (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%).

[0200] (ii) Fucosylation In some embodiments, the anti-CD20 antibody compositions provided herein comprise 20% to 40% fucosylated glycan, 23% to 36% fucosylated glycan, 20% to 35% fucosylated glycan, 28% to 33% fucosylated glycan, or about 33% fucosylated glycan (where “about” means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). The fucosylated glycan is the N-glycan shown in FIG. 1 that bears a fucose residue (shown as white triangles in FIG. 1). Assays for determining the percentage of fucosylation (or fucosylated N-glycan) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is subjected to enzymatic deglycosylation, whereby all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percent of fucosylated N-glycan is the percent of fucosylated N-glycan among the N-glycans cleaved using the enzymatic digestion.

[0201] 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 glycan.

[0202] (iii) Ratio of galactosylation to fucosylation In some embodiments, the provided anti-CD20 antibody composition is characterized by a specified ratio of total fucosylated glycan to total galactosylated glycan (or “fucose / galactose ratio”). The fucose / galactose ratio can be 1.5 to 2.1; 1.5 to 2; 1.5 to 1.9; 1.5 to 1.8; 1.6 to 2.1; 1.7 to 2.1; 1.8 to 2.8; 1.6 to 2.0; 1.7 to 1.9; 1.6 to 1.8, or the fucose / galactose ratio can be about 1.75 (where “about” means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). The fucosylated glycan is the N-glycan shown in FIG. 1 that bears a fucose residue (shown as a white triangle in FIG. 1). The galactosylated glycan is the N-glycan shown in FIG. 1 that bears a galactose residue (shown as a white circle in FIG. 1). Assays for determining the percentage of fucosylation (or fucosylated N-glycan) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is subjected to enzymatic deglycosylation, whereby 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-glycan is the percentage of fucosylated N-glycan among the N-glycans cleaved using the enzymatic digestion.

[0203] (iv) Bifurcated N-glycan In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least 10%, 15%, 20%, 25%, or at least 30% biantennary 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% biantennary N-glycans. The biantennary N-glycans are the N-glycans shown in Figure 1 that have a third GlcNAc (shown as a white triangle in Figure 1) attached to the mannose residue closest to the protein backbone. Assays for determining the percentage of biantennary N-glycans are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is subjected to enzymatic deglycosylation, whereby 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 biantennary N-glycans among the N-glycans cleaved using enzymatic digestion.

[0204] (v) Sialylation In some embodiments, the anti-CD20 antibody compositions provided herein comprise 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 comprise 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 contain no detectable amount 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 protein is subjected to enzymatic deglycosylation, whereby 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 the N-glycans cleaved using enzymatic digestion.

[0205] In some embodiments, the anti-CD20 antibody compositions provided herein contain sialylated glycans in an amount of at least undetectable, 0.5%, 1%, 2%, 3%, 4%, or at least 5%, and at most 0.5%, 1%, 2%, 3%, 4%, 5%, or at most 10%. In some embodiments, the anti-CD20 antibody compositions provided herein do not contain detectable amounts of sialylated glycans.

[0206] (vi) G0B N-glycan In some embodiments, the anti-CD20 antibody compositions provided herein contain 5% - 15% G0B N-glycan, 9% - 11% G0B N-glycan, or approximately 10% G0B N-glycan (where "approximately" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). G0B N-glycan is shown in Figure 1. Assays for determining the percentage of G0B N-glycan are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is subjected to enzymatic deglycosylation, whereby all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of G0B N-glycan is the percentage of G0B N-glycan among the N-glycans cleaved using enzymatic digestion.

[0207] In some embodiments, the anti-CD20 antibody compositions provided herein contain 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% G0B N-glycan.

[0208] (vii) Man5 N-glycan In some embodiments, the anti-CD20 antibody compositions provided herein comprise 0.1% to 1.5% Man5 N-glycan, 0.4% to 0.7% G0B N-glycan, or about 0.6% Man5 N-glycan (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). Man5 N-glycan is shown in Figure 1. The assay for determining the percentage of Man5 N-glycan is described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is subjected to enzymatic deglycosylation, whereby 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-glycan is the percentage of Man5 N-glycan among the N-glycans cleaved using enzymatic digestion.

[0209] 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-glycan.

[0210] In some embodiments, Man5 N-glycan is the only high-mannose species in the N-glycan profile.

[0211] (viii) G0 N-glycan In some embodiments, the anti-CD20 antibody compositions provided herein contain from about 42% to 52.8% G0 N-glycan (where "about" means + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). The G0 N-glycan is shown in Figure 1. The assay for determining the percentage of G0 N-glycan is described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is subjected to enzymatic deglycosylation, whereby all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percent of G0 N-glycan is the percent of G0 N-glycan among the N-glycans cleaved using the enzymatic digestion.

[0212] In some embodiments, the anti-CD20 antibody compositions provided herein contain 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% G0 N-glycan.

[0213] (ix) The ratio of G1 N-glycan to G0 N-glycan In some embodiments, the provided anti-CD20 antibody composition is characterized by a specified abundance ratio of G1 N-glycan to G0 N-glycan. The abundance ratio of G1 N-glycan to G0 N-glycan may be 0.02-0.3; 0.05-0.25; 0.08-0.22; 0.09-0.2; 0.1-0.19; 0.1-0.18; 0.1-0.17; 0.1-0.16, or the abundance ratio of G1 N-glycan to G0 N-glycan may be 0.1-0.15. G1 N-glycan and G1 N-glycan are shown in Figure 1. Assays for determining the percentage of G1 or G0 glycan are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is subjected to enzymatic deglycosylation, whereby 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 glycan is the percentage of each G1 or G0 glycan among the N-glycans cleaved using enzymatic digestion.

[0214] (x) Ratio of G1F N-glycan to G1 N-glycan In some embodiments, the provided anti-CD20 antibody composition is characterized by a specified abundance ratio of G1F N-glycan to G1 N-glycan. The abundance ratio of G1F N-glycan to G1 N-glycan may be 0.1-1.2; 0.2-1.1; 0.3-1; 0.4-1; 0.5-1, or the abundance ratio of G1 N-glycan to G0 N-glycan may be 0.5-0.9. G1F N-glycan and G1 N-glycan are shown in Figure 1. Assays for determining the percentage of G1F or G1 glycan are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is subjected to enzymatic deglycosylation, whereby 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 glycan is the percentage of each G1F or G1 glycan among the N-glycans cleaved using enzymatic digestion.

[0215] (b) Deamidation In some embodiments, the population of anti-CD20 antibody proteins described herein includes deamidation of asparagine at one or more asparagine residues present in the heavy chain. In some embodiments, one or more deamidated asparagine residues present in the heavy chain are selected from Asn-33 (shown in SEQ ID NO: 18), Asn-55 (shown in SEQ ID NO: 19), Asn-61 (shown in SEQ ID NO: 19), Asn-160 (shown in SEQ ID NO: 22), Asn-202 (shown in SEQ ID NO: 22), Asn-204 (shown in SEQ ID NO: 22), Asn-277 (shown in SEQ ID NO: 25), Asn-287 (shown in SEQ ID NO: 25), Asn-362 (shown in SEQ ID NO: 27), or Asn-385 (shown in SEQ ID NO: 28). In some embodiments, the population of anti-CD20 antibody proteins described herein includes asparagine deamidation at one or more asparagine residues present in the light chain. In some embodiments, one or more deamidated asparagine residues present in the light chain are selected from Asn-136 (shown in SEQ ID NO: 31), Asn-137 (shown in SEQ ID NO: 31), Asn-151 (shown in SEQ ID NO: 32), or Asn-157 (shown in SEQ ID NO: 32).

[0216] (c) Oxidation In some embodiments, the population of anti-CD20 antibody proteins includes oxidation of methionine at one or more methionine residues present in the heavy chain. In some embodiments, one or more methionine residues present in the heavy chain are each selected from Met-20, Met-34, Met-81, Met-253, or Met-428 shown in SEQ ID NOs: 17, 18, 21, 24, or 29, respectively. In some embodiments, the population of anti-CD20 antibody proteins includes oxidation of methionine at one or more methionine residues present in the light chain. In some embodiments, one or more methionine residues present in the light chain are selected from Met-21 or Met-32 shown in SEQ ID NO: 30.

[0217] (d) Pyroglutamination In some embodiments, the population of anti-CD20 antibody proteins comprises pyroglutamination at the N-terminal glutamine residue present in the heavy or light chain. In some embodiments, pyroglutamination at the N-terminal glutamine residue is present in the heavy chain, such as pGlu-1, as shown in SEQ ID NO: 13. In some embodiments, pyroglutamination at the N-terminal glutamine residue is present in the light chain, such as pGlu-1, as shown in SEQ ID NO: 14. In some embodiments, the glutamate at position 1 of the heavy chain is pyroglutamate and the glutamate at position 1 of the light chain is pyroglutamate.

[0218] (e) Truncation of lysine In some embodiments, the population of anti-CD20 antibody proteins comprises deletion of the C-terminal lysine amino acid residue present in the heavy or light chain. In some embodiments, the C-terminal lysine amino acid residue of the heavy chain of the anti-CD20 antibody in the population is truncated. In some embodiments, the population of anti-CD20 antibody proteins comprises deletion of the C-terminal lysine of 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.

[0219] (f) Conformation The three-dimensional conformation or protein folding can be determined using the assays described in Section 7.5(e).

[0220] In some embodiments, the compositions or populations of anti-CD20 antibody proteins provided herein (e.g., expressed from 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) have a specified three-dimensional folding pattern or conformation. In some embodiments, the population of anti-CD20 antibody proteins comprises two or more of the following secondary structures as determined by a circular dichroism (CD) spectrum from 205 nm to 260 nm. Alpha-helix range 3.0% - 15.0%; anti-parallel beta-sheet range 25.0% - 40.0%; parallel beta-sheet range 0.5% - 12.0%; beta-turn range 10.0% - 25.0%; and random coil range 30.0% - 42.0%.

[0221] In some embodiments, the population of anti-CD20 antibody proteins comprises two or more of the following secondary structures as determined by a circular dichroism (CD) spectrum from 205 nm to 260 nm. Alpha-helix range 8.0% - 10.0%; anti-parallel beta-sheet range 32.0% - 36.0%; parallel beta-sheet range 5.0% - 6.0%; beta-turn range 16.0% - 18.0%; and random coil range 35.0% - 36.0%.

[0222] In some embodiments, the population of anti-CD20 antibody proteins comprises two or more of the following secondary structures as determined by a circular dichroism (CD) spectrum from 205 nm to 260 nm. Alpha-helix approximately 9.0%; anti-parallel beta-sheet approximately 33.0%; parallel beta-sheet approximately 5.6%; beta-turn approximately 17.5%; and random coil approximately 35.2%. The term "approximately" means ±5%.

[0223] 7.5 Assays for Quantifying Post-Translational Modifications In some embodiments, methods are provided herein for determining the amount of post-translational modification in a population of anti-CD20 antibody proteins. In some embodiments, the post-translational modification is selected from asparagine deamidation, methionine oxidation, glycosylation, pyroglutamate formation, and lysine truncation.

[0224] (a) General digestion In some embodiments, a method for determining the amount of post-translational modification in a population of anti-CD20 antibody proteins includes digesting the population of anti-CD20 antibody proteins with an endoprotease. In some embodiments, the population of anti-CD20 antibody proteins is reduced prior to digestion. In some embodiments, the population of anti-CD20 antibody proteins 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 population of anti-CD20 antibody proteins is digested overnight at 37 °C with Asp-N or Lys-C. In some embodiments, the population of anti-CD20 antibody proteins is digested overnight at 37 °C with trypsin at a ratio of 50:1 (w:w). In some embodiments, the digested population of anti-CD20 antibody proteins is purified from the digestion reaction components and / or undigested anti-CD20 antibody proteins.

[0225] (b) Method for determining N-glycosylation Exemplary assays and their results are discussed in Examples in Sections 8.1 and 8.2 below.

[0226] In some embodiments, the method includes steps for deglycosylating a population of anti-CD20 antibody proteins, whereby released N-glycans are produced for labeling. In some embodiments, deglycosylation includes the step of breaking the glycosidic bond between one or more or all of the N-glycans from the population of anti-CD20 antibody proteins. In some embodiments, deglycosylation includes the step of releasing some or most or substantially all of the N-glycans from the population of anti-CD20 antibody proteins. In some embodiments, deglycosylation releases more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 97%, more than 98%, more than 99%, or 100% of the N-glycans present on the population of anti-CD20 antibody proteins.

[0227] In some embodiments, deglycosylation includes the step of contacting the population of anti-CD20 antibody proteins with one or more deglycosylating agents, which cleave N-glycans or N-linked oligosaccharides. In some embodiments, the deglycosylating agent is a deglycosylating enzyme or a chemical agent. In some embodiments, the deglycosylating enzyme is PNGase F.

[0228] In some embodiments, deglycosylation comprises contacting a population of anti-CD20 antibody proteins with one or more deglycosylating enzymes or chemical agents 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 rate of deglycosylation can be increased by increasing the deglycosylation temperature. In some embodiments, deglycosylation comprises contacting a population of anti-CD20 antibody proteins with one or more deglycosylating enzymes or chemical agents for a time sufficient to release some or most or substantially all of the N-glycans from the population of anti-CD20 antibody proteins. In some embodiments, deglycosylation comprises contacting a population of anti-CD20 antibody proteins with the one or more deglycosylating enzymes or chemical agents 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 a population of anti-CD20 antibody proteins with the one or more deglycosylating enzymes or chemical agents for 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 hours to about 30 hours, greater than about 30 hours, and / or less than 48 hours. In some embodiments, deglycosylation comprises contacting a population of anti-CD20 antibody proteins with one or more deglycosylating enzymes or chemical agents for about 12 to about 20 hours. In some embodiments, deglycosylation comprises contacting a population of anti-CD20 antibody proteins with PNGase F at about 37°C for about 12 to about 20 hours. In some embodiments, deglycosylation comprises contacting an anti-CD20 antibody with PNGase F at 37°C for 12 to 20 hours. In some embodiments, deglycosylation with PNGase F is performed in the presence of a nonionic surfactant (e.g., NP-40).

[0229] In some embodiments, deglycosylation includes the step of denaturing the population of anti-CD20 antibody proteins prior to contacting the population with the one or more deglycosylating enzymes or chemical agents. In some embodiments, denaturing includes thermal denaturation, chemical denaturation, or a combination of both. In some embodiments, thermal denaturation includes incubating the population of anti-CD20 antibody proteins at a denaturation temperature and for a denaturation time sufficient to unfold some or most or all of the immunoglobulin folding domains of the anti-CD20 antibody proteins of the 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 includes incubating the population of anti-CD20 antibody proteins at a denaturation temperature of at least 25°C, at least 30°C, at least 37°C, or a high temperature (i.e., the thermal denaturation temperature). 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 population of anti-CD20 antibody proteins with one or more chemical denaturing agents. In some embodiments, the one or more chemical denaturing agents are selected from ionic surfactants, nonionic surfactants, zwitterionic surfactants, chaotropic surfactants, or reducing agents.In some embodiments, the chemical denaturant is selected from sodium dodecyl sulfate (SDS), urea, or dithiothreitol (DDT). The effects of certain chemical denaturants interfere with deglycosylation (i.e., enzymatic deglycosylation) and may be disrupted in solution. In some embodiments, the chemical denaturant is disrupted by the addition of a nonionic surfactant to the solution. In some embodiments, the chemical denaturant is SDS and the additional nonionic surfactant is NP-40. In some embodiments, the denaturation of the anti-CD20 antibody protein population includes heat denaturation at 70°C for 10 minutes.

[0230] In some embodiments, the method includes purifying the released N-glycans of the anti-CD20 antibody protein population from the deglycosylation reaction mixture. In some embodiments, the released N-glycans are substantially free of deglycosylating agent and deglycosylated or unreacted anti-CD20 antibody. In some embodiments, the released N-glycans are substantially free of salts and / or surfactants. In some embodiments, the released N-glycans are purified by hydrophilic interaction. In some embodiments, the released N-glycans are purified by chromatography comprising a hydrophilic stationary phase and a reverse phase 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 using 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 equivalent to the purity level obtained when purified using a Waters HILIC MassPrep μElution plate according to the manufacturer's protocol.

[0231] In some embodiments, the method includes labeling the released N-glycans of a population of anti-CD20 antibody proteins, thereby producing labeled N-glycans for detection. In some embodiments, the labeling of the released N-glycans includes chemical derivatization to provide, for example, a detectable charge, ultraviolet activity, or fluorescence characteristic of the released N-glycans. In some embodiments, the labeling of the released N-glycans includes reductive amination, hydrazide labeling, methylation, Michael addition, or permethylation. In some embodiments, the labeling of 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-aminonaphthalenetrisulfonic acid (ANTS), or 1-aminopyrene-3,6,8-trisulfonic acid (APTS). In some embodiments, the label is 2-AB. In some embodiments, the labeling of the released N-glycans by reductive amination includes the use of a reducing agent. In some embodiments, the reducing agent is selected from sodium cyanoborohydride or 2-picolyl borane. In some embodiments, the reducing agent is cyanoborohydride. In some embodiments, the labeling of the released N-glycans by reductive amination includes contacting the released N-glycans with a label and a reducing agent suitable for reductive amination at a reaction temperature and for a reaction time sufficient to produce the label. 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, the labeling of released N-glycans from a population of anti-CD20 antibody proteins involves reductive amination at 65° C. for 3 hours using 2-AB and cyanoborohydride, whereby labeled N-glycans are produced.

[0232] In some embodiments, the method includes the step of purifying labeled N-glycans of a population of anti-CD20 antibody proteins from the labeling reaction mixture. In certain embodiments, the labeled N-glycans are substantially free of unreacted label. 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 agent (i.e., cyanoborohydride). In certain embodiments, the labeled N-glycans are purified by hydrophilic interaction. In certain embodiments, the labeled N-glycans are purified by chromatography comprising a hydrophilic stationary phase and a reverse phase 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 using a Waters HILIC MassPrep μElution plate according to the manufacturer's protocol. In some embodiments, the labeled N-glycans are purified to a purity level equivalent to the purity level obtained when purified using a Waters HILIC MassPrep μElution plate according to the manufacturer's protocol.

[0233] In some embodiments, the method includes separating the labeled N-glycans of a population of anti-CD20 antibody proteins and determining the N-glycan profile of the population. In some embodiments, the separation of the labeled N-glycans includes separation by hydrophilicity. In some embodiments, the separation of the labeled N-glycans includes chromatography, which includes a hydrophilic stationary phase and a reverse-phase eluent. In certain embodiments, the chromatography is hydrophilic interaction liquid chromatography (HILIC). In some embodiments of the method, the separation of the labeled N-glycans includes chromatography including an amide stationary phase. In some embodiments, the amide stationary phase is a Glycan BEH Amide column. In certain embodiments, the reverse-phase eluent includes one or more mobile phases, which include an acidic ammonium buffer and / or acetonitrile.

[0234] In some embodiments, chromatography is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time, and the combined parameters are sufficient to separate the labeled N-glycans. In some embodiments, chromatography is performed using a chromatography system (i.e., an HPLC or UPLC system) that controls these parameters. In some embodiments, the chromatography system is coupled to a detector for detecting the labeled N-glycans of a population of anti-CD20 antibody proteins. 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 a population of anti-CD20 antibody proteins in the labeling step). In some embodiments, the label is 2-AB. In some embodiments, the detection of fluorescence includes the step of exciting with light at a wavelength of 360 nm and measuring the fluorescence emission at a wavelength of 428 nm. In some embodiments, the chromatography system is a HILIC-UPLC system. In some embodiments, the separation and detection of the labeled N-glycans are performed using Waters UPLC equipped with a hydrophilic interaction stationary phase, a reverse phase eluent, and a fluorescence detector. In some embodiments, the hydrophilic interaction stationary phase includes a Glycan BEH Amide column (130 Å, 1.7 μm, 2.1 mm × 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 phase eluent includes one or more mobile phases including a first mobile phase having a pH of about 4.4 containing about 250 mM ammonium formate and a second mobile phase containing acetonitrile. In some embodiments, the mobile phase gradient includes an increase from 22.0% to 44.1% of the first mobile phase over 38.5 minutes.

[0235] In some embodiments, the detection of fluorescence includes the generation of a chromatogram, and the dependent variable is selected from mobile phase volume, volume of eluate passing through the chromatography column, or time, and what can be measured is fluorescence. In certain embodiments, the determination of the N-glycan profile of a population of anti-CD20 antibody proteins includes the step of quantifying the relative amounts of the labeled G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1’, G1B, G1F, G1F’, G1FB, G2, and G2F N-glycans of said population. In certain embodiments, chromatography (i.e., HILIC-UPLC) is performed using parameters for a sufficient time to separate the G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1’, G1B, G1F, G1F’, G1FB, G2, and G2F N-glycans in terms of flow rate, column temperature, mobile phase gradient, and quantification. In certain embodiments, the amount of N-glycan is quantified by calculating the area under the curve including the labeled G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1’, G1B, G1F, G1F’, G1FB, G2, and G2F N-glycans in the chromatograph that separates said 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 said N-glycan with respect to the total peak areas of the G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1’, G1B, G1F, G1F’, G1FB, G2, and G2F N-glycans in the chromatograph that separates said N-glycans. In certain embodiments, the peak area of the N-glycan in the chromatograph is 0.25% or more. In certain embodiments, the peak area of the N-glycan in the chromatograph has a signal / noise ratio of 3.0 or more.

[0236] The N-glycan profile of a population of anti-CD20 antibody proteins may determine the relative amount of glycosylation of the population by, for example, summing the results of site-specific glycosylation of a digested population of anti-CD20 antibody proteins by liquid chromatography-linked mass spectrometry (LC-MS) peptide mapping.

[0237] In some embodiments, LC-MS peptide mapping includes determining the molecular weight of peptides derived from a Lys-C digested population of anti-CD20 antibody proteins and determining the presence and amount of glycosylated residues (i.e., G0, G0F, G0B, G0FB, G1, or G1F). In some embodiments, the population of anti-CD20 antibody proteins is reduced and alkylated prior to digestion. In certain embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time, and the combined parameters are sufficient to separate the peptides of the Lys-C digestion. In certain embodiments, the relative abundance of the peptides of the Lys-C digestion is calculated by integrating the area under its curve compared to the total area.

[0238] In some embodiments, the measured molecular weight is compared to the theoretical molecular weight for a glycosylated peptide selected from column 2 of Table 1. In certain embodiments, the measured molecular weight is compared to the theoretical molecular weight selected from column 4 of Table 1.

[0239] [Table 9]

[0240] In some embodiments, the population of anti-CD20 antibody proteins includes glycosylation at residue Asn-298 of SEQ ID NO: 33 present in the heavy chain.

[0241] (c) Method for determining deamidation (i) Deamidation in general Also provided herein is a method for determining the amount of asparagine deamidation in a population of anti-CD20 antibody proteins. In some embodiments, the amount of asparagine deamidation in a population of anti-CD20 antibody proteins is the amount of isoaspartate residues in the population. In some embodiments, the method includes detecting the amount of isoaspartate residues in a digested population of anti-CD20 antibody proteins.

[0242] (ii) Deamidation by Isoquant The amount of asparagine deamidation or isoaspartate in a population or digested population of anti-CD20 antibody proteins can be determined by an enzymatic method, for example, by the addition of a methyltransferase that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to isoaspartic acid (i.e., protein isoaspartyl methyltransferase (PIMT)). The reaction catalyzed by this PIMT produces S-adenosylhomocysteine (SAH) in an amount that is directly proportional to the level of isoaspartate present in the sample (approximate 1:1 stoichiometry). SAH can be directly measured by separating the SAH (i.e., by ultra-performance liquid chromatography (UPLC) or reverse-phase high-performance liquid chromatography (RP-HPLC)), detecting it at 260 nm, and quantifying the amount of SAH by comparing it to a standard curve of SAH (i.e., the Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu peptide).

[0243] In some embodiments, the method includes a derivatization step that includes contacting a population of anti-CD20 antibody proteins with PIMT and SAM. In some embodiments, the population of anti-CD20 antibody proteins is contacted with PIMT and SAM at a temperature and for a time sufficient for substantially all derivatization (i.e., methylation) of the aspartate residues present in the population. In some embodiments, the method optionally includes a step of stopping the derivatization reaction. In certain embodiments, the method includes a step of separating SAH by hydrophobicity for the quantification of isoaspartate. In some embodiments, the step of separating SAH includes chromatography with a hydrophobic interaction stationary phase and a reverse phase eluent. In some embodiments, the step of separating SAH includes chromatography using a C18 stationary phase. In some embodiments, the C18 stationary phase is a Hydro-RP column. In certain embodiments, the reverse phase eluent includes one or more mobile phases (i.e., a weakly acidic phosphate buffer and / or methanol). In certain embodiments, the chromatography is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time, and the combined parameters are sufficient to separate SAH from the derivatization reaction components. In some embodiments, the 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 (i.e., absorbance at 260 nm) for detecting SAH after derivatization. In some embodiments, the separation and detection of SAM is performed using an RP-HPLC or UPLC system equipped with a hydrophilic interaction stationary phase, a reverse phase eluent, and a detector for the absorbance of SAH at 260 nm. In some embodiments, the hydrophobic interaction stationary phase includes Synergi Hydro-RP (4.6 mm × 150 mm). In some embodiments, the stationary phase (i.e., column) temperature is about 25 °C or room temperature. In certain embodiments, the flow rate is 1 mL / min.In some embodiments, the reverse-phase eluent comprises one or more mobile phases comprising a first mobile phase of about 6.2 pH containing about 50 mM potassium phosphate and a second mobile phase containing methanol. In some embodiments, the gradient of the 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 of the second mobile phase. In some embodiments, the amount of SAH is quantified by comparing its integrated curve area to an 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 protein is determined using the Promega Isoquant kit according to the manufacturer's protocol together with an Isoasp-DSIP standard.

[0244] (iii) Deamidation by LC-MS The amount of asparagine deamidation or isoaspartate in a population of anti-CD20 antibody protein or a digested population may be determined by liquid chromatography coupled mass spectrometry (LC-MS) peptide mapping, for example, by summing the results of site-specific deamidation of a digested population of anti-CD20 antibody protein.

[0245] In some embodiments, LC-MS peptide mapping includes determining the molecular weight and / or relative abundance of peptides derived from a Lys-C digested population of anti-CD20 antibody protein, and determining the presence and amount of deamidated or isoaspartate residues. In some embodiments, the population of anti-CD20 antibody protein is reduced and alkylated prior to digestion. In certain embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time, and the combined parameters are sufficient to separate the peptides of the Lys-C digestion. In certain embodiments, the relative abundance of the peptides of the Lys-C digestion is calculated by integrating its curve under area compared to the total area.

[0246] In some embodiments, the measured molecular weight is compared to the theoretical molecular weight for a deamidated peptide selected from column 2 of Table 2. In certain embodiments, the measured molecular weight is compared to the theoretical molecular weight selected from column 3 of Table 2.

[0247]

Table 10

[0248] In some embodiments, LC-MS peptide mapping includes determining the molecular weight and / or relative abundance of peptides derived from an Asp-N digested population of anti-CD20 antibody protein, and determining the presence and amount of deamidated or isoaspartate residues. In some embodiments, the population of anti-CD20 antibody protein is reduced and alkylated prior to digestion. In certain embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time, and the combined parameters are sufficient to separate the peptides of the Asp-N digestion. In certain embodiments, the relative abundance of the peptides of the Asp-N digestion is calculated by integrating its curve under area compared to the total area.

[0249] In certain embodiments, the measured molecular weight is compared to the theoretical molecular weight for deamidated peptides selected from column 2 of Table 3. In certain embodiments, the measured molecular weight is compared to the theoretical molecular weight selected from column 3 of Table 3.

[0250] [Table 11]

[0251] (d) Methods for determining oxidation (i) Oxidation in general Also provided herein are methods for determining the level of methionine oxidation in a population of anti-CD20 antibody proteins described herein. In some embodiments, the amount of methionine oxidation in a population of anti-CD20 antibody proteins is the amount of Met sulfoxide (MetO) residues in the population. In some embodiments, the method includes the step of detecting the amount of Met sulfoxide (MetO) residues in the population.

[0252] (ii) Oxidation by LC-MS The amount of methionine oxidation or Met sulfoxide (MetO) residues in a population of anti-CD20 antibody proteins or a digested population may be determined by liquid chromatography coupled mass spectrometry (LC-MS) peptide mapping, for example, by summing the results of site-specific oxidation of a digested population of anti-CD20 antibody proteins.

[0253] In some embodiments, LC-MS peptide mapping includes determining the molecular weights of peptides derived from a Lys-C digested population of anti-CD20 antibody protein and determining the presence and amount of oxidized or MetO residues. In some embodiments, the population of anti-CD20 antibody protein is reduced and alkylated prior to digestion. In some embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time, and the combined parameters are sufficient to separate the peptides of the Lys-C digestion. In some embodiments, the relative abundance of the peptides of the Lys-C digestion is calculated by integrating the area under its curve compared to the total area.

[0254] In some embodiments, the measured molecular weight is compared to the theoretical molecular weight for an oxidized peptide selected from column 2 of Table 4. In some embodiments, the measured molecular weight is compared to the theoretical molecular weight selected from column 3 of Table 4.

[0255] [Table 12]

[0256] In some embodiments, the population of anti-CD20 antibody protein includes 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 each selected from Met-20, Met-34, Met-81, Met-253, or Met-428 shown in SEQ ID NO: 17, 18, 21, 24, or 29. In certain embodiments, the population of anti-CD20 antibody protein includes 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 shown in SEQ ID NO: 30.

[0257] (e) Assay for determining the conformation of a protein - Circular dichroism In some embodiments, the secondary structure of the anti-CD20 antibody protein is analyzed by using a circular dichroism (CD) spectrum to measure the difference in absorbance between left and right circularly polarized light due to structural asymmetry. The CD spectrum uses the far-ultraviolet spectrum at wavelengths of about 170 - 260 nm. At these wavelengths, different secondary structures commonly found in proteins, such as α-helix, parallel and antiparallel β-sheets, β-turns, and random coils, each produce characteristic spectra, allowing the analysis of different secondary structures in the protein. Using the spectrum of a given protein, its percentage content in the secondary structure can be inferred.

[0258] 7.6 Single batch composition Compositions or populations in scaled-up amounts of the anti-CD20 antibody protein described in Section 7.4 are provided herein. In certain embodiments, these scaled-up amounts are present in a composition derived from a single batch, i.e., a single run of a specified volume of a single bioreactor. For example, an anti-CD20 antibody protein obtained from a single run of a 15,000 L bioreactor may be referred to as a single batch. In certain embodiments, the bioreactor has a capacity of 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. 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 - 35 mg / ml, 10 - 30 mg / ml, 10 - 25 mg / ml, 10 - 20 mg / ml, 10 - 15 mg / ml, 15 - 35 mg / ml, 15 - 30 mg / ml, 15 - 25 mg / ml, 15 - 20 mg / ml, 20 - 35 mg / ml, 20 - 30 mg / ml, 20 - 25 mg / ml, 25 - 35 mg / ml, or 25 - 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%).

[0259] In some aspects, the anti-CD20 antibody proteins disclosed herein are produced at commercial scale. In certain embodiments, the commercial scale is 10,000 - 25,000 L.

[0260] (a) Total protein In some embodiments, the amount of total protein is measured using spectrophotometry. In certain embodiments, the amount of total protein is measured by absorbance at 280 nm.

[0261] In some embodiments, the amount of total protein can be measured using the following assay procedure. This method determines the concentration of anti-CD20 antibody protein using ultraviolet absorbance measurement at 280 nm with correction for light scattering at 320 nm. The concentration calculation is based on Beer-Lambert's law. The extinction coefficient of the anti-CD20 antibody protein at 280 nm is determined based on the amino acid composition and is 1.61 mL / mg / cm. The extinction coefficient can be used. This method is suitable for use in in-process sample testing, release testing, and stability testing after purification by Protein A. The test sample is diluted three times with 0.9% sodium chloride by weight to a target protein concentration of 0.4 mg / mL. Physiological saline is used as the instrument blank. Bovine serum albumin is used as a system suitability control and is tested before and after measurement of the sample. The test sample is loaded into a quartz cuvette with a 1 cm optical path length, and the absorbance at 280 and 320 nm is measured. The test 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 system suitability criteria for protein concentration listed in Table 6.

[0262] [Table 13]

[0263] [Table 14]

[0264] In some embodiments, the population of anti-CD20 antibody proteins provided herein has a total protein amount of 25.5 - 25.8 mg / mL. In certain embodiments, the population has a total protein amount of about 25.6 mg / mL.

[0265] 7.7 Biological properties In some embodiments, the population of anti-CD20 antibody proteins provided herein retains 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).

[0266] (a) Assays (i) Cell line antibody-dependent cell-mediated cytotoxicity (ADCC) In some embodiments, the cell line ADCC assay uses Raji cells as target cells. In certain embodiments, Raji cells express CD20. In certain embodiments, the cell line ADCC assay uses effector cells selected from CD16 effector cells and primary NK cells. In certain embodiments, the cell line ADCC assay uses Raji cells as target cells and KILR CD16a effector cells. In certain embodiments, the cell line ADCC assay uses Raji cells as target cells and primary NK cells as effector cells. In certain embodiments, KILR CD16a effector cells are human CD8+ T lymphocytes from a single donor engineered to express CD16 / FcγRIII on the surface of its plasma membrane.

[0267] In some embodiments, the cell-based ADCC assay uses target cell lysis as a readout. In some embodiments, target cell lysis is quantified using CytoTox Glo™ (Promega). In some embodiments, the cell-based ADCC assay shows the relative potency of the population against a commercially available reference standard. In some embodiments, the cell-based ADCC assay yields a dose-response curve and an EC50 value. In some embodiments, the compositions or populations of anti-CD20 antibody proteins provided herein function in cell-based ADCC using CD16 effector cells at more than 100% of that of the commercially available reference standard RS-117808.

[0268] In some embodiments, ADCC activity can be measured using a CD16 activity assay. In some embodiments, the CD16 activity assay evaluates ADCC activity using an alternative readout. In some embodiments, the CD activity assay uses WIL2-S as the target cell. In certain embodiments, the effector cells used are stably transformed Jurkat cell lines expressing a chimeric molecule consisting of the extracellular domain of FcγRIIIa conjugated to the transmembrane and intracellular domains of the gamma chain of the mast cell / basophil Fc receptor for IgE. In certain embodiments, the cells are treated in the presence of PMA (phorbol 12-myristate 13-acetate) in combination with a serial dilution of the anti-CD20 antibody. In certain embodiments, activation of the effector cells induces the release of IL-2, which is measured using a commercially available ELISA kit. In certain embodiments, potency is reported as a percentage relative to a reference standard.

[0269] In some embodiments, the population has a relative potency of 106-126% compared to a commercially available reference standard in the CD16 activity assay. In some embodiments, the population has a relative potency of about 115% compared to a commercially available reference standard in the CD16 activity assay. In some embodiments, the commercially available reference standard is RS-117808.

[0270] In some embodiments, the cellular ADCC activity uses the following assay procedure. An eight-point dilution series of reference standards, QC reference controls, and test materials is used with concentration ranges of 250.00 pg / mL to 0.04 pg / mL (250, 50, 16.7, 5.6, 1.9, 0.6, 0.2, 0.04 pg / mL). Two independent preparations of the material are prepared and assayed across duplicate measurement plates. The assay controls are prepared in triplicate. This includes a target cell alone control, a target cell death control, an effector cell alone control, and an effector cell and target cell control. KILR cells are obtained from Eurofins. These are obtained from a master cell bank made from specific human donors. The master and working cell banking systems and qualification protocols enable the vendor to create thousands of vials in a reproducible manner, and TG maintains a secured supply of this important agent. Procedures such as the qualification process for incoming materials ensure that highly reproducible cells are used in all assays. The KILR cells are thawed and cultured at 1 × 10 6 cells / mL in medium supplemented with IL-2, rested for at least 6 days before use, and not used beyond 14 days after thawing. To perform the KILR ADCC assay, Raji cells, also managed by the master and working cell banking systems, are seeded at 1 × 10 5 cells / mL, and reference standards, internal assay controls, and test sample dilutions are added to the Raji cells. KILR effector cells are added at 5 × 10 5Seed at cells / mL and set the final effector:target (E:T) ratio to 5:1. Incubate the cells at 36 ± 1 °C and 5 ± 1% CO2 for 18 - 22 hours. At the end of the incubation, add the CytoTox GLo™ preparation and incubate the plate for 30 ± 10 minutes. Read the plate using a SpectraMax plate reader. Analyze the data by weighted non-linear regression using a four-parameter logistic fit with SoftMax Pro. Evaluate the resulting data for potency against a reference standard using SoftMax Pro software. Representative dose-response curves for the reference standard and samples are shown. Report the results as a percentage of the potency against the primary reference standard or its derivative.

[0271] In some embodiments, the cell-based ADCC assay is used as a potency assay for batch release. In some embodiments, the cell-based ADCC assay is used as a potency assay for stability testing. In some embodiments, the cell-based ADCC assay is used as a potency assay in manufacturing quality control and processes. In some embodiments, the cell-based ADCC assay is used in comparative studies (e.g., in research and development or clinical trials).

[0272] In some embodiments, the high potency of a population in the cell-based ADCC assay is related to a low level of fucose content in the N-glycan profile of the population. In some embodiments, the high potency of a population in the cell-based ADCC assay is due to a low level of fucose content in the N-glycan profile of the population.

[0273] (ii) Antibody-dependent cellular phagocytosis (ADCP) In some embodiments, antibody-dependent cellular phagocytosis (ADCP) activity can be measured using Daudi cells expressing CD20 as target cells (labeled with PKH26). In some embodiments, human monocytes are isolated from PBMCs and differentiated in vitro using GM-CSF to obtain macrophages. In some embodiments, macrophages are co-cultured with PKH26-labeled target cells pre-incubated with serially diluted anti-CD20 antibody samples. In some embodiments, target cell phagocytosis is evaluated by flow cytometry. In some embodiments, a dose-response curve and the calculated IC50 of the tested samples can be shown.

[0274] (iii) Complement-dependent cytotoxicity (CDC) In some embodiments, the cell-based CDC assay uses Jeko-1 cells as target cells and a rabbit complement system. In certain embodiments, the cell-based CDC assay uses Raji cells as target cells and a human complement system. In some embodiments, the cell-based CDC assay uses target cell lysis as a readout.

[0275] In some embodiments, the cell-based CDC assay uses the following assay procedure. A 9-point dilution series of reference standards, QC reference controls, and test materials is used with 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 of the material are prepared and assayed across duplicate plates. The assay negative control is prepared in triplicate. This includes target cell and complement controls and target cell alone controls. In this assay, Jeko-1 cells obtained from ATCC and maintained through a master banking system are seeded at 3×10 5Seed at cells / mL and incubate for 60 - 90 minutes. Add duplicate test samples diluted with reference standard, internal assay control, and cell culture medium. Then add complement to the wells and incubate the plate at 36°C ± 1°C for approximately 2 hours, followed by incubation at room temperature for 25 ± 5 minutes. Controls include target cells with complement-only control and target cells with control-only, providing a baseline level of target cell viability over the course of the assay. Then add Cell Titer-Glo reagent and incubate at room temperature for an additional 30 ± 10 minutes. At the end of the assay, read the plate using a SpectraMax M5 plate reader. Analyze the data by weighted non-linear regression using a four-parameter logistic fit with SoftMax Pro. Evaluate the resulting data for potency against the reference standard using SoftMax Pro software. Report the results as a percentage of potency against the reference standard. Representative dose-response curves for reference standard and samples are shown.

[0276] (iv) CD20 binding activity In some embodiments, the cell line CD20 binding assay uses Jeko-1 cells and an MSD assay. In some embodiments, the cell line CD20 binding assay yields a dose-dependent binding curve and an EC50 value.

[0277] In some embodiments, the cell line CD20 binding assay uses the following assay procedure. Prepare an 8-point dilution series of reference standard, QC reference control, and test material with concentration ranges from 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). Prepare two independent preparations of the test material for each 2-plate test material evaluation. Assay controls include no cell control (reference standard / test material dilution plus detection reagent, excluding cells) and cells-only control (cells plus detection reagent, excluding reference standard / test material). Seed Jeko-1 cells in an MSD high-binding plate in PBS at 3×10 5Seed at a density of cells / mL and a final volume of 100 μL / well, and incubate at 35 - 37 °C for 2 hours ± 10 minutes. Remove unbound cells by PBS washing, and block the plate with 45% FBS. Add 50 μL of reference standard, QC reference control, or test material dilution, and incubate the plate for 1 hour ± 10 minutes at room temperature with shaking. Following incubation and three PBS washes, add 50 μL of anti-human Fc detection antibody conjugated with STREP-SULFOTAG, and incubate for 1 hour ± 5 minutes at room temperature with shaking. Wash the plate again with PBS, and add 150 μL of MesoScale read buffer containing tripropylamine (TPA) as the chemiluminescent co-reactant for electrochemiluminescence reading. Read the plate immediately with an MSD reader using Workbench 4.0. Evaluate the resulting data using PLA software and analyze it using a constrained four-parameter logistic model to generate relative binding, 95% confidence intervals, and results relative to the reference standard. Report the binding activity results as a percentage of the potency relative to the reference standard. Calculate the relative potency test results from the dose-response curves of representative reference standards and test samples.

[0278] (v) Binding activity to FcγRIIIa 158V and FcγRIIIa 158F In some embodiments, the FcγRIIIa binding assay uses surface plasmon resonance (SPR). In certain embodiments, the FcγRIIIa binding assay yields a sensorgram that shows dose-dependent binding, saturation, and dissociation. In certain embodiments, the FcγRIIIa binding assay calculates the dissociation constant by on-off rates and steady-state kinetics. In certain embodiments, the binding affinity of the population for FcγRIIIa 158V is approximately one order of magnitude greater than the binding affinity of the population for FcγRIIIa 158F. In certain embodiments, the binding affinity of the population for FcγRIIIa 158V is significantly greater than rituximab. In certain embodiments, the binding affinity of the population for FcγRIIIa 158F is significantly greater than rituximab. In certain embodiments, the binding affinity of the population for both FcγRIIIa 158V and FcγRIIIa 158F is significantly greater than rituximab.

[0279] 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 linkage chemistry. An 8-point dilution series of the reference standard, QC reference control, and test material is prepared with a concentration range of 2000 nM to 15.6 nM and a dilution factor of 2. Independent duplicate sample dilutions are injected onto the chip, followed by surface regeneration between each cycle. Binding is measured in response units (RU). The kinetics of the binding reaction are determined by measuring the change in SPR due to the increase in mass near the biosensor chip surface. The change in the mass of the complex as a function of time is visualized as a sensorgram. The equilibrium dissociation constant (K D ) and the relative affinity of each sample for the reference standard are determined for each receptor. The rate of change of the SPR signal is analyzed for the FcγRIIIa 158V variant using a 1:1 Langmuir model to obtain apparent rate constants for the association and dissociation phases of the reaction, and the equilibrium dissociation constant (K D) is determined using the steady-state affinity for the FcγRIIIa 158F variant. The binding signal is exported to the PLA and the relative binding response is determined. The results are reported as % potency relative to a reference standard for each of the FcγRIIIa variants, i.e., 158V and 158F.

[0280] (vi) C1q binding activity 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. Prepare a 7-point dilution series of the reference standard, QC reference control, and test material with a concentration range of 15.00 μg / mL to 0.12 μg / mL. Coat the reference standard, QC reference control, and test material dilutions onto an ELISA plate and incubate the plate at room temperature for 1 hour ± 30 minutes (shaking at 150 - 200 rpm). After coating, wash the plate (3 × PBS / 0.05% Tween), block (with 1% BSA, at room temperature, incubate for 1 hour ± 10 minutes while shaking at 150 - 200 rpm), and wash (3 × PBS / 0.05% Tween). Then add C1q conjugated to peroxidase and incubate the plate at room temperature for 1.5 hours ± 30 minutes (shaking at 150 - 200 rpm). After incubation and washing, add the tetramethylbenzidine (TMB) substrate solution and incubate the plate at room temperature for 7 minutes (-1 minute / +30 seconds). This results in a colorimetric reaction proportional to the level of bound C1q. Stop the reaction by adding 1M sulfuric acid and measure the color development at 450 nm using a Molecular Devices SpectraMax microplate reader. Analyze the data by weighted non-linear regression using a 4-parameter logistic fit with SoftMax Pro. Evaluate the resulting data for potency relative to the reference standard using SoftMax Pro software. The results of the C1q binding assay are reported as a percentage of potency relative to the reference standard. A representative dose-response curve from one assay can be shown.

[0281] (vii) B cell depletion activity In some embodiments, the B cell depletion activity can be measured in a human whole blood B cell depletion assay. In certain embodiments, the B cell depletion activity can be measured in an autologous normal human whole blood B cell depletion assay. In certain embodiments, the depletion of B cells can be measured by presenting cells in the CD45 positive lymphocyte gate and counting CD3 positive T cells, CD19 positive B cells, and CD20 positive B cells. In certain embodiments, the percentage of B cell depletion (100 - ([100 / B / T cell ratio in sample without antibody] × [B / T cell ratio in sample with antibody])) can be calculated and plotted against the sample concentration.

[0282] In some embodiments, the B cell depletion activity can be measured using blood from three healthy donors. In certain embodiments, the depletion of B cells can be measured by presenting cells in the CD45 positive lymphocyte gate and counting CD3 positive T cells, CD19 positive B cells, and CD20 positive B cells.

[0283] (b) Biological properties In some embodiments, the biological properties of the compositions or populations of anti-CD20 antibody proteins provided herein can be measured and described by using a comparison with a reference standard in the assays described in section 7.7(a). In certain embodiments, the reference standard is a commercially available reference standard.

[0284] In some embodiments, the reference standard is an anti-CD20 antibody. In certain embodiments, the reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab).

[0285] In some embodiments, the commercially available reference standard is RS-117808. In certain embodiments, the population of anti-CD20 antibody proteins provided herein retains the biological properties shown in Table 7. RS-117808 (“antibody obinutuzumab (TG-1101)”) was deposited with the American Type Culture Collection (ATCC) located at 10801 University Boulevard, Manassas, VA 20110 in accordance with the terms of the Budapest Treaty, accepted by the ATCC on April 15, 2022, and has been assigned the unofficial patent deposit number PTA-127294.

[0286]

Table 15

[0287] (i) Cell line antibody-dependent cell-mediated cytotoxicity (ADCC) In some embodiments, the compositions or populations of anti-CD20 antibody proteins provided herein function with an appropriate potency of at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200% of the commercially available reference standard RS-117808 in cell line ADCC using the CD16 effector cell assay (see Section 7.7(a)(i)). In certain embodiments, the compositions or populations of anti-CD20 antibody proteins provided herein function with an appropriate 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 of the commercially available reference standard Rituxan® (Genentech / Biogen) in cell line ADCC using the CD16 effector cell assay (see Section 7.7(a)(i)).

[0288] In some embodiments, the population induces cytotoxicity in a cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay. In certain embodiments, the population induces greater cytotoxicity compared to a commercially available reference standard in an ADCC assay.

[0289] In some embodiments, the population induces greater cytotoxicity compared to an anti-CD20 antibody in an ADCC assay. In certain embodiments, the population induces greater cytotoxicity compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutifan (IMMU-106), ZEVALIN (ibritumomab tiuxetan), and / or OCREVUS (ocrelizumab) in an ADCC assay. In certain embodiments, the population induces greater cytotoxicity compared to GAZYVA (obinutuzumab) in an ADCC assay. In certain embodiments, the population induces greater cytotoxicity compared to ARZERRA (ofatumumab) in an ADCC assay. In certain embodiments, the population induces greater cytotoxicity compared to RITUXAN (rituximab) in an ADCC assay. In certain embodiments, the population induces greater cytotoxicity compared to belzutifan (IMMU-106) in an ADCC assay. In certain embodiments, the population induces greater cytotoxicity compared to ZEVALIN (ibritumomab tiuxetan) in an ADCC assay. In certain embodiments, the population induces greater cytotoxicity compared to OCREVUS (ocrelizumab) in an ADCC assay.

[0290] In some embodiments, the population has a relative potency of at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, or at least 25% compared to a commercially available reference standard in a cell-based ADCC assay. 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 commercially available reference standard in a cell-based ADCC assay. In certain embodiments, the population has a relative potency of greater than at least 100% compared to a commercially available reference standard in a cell-based ADCC assay. In certain embodiments, the population has a relative potency greater than 100% compared to a commercially available reference standard in a cell-based ADCC assay.

[0291] In some embodiments, the population has a relative potency 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 compared to a commercially available reference standard in a cell-based ADCC assay. In certain embodiments, the population has a relative potency 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 commercially available reference standard in a cell-based ADCC assay.

[0292] In some embodiments, the commercially available reference standard is an anti-CD20 antibody. In certain embodiments, the commercially available reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), bertuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab). In certain embodiments, the population has a relative potency of about 38% to about 3-fold compared to GAZYVA (obinutuzumab) in a cell-based ADCC assay. In certain embodiments, the population has a relative potency of about 10-fold to about 31-fold compared to ARZERRA (ofatumumab) in a cell-based ADCC assay. In certain embodiments, the population has a relative potency of about 28-fold to about 2250-fold compared to RITUXAN (rituximab) in a cell-based ADCC assay. In certain embodiments, the population has a relative potency of about 25-fold compared to OCREVUS (ocrelizumab) in a cell-based ADCC assay.

[0293] In some embodiments, the population has a relative potency of 60 - 200%, 70 - 190%, 80% - 180%, 85 - 170%, or 90 - 163% compared to the commercially available reference standard in a cell-based ADCC assay. In certain embodiments, the population has a relative potency of 90 - 163% compared to the commercially available reference standard in a cell-based ADCC assay. In certain embodiments, the population has a relative potency of about 117% compared to the commercially available reference standard in a cell-based ADCC assay. In certain embodiments, the commercially available reference standard is RS-117808.

[0294] In some embodiments, the EC50 value of the ADCC potency measured in a population cell line ADCC assay is 2 - 6 pg / mL. In certain embodiments, the EC50 value of the ADCC potency measured in a population cell line ADCC assay is 0.2 - 20 pg / mL, 0.3 - 18 pg / mL, 0.4 - 15 pg / mL, 0.5 - 12 pg / mL, 0.6 - 10 pg / mL, 0.7 - 9 pg / mL, 0.8 - 8 pg / mL, 0.9 - 7 pg / mL, or 1 - 6 pg / mL. In certain embodiments, the EC50 value of the ADCC potency is the average EC50 value calculated from the EC50 values obtained in a cell line ADCC assay repeated 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, or more times. In certain embodiments, the EC50 value of the ADCC potency assay measured in a population cell line ADCC assay (e.g., using Eurofins / DiscoverX KILR CD16a effector cells) is about 5.45 pg / mL. In certain embodiments, the EC50 value of the ADCC potency assay measured in a population cell line ADCC assay (e.g., using Eurofins / DiscoverX KILR CD16a effector cells) is about 2.42 pg / mL.

[0295] (ii) Antibody-dependent cellular phagocytosis (ADCP) In some embodiments, the population induces antibody-dependent cellular phagocytosis (ADCP). In certain embodiments, the population induces greater phagocytosis compared to a commercially available reference standard in an ADCP assay. In certain embodiments, the population induces greater phagocytosis compared to an anti-CD20 antibody in an ADCP assay.

[0296] In some embodiments, the population induces greater phagocytosis compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutifan (IMMU-106), ZEVALIN (ibritumomab tiuxetan), and / or OCREVUS (ocrelizumab) in an ADCP assay. In certain embodiments, the population induces phagocytosis compared to GAZYVA (obinutuzumab) in an ADCP assay. In certain embodiments, the population induces greater cytotoxicity compared to ARZERRA (ofatumumab) in a CDC assay. In certain embodiments, the population induces greater phagocytosis compared to RITUXAN (rituximab) in an ADCP assay. In certain embodiments, the population induces greater cytotoxicity compared to belzutifan (IMMU-106) in a CDC assay. In certain embodiments, the population induces greater phagocytosis compared to ZEVALIN (ibritumomab tiuxetan) in an ADCP assay. In certain embodiments, the population induces greater phagocytosis compared to OCREVUS (ocrelizumab) in an ADCP assay.

[0297] In some embodiments, the population has a relative potency of at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, or at least 25% compared to a commercially available reference standard in an ADCP assay. In certain embodiments, the population has a relative potency of at least 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 commercially available reference standard in an ADCP assay. In certain embodiments, the population has a relative potency of greater than at least 100% compared to a commercially available reference standard in an ADCP assay. In certain embodiments, the population has a relative potency of greater than 100% compared to a commercially available reference standard in an ADCP assay.

[0298] In some embodiments, the population has a relative potency 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 compared to a commercially available reference standard in an ADCP assay. In certain embodiments, the population has a relative potency of 5% - 50%, 50% - 100%, 100% - 500%, 500% - 1000%, 10-fold - 50-fold, 50-fold - 100-fold, 100-fold - 150-fold, or 150-fold - 300-fold compared to a commercially available reference standard in an ADCP assay.

[0299] In some embodiments, the commercially available reference standard is an anti-CD20 antibody. In certain embodiments, the commercially available reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab). In certain embodiments, the population has a relative potency of about 7-fold compared to ARZERRA (ofatumumab) in an ADCP assay. In certain embodiments, the population has a relative potency of about 15-fold compared to RITUXAN (rituximab) in an ADCP assay. In certain embodiments, the population has a relative potency of about 23-fold compared to GAZYVA (obinutuzumab) in an ADCP assay.

[0300] In some embodiments, the EC50 value of the ADCP potency measured in the population ADCP assay is 0.1 - 1 ng / mL. In certain embodiments, the EC50 value of the ADCP potency measured in the population ADCP assay is 1 - 10, 2 - 9, 3 - 8, 4 - 7, or 5 - 6 ng / mL. In certain embodiments, the EC50 value of the ADCP potency measured in the population ADCP assay is 0.05 - 20 ng / mL, 0.1 - 19 ng / mL, 0.15 - 18 ng / mL, 0.2 - 18 ng / mL, 0.25 - 15 ng / mL, 0.3 - 12 ng / mL, 0.3 - 10 ng / mL, 0.3 - 9 ng / mL, 0.3 - 8 ng / mL, 0.3 - 7 ng / mL, or 0.3 - 6 ng / mL. In certain embodiments, the EC50 value of the ADCP potency is the average EC50 value calculated from the EC50 values obtained in 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeated ADCP assays. In certain embodiments, the EC50 value of the ADCP potency measured in the population ADCP assay is about 5.50 ng / mL.

[0301] (iii) Complement-dependent cytotoxicity (CDC) In some embodiments, the population induces complement-dependent cytotoxicity (CDC). In certain embodiments, the population induces significant cytotoxicity compared to a commercially available reference standard in a CDC assay. In certain embodiments, the population induces significant cytotoxicity compared to an anti-CD20 antibody in a CDC assay.

[0302] In some embodiments, the population induces greater cytotoxicity compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutifan (IMMU-106), ZEVALIN (ibritumomab tiuxetan), and / or OCREVUS (ocrelizumab) in a CDC assay. In certain embodiments, the population induces greater cytotoxicity compared to GAZYVA (obinutuzumab) in a CDC assay. In certain embodiments, the population induces greater cytotoxicity compared to ARZERRA (ofatumumab) in a CDC assay. In certain embodiments, the population induces greater cytotoxicity compared to RITUXAN (rituximab) in a CDC assay. In certain embodiments, the population induces greater cytotoxicity compared to belzutifan (IMMU-106) in a CDC assay. In certain embodiments, the population induces greater cytotoxicity compared to ZEVALIN (ibritumomab tiuxetan) in a CDC assay. In certain embodiments, the population induces greater cytotoxicity compared to OCREVUS (ocrelizumab) in a CDC assay.

[0303] 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% compared to a commercially available reference standard in a cell line CDC assay. 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 commercially available reference standard in a cell line CDC assay. In certain embodiments, the population has a relative potency of at least greater than 100% compared to a commercially available reference standard in a cell line CDC assay. In certain embodiments, the population has a relative potency greater than 100% compared to a commercially available reference standard in a cell line CDC assay.

[0304] In some embodiments, the population has a relative potency 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 compared to a commercially available reference standard in a cell-based CDC assay. In certain embodiments, the population has a relative potency 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 commercially available reference standard in a cell-based CDC assay.

[0305] In some embodiments, the commercially available reference standard is an anti-CD20 antibody. In certain embodiments, the commercially available reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab). In certain embodiments, the population has a relative potency of about 50% compared to ARZERRA (ofatumumab) in a cell-based CDC assay. In certain embodiments, the population has a relative potency of about 37% compared to RITUXAN (rituximab) in a cell-based CDC assay. In certain embodiments, the population has a relative potency of about 1.8-fold compared to OCREVUS (ocrelizumab) in a cell-based CDC assay.

[0306] In some embodiments, the population has a relative potency of 50-150%, 60-140%, 70-130%, 75-120%, or 78-116% compared to that of a commercially available reference standard in a cell-based CDC assay. In certain embodiments, the population has a relative potency of 78-116% compared to that of a commercially available reference standard in a cell-based CDC assay. In certain embodiments, the population has a relative potency of 73-128% or 74-127% compared to the commercially available reference standard in a cell-based CDC assay. In certain embodiments, the population has a relative potency of about 91% compared to that of a commercially available reference standard in a cell-based CDC assay. In certain embodiments, the commercially available reference standard is RS-117808.

[0307] In some embodiments, the EC50 value of the CDC potency measured by the cell-based CDC assay of the population is 0.4-0.7 μg / mL or 0.4-0.6 μg / mL. In certain embodiments, the EC50 value of the CDC potency measured by the cell-based CDC assay of the population is 0.05-5 μg / mL, 0.1-4 μg / mL, 0.15-3 μg / mL, 0.2-2 μg / mL, 0.25-1 μg / mL, 0.3-0.9 μg / mL, 0.3-0.8 μg / mL, or 0.3-0.7 μg / mL. In certain embodiments, the EC50 value of the CDC potency is the average EC50 value calculated from the EC50 values obtained in 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeated cell-based CDC assays. In certain embodiments, the EC50 value of the CDC potency measured by the cell-based CDC assay of the population is about 0.5 μg / mL.

[0308] (iv) CD20 binding activity In some embodiments, the population retains CD20 binding activity in a cell-based CD20 binding assay. In certain embodiments, the population retains greater CD20 binding activity compared to a commercially available reference standard in a cell-based CD20 binding assay.

[0309] In some embodiments, the population retains greater CD20 binding activity compared to an anti-CD20 antibody in a cell-based CD20 binding assay. In certain embodiments, the population retains greater CD20 binding activity compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutifan (IMMU-106), ZEVALIN (ibritumomab tiuxetan), and / or OCREVUS (ocrelizumab) in a cell-based CD20 binding assay. In certain embodiments, the population retains greater CD20 binding activity compared to GAZYVA (obinutuzumab). In certain embodiments, the population retains greater CD20 binding activity compared to ARZERRA (ofatumumab). In certain embodiments, the population retains greater CD20 binding activity compared to RITUXAN (rituximab). In certain embodiments, the population retains greater CD20 binding activity compared to belzutifan (IMMU-106). In certain embodiments, the population retains greater CD20 binding activity compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population retains and induces greater CD20 binding activity compared to OCREVUS (ocrelizumab).

[0310] 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% compared to a commercially available reference standard in a CD20 binding assay. 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 commercially available reference standard in a CD20 binding assay. In certain embodiments, the population has a relative potency of greater than at least 100% compared to a commercially available reference standard in CD20 binding. In certain embodiments, the population has a relative potency of greater than 100% compared to a commercially available reference standard in CD20 binding.

[0311] In some embodiments, the population has a relative potency between 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 compared to a commercially available reference standard in a CD20 binding assay. In certain embodiments, the population has a relative potency between 5% and 50%, 50% and 100%, 100% and 500%, 500% and 1000%, 10-fold and 50-fold, 50-fold and 100-fold, 100-fold and 150-fold, or 150-fold and 300-fold compared to a commercially available reference standard in a CD20 binding assay.

[0312] In some embodiments, the commercially available reference standard is an anti-CD20 antibody. In certain embodiments, the commercially available reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab). In certain embodiments, the population has a relative potency between about 1.6-fold and about 5.8-fold compared to GAZYVA (obinutuzumab) in a CD20 binding assay. In certain embodiments, the population has a relative potency between about 81% and about 4.3-fold compared to ARZERRA (ofatumumab) in a CD20 binding assay. In certain embodiments, the population has a relative potency between about 1.6-fold and about 4.1-fold compared to RITUXAN (rituximab) in a CD20 binding assay.

[0313] In some embodiments, the population has a relative potency of 50-150%, 60-140%, 70-130%, 80-120%, 90-120%, or 92-118% compared to that of a commercially available reference standard in a cell-based CD20 binding activity bioassay. In certain embodiments, the population has a relative potency of 92-118% compared to that of a commercially available reference standard in a cell-based CD20 binding activity bioassay. In certain embodiments, the population has a relative potency of 82-138% compared to that of a commercially available reference standard in a cell-based CD20 binding activity bioassay. In certain embodiments, the population has a relative potency of about 109% compared to that of a commercially available reference standard in a cell-based CD20 binding activity bioassay. In certain embodiments, the commercially available reference standard is RS-117808.

[0314] In some embodiments, the EC50 value of the CD20 binding potency measured in the cell-based CD20 binding activity bioassay of the population is 0.05-0.1 μg / mL. In certain embodiments, the EC50 value of the CD20 binding potency measured in the cell-based CD20 binding activity bioassay of the population is 0.01-0.5 μg / mL, 0.02-0.4 μg / mL, 0.03-0.3 μg / mL, 0.04-0.2 μg / mL, or 0.05-0.1 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 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeated cell-based CD20 binding activity bioassays. In certain embodiments, the EC50 value of the CD20 binding potency measured in the cell-based CD20 binding activity bioassay of the population is about 0.093 μg / mL. In certain embodiments, the EC50 value of the CD20 binding potency measured in the cell-based CD20 binding activity bioassay of the population is about 0.063 μg / mL.

[0315] (v) Binding activity to FcγRIIIa 158V and FcγRIIIa 158F In some embodiments, the population retains FcγRIIIa 158V binding activity in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158V binding activity compared to a commercially available reference standard in an FcγRIIIa binding assay.

[0316] In some embodiments, the population retains greater FcγRIIIa 158V binding activity compared to an anti-CD20 antibody in an FcγRIIIa binding assay. In certain embodiments, the population retains greater CD20 binding activity compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), and / or OCREVUS (ocrelizumab) in a cell line CD20 binding assay. In certain embodiments, the population retains greater FcγRIIIa 158V binding activity compared to GAZYVA (obinutuzumab) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158V binding activity compared to ARZERRA (ofatumumab) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158V binding activity compared to RITUXAN (rituximab) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158V binding activity compared to belzutuzumab (IMMU-106) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158V binding activity compared to ZEVALIN (ibritumomab tiuxetan) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158V binding activity compared to OCREVUS (ocrelizumab) in an FcγRIIIa binding assay.

[0317] In some embodiments, the population has at least 1000%, 750%, 500%, 250%, 100%, 75%, or at least 50% relative FcγRIIIa 158V binding activity compared to a commercially available reference standard in an FcγRIIIa binding assay. 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 compared to a commercially available reference standard in an FcγRIIIa binding assay. In certain embodiments, the population has at least greater than 100% relative FcγRIIIa 158V binding activity compared to a commercially available reference standard in a cell-based ADCC assay. In certain embodiments, the population has greater than 100% relative FcγRIIIa 158V binding activity compared to a commercially available reference standard in an FcγRIIIa binding assay.

[0318] 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 relative FcγRIIIa 158V binding activity compared to a commercially available reference standard in an FcγRIIIa binding assay. In certain embodiments, the population has at least 50% - 100%, 100% - 500%, 500% - 1000%, 10-fold - 50-fold, 50-fold - 100-fold, 100-fold - 150-fold, or 150-fold - 300-fold relative FcγRIIIa 158V binding activity compared to a commercially available reference standard in an FcγRIIIa binding assay.

[0319] In some embodiments, the commercially available reference standard is an anti-CD20 antibody. In certain embodiments, the commercially available reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutifan (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab). In certain embodiments, the population has a relative FcγRIIIa 158V binding activity that is about 3.8-fold compared to GAZYVA (obinutuzumab) in an FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158V binding activity that is about 25.6-fold compared to ARZERRA (ofatumumab) in an FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158V binding activity that is about 18.7-fold compared to RITUXAN (rituximab) in an FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158V binding activity that is about 16-fold compared to OCREVUS (ocrelizumab) in an FcγRIIIa binding assay.

[0320] In some embodiments, the population retains FcγRIIIa 158F binding activity in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158F binding activity compared to a commercially available reference standard in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158F binding activity compared to an anti-CD20 antibody in an FcγRIIIa binding assay. In certain embodiments, the population retains greater CD20 binding activity compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutifan (IMMU-106), ZEVALIN (ibritumomab tiuxetan), and / or OCREVUS (ocrelizumab) in a cell line CD20 binding assay. In certain embodiments, the population retains greater FcγRIIIa 158F binding activity compared to GAZYVA (obinutuzumab) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158F binding activity compared to ARZERRA (ofatumumab) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158F binding activity compared to RITUXAN (rituximab) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158F binding activity compared to belzutifan (IMMU-106) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158F binding activity compared to ZEVALIN (ibritumomab tiuxetan) in an FcγRIIIa binding assay. In certain embodiments, the population retains greater FcγRIIIa 158F binding activity compared to OCREVUS (ocrelizumab) in an FcγRIIIa binding assay.

[0321] In some embodiments, the population has a relative FcγRIIIa 158F binding activity that is at least 1000%, 750%, 500%, 250%, 100%, 75%, or at least 50% compared to a commercially available reference standard in an FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158F binding activity that is 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 commercially available reference standard in an FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158F binding activity that is greater than 100% compared to a commercially available reference standard in a cellular FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158F binding activity that is greater than 100% compared to a commercially available reference standard in an FcγRIIIa binding assay.

[0322] In some embodiments, the population has a relative FcγRIIIa 158F binding activity 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 compared to a commercially available reference standard in an FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158F binding activity that is at least 5% - 50%, 50% - 100%, 100% - 500%, 500% - 1000%, 10-fold - 50-fold, 50-fold - 100-fold, 100-fold - 150-fold, or 150-fold - 300-fold compared to a commercially available reference standard in an FcγRIIIa binding assay.

[0323] In certain embodiments, the commercially available reference standard is an anti-CD20 antibody. In certain embodiments, the commercially available reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab). In certain embodiments, the population has a relative FcγRIIIa 158F binding activity that is about 2.6-fold higher compared to GAZYVA (obinutuzumab) in an FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158F binding activity that is about 21.8-fold higher compared to ARZERRA (ofatumumab) in an FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158F binding activity that is about 10.2-fold higher compared to RITUXAN (rituximab) in an FcγRIIIa binding assay. In certain embodiments, the population has a relative FcγRIIIa 158F binding activity that is about 9.9-fold higher compared to OCREVUS (ocrelizumab) in an FcγIIIa binding assay. In certain embodiments, the population has a significantly higher binding affinity for FcγRIIIa 158V or FcγRIIIa 158F than rituximab.

[0324] In some embodiments, the population has a relative potency of 20 - 300%, 30 - 250%, 40% - 220%, or 50 - 200% compared to a commercially available reference standard in a cell-based FcγRIIIa binding assay. In certain embodiments, the population has a relative potency of 50 - 200% compared to a commercially available reference standard in a cell-based FcγRIIIa binding assay. In certain embodiments, the population has a relative potency of 76 - 130% or 82 - 130% compared to a commercially available reference standard in a cell-based FcγRIIIa 158V binding assay. In certain embodiments, the commercially available reference standard is RS-117808.

[0325] In some embodiments, the population has a K of 55 - 70 nM as measured by surface plasmon resonance in an FcγRIIIa-158V binding assay Dhas a value. In certain embodiments, the population has a K value of 10-100 nM, 15-90 nM, 10-80 nM, or 30-70 nM as measured by surface plasmon resonance in an FcγRIIIa-158V binding assay D has a value. In certain embodiments, the population has a K value of 30-70 nM as measured by surface plasmon resonance in an FcγRIIIa-158V binding assay D has a value. In certain embodiments, K D The value is the average K value calculated from K values obtained in two, three, four, five, six, seven, eight, nine, ten, or more repeated FcγRIIIa-158V binding assays D value D has a value. In certain embodiments, the population has a K value of about 59 nM as measured by surface plasmon resonance in an FcγRIIIa-158V binding assay D has a value. In certain embodiments, the population has a K value of about 64.1 nM as measured by surface plasmon resonance in an FcγRIIIa-158V binding assay D has a value

[0326] In some embodiments, the population has a K value of 600-800 nM as measured by surface plasmon resonance in an FcγRIIIa 158F binding assay D has a value. In certain embodiments, the population has a K value of 100-2000 nM, 200-1800 nM, 300-1700 nM, 400-1600 nM, 500-1500 nM, 500-1200 nM, 600-1000 nM, or 600-800 nM as measured by surface plasmon resonance in an FcγRIIIa 158F binding assay D has a value. In certain embodiments, the population has a K value of 500-1000 nM as measured by surface plasmon resonance in an FcγRIIIa 158F binding assay D has a value. In certain embodiments, K D The value is the average K value calculated from K values obtained in two, three, four, five, six, seven, eight, nine, ten, or more repeated FcγRIIIa-158V binding assays D value Dvalue. In certain embodiments, the population has a K value of 760 nM as measured by surface plasmon resonance in an FcγRIIIa 158F binding assay D value. In certain embodiments, the population has a K value of 680.3 nM as measured by surface plasmon resonance in an FcγRIIIa 158F binding assay D value.

[0327] (vi) C1q binding activity In some embodiments, the population retains C1q binding activity as measured by ELISA. In certain embodiments, the population retains greater C1q binding activity as measured by ELISA compared to a commercially available reference standard

[0328] In some embodiments, the population retains greater C1q binding activity as measured by ELISA compared to an anti-CD20 antibody. In certain embodiments, the population retains greater C1q binding activity as measured by ELISA compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), and / or OCREVUS (ocrelizumab). In certain embodiments, the population retains greater C1q binding activity as measured by ELISA compared to GAZYVA (obinutuzumab). In certain embodiments, the population retains greater C1q binding activity as measured by ELISA compared to ARZERRA (ofatumumab). In certain embodiments, the population retains greater C1q binding activity as measured by ELISA compared to RITUXAN (rituximab). In certain embodiments, the population retains greater C1q binding activity as measured by ELISA compared to belzutuzumab (IMMU-106). In certain embodiments, the population retains greater C1q binding activity as measured by ELISA compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population retains greater induced C1q binding activity as measured by ELISA compared to OCREVUS (ocrelizumab).

[0329] In some embodiments, the population has a relative potency of at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, or at least 25% as measured by ELISA in a C1q binding assay and compared to a commercially available 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 as measured by ELISA in a C1q binding assay and compared to a commercially available reference standard. In certain embodiments, the population has a relative potency of greater than at least 100% as measured by ELISA in a C1q binding assay and compared to a commercially available reference standard. In certain embodiments, the population has a relative potency of greater than 100% as measured by ELISA in a C1q binding assay and compared to a commercially available reference standard.

[0330] In some embodiments, the population has a relative potency 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 as measured by ELISA in a C1q binding assay and compared to a commercially available reference standard. In certain embodiments, the population has a relative potency of 5% - 50%, 50% - 100%, 100% - 500%, 500% - 1000%, 10-fold - 50-fold, 50-fold - 100-fold, 100-fold - 150-fold, or 150-fold - 300-fold as measured by ELISA in a C1q binding assay and compared to a commercially available reference standard.

[0331] In some embodiments, the commercially available reference standard is an anti-CD20 antibody. In certain embodiments, the commercially available reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (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).

[0332] In some embodiments, the population has a relative potency of 30 - 180%, 40 - 170%, 50 - 160%, 60 - 150%, 70 - 140%, 80 - 130%, 85% - 120%, or 88 - 113% as measured by ELISA in a C1q binding assay compared to the commercially available reference standard. In certain embodiments, the population has a relative potency of 88 - 113% or 86 - 117% as measured by ELISA in a C1q binding assay compared to the commercially available reference standard. In certain embodiments, the population has a relative potency of 86 - 116% as measured by ELISA in a C1q binding assay compared to the commercially available reference standard. In certain embodiments, the population has a relative potency of about 99% as measured by ELISA in a C1q binding assay compared to the commercially available reference standard. In certain embodiments, the commercially available reference standard is RS-117808.

[0333] In some embodiments, the EC50 value of the C1q binding activity of the population measured by ELISA is 1.5 - 3 μg / mL. In certain embodiments, the EC50 value of the C1q binding activity of the population measured by ELISA is 0.2 - 9 μg / mL, 0.3 - 8 μg / mL, 0.4 - 7 μg / mL, 0.5 - 6 μg / mL, 0.6 - 5 μg / mL, 0.7 - 4 μg / mL, 0.8 - 3 μg / mL, 0.9 - 2.9 μg / mL, or 1 - 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 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeated ELISA experiments. In certain embodiments, the EC50 value of the C1q binding activity of the population measured by ELISA is approximately 1.92 μg / mL. In certain embodiments, the EC50 value of the C1q binding activity of the population measured by ELISA is approximately 2.6 μg / mL.

[0334] (vii) B cell depletion activity In some embodiments, the population retains B cell depletion activity as measured by a human whole blood B cell depletion assay. In certain embodiments, the population retains significant B cell depletion activity as measured by a human whole blood B cell depletion assay compared to a commercially available reference standard.

[0335] In some embodiments, the population retains greater B cell depletion activity as measured by a human whole blood B cell depletion assay compared to an anti-CD20 antibody. In certain embodiments, the population retains greater B cell depletion activity as measured by a human whole blood B cell depletion assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutifan (IMMU-106), ZEVALIN (ibritumomab tiuxetan), and / or OCREVUS (ocrelizumab). In certain embodiments, the population retains greater B cell depletion activity as measured by a human whole blood B cell depletion assay compared to GAZYVA (obinutuzumab). In certain embodiments, the population retains greater B cell depletion activity as measured by a human whole blood B cell depletion assay compared to ARZERRA (ofatumumab). In certain embodiments, the population retains greater B cell depletion activity as measured by a human whole blood B cell depletion assay compared to RITUXAN (rituximab). In certain embodiments, the population retains greater B cell depletion activity as measured by a human whole blood B cell depletion assay compared to belzutifan (IMMU-106). In certain embodiments, the population retains greater B cell depletion activity as measured by a human whole blood B cell depletion assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population retains greater B cell depletion activity as measured by a human whole blood B cell depletion assay compared to OCREVUS (ocrelizumab).

[0336] 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% compared to a commercially available reference standard in a human whole blood B cell depletion assay. 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 commercially available reference standard in a human whole blood B cell depletion assay. In certain embodiments, the population has a relative potency of more than at least 100% compared to a commercially available reference standard in a human whole blood B cell depletion assay. In certain embodiments, the population has a relative potency of more than 100% compared to a commercially available reference standard in a human whole blood B cell depletion assay.

[0337] In some embodiments, the population has a relative potency between 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 compared to a commercially available reference standard in a human whole blood B cell depletion assay. In certain embodiments, the population has a relative potency of 5% - 50%, 50% - 100%, 100% - 500%, 500% - 1000%, 10-fold - 50-fold, 50-fold - 100-fold, 100-fold - 150-fold, or 150-fold - 300-fold compared to a commercially available reference standard in a human whole blood B cell depletion assay.

[0338] In certain embodiments, the commercially available reference standard is an anti-CD20 antibody. In certain embodiments, the commercially available reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belimumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab).

[0339] 7.8 Methods of treating and preventing medical conditions Methods of treating multiple sclerosis Provided herein is a method of treating relapsing multiple sclerosis (RMS) in a subject in need thereof by administering to the subject an effective amount of the anti-CD20 antibody composition described herein. In some embodiments, the relapsing multiple sclerosis (RMS) is selected from clinically isolated syndrome (“CIS”), relapsing-remitting (“RRMS”), or active secondary progressive disease (“SPMS”). In some embodiments, the RMS is CIS. In some embodiments, the RMS is RRMS. In some embodiments, the RMS is SPMS. In some embodiments, the subject is diagnosed as having RMS according to the McDonald criteria (2010) or another suitable method known to those of skill in the art.

[0340] In some embodiments, the method includes administering to a subject the anti-CD20 antibody composition described herein in a multiple dosing regimen. In certain embodiments, the method includes administering to a subject the anti-CD20 antibody composition described herein by intravenous infusion. For example, the anti-CD20 antibody composition described herein can be administered to a subject by intravenous infusion in a multiple infusion dosing regimen. The anti-CD20 antibody composition described herein can be administered to a subject by intravenous infusion in a multiple infusion dosing regimen over 48 weeks. Additionally or alternatively, the anti-CD20 antibody composition described herein can be administered to a subject by intravenous infusion in a multiple infusion dosing regimen over 96 weeks. In some embodiments, the multiple infusion dosing regimen includes the first, second, and subsequent intravenous infusions of obinutuzumab. In some embodiments, the "subsequent infusions" of obinutuzumab are any number of infusions after the second infusion.

[0341] In some embodiments, the intravenous infusion comprises a multi - dosing regimen (e.g., a multi - injection dosing regimen) comprising: a) a first infusion on day 1 with about 100 to about 200 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan); b) a second infusion about 2 weeks after the first infusion with about 400 to about 500 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan); c) a first subsequent infusion about 24 weeks or about 6 months after the first infusion with about 400 to about 500 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan); and d) one or more subsequent infusions about 24 weeks or about 6 months after the previous infusion with about 400 to about 500 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan).

[0342] In some embodiments, the intravenous infusion comprises a multi-dose regimen (e.g., a multi-injection dosing regimen) comprising: a) a first infusion on day 1 with 150 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan); b) a second infusion about 2 weeks after the first infusion with 450 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan); c) a first subsequent infusion about 24 weeks or about 6 months after the first infusion with 450 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan); and d) one or more subsequent infusions about 24 weeks or about 6 months after the previous infusion with 450 mg of the anti-CD antibody composition described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan). In some embodiments, the first infusion is administered over 4 hours, the second infusion is administered over 1 hour, and subsequent infusions (e.g., the first subsequent infusion and / or one or more subsequent infusions) are administered over 1 hour.

[0343] In some embodiments, the method comprises a first administration to a subject by intravenous infusion of an anti-CD20 antibody composition (e.g., GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belimumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab)) and a second administration to the subject of an anti-CD20 antibody as described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan).In some embodiments, the intravenous infusion comprises a multi - infusion dosing regimen comprising: a) a first infusion of the prescribed dosage of the anti - CD20 antibody composition of the present specification (e.g., GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU - 106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab)) on day 1; b) a second infusion about 2 weeks after the first infusion, comprising about 400 - about 500 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan); c) a first subsequent infusion about 24 weeks or about 6 months after the first infusion, comprising about 400 - about 500 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan); and d) one or more subsequent infusions about 24 weeks or about 6 months after the previous infusion, comprising about 400 - about 500 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan).

[0344] In some embodiments, the intravenous infusion comprises a multi - infusion dosing regimen comprising: a) a first infusion of the prescribed dosage of the anti - CD20 antibody composition of the present specification on day 1 (e.g., GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), belzutuzumab (IMMU - 106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab)); b) a second infusion of 450 mg of the anti - CD20 antibody composition described herein approximately 2 weeks after the first infusion (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and approximately 20 - 40% fucosylated glycan); c) a first subsequent infusion of 450 mg of the anti - CD20 antibody composition described herein approximately 24 weeks or approximately 6 months after the first infusion (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and approximately 20 - 40% fucosylated glycan); and d) one or more subsequent infusions of 450 mg of the anti - CD20 antibody composition described herein approximately 24 weeks or approximately 6 months after the previous infusion (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and approximately 20 - 40% fucosylated glycan). In some embodiments, the first infusion is administered over 4 hours, the second infusion is administered over 1 hour, and subsequent infusions (e.g., the first subsequent infusion and / or one or more subsequent infusions) are administered over 1 hour.

[0345] In some embodiments, the method includes a treatment period of at least 48 weeks. In some embodiments, the method includes a treatment period of at least 96 weeks.

[0346] 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 recurrence in the year prior to treatment or two recurrences in the two years prior to treatment. In some embodiments, the subject has experienced or had the presence of T1 gadolinium (Gd)-enhancing lesions in the year prior to treatment with the anti-CD20 antibody composition described herein. In some embodiments, the subject has an Expanded Disability Status Scale (EDSS) score of 0 to 5.5 prior to treatment with the anti-CD20 antibody composition described herein.

[0347] In some embodiments of the method, the subject has not been treated with non-steroidal therapy for MS in the five years prior to treatment with the anti-CD20 antibody composition described herein. In some embodiments, the subject has no experience with treatment for MS. In certain embodiments, the subject is negative for hepatitis B virus (HBV). In some embodiments, the subject is negative for hepatitis B surface antigen (HBsAg). In some embodiments, the subject is negative for anti-hepatitis B virus core antibody. In certain embodiments, the subject has not been immunized with a vaccine for at least two weeks or at least four weeks prior to treatment with the anti-CD20 antibody composition described herein.

[0348] In some embodiments of the method, the subject has received a pre - dose of an amount of corticosteroid about 30 to about 60 minutes prior to administration of the anti - CD20 antibody composition described herein. In some embodiments, the corticosteroid is methylprednisolone or a corticosteroid biological equivalent thereof. In some embodiments, the amount of corticosteroid is about 100 mg of methylprednisolone. In some embodiments, the corticosteroid is dexamethasone or a corticosteroid biological equivalent thereof. In some embodiments, the amount of corticosteroid is about 10 - 20 mg of dexamethasone. In certain embodiments, the subject has received a pre - dose of an amount of antipyretic about 30 to about 60 minutes prior to administration of the anti - CD20 antibody composition described herein. In some embodiments, the antipyretic is acetaminophen or an antipyretic biological equivalent thereof. In some embodiments, the subject has received a pre - dose of an amount of antihistamine about 30 to about 60 minutes prior to administration of the anti - CD20 antibody composition described herein. In some embodiments, the antihistamine is diphenhydramine HCl or an antihistamine biological equivalent thereof. In some embodiments, the amount of antihistamine is about 25 - 50 mg of diphenhydramine HCl. In some embodiments, the subject has received a pre - dose of an amount of corticosteroid (as above) and an amount of antihistamine (as above) about 30 to about 60 minutes prior to administration of the anti - CD20 antibody composition described herein. In some embodiments, the corticosteroid and / or antihistamine is administered orally to the subject.

[0349] In some embodiments, the method reduces or delays the progression of one or more symptoms of MS in a subject. In certain embodiments, the method reduces the annualized relapse rate (ARR) in a subject following administration of an anti-CD20 antibody (e.g., ocrelizumab) composition described herein. In some embodiments, the ARR is the total number of relapses for the subject divided by the total treatment period (i.e., this is the ratio of the total RMS relapse count for the subject divided by the total treatment period (years) for the subject). In some embodiments, the ARR is the number of relapses confirmed by an independent relapse assessment panel (IRAP) per year for the subject. In some embodiments, the reduction in ARR is evaluated at about 96 weeks after the first infusion. In some embodiments, the subject achieves a significantly reduced ARR following administration of a multiple-infusion dosing regimen of an anti-CD20 antibody composition described herein as compared to a subject receiving daily oral administration of 14 mg of teriflunomide for the same treatment period. For example, administration of an anti-CD20 antibody composition described herein can significantly reduce the ARR as compared to administration of teriflunomide, and the relative reduction in relapse rate is 59%. Additionally or alternatively, administration of an anti-CD20 antibody composition described herein can significantly reduce the ARR as compared to administration of teriflunomide, and the relative reduction in relapse rate is 49%.In some embodiments, treatment with the anti-CD20 antibody composition described herein results in an ARR of from about 0.100 to about 0.050, such as from about 0.100 to about 0.090, from about 0.090 to about 0.080, from about 0.080 to about 0.070, from about 0.070 to about 0.060, or from 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, 0.084, 0.083, 0.082, 0.081, 0.080, 0.079, 0.078, 0.077, 0.076, 0.075, 0.074, 0.073, 0.072, 0.071, 0.070, 0.069, 0.068, 0.067, 0.066, 0.065, 0.064, 0.063, 0.062, 0.061, 0.060, 0.059, 0.058, 0.057, 0.056, 0.055, 0.054, 0.053, 0.052, 0.051, or 0.050).

[0350] In some embodiments, the method reduces the total number of T1 gadolinium (Gd)-enhancing lesions in a subject after administration of the anti-CD20 antibody composition described herein. In some embodiments, the reduction of Gd-enhancing T1 lesions is evaluated by MRI scan. In some embodiments, the reduction of Gd-enhancing T1 lesions is evaluated at about 96 weeks after the first infusion. In some embodiments, subjects who received a multiple-injection dosing regimen of an anti-CD20 antibody achieved a reduction in the total number of Gd-enhancing T1 lesions by MRI scan compared to subjects who received oral administration of 14 mg of teriflunomide daily during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., ofatumumab) significantly reduced the total number of gadolinium-enhancing T1 lesions compared to administration of teriflunomide, with a relative reduction of about 97%.

[0351] In some embodiments, the method reduces the number of new or enlarging T2 hyperintense lesions in a subject after administration of the anti-CD20 antibody composition described herein. In some embodiments, the reduction in the number of new or enlarging T2 hyperintense lesions is evaluated by MRI scan. In some embodiments, the reduction in the number of new or enlarging T2 hyperintense lesions is evaluated at about 96 weeks after the first infusion. In some embodiments, subjects who received a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., ocrelizumab) achieved a reduction in the total number of new and enlarging T2 hyperintense lesions by MRI scan compared to subjects who received daily oral administration of 14 mg of teriflunomide during the same treatment period. In some embodiments, administration of the anti-CD20 antibody (e.g., ocrelizumab) reduced the total number of new or enlarging T2 hyperintense lesions compared to administration of teriflunomide, and the relative reduction was about 90-92%.

[0352] In some embodiments, the method achieves progression of a confirmed disability in a subject after administration of the anti-CD20 antibody composition described herein. In some embodiments, confirmation of progression of the disability includes an increase of 1.0 point or more from the baseline EDSS score of the subject due to MS, wherein the baseline EDSS score is 5.5 or less. In some embodiments, confirmation of progression of the disability includes an increase of 0.5 point or more from the baseline EDSS score of the subject due to MS, wherein the baseline EDSS score is greater than 5.5.

[0353] In some embodiments, the method results in no evidence of disease activity (NEDA) in a subject. In certain cases, NEDA includes one or more of no confirmed relapses, no gadolinium-enhanced (Gd+) T1 lesions, no new and / or enlarging T2 lesions, and no progression of disability confirmed at 12 weeks. In some embodiments, the methods of the present disclosure result in NEDA in a subject at about 24 weeks after administration of a pharmaceutical formulation (e.g., an anti-CD20 antibody composition) described herein. For example, administration of a pharmaceutical formulation described herein can result in NEDA in a subject at about 24 to 96 weeks (e.g., 24 to 48 weeks, 24 to 72 weeks, 48 to 72 weeks, 72 to 96 weeks, or 48 to 96 weeks (e.g., 24 weeks, 36 weeks, 48 weeks, 60 weeks, 72 weeks, 84 weeks, 96 weeks, or any range therebetween)) after administration. In some embodiments, subjects who received a multi-infusion dosing regimen of an anti-CD20 antibody (e.g., ofatumumab) achieved an increase in NEDA status compared to subjects who received daily oral administration of 14 mg of teriflunomide during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., ofatumumab) increased the NEDA status by 197% compared to administration of teriflunomide. In some embodiments, administration of an anti-CD20 antibody (e.g., ofatumumab) increased the NEDA status by 277% compared to administration of teriflunomide.

[0354] In some embodiments, subjects who received a multi-infusion dosing regimen of an anti-CD20 antibody (e.g., ofatumumab) achieved an increase in improvement in disability (CDI) compared to subjects who received daily oral administration of 14 mg of teriflunomide during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., ofatumumab) increased the CDI by 116% at 12 weeks compared to teriflunomide. In some embodiments, administration of an anti-CD20 antibody (e.g., ofatumumab) increased the CDI by 103% at 24 weeks compared to administration of teriflunomide.

[0355] In some embodiments, subjects administered multiple infusions of an anti-CD20 antibody (such as ocrelizumab) achieve an increase in the Multiple Sclerosis Functional Composite (MSFC) score compared to subjects receiving daily oral administration of 14 mg of teriflunomide during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (such as ocrelizumab) increased the MSFC score by about 76% to 90% compared to administration of teriflunomide.

[0356] In some embodiments, subjects administered multiple infusions of an anti-CD20 antibody (such as ocrelizumab) achieve improvement in the Timed 25-Foot Walk (T25FW) score compared to subjects receiving daily oral administration of 14 mg of teriflunomide during the same treatment period. In some embodiments, subjects administered multiple infusions of an anti-CD20 antibody (such as ocrelizumab) achieve improvement in the 9-Hole Peg Test (9-HPT) score compared to subjects receiving daily oral administration of 14 mg of teriflunomide during the same treatment period.

[0357] In some embodiments, subjects administered multiple infusions of an anti-CD20 antibody (such as ocrelizumab) achieve a significant reduction in the volume and number of new T1 hypointense lesions on MRI scans compared to subjects receiving daily oral administration of 14 mg of teriflunomide during the same treatment period.

[0358] In some embodiments, the treatment of MS (e.g., RMS) disclosed herein further comprises administering an anti-CD20 antibody in combination with one or more additional therapeutic agents. For example, the anti-CD20 antibody used in the method of treating RMS can be administered in combination with other compounds, drugs, and / or agents useful for the treatment of RMS. Such compounds, drugs, and / or agents 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 current standard of care MS treatments. In some cases, the anti-CD20 antibody is used in combination with a Bruton's tyrosine kinase (BTK) inhibitor for treating RMS, including, for example, fenebrutinib, evobrutinib, trebrutinib, orelabrutinib BIIB091, AC0058, PRN473 (Dolgin, Nature Biotechnology 39:3-12 (2021)), and the compounds described in U.S. Patent No. 9,951,077. In some cases, the anti-CD20 antibody is used in a combination regimen that takes advantage of the non-overlapping mechanisms of ublituximab for B cell depletion.

[0359] An anti-CD20 antibody (e.g., rituximab) can be combined with an injectable pharmaceutical, an oral pharmaceutical, or an infused pharmaceutical. Injectable pharmaceuticals for combination therapy with an anti-CD20 antibody include, but are not limited to, interferons such as AVONEX (interferon beta-1a), BETASERON (interferon beta-1b), EXTAYIA (interferon beta-1b), PLEGRIDY (peginterferon beta-1a), and REBIF (interferon beta-1a). Other injectable pharmaceuticals include COPAXONE (glatiramer acetate). Orally available pharmaceuticals for combination with rituximab include fumarate-based compositions such as AUBAGIO (teriflunomide), BAFIERTAM, dimethyl fumarate, TECFIDERA, and VUMERITY. Additionally or alternatively, an anti-CD20 antibody (e.g., rituximab) can be used in combination with the monoclonal antibody Tysabri (natalizumab). Oral dosage agents that can be used in combination with an anti-CD20 antibody include GILENYI (fingolimod), MAYZENT (siponimod), ZEPOSIA (ozanimod), and PONVORY (ponesimod). Further oral dosage agents include MAVENCLAD (cladribine).

[0360] The anti-CD20 antibody (e.g., rituximab) can also be used in combination with an infused pharmaceutical including LEMTRADE (alemtuzumab) and NOVANTRONE (mitoxantrone).

[0361] In some embodiments, in a subject (e.g., a patient) having known resistance to one or more anti-CD20 antibodies, the anti-CD20 antibody of the present disclosure (e.g., rituximab or an anti-CD20 antibody that binds to the same epitope as rituximab) can be administered. This is because rituximab targets a unique epitope region on the CD20 antigen that is not targeted by other anti-CD20 monoclonal antibodies.

[0362] In some embodiments, the additional therapeutic agent may include a B cell depleting agent that is not an anti-CD20 antibody. In some embodiments, the B cell depleting agent is a PI3K inhibitor.

[0363] In some embodiments, the additional therapeutic agent includes a B cell depleting agent that is an anti-CD20 antibody, similar to the anti-CD20 antibodies described herein (e.g., ocrelizumab). For example, ocrelizumab can be used in combination with additional anti-CD20 antibody compositions such as, for example, OCREVUS® (ocrelizumab), KESIMPTA® (ofatumumab), and Rituxan® (rituximab).

[0364] The additional therapeutic agent can be administered orally, parenterally, intravenously, or subcutaneously.

[0365] In some embodiments, the methods described herein may result in treatment-emergent adverse events (TEAEs). In some cases, TEAEs include cytopenia or a reduction in blood cell counts in a subject. Blood cell counts can be evaluated by a blood test such as a complete blood count (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 hemocytometer) and automated methods (e.g., by using an automated cell counter). In some cases, cytopenia includes a reduction or decrease of about 20-100% (e.g., about 20-30%, about 20-40%, about 30-40%, about 30-50%, about 40-50%, about 40-60%, about 50-60%, about 50-70%, about 60-70%, about 60-80%, about 70-80%, about 70-90%, about 80-90%, about 80-100%, about 90-100%, 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)) in the subject's blood cell counts compared to a control or normal blood cell count. A control or normal blood cell count may include the blood cell counts of healthy subjects not administered the pharmaceutical formulations of the present disclosure. Additionally or alternatively, the blood cell counts of the subject prior to treatment by the method may be used as the control or normal blood cell count. Cytopenia or a reduction in blood cell counts may include one or more of lymphopenia, neutropenia, leukopenia, and anemia.

[0366] In some cases, administration of the pharmaceutical formulations (e.g., anti-CD20 antibody compositions) described herein results in cytopenia in a subject about 1 to 3 days (e.g., about 1 day, about 2 days, or about 3 days) after administration. For example, administration of the pharmaceutical formulation may result in lymphopenia in the subject about 2 days after administration. In certain embodiments, the cytopenia (e.g., lymphopenia, neutropenia, leukopenia, and / or anemia) is transient, and the blood cell count in the subject normalizes (e.g., becomes the same or similar to a control or normal blood cell count) by about 15 days (e.g., about 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, the lymphocyte count in the subject can normalize in the subject by 8 days after administration of the pharmaceutical formulation.

[0367] Method for treating cancer Also provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the anti-CD20 antibody composition described herein. In some embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, gynecological cancer (i.e., cancer of the cervix, ovary, uterus, vagina, or vulva), head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, mesothelioma, multiple myeloma, prostate cancer, skin cancer, or thyroid cancer.

[0368] A method of treating a disease or disorder associated with the overproliferation of B cells in a subject in need thereof, the method comprising administering to the subject an effective amount of the anti-CD20 antibody composition described herein is also provided herein. In some embodiments, the disease or disorder associated with the overproliferation of B cells is a blood cancer. In some embodiments, the blood cancer is lymphoma, leukemia, or myeloma. In some embodiments, the blood cancer is selected from B cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenström macroglobulinemia (WM), diffuse large B cell lymphoma (DLBCL), marginal zone lymphoma (MZL), hairy cell leukemia (HCL), Burkitt lymphoma (BL), Richter transformation, or primary central nervous system lymphoma (PCNSL). In various aspects of the method, the anti-CD20 antibody composition described herein comprises an anti-CD20 antibody, which is an IgG1 antibody comprising 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, respectively.

[0369] In some embodiments, the blood cancer is B-cell lymphoma. In some embodiments, the B-cell lymphoma is relapsed or refractory. In certain embodiments, the blood cancer is non-Hodgkin lymphoma (NHL). In some embodiments, the NHL is relapsed or refractory. In certain embodiments, the blood cancer is Waldenström macroglobulinemia (WM). In some embodiments, the WM is relapsed or refractory. In certain embodiments, the blood cancer is marginal zone lymphoma (MZL). In some embodiments, the MZL is relapsed or refractory. In certain embodiments, the blood cancer is chronic lymphocytic leukemia (CLL). In some embodiments, the CLL is relapsed or refractory. In certain embodiments, the blood cancer is small lymphocytic lymphoma (SLL). In some embodiments, the SLL is relapsed or refractory. In certain embodiments, the blood cancer is primary central nervous system lymphoma (PCNSL). In some embodiments, the PCNSL is relapsed or refractory.

[0370] In some embodiments, the method of treating a blood cancer comprises administering to a subject the anti-CD20 antibody composition described herein by intravenous infusion. In some embodiments, the intravenous infusion consists of infusions comprising about 900 mg of the anti-CD20 antibody composition described herein on day 1 and day 2 (split into a 150 mg dose on day 1 and a 750 mg dose on day 2), day 8, and day 15 of cycle 1 (each cycle being 28 days), day 1 of cycles 2-6, and day 1 every 3 cycles after cycle 6 (e.g., cycles 9, 12, 15, etc.). In some embodiments, the blood cancer is CLL.

[0371] In some embodiments, the methods described herein may result in treatment-emergent adverse events (TEAEs). In some cases, TEAEs include cytopenia or a reduction in blood cell counts in a subject. Blood cell counts can be evaluated by a blood test such as a complete blood count (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 hemocytometer) and automated methods (e.g., by using an automated cell counter). In some cases, cytopenia includes a reduction or decrease of about 20-100% (e.g., about 20-30%, about 20-40%, about 30-40%, about 30-50%, about 40-50%, about 40-60%, about 50-60%, about 50-70%, about 60-70%, about 60-80%, about 70-80%, about 70-90%, about 80-90%, about 80-100%, about 90-100%, 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)) in the subject's blood cell counts as compared to a control or normal blood cell count. A control or normal blood cell count may include the blood cell counts of healthy subjects not administered the pharmaceutical formulations of the present disclosure. Additionally or alternatively, the blood cell counts of the subject prior to treatment by the methods may be used as the control or normal blood cell count. Cytopenia or a reduction in blood cell counts may include one or more of lymphopenia, neutropenia, leukopenia, and anemia.

[0372] Multiple infusion dosing In any of the embodiments described herein, the intravenous infusion comprises a multi-injection dosing regimen comprising: a) a first infusion on day 1 of from about 400 to about 500 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan), b) a second infusion about 1 week after the first infusion of from about 500 to about 700 mg, from about 800 to about 1000 mg, or from about 1100 to about 1300 mg of the anti-CD20 antibody composition, c) a third infusion about 2 weeks after the first infusion of from about 500 to about 700 mg, from about 800 to about 1000 mg, or from about 1100 to about 1300 mg of the anti-CD20 antibody composition, d) a fourth infusion about 3 weeks after the first infusion of from about 500 to about 700 mg, from about 800 to about 1000 mg, or from about 1100 to about 1300 mg of the anti-CD20 antibody composition, and e) one or more subsequent infusions about 1 month after the previous infusion of from about 400 to about 500 mg, from about 500 to about 700 mg, from about 800 to about 1000 mg, or from about 1100 to about 1300 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered over 4 hours, the second infusion is administered over 1 hour, the third infusion is administered over 1 hour, the fourth infusion is administered over 1 hour, and the one or more subsequent infusions are administered over 1 hour.

[0373] In some embodiments, the intravenous infusion comprises a multi-infusion dosing regimen comprising: a) a first infusion on day 1 of 450 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan), b) a second infusion about 1 week after the first infusion of 600, 900, or 1200 mg of the anti-CD20 antibody composition, c) a third infusion about 2 weeks after the first infusion of 600, 900, or 1200 mg of the anti-CD20 antibody composition, d) a fourth infusion about 3 weeks after the first infusion of 600, 900, or 1200 mg of the anti-CD20 antibody composition, and e) one or more subsequent infusions of 450, 600, 900, or 1200 mg of the anti-CD20 antibody composition about 1 month after the previous infusion. In some cases, the first infusion is administered over 4 hours, the second infusion is administered over 1 hour, the third infusion is administered over 1 hour, the fourth infusion is administered over 1 hour, and one or more subsequent infusions are administered over 1 hour.

[0374] In some embodiments, the intravenous infusion comprises a multi-infusion dosing regimen comprising: a) a first infusion on day 1 with about 400 to about 500 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan), b) a second infusion about 1 week after the first infusion with about 500 to about 700 mg of the anti-CD20 antibody composition, c) a third infusion about 2 weeks after the first infusion with about 800 to about 1000 mg of the anti-CD20 antibody composition, and d) one or more subsequent infusions about 1 month after the previous infusion with about 400 to about 1000 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered over 4 hours, the second infusion is administered over 1 hour, the third infusion is administered over 1 hour, and the one or more subsequent infusions are administered over 1 hour.

[0375] In some embodiments, the intravenous infusion comprises a multi-infusion dosing regimen comprising: a) a first infusion on day 1 with 450 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan), b) a second infusion about 1 week after the first infusion with 600 mg of the anti-CD20 antibody composition, c) a third infusion about 2 weeks after the first infusion with 900 mg of the anti-CD20 antibody composition, and d) one or more subsequent infusions about 1 month after the previous infusion with 450, 600, or 900 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered over 4 hours, the second infusion is administered over 1 hour, the third infusion is administered over 1 hour, and the one or more subsequent infusions are administered over 1 hour.

[0376] In some embodiments, the intravenous infusion comprises a multi - infusion dosing regimen that includes: a) a first infusion on day 1 of about 5 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan), and b) one or more subsequent infusions, about 1 week after the previous infusion, of about 5 - about 450 mg of the anti - CD20 antibody composition, each subsequent infusion being at a higher dose than the previous infusion. In some cases, the first infusion is administered over 4 hours and the one or more subsequent infusions are administered over 1 hour.

[0377] In some embodiments, the intravenous infusion comprises a multi - infusion dosing regimen that includes: a) a first infusion on day 1 of about 100 - about 200 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan), and b) one or more subsequent infusions, about 1 week after the previous infusion, of about 400 - about 500 mg of the anti - CD20 antibody composition. In some cases, the first infusion is administered over 4 hours and the one or more subsequent infusions are administered over 1 hour.

[0378] In some embodiments, the intravenous infusion comprises a multi-infusion dosing regimen that includes a) a first infusion on day 1 comprising 150 mg of the anti-CD20 antibody composition, and b) one or more subsequent infusions, about 1 week after the previous infusion, comprising 450 mg of the anti-CD20 antibody composition. In some embodiments, the method includes 7 or more subsequent infusions. In some embodiments, the method includes 7 subsequent infusions. In some cases, the first infusion is administered over 4 hours and the one or more infusions are administered over 1 hour.

[0379] In some embodiments, the intravenous infusion comprises a multi-infusion dosing regimen that includes a) a first infusion on day 1 comprising about 500 to about 1000 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the population is expressed from 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 the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10-20% galactosylated glycan and about 20-40% fucosylated glycan), 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 1 month after the previous infusion, comprising about 500 to about 1000 mg of the anti-CD20 antibody composition. In certain embodiments, the one or more subsequent infusions each comprise 600 mg of the anti-CD20 antibody composition described herein. In certain embodiments, the first infusion, the second infusion, the third infusion, and the one or more subsequent infusions each comprise 600 mg of the anti-CD20 antibody composition described herein. In some embodiments, the first infusion, the second infusion, the third infusion, and the one or more subsequent infusions each comprise 900 mg of the anti-CD20 antibody composition described herein. In some cases, the first infusion is administered over 4 hours, the second infusion is administered over 1 hour, the third infusion is administered over 1 hour, and the one or more subsequent infusions are administered over 1 hour.

[0380] In some embodiments, the intravenous infusion comprises a multi - infusion dosing regimen that includes: a) a first infusion on day 1 of about 100 to about 200 mg of the anti - CD20 antibody composition described herein (i.e., the anti - CD20 antibody in the population is expressed from 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 the population of anti - CD20 antibody proteins has an N - glycan profile comprising 10 - 20% galactosylated glycan and about 20 - 40% fucosylated glycan), b) a second infusion about 1 week after the first infusion of about 700 to about 800 mg of the anti - CD20 antibody composition, c) a third infusion about 2 weeks after the first infusion of about 850 to about 950 mg of the anti - CD20 antibody composition, and d) one or more subsequent infusions of about 850 to about 950 mg of the anti - CD20 antibody composition about 1 month after the previous infusion. 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 over 4 hours, the second infusion is administered over 1 hour, the third infusion is administered over 1 hour, and the one or more subsequent infusions are administered over 1 hour.

[0381] Subject In some embodiments, the subject is human. In some embodiments, the human subject is an adult. In some embodiments, the subject has relapsed or refractory B-cell lymphoma. In some embodiments, the subject has been treated in a prior treatment course with at least one rituximab or rituximab-based therapy. In some embodiments, the subject has been treated in a prior treatment course with at least one fludarabine or fludarabine-based therapy. In some embodiments, the subject has no experience with 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, the subject has an Eastern Cooperative Oncology Group (ECOG) score of 0-2 prior to treatment with the anti-CD20 antibody composition described herein. In some embodiments, the subject has a peripheral blood lymphocyte count greater than 5,000 / μL prior to treatment with the anti-CD20 antibody composition described herein.

[0382] In some embodiments, the anti-CD20 antibody composition provided herein can be used for the treatment and / or prevention of 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 glomerulonephritis, rheumatoid arthritis (RA), interstitial lung disease, myasthenia gravis (MG), subepidermal autoimmune blistering disease, pulmonary infectious disease, acquired hemophilia, refractory mixed cryoglobulinemia, idiopathic 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-transplant lymphoproliferative disorder, autoimmune hemolytic anemia, cerebral vasculitis, microscopic polyangiitis (MPA), or idiopathic nephrotic syndrome.

[0383] 7.9 Pharmacokinetic properties Also provided herein is a method of treating a human patient suffering from a disease (e.g., an autoimmune disease), the method comprising administering to the patient an anti-CD20 antibody provided herein.

[0384] In some embodiments, the anti-CD20 antibody is administered as: i) a first infusion at a dose of about 150 mg, ii) a second infusion at a dose of about 450 mg two weeks later, and iii) subsequent infusions at a dose of about 450 mg every six months.

[0385] In some embodiments, administration of the anti-CD20 antibody results in 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 the steady state AUC.

[0386] In some embodiments, administration of the anti-CD20 antibody results in 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 the steady state Cmax.

[0387] In some embodiments, administration of the anti-CD20 antibody results in 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 the steady state Cmin.

[0388] In some embodiments, administration of the anti-CD20 antibody results in 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 the steady state Cavg.

[0389] 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 (organs, bone marrow, stem cells, and other cells and tissues), graft rejection, graft-versus-host disease, systemic 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 recurrent hepatitis, primary biliary cirrhosis, allergic conjunctivitis, atopic dermatitis, chronic obstructive pulmonary disease, glomerulonephritis, neuroinflammatory diseases, and uveitis.

[0390] In some embodiments, the autoimmune disease is multiple sclerosis. In certain embodiments, the multiple sclerosis is relapsing multiple sclerosis. In certain embodiments, the relapsing 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 MS is clinically isolated syndrome (CIS). In certain embodiments, the relapsing MS is relapsing-remitting multiple sclerosis (RRMS). In certain embodiments, the relapsing MS is active secondary progressive multiple sclerosis (SPMS). In certain embodiments, the relapsing MS is primary progressive MS (PPMS).

[0391] In certain embodiments, the anti-CD20 antibody is administered intravenously.

[0392] 7.10 Preparation Method Also provided herein are methods of making a population of anti-CD20 antibody proteins comprising the post-translational modifications specified herein within a given range. Example 15 provides an exemplary method of making a population of anti-CD20 antibody proteins provided herein.

[0393] In some embodiments, a method of making a population of anti-CD20 antibody proteins comprising post-translational modifications within a specified range described herein includes: 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 the 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 the second culture pH of about 6.5 to about 6.99 from day 3 of the culture to day 14 of the cell culture; and iv) controlling the culture pCO2 level to less than about 200 mmHg throughout the culture period. In some embodiments, the second culture pH is from about 6.60 to about 6.96 (e.g., the second culture pH is 6.8).

[0394] In some embodiments, the second culture pH results in a high integrated viable cell density (IVCD) and a high titer at harvest.

[0395] In some embodiments, the second culture pH results in a low percentage of fucosylation.

[0396] In some embodiments, rat hybridoma cells expressing a recombinant protein are cultured in a basal medium that is a chemically defined, animal-derived component-free (ADCF) culture medium.

[0397] In some embodiments, a feeding medium is added to the basal medium.

[0398] In some embodiments, the method further includes an initial temperature set point of about 37°C, which is set from day 0 to day 1 of the culture.

[0399] In some aspects, the method further includes a second temperature set point of about 35°C, which is set from the end of day 1 to day 3 of the culture.

[0400] In some embodiments, the method further includes a step of including a third temperature set point of about 32 °C to about 33 °C, where the third temperature set point is set on the third day of the culture and maintained through the recovery. The term "recovery" refers to the point in the mammalian cell culture process when cells containing recombinant protein are separated, removed from the cell culture medium, and subjected to further processing such as centrifugation, filtration, or purification.

[0401] In some embodiments, cell recovery is performed on the 12th, 13th, or 14th day of the cell culture process, or earlier when the cell viability drops below 20%.

[0402] In some embodiments, the method further includes a step of recovering a recombinant protein produced by rat hybridoma cells.

[0403] In some embodiments, the method further includes a step of purifying the recombinant protein by affinity chromatography and / or ion exchange chromatography. In some embodiments, the affinity chromatography includes purification by Protein A.

[0404] In some embodiments, the method results in an increase in the yield of the recombinant protein. For example, the recombinant protein is 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% greater than the recombinant protein produced by a culture process that does not employ the culture conditions cited above.

[0405]

Table 16-1

[0406]

Table 16-2

[0407]

Table 16-3

[0408]

Table 16-4

[0409]

Table 16-5

[0410]

Table 16-6

[0411] 7.11 Preparation of Ublituximab for Intravenous Injection in the Disclosed Treatment Methods In some embodiments, the injection of ublituximab is prepared in 250 mL of 0.9% sodium chloride for injection. For example, the injection of ublituximab can be prepared in an infusion bag containing 0.9% sodium chloride for injection.

[0412] In some embodiments, the first infusion of 150 mg of obinutuzumab is prepared by: (1) inspecting one vial of 150 mg / 6 mL obinutuzumab solution for particulate matter or discoloration and not using the solution if it contains individual foreign particles; (2) if there is no particulate matter in the vial, preparing a 250 mL infusion bag for the first infusion by using one vial of 150 mg / 6 mL obinutuzumab solution; (3) removing and discarding 6 mL of 0.9% sodium chloride for injection from the infusion bag; (4) removing 6 mL of obinutuzumab from the vial; (5) diluting 6 mL (150 mg) of obinutuzumab immediately for administration into the 250 mL infusion bag containing 0.9% sodium chloride for injection; and (6) mixing the infusion bag gently by inversion without shaking.

[0413] In some embodiments, the second or subsequent infusion of 450 mg of obinutuzumab is prepared by: (1) inspecting three vials of 150 mg / 6 mL obinutuzumab solution for particulate matter or discoloration and not using the solution if it contains individual foreign particles; (2) if there is no particulate matter in the vials, preparing a 250 mL infusion bag for the second or subsequent infusion by using three vials of 150 mg / 6 mL obinutuzumab solution; (3) removing and discarding 18 mL of 0.9% sodium chloride for injection from the infusion bag; (4) removing 18 mL of obinutuzumab from the vials; (5) diluting 18 mL (450 mg) of obinutuzumab immediately for administration into the 250 mL infusion bag containing 0.9% sodium chloride for injection; and (6) mixing the infusion bag gently by inversion without shaking.

[0414] In some embodiments, prior to initiation of intravenous infusion of obinutuzumab, the contents of the infusion bag are at room temperature.

[0415] 7.12 Administration of obinutuzumab in the methods of treatment disclosed herein In some embodiments, prior to initiation of administration of obinutuzumab, the subject is pre-screened for hepatitis B virus (HBV). If the subject's test is positive for hepatitis B surface antigen (HbsAg), obinutuzumab should not be administered. In some embodiments, the subject to be administered obinutuzumab is negative for hepatitis B virus surface antigen (HBsAg).

[0416] In some embodiments, the subject is pre-medicated with corticosteroids and antihistamines 30 to 60 minutes prior to administration of obinutuzumab or an anti-CD20 antibody that binds to the same epitope as obinutuzumab, and both of these may be administered orally or intravenously. In some embodiments, the corticosteroids and / or antihistamines are administered orally to the subject. In some cases, the pre-medication dose of corticosteroid is about 100 mg of methylprednisolone, 10 to 20 mg of dexamethasone, or an equivalent corticosteroid.

[0417] In some embodiments, obinutuzumab is administered in a multi-dose regimen. In certain cases, obinutuzumab is administered by intravenous infusion. For example, obinutuzumab can be administered by intravenous infusion in a multi-infusion dosing regimen.

[0418] In some embodiments, obinutuzumab is administered by intravenous infusion to patients with RMS in a multi-infusion dosing regimen over 48 weeks. In some embodiments, obinutuzumab is administered by intravenous infusion to patients with RMS in a multi-infusion dosing regimen over 96 weeks.

[0419] The multi-infusion dosing regimen may include the first, second, and subsequent intravenous infusions of obinutuzumab. In some embodiments, "subsequent infusions" of obinutuzumab may be any number of infusions after the first and second infusions.

[0420] In some embodiments, the multiple - injection dosing regimen includes an initial and second intravenous infusion of 150 mg of obinutuzumab (first infusion), followed two weeks later by an intravenous infusion of 450 mg of obinutuzumab (second infusion). In some embodiments, the multiple - injection dosing regimen further includes subsequent infusions by intravenous infusion of 450 mg of obinutuzumab every six months.

[0421] In some embodiments, the duration of the first infusion of obinutuzumab or an anti - CD20 antibody that binds to the same epitope as obinutuzumab is about 4 hours. For example, the infusion rate of the first infusion of obinutuzumab or an anti - CD20 antibody that binds to the same epitope as obinutuzumab can be 10 mL per hour for the first 30 minutes, 20 mL per hour for the next 30 minutes, 35 mL per hour for the next 1 hour, and 100 mL per hour for the remaining 2 hours. In some embodiments, the duration of the second and subsequent infusions of obinutuzumab is about 1 hour. For example, the infusion rate of the second and / or subsequent infusions of obinutuzumab can be 100 mL per hour for the first 30 minutes and 400 mL per hour for the remaining 30 minutes. If the infusion is interrupted or delayed, the duration of the infusion may be longer.

[0422] In some embodiments, the subject is monitored for at least 1 hour after the first two infusions of obinutuzumab are completed. If no infusion - related reactions (IRR) and / or hypersensitivity are observed, subsequent infusions do not require post - infusion monitoring.

[0423] In some embodiments, if the subject has symptoms of an infusion-related reaction that is life-threatening, the intravenous infusion of ublituximab is stopped and permanently discontinued. In some embodiments, if the subject has symptoms of a severe infusion-related reaction, the intravenous infusion of ublituximab is discontinued and restarted if the subject's symptoms of the infusion-related reaction resolve. In some embodiments, the intravenous infusion of ublituximab is restarted at half the infusion rate at the onset of the infusion-related reaction. If the subject tolerates the half infusion rate, the infusion rate of ublituximab can be increased to the original infusion rate. In some embodiments, if the subject has symptoms of a mild to moderate infusion-related reaction, the infusion rate of ublituximab is reduced by half and the half infusion rate is maintained for at least 30 minutes. If the subject tolerates the half infusion rate, the infusion rate of ublituximab can be increased to the original infusion rate.

[0424] In some embodiments, administration of ublituximab by intravenous infusion results in a geometric mean steady-state AUC of 3000 mcg / mL (CV = 28%) per day and a mean maximum concentration of 139 mcg / mL (CV = 15%).

[0425] In some embodiments, administration of ublituximab may be via a route of administration other than intravenous infusion (e.g., subcutaneous injection, intramuscular injection, oral, epidermal, spinal, or inhalation).

[0426] 7.13 Pharmaceutical Composition "Pharmaceutical composition" can refer to, for example, a composition that is acceptable for the administration of a medicament to humans. Such a composition may contain substances that are impurities at levels not exceeding levels acceptable for the administration of the medicament (such levels include the absence of such impurities), and in addition to any active agent, may contain pharmaceutically acceptable excipients, vehicles, carriers, stabilizers, and other inert components, for example, for formulating such a composition to facilitate administration.

[0427] The present disclosure provides a pharmaceutical composition for use in the treatment of a subject having relapsing multiple sclerosis by any of the methods disclosed herein, the composition comprising ocrelizumab or an antibody that binds to the same epitope as ocrelizumab.

[0428] In some embodiments, the pharmaceutical composition comprises ocrelizumab. In some embodiments, a pharmaceutical composition comprising ocrelizumab is formulated with a pharmaceutical carrier. Suitable pharmaceutical carriers are known to those of skill in the art (Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (1990)).

[0429] The pharmaceutical composition may include any number of excipients. Excipients that can be used include carriers, surfactants, thickening or emulsifying agents, solid binders, aids for dispersion or suspension, solubilizing agents, coloring agents, flavoring agents, coatings, disintegrants, lubricants, sweetening agents, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0430] The pharmaceutical compositions described herein may be suitable for administration by intravenous, intramuscular, subcutaneous, parenteral, intraspinal, or topical routes (e.g., by injection or infusion). Depending on the route of administration, the active compound can be coated with a material that protects the active compound from the action of acids and other natural conditions that may inactivate the active compound. As used herein, "parenteral administration" means a mode of administration usually by injection, other than enteric and topical administration, and can include without limitation intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, intradural, and intrasternal injections and infusions. Further or alternatively, the pharmaceutical compositions described herein can be administered via non-parenteral routes, such as topical, epidermal, or mucosal routes of administration, e.g., intranasally, orally, vaginally, rectally, sublingually, or topically.

[0431] In some embodiments, the pharmaceutical composition comprising ublituximab is for administration to a subject by intravenous infusion.

[0432] The following examples are provided for illustration and not for limitation.

Examples

[0433] 8. Examples [Example 1] 8.1 Example 1 - Glycosylation Profile The glycosylation profile of the anti-CD20 antibody protein sample provided herein was determined by measuring fluorescently labeled N-glycans (the fluorescent label is 2-aminobenzamide) enzymatically cleaved from the anti-CD20 antibody protein using PNGase F. The labeled glycans were separated using a pre-packed hydrophilic interaction column. The glycans passed through a fluorescence detector after separation. The identification of peaks from the chromatogram of the test sample was identified relative to the peaks of glycan standards confirmed by mass spectrometry based on the retention time. The relative percentage of each N-glycan was calculated based on the peak area of the N-glycan divided by the total peak area of all N-glycans. The glycosylation profile is shown in Figure 2.

[0434] [Example 2] 8.2 Example 2 - Intact Mass Method The glycosylation profile of the anti-CD20 antibody protein provided herein was evaluated by intact mass analysis (LC-MS) under non-reducing conditions. Samples of the anti-CD20 antibody protein provided herein were first exchanged into a buffer suitable for MS during a chromatography step using SEC and a mobile phase (containing TFA, acetonitrile, and water). The samples were then introduced into an ESI-QTOF for intact mass analysis. The mass spectra were deconvoluted and peaks were assigned based on mass. The relative abundance of each anti-CD20 antibody protein provided herein containing N-glycans was calculated by taking the abundance of the N-glycans and dividing by the total abundance of all identified peaks. The results are provided in the following tables and Figure 3.

[0435]

Table 17-1

[0436]

Table 17-2

[0437] [Example 3] 8.3 Example 3 - Cell Line ADCC Assay (a) Materials and Methods Antibody-dependent cellular cytotoxicity (ADCC) is mediated by the binding of the Fc portion of TG-1101 (TG Therapeutics, Inc.) to the FcγIIIA receptor on effector cells. The assay used for this analysis employs Eurofins-DiscoverX's "KILR CD16a effector cells", which are human CD8+ T lymphocytes from a single donor engineered to express CD16 (FcγRIII) on the surface of their plasma membrane. These cytotoxic T cells provide reduced background killing, increased precision, and reproducibility compared to PMBC preparations isolated from fresh blood. Raji cells are used as target cells, and ADCC activity is determined from the lysis of the target cells.

[0438] KILR cells are obtained from Eurofins and Raji cells are obtained from ATCC. To ensure the quality of the drugs, master and working cell bank systems were used. Raji cells were seeded at 1×10 5 cells / mL, and KILR effector cells were seeded at 5×10 5Cells were seeded at cells / mL and the final effector:target (E:T) ratio was set to 5:1. An 8-point dilution series of samples with a concentration range of 250.00 pg / ml to 0.04 pg / ml (250, 50, 16.7, 5.6, 1.9, 0.6, 0.2, 0.04 pg / ml) was used. The cell mixture and test samples were cultured at 36 ± 1 °C, 5 ± 1% CO2 for 18 - 22 hours. At the end of the incubation, CytoTox GLo™ preparation was added and the plates were incubated for 30 ± 10 minutes. The plates were read using a SpectraMax plate reader. Two independent preparations of the material were prepared and assayed across duplicate plates. Assay controls were prepared in triplicate and included a control of target cells only, a control of target cell death, a control of effector cells only, and a control of effector and target cells. Using SoftMax Pro, the data was analyzed by weighted non-linear regression using a 4-parameter logistic fit. The results are reported as the percentage of ADCC activity compared to the obinutuzumab reference standard. The EC50 of each test sample based on the 4-parameter logistic fit curve was generated for further information. This test method is the validated assay CTSOP482 used for the release and stability testing of the drug substance and drug product of TG-1101 (TG Therapeutics, Inc.).

[0439] (b) Results The tested samples included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. RS-117808 (117808), a commercial reference standard of TG-1101 (TG Therapeutics, Inc.), was used as a control. As shown in Figure 4, 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. Table 9 shows the ADCC activity expressed as a percentage relative to the TG-1101 (TG Therapeutics, Inc.) reference standard. Compared to TG-1101 (TG Therapeutics, Inc.), Gazyva had relatively similar ADCC activity, while the ADCC activities of Rituxan, Ocrevus, and Arzerra were significantly lower. The ED50 of each anti-CD20 is also shown in Table 9. Overall, TG-1101 (TG Therapeutics, Inc.) and Gazyva had lower EC50s than Arzerra, Rituxan, and Ocrevus, and the EC50 of TG-1101 (TG Therapeutics, Inc.) was approximately 1 / 25 of that of Ocrevus.

[0440]

Table 18

[0441] [Example 4] 8.4 Example 4 - Cell Line ADCC Assay Using Primary NK Cells (a) Materials and Methods This ADCC assay was performed using Raji cells expressing CD20 as target cells, primary NK cells as effector cells, and LDH as a readout of target cell lysis. Raji cells (ATCC, Cat#CCL-86TM) were seeded on plates at 1×105 cells / well. Primary NK cells were isolated from human donor PBMC using the Miltenyibiotec kit (Cat#130-092-657). An E / T ratio of 5:1 was used for NK92 / CD16a cells and primary NK cells in the assay. An 8-point dilution series of samples with a concentration range of 0.01 μg / ml to 0 μg / ml and a dilution factor of 10 was used in triplicate measurements. Target cells were incubated with the test sample diluent in an incubator at 37°C for 30 minutes. Effector cells were added to the target cell culture and subsequently incubated for 6 hours, after which the supernatant was collected. The release of LDH was calculated using the OD492nm data minus the background (OD650nm). 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).

[0442] (b) Results The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, and Arzerra. As shown in Figure 5, 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.

[0443]

Table 19

[0444] [Example 5] 8.5 Example 5 - Cell Line ADCP Assay (a) Materials and Methods Antibody-dependent cellular phagocytosis (ADCP) is another possible mechanism of action (MOA) of anti-CD20. ADCP activity was evaluated using an assay with Daudi cells expressing CD20 as target cells (labeled with ATCC, Cat#CCL-213, PKH26). Human monocytes were isolated from the PBMC 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 obtain macrophages. In this assay, ADCP was evaluated by flow cytometry using an E / T ratio of 5:1. An 8-point dilution series of samples with a concentration range of 100 μg / ml to 0 μg / ml and a dilution factor of 10 was incubated with PKH26-labeled target cells. Macrophages were then co-cultured with PKH26-labeled target cells for 22 hours. Phagocytosis of target cells was evaluated by flow cytometry. Controls in the assay included a target cell control of only PKH26-stained Daudi cells and an effector cell control of only PKH67-stained MDM.

[0445] Effector cell and target cell controls with non-specific IgG1 antibody, effector cell and target cell controls (background controls). ADCP was determined by FACS as the percentage of PKH26 / PKH67 double-positive cells / PKH26.

[0446] (b) Results The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, and Arzerra. As shown in Figure 6, all anti-CD20 samples exhibited dose-dependent ADCP activity. The calculated EC50 values are summarized in Table 11. The EC50 is in the range of ng / ml, and considering the nature of the test method, the ADCP activity of all samples is considered similar.

[0447]

Table 20

[0448] [Example 6] 8.6 Example 6 - Cell Line CDC Assay (a) Materials and Methods Complement-dependent cytotoxicity (CDC) is mediated by the 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 line assay using the human mantle cell lymphoma cell line Jeko-1 that expresses CD20 and rabbit serum as a complement source. Cell lysis mediated by CDC is measured by the Cell Titer-Glo™ reagent (Promega). A 9-point dilution series of samples with concentration ranges from 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) was used. Two independent preparations of each sample were prepared and assayed across duplicate plates. The assay negative control was prepared in triplicate and included controls for target cells and complement, as well as a control for target cells alone.

[0449] Jeko-1 cells obtained from ATCC and maintained by the master banking system were 3×10 5Seeded at cells / mL and incubated for 60 - 90 minutes. Diluted the sample, then added complement and incubated the plate at 37 °C for about 2 hours and at room temperature for 25 minutes. Controls of target cells and complement only, and target cells only provided the baseline levels of target cell viability over the course of the assay. Then added Cell Titer-Glo reagent and incubated for an additional 30 minutes at room temperature. Read the plate at the end of the assay using a SpectraMax M5 plate reader. Analyze the data by weighted non-linear regression using a 4-parameter logistic fit with SoftMax Pro. Evaluate the resulting data for parallelism and potency against a reference standard using PLA software. Report the results as % potency against the reference standard. Generated the EC50 for each test sample based on the 4-parameter logistic fit curve for further information. This test method is the validated assay CTSOP463 used for the release and stability testing of the drug substance and drug product of TG-1101 (TG Therapeutics, Inc.).

[0450] (b) Results The tested samples included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. RS-117808 (117808), a commercial reference standard of TG-1101 (TG Therapeutics, Inc.), was used as a control. As shown in Figure 7, all samples except Gazyva, which is known to have reduced CDC activity, exhibited dose-dependent CDC activity. Rituxan and Arzerra had comparable CDC activity, and Ubli and Ocrevus had comparable CDC activity. Table 12 shows the CDC activity expressed as a percentage relative to the TG-1101 (TG Therapeutics, Inc.) reference standard compared to TG-1101 (TG Therapeutics, Inc.). While Gazyva had relatively comparable ADCC activity to TG-1101 (TG Therapeutics, Inc.), the ADCC activity of Rituxan, Ocrevus, and Arzerra was significantly lower. The ED50 of each anti-CD20 is also shown in Table 12. This shows a comparison similar to the level of CDC activity.

[0451]

Table 21

[0452] [Example 7] 8.7 Example 7 - Cell Line CD20 Binding Assay (a) Materials and Methods The CD20 binding used for this analysis is a cell line binding assay using the human mantle cell lymphoma cell line Jeko-1 that expresses CD20 and the MesoScale Discovery (MSD) assay format. Jeko-1 target cells are seeded onto MSD plates, and test samples are incubated to bind to the Jeko-1 cells. An anti-human Fc detection antibody conjugated to streptavidin-SULFOTAG™ is used to emit an electrochemiluminescence signal. An 8-point dilution series of test samples with concentration ranges from 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) is used. Two independent preparations of test materials are prepared for the evaluation of two plates each. Assay controls include a cell-free control (reference standard / test material dilution plus detection reagent, excluding cells) and a cell-only control (cells plus detection reagent, excluding reference standard / test material).

[0453] Jeko-1 cells obtained from ATCC and managed by a master banking system were resuspended in PBS at 3 × 10 5Seed the MSD high-binding plates at a final volume of 100 μL / well at cells / mL and incubate at 35 - 37 °C for 2 hours ± 10 minutes. Remove unbound cells by PBS washing, block the plates and then wash. Add 50 μL of sample diluent and incubate the plates for 1 hour ± 10 minutes at room temperature with shaking. After incubation and three washes, add 50 μL of anti-human Fc detection antibody conjugated to STREP-SULFOTAG and incubate for 1 hour ± 10 minutes at room temperature with shaking. Wash the plates again and add 150 μL of MesoScale read buffer containing tripropylamine (TPA) as a co-reactant for luminescence for electrochemical luminescence reading. Immediately read the plates on an MSD reader using Workbench 4.0. Evaluate the resulting data using PLA software and analyze using a constrained four-parameter logistic model. Report the results of binding activity as a percentage of potency relative to a reference standard. Generate the EC50 of each test sample based on the four-parameter logistic fit curve for further information. This test method is the validated assay CTSOP466 used for the release and stability testing of the drug substance and drug product of TG-1101 (TG Therapeutics, Inc.).

[0454] (b) Results The tested samples included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. RS-117808 (117808), the commercial reference standard of TG-1101 (TG Therapeutics, Inc.), was used as a control. As shown in Figure 8, all samples exhibited dose-dependent CD20 binding. Except for Gazyva, which is a type II anti-CD20 known to have a target occupancy of approximately 50%, obinutuzumab, Rituxan, Ocrevus, and Arzerra had similar maximum binding. Table 13 shows the CD20 binding activity and the EC50 of the binding, expressed as a percentage relative to the TG-1101 (TG Therapeutics, Inc.) reference standard. The CD20 binding affinities of the four anti-CD20s were similar.

[0455]

Table 22

[0456] The listed EC50 values are negative and result from the PLA software logarithmically transforming the concentration at the bottom 2. The actual ED50 (μg / ml) is 2 n where n = the number listed.

[0457] [Example 8] 8.8 Example 8 - CD20 Binding to the Cell Surface by FACS (a) Materials and Methods The binding of TG-1101 (TG Therapeutics, Inc.) to cell surface CD20 on Raji cells and Daudi cells was characterized by FACS analysis at LakePharma. A 6-point dilution series of samples with a concentration range of 40 μg / ml to 0 μg / ml and a dilution factor of 5 was used in duplicate measurements. The cells were incubated with the sample diluent, and binding was detected using a PE-conjugated anti-human secondary antibody.

[0458] (b) Results The tested samples included TG-1101 (TG Therapeutics, Inc.), Gazyva, Arzerra, and Rituxan. As shown in Figure 9, all anti-CD20 samples exhibited dose-dependent CD20 binding to both Daudi cells and Raji cells. The binding characteristics evaluated by FACS assay were similar to those evaluated by MSD assay. Except for Gazyva, which is a type II anti-CD20 known to have a target occupancy of nearly 50%, obinutuzumab, Rituxan, and Arzerra had similar maximum binding. The calculated EC50 values are summarized in Table 14. The CD20 binding affinities of the four anti-CD20 antibodies were similar.

[0459] [Table 23]

[0460] [Example 9] 8.9 Example 9 - FcγRIIIA Binding Assay (a) Materials and Methods The assay used in this analysis is a surface plasmon resonance (SPR)-based method that measures binding to the FcγRIIIa 158V receptor and the FcγRIIIa 158F receptor. This method follows a direct binding assay methodology in which the FcγRIIIa receptor is directly immobilized in a flow cell on the sensor chip surface and the sample is injected onto the chip and binding is evaluated. The FcγRIIIa 158V receptor (3 μg / ml) or the FcγRIIIa 158F receptor (6 μg / ml) is immobilized on the chip surface using covalent amine coupling chemistry. An 8-point dilution series of the test sample is prepared with a concentration range of 1000 nM to 15.6 nM and a dilution factor of 2. Independent duplicate sample dilutions are injected onto the chip, followed by surface regeneration between each cycle. Binding is measured in response units (RU). The kinetics of the binding reaction are determined by measuring the change in SPR due to the increase in mass close to the surface of the biosensor chip. The change in the mass of the complex as a function of time is visualized as a sensorgram.

[0461] The equilibrium dissociation constant (K D ) of each sample is determined for each receptor. The rate of change of the SPR signal is analyzed for the FcγRIIIa 158V variant using a 1:1 Langmuir model to obtain the apparent rate constants and the equilibrium dissociation constant for the association and dissociation phases of the reaction. K D is determined using the steady-state affinity for the FcγRIIIa 158F variant. The binding signal is exported to PLA to determine the relative binding response. The relative affinity and relative binding for the samples are also reported relative to the TG-1101 (TG Therapeutics, Inc.) reference standard. This test method is the validated assay CTSOP477 used for the public testing of the formulation ingredients of TG-1101 (TG Therapeutics, Inc.).

[0462] (b) Results The samples tested included 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 greatest binding affinity to both the FcγRIIIa 158V receptor and the FcγRIIIa 158F receptor. Gazyva is ranked second in binding affinity. For the high-affinity receptor FcγRIIIa158V, TG-1101 (TG Therapeutics, Inc.) has an affinity approximately 15 times greater than Ocrevus, and for the low-affinity receptor FcγRIIIa158F, TG-1101 (TG Therapeutics, Inc.) has an affinity approximately 10 times higher than Ocrevus. Table 16 shows the relative affinity and relative binding values using the TG-1101 (TG Therapeutics, Inc.) reference standard as a reference. The results indicate that TG-1101 (TG Therapeutics, Inc.) has greater relative binding and relative affinity than all other anti-CD20s.

[0463]

Table 24

[0464]

Table 25

[0465] [Example 10] 8.10 Example 10 - Fc Receptor Binding by Octet This analysis was performed by LakePharma. The binding characterization was performed at 25 °C using an Octet HTX instrument. The human Fc receptor panel was loaded onto an Anti-Penta His (H1S1K) biosensor. The loaded sensor was dipped into serial dilutions of the test sample (starting at 300 nM, 1:3 dilution, 7 points). The kinetic constants were calculated using a monovalent (1:1) binding model.

[0466] (b) Results The samples tested included 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 the FcγRIIIa 158V receptor and the FcγRIIIa 158F receptor, corroborating the SPR data. The binding affinity to FCRN, which may affect PK, is similar among all anti-CD20s. Compared to Ocrevus, TG-1101 (TG Therapeutics, Inc.) also has a high affinity for FcγRIIA and FcγRIIIB.

[0467]

Table 26

[0468] [Example 11] 8.11 Example 11 - C1q Binding Assay 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 on the plate together with the sample. The bound HRP generates a colorimetric signal in the presence of the substrate TMB. A 7-point dilution series of the test material was prepared with a concentration range of 15.00 μg / ml to 0.12 μg / ml and 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 results in a colorimetric reaction proportional to the level of bound C1q. The reaction is stopped by the addition of 1 M sulfuric acid, and the coloration is measured at 450 nm using a Molecular Devices SpectraMax microplate reader. The data is analyzed by weighted non-linear regression using SoftMax Pro with a 4-parameter logistic fit. The results of the binding activity are reported as a percentage of the potency relative to the TG-1101 (TG Therapeutics, Inc.) reference standard. The EC50 of each test sample based on the 4-parameter logistic fit curve was generated for further information. This test method is the validated assay CTSOP455 used for the release and stability testing of the drug substance and drug product of TG-1101 (TG Therapeutics, Inc.).

[0469] (b) Results The tested samples 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 shown in Figure 10, Gazyva had the lowest C1q binding (as expected), and the other four anti-CD20s exhibited dose-dependent C1q binding. TG-1101 (TG Therapeutics, Inc.) had a slightly larger maximum binding. Table 18 shows the C1q binding activity expressed as a percentage relative to the TG-1101 (TG Therapeutics, Inc.) reference standard. The ED50 of each anti-CD20 is also shown in Table 18. The EC50 of TG-1101 (TG Therapeutics, Inc.) was slightly smaller than that of all the anti-CD20s, suggesting that TG-1101 (TG Therapeutics, Inc.) has a high affinity for C1q.

[0470]

Table 27

[0471] [Example 12] 8.12 Example 12 - Human Whole Blood B Cell Depletion Assay (a) Materials and Methods TG-1101 (TG Therapeutics, Inc.) was characterized in an autologous normal human whole blood B cell depletion assay and compared to Gazyva, Rituxan, Ocrevus, and Arzerra. Whole blood from three human donors was used and donors were selected to have the 158V / 158V genotype for the FCGR3A_SNP target (rs396991). Sample dilutions in the concentration range of 0.000001 - 100 μg / ml were added to the whole blood and incubated at 37°C for 24 hours in a humidified cell incubator. Aliquots of blood were stained for markers including CD45 (lymphocyte population), CD3 (T cells), CD19 (B cells), and CD20 (B cells). B cell depletion was evaluated by presenting cells in the CD45 positive lymphocyte gate and counting CD3 positive T cells, CD19 positive B cells, and CD20 positive B cells. The percent B cell depletion (100 - ([100 / B / T cell ratio in sample without antibody] × [B / T cell ratio in sample with antibody])) was calculated and plotted against sample concentration.

[0472] (b) Results The tested samples included TG-1101 (TG Therapeutics, Inc.) commercial reference standard RS-117808 (117808), the drug substance batch (PPQ1) manufactured by the commercial process (C2) at Samsung Biologics, Gazyva, Rituxan, Ocrevus, and Arzerra. As shown in FIGS. 11A to 11C, all samples exhibited dose-dependent B-cell depletion activity, but there were minor differences among donors. Overall, TG-1101 (TG Therapeutics, Inc.) and Gazyva had greater B-cell depletion activity than Arzerra, Rituxan, and Ocrevus. Three anti-CD20 antibodies and one anti-CD19 antibody were used in this experiment. The ED50 of B-cell depletion calculated using each of the antibodies for B-cell labeling is summarized in Table 19. Overall, TG-1101 (TG Therapeutics, Inc.) and Gazyva also had smaller EC50s than Arzerra, Rituxan, and Ocrevus. On average, the EC50 of TG-1101 (TG Therapeutics, Inc.) was less than one-tenth of that of Ocrevus.

[0473]

Table 28

[0474] [Example 13] 8.13 Example 13 - Calculation of Pharmacokinetic (PK) Values The relevant steady-state PK parameters were calculated according to methods known in the art. Steady state is reached when the amount of drug excreted 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 to reduce the plasma concentration of the drug that has reached steady state by 50%. Here, t = time, Vd = volume of distribution, and Cl = clearance. The half-life was calculated using the following formula.

[0475]

Equation

[0476] AUC represents the total dose of drug exposure over time. AUC is used as a measure in determining the prescription of equivalent doses and the resulting tissue or plasma exposure. AUC is equivalent to the average concentration over a time interval. Here, t = time and Cpt is the drug concentration last measured with respect to time. AUC was calculated by the following formula.

[0477] [Number]

[0478] Cmax was determined by measuring the highest point during the actual measurement period of drug concentration from the end of the absorption phase to the start of the excretion phase. Cmin was determined by measuring the highest point during the actual measurement period of drug concentration from the end of the absorption phase to the start of the excretion phase.

[0479] [Example 14] 8.14 Example 14 - Method for Determining the Population Pharmacokinetic (PPK) Values of TG-1101 for the Treatment of Autoimmune Disorders Serum concentration-time, dose, demographics, and covariate data of TG-1101 from one Phase 2 trial (TG1101-RMS201) and two Phase 3 trials (TG1101-RMS301 and TG1101-RMS302) in subjects with RMS were pooled for Pop PK analysis of TG-1101. The dataset was combined with a previous dataset of TG-1101 in subjects with hematological malignancies.

[0480] All subjects who received at least one dose of TG-1101 in the trials TG1101-RMS201, TG1101-RMS301, and TG1101-RMS302 were included in the dataset for PK analysis. Subjects without at least one quantifiable post-dose TG-1101 concentration were included in the dataset and flagged. Exposure for these subjects was determined based on typical population PK parameters.

[0481] In the Phase 2a clinical trial TG1101 - RMS201, TG - 1101 was administered as a single agent to examine the level of B - cell depletion by TG - 1101 compared to placebo and to determine the optimal dose and infusion time of TG - 1101 in subjects with RMS. Based on the results of this trial, a dose of 150 mg 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 1 hour), resulted in a median B - cell depletion of over 99%, which was achieved by Week 4 and persisted until Week 24. The dosing regimen was well - tolerated by the subjects, and infusion - related reactions (Grades 1 and 2) were generally reported adverse events. Two Phase 3 trials in subjects with RMS were completed. Two Phase 3 trials, namely TG1101 - RMS301 (also known as ULTIMATE I) and TG1101 - RMS302 (ULTIMATE II), were randomized, double - blind, double - dummy, active - controlled trials of TG - 1101 compared to oral teriflunomide to evaluate ARR, safety, and tolerability in subjects with RMS.

[0482] 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, and records with no information or outliers (more than 10 times the standard deviation from the mean TG - 1101 concentration during the nominal dosing period) accounted for 0.20%, which were excluded. Post - dose samples that were below the limit of quantification (BLQ) accounted for 3.47% of the data. Exclusions resulted in no quantifiable post - dose PK samples for 36 subjects. Thus, the PK analysis dataset included 7485 PK samples from 895 subjects, of which 5624 PK samples were from 591 subjects with RMS.

[0483] The final PopPK parameter estimates are presented in Table 20. The PK parameter estimates for a typical subject (defined as a 73-kg ADA-negative male subject from North America or Western Europe) were as follows. CL was estimated to be 11.6 mL / h, with an IIV of 38.1%. Ve was estimated to be 3.18 L (IIV = 15.0%). Vp was estimated to be 3.60 L (IIV = 21.3%). Q was estimated to be 11.6 mL / h.

[0484] Body weight and ADA were found to be statistically significant predictors of TG-1101 CL. TG-1101 CL increased moderately by 14% in ADA-positive subjects compared to those without quantifiable ADA. For a wide range of body weights (45.1–154 kg) in the RMS subpopulation, CL ranged from 22% lower to 48% higher than that of a typical 73-kg subject. Further, at a later time point (417 days after treatment initiation), CL was reduced by a median of 12.5%.

[0485] Body weight, gender, and region were found to be statistically significant predictors of Vc. Subjects from Eastern Europe were found to have a slightly larger (10%) Vc than Western Europeans and North Americans, and females had a slightly smaller (7%) Vc than males. For a wide range of body weights (45.1–154 kg) in the RMS subpopulation, Vc ranged from 19% lower to 38% higher than that of a typical 73-kg subject.

[0486] After including body weight in the model, there was no effect of age, hemoglobin concentration, platelet count, white blood cell count, renal impairment, or hepatic impairment on the PK of TG-1101.

[0487]

Table 29

[0488] In the evaluation of the final TG-1101 PopPK model, the standard errors were obtained and presented in Table 20 along with the parameter estimations. The model parameters were accurately estimated with RSE less than 20% for the structural and covariate model parameters and less than 30% for the variable effect estimations. The shrinkage rates were acceptable for CL and Vc (5.32% and 32.6%, respectively), and there was a large shrinkage rate (40%) for Vp.

[0489] Important goodness-of-fit (GOF) diagnostics for the final PopPK model of TG-1101 suggested a satisfactory fit with minimal bias in the residuals between the observed and predicted concentration values over time and showed a good agreement between the predicted and measured concentrations. For the base model, the relationship between ETA and covariates was resolved, and in the RMS subpopulation, there was no clear further trend between ETA (in CL and Vc) and covariates, suggesting that the model appropriately captured the significant covariate relationships. Overall, the predicted-corrected visual predictive check (pcVPC) plots suggested that the model well predicted the central tendency of the measured TG-1101 concentrations and appropriately captured the range of the data.

[0490] The relative importance of the covariates effects included in the final PoPK model was evaluated by the forest plots of the relative changes (Cmax,ss, Cmin,ss, and AUCss) in exposure when changing the covariates one by one (i.e., univariate analysis). The effects of these covariates, including the change rates of body weight, gender, region, ADA, and late CL on the exposure to TG-1101, fell within the range of 0.8 to 1.25 compared to the reference exposure (defined as the exposure for male subjects from North America / Western Europe with a body weight of 73 kg, ADA negative, and treated for less than 416 days). Therefore, none of the covariates were considered clinically relevant. Furthermore, the covariates had no significant effect on the magnitude of the IIV of CL or Vc. The combined effect of body weight and ADA only reduced the IIV by 2.8% from 39.2% in the basic model to 38.1% in the final model (Table 20). Similarly, body weight, gender, and region reduced the IIV of Vc by 18.5% from 18.4% in the basic model to 15.0% in the final model.

[0491] Figure 12 shows the goodness-of-fit (GOF) diagnosis of the final model of TG-1101. Figure 13 shows the pcVPC for the final PK model of TG-1101 from the clinical trial. Figure 14 shows the forest plot of the covariate effects on the TG-1101 drug exposure.

[0492] The final PopPK model was re-estimated using the dataset including the excluded outliers. All the structural parameters were accurately estimated without the associated changes in these estimations, while the IIV terms were expanded because the outliers were included. Therefore, it can be expected that by excluding outliers during model development, the influence of these outliers can be eliminated and the false findings in the covariate analysis can be minimized. The final PopPK model was also re-estimated including only the RMS subpopulation. The model fit to the RMS data resulted in similar structural parameters and variable effects, with the exception of a slightly lower IIV in CL. The accuracy of the parameter estimation was also similar.

[0493] Conclusion: PPK Analysis of TG-1101

[0494] Using the final model of TG-1101, individual post hoc estimates of PK parameters were obtained. For each subject enrolled in the clinical trials TG1101-RMS201, TG1101-RMS301, and TG1101-RMS302, PK parameters (AUCss, Cavg,ss, Cmax,ss, and Cmin,ss) at 48 weeks were estimated based on the post hoc PK parameters.

[0495] The geometric mean 1 1 / 2 (90% confidence interval [CI]) was calculated to be 21.8 days (21.4 days, 22.1 days). The median time to reach steady state was determined to be 15.5 weeks. Thus, there was no accumulation for subjects who received 150 mg of TG-110 on day 1, followed by the protocol-specified regimen of 450 mg on days 15, 24 weeks, and 48 weeks. The median Cmax ratio to Cmax on day 1 at 24 weeks was 3.04 (range 3.00 - 3.42), consistent with a three-fold increase in dose and indicating no accumulation. Similarly, the ratio of Cmax at 48 weeks to that at 24 weeks was 1, indicating no accumulation.

[0496] The geometric mean AUCss, Cavgss, Cmaxss, and Cminss predicted from the model were 3000 μg·d / mL (±28%), 8940 ng / mL (±28%), 139000 (±15.1%), and 139 (±170%), respectively.

[0497] [Example 15] 8.15 Example 15 - Commercial-scale manufacturing process for producing TG-1101 in YB2 / 0 rat hybridoma cells In this example, a process for manufacturing TG-1101 expressed in YB2 / 0 rat hybridoma cells at 15,000 L is described. An overview of the manufacturing process of TG-1101 is illustrated in the flow diagram of Table 21.

[0498]

Table 30

[0499] In summary, the production of each batch of TG-1101 began with the thawing of Working Cell Bank (WCB) vials as further described below. Cultivation was expanded by seeding bioreactors that ...

Claims

1. A composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are expressed from 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 the anti-population of CD20 antibody proteins has an N-glycan profile comprising about 20-40% fucosylated glycan and optionally about 10-20% galactosylated glycan.

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

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

4. The N-glycan profile comprises 12%-30% biantennary N-glycan, optionally about 18% biantennary N-glycan, The composition according to any one of claims 1 to 3.

5. The biantennary N-glycan comprises one or more of G0B, G0FB, G1FB, G2FBS1, and G2FBS2, The composition according to claim 4.

6. The population of anti-CD20 antibody proteins has an N-glycan profile comprising less than 5% sialylated glycan, Optionally the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1%, or 0.5% sialylated glycan, Optionally the N-glycan profile does not contain a detectable amount of sialylated glycan, The composition according to any one of claims 1 to 5.

7. The population of anti-CD20 antibody proteins has an N-glycan profile comprising 0.1%-1.5% Man5 N-glycan, Optionally the N-glycan profile comprises 0.4%-0.7% Man5 N-glycan, Optionally the N-glycan profile comprises about 0.6% Man5 N-glycan, The composition according to any one of claims 1 to 6, wherein optionally 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 population of anti-CD20 antibody proteins comprises 0.20 to 0.40 moles of isoaspartate per mole of protein, optionally 0.25 to 0.35 moles of isoaspartate per mole of protein.

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

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

9.

11. The population of anti-CD20 antibody proteins is as follows: (a) G0-GN 0.3% to 2%; (b) G0F-GN 0.1% to 2%; (c) G0 30% to 60%; (d) G1-GN 0.1% to 1%; (e) G0B 5% to 20%; (f) G0F 5% to 30%; (g) Man5 0.1% to 1.5%; (h) G0FB 1% to 15%; (i) G1 1% to 13%; (j) G1' 0.5% to 10%; (k) G1B 0.5% to 6%; (l) G1F 0.5% to 12%; (m) G1F' 0.1% to 3%; (n) G1FB 0.1% to 3%; (o) G2 0.1% to 2%; and (p) G2F 0.1% to 2% and further comprises at least two N-glycans within the range of, Optionally, the population of anti-CD20 antibody proteins is as follows: (a) G0-GN 0.8% to 1.1%; (b) G0F-GN 0.5% to 1.1%; (c) G0 42.5% to 48.8%; (d) G1-GN 0.3% to 0.6%; (e) G0B 9.5% to 14.1%; (f) G0F 12.8% to 19.7%; (g) Man5 0.4% to 0.7%; (h) G0FB 5.1% to 7.0%; (i) G1 5.7% to 6.4%; (j) G1' 2.7% to 3.3%; (k) G1B 1.4% to 2.0%; (l) G1F 2.6% to 4.2%; (m) G1F' 1.1% to 1.6%; (n) G1FB 1.1% to 1.8%; (o) G2 0.5% to 0.7%; and (p) G2F 0.3% to 0.5% (q) further comprising at least two N-glycans in the range of; (r) optionally, the population of anti-CD20 antibody proteins has the following relative abundances: (a) G0-GN 0.9%; (b) G0F-GN 0.8%; (c) G0 46.1%; (d) G1-GN 0.5%; (e) G0B 10.9%; (f) G0F 17.0%; (g) Man5 0.6%; (h) G0FB 6.0%; (i) G1 6.1%; (j) G1' 2.9%; (k) G1B 1.6%; (l) G1F 3.2%; (m) G1F' 1.3%; (n) G1FB 1.3%; (o) G2 0.5%; and (p) G2F 0.3% (q) further comprising at least two N-glycans in the range of, the composition according to any one of claims 1 to 10.

12. (r) the population of anti-CD20 antibody proteins has the following relative abundances: (a) G0-GN 0.3% to 2%; (b) G0F-GN 0.1% to 2%; (c) G0 30% to 60%; (d) G1-GN 0.1% to 1%; (e) G0B 5% to 20%; (f) G0F 5% to 30%; (g) Man5 0.1% to 1.5%; (h) G0FB 1% to 15%; (i) G1 1% to 13%; (j) G1' 0.5% to 10%; (k) G1B 0.5% to 6%; (l) G1F 0.5% to 12%; (m) G1F' 0.1% to 3%; (n) G1FB 0.1% to 3%; (o) G2 0.1% to 2%; and (p) G2F 0.1% to 2% (q) further comprising at least three, four, or five N-glycans within the range of, the composition according to claim 11.

13. (r) the N-glycan profile of the population of anti-CD20 antibody proteins is (a) incubating the population of anti-CD20 antibody proteins with an enzyme, wherein the enzyme catalyzes the release of the N-glycans from the anti-CD20 antibody, (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 (q) determined using a method comprising; (r) optionally, after step (a) and before step (b), the following steps: (c) purifying the N-glycans, and Step (d) of labeling the N-glycan with a fluorescent compound further comprising Optionally, the enzyme is PNGase F and / or the fluorescent compound is 2-aminobenzamide (2-AB), the composition according to any one of claims 1 to 12.

14. Less than 10% of the anti-CD20 antibody protein in the population is not glycosylated, Optionally, less than 5% of the anti-CD20 antibody protein in the population is not glycosylated, Optionally, less than 1% of the anti-CD20 antibody protein in the population is not glycosylated, the composition according to any one of claims 1 to 13.

15. The population of anti-CD20 antibody proteins is as follows: (a) α-helix 8.0% - 10.0%; (b) Antiparallel β-sheet 32.0% - 36.0%; (c) Parallel β-sheet 5.0% - 6.0%; (d) β-turn 16.0% - 18.0%; and (e) Random coil 35.0% - 36.0% comprising two or more secondary structures determined by circular dichroism at 205 nm to 260 nm, Optionally, the population of CD20 antibody proteins is as follows: (a) α-helix 8.0% - 10.0%; (b) Antiparallel β-sheet 32.0% - 36.0%; (c) Parallel β-sheet 5.0% - 6.0%; (d) β-turn 16.0% - 18.0%; and (e) Random coil 35.0% - 36.0% comprising a secondary structure determined by circular dichroism at 205 nm to 260 nm, Optionally, the population of anti-CD20 antibody proteins is as follows: (a) α-helix approximately 9.0%; (b) Antiparallel β-sheet approximately 33.0%; (c) Parallel β-sheet approximately 5.6%; (d) β-turn approximately 17.5%; and (e) Random coil approximately 35.2% comprising two or more secondary structures determined by circular dichroism at 205 nm to 260 nm, the composition according to any one of claims 1 to 14.

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

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

18. The composition according to any one of claims 1 to 17, wherein the anti-CD20 antibody protein in the population induces greater cytotoxicity compared to obinutuzumab, ofatumumab, rituximab, belimumab, ibritumomab tiuxetan, and / or ocrelizumab in a cell-based antibody-dependent cell cytotoxicity (ADCC) assay.

19. The population has a relative potency of 90 to 163% compared to a commercially available reference standard in a cell-based ADCC assay, a relative potency of 78% to 116% or 73% to 128% compared to that of a commercially available reference standard in a cell-based complement-dependent cell cytotoxicity (CDC) assay, a relative potency of 92 to 118% or 82 to 138% compared to that of a commercially available reference standard in a cell-based CD20 binding activity bioassay, In the FcγRIIIa-158V binding assay, measured by surface plasmon resonance, a K value of 30 to 70 nM Ｄ value In the FcγRIIIa 158F binding assay, measured by surface plasmon resonance, a K value of 500-1000 nM Ｄ value a binding affinity for FcγRIIIa 158V or FcγRIIIa 158F that is significantly higher than that of rituximab, a relative potency of 88 to 113% or 86 to 116% compared to a commercially available reference standard as measured by ELISA in a C1q binding assay, and / or a relative potency of 106 to 126% compared to a commercially available reference standard in a CD16 activity assay The composition according to any one of claims 1 to 18.

20. The composition according to any one of claims 1 to 19, wherein the population has 99.2 to 99.9% monomer as detected by size exclusion chromatography (SEC), 0.1 to 0.8% dimer as detected by SEC, aggregates at a non-detectable level as detected by SEC, and / or fragments at a non-detectable level as detected by SEC.

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

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

2.

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

24. A pharmaceutical formulation comprising the composition according to 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, and optionally the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg / mL.

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

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

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

28. (i) The composition according to any one of claims 1 to 23, comprising the population of anti-CD20 antibody protein in a single-dose form, 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 anhydrous, (iv) about 0.7 mg / mL of polysorbate 80, and (v) about 0.4 mg / mL of hydrochloric acid A pharmaceutical formulation comprising.

29. A preparation of a single batch of a population of anti-CD20 antibody proteins cited in the method according to any one of claims 1 to 23 or a pharmaceutical preparation 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 said anti-CD20 antibody protein, a preparation of a single batch.

30. A population of anti-CD20 antibody proteins cited in the method according to any one of claims 1 to 23 or a pharmaceutical preparation according to any one of claims 24 to 28 produced in a 15,000 L or 20,000 L bioreactor.

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

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

33. The method according to claim 31 or 32, wherein the composition or the pharmaceutical preparation is administered as: i) a first infusion of about 150 mg of the anti-CD20 antibody protein, ii) a second infusion of about 450 mg of the anti-CD20 antibody protein 2 weeks later, and iii) subsequent infusions of about 450 mg of the anti-CD20 antibody protein every 24 weeks or every 6 months.

34. Administration of the composition or the pharmaceutical preparation 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) Cmin of 40 ng / mL to 375 ng / mL; and (d) Cavg of 6,437 ng / mL to 11,443 ng / mL and optionally administration of the composition or the pharmaceutical preparation to the subject results in one or more of the following pharmacokinetic parameters: (a) AUC of about 3,000 μg / mL; (b) Cmax of about 139,000 ng / mL; (c) Cmin of about 139 ng / mL; and (d) Cavg of about 8,940 ng / mL ​ The method according to any one of claims 31 to 33, which results in one or more of

35. The method according to any one of claims 31 to 34, comprising a treatment period of at least 96 weeks.

36. The subject has received a pre-medication of corticosteroid 30 to 60 minutes before administration of the composition or the pharmaceutical formulation, optionally the corticosteroid is methylprednisolone or dexamethasone, optionally the methylprednisolone is administered at a dose of about 100 mg, and / or the dexamethasone is administered at a dose of about 10 to 20 mg. The method according to any one of claims 31 to 35.

37. The subject has received a pre-medication of antihistamine 30 to 60 minutes before administration of the composition or the pharmaceutical formulation, optionally the antihistamine is diphenhydramine HCl, optionally the diphenhydramine HCl is administered at a dose of about 25 to 50 mg. The method according to any one of claims 31 to 36.

38. The subject has received a pre-medication of antipyretic 30 to 60 minutes before administration of the composition or the pharmaceutical formulation, optionally the antipyretic is acetaminophen or its antipyretic biological equivalent. The method according to any one of claims 31 to 37.

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

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

41. A method for treating multiple sclerosis (MS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition according to any one of claims 1 to 23 or the 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 the administration.

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

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

44. The method according to claim 43, wherein the number of lymphocytes normalizes by the 8th day of the administration.

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

46. A method of reducing the annualized relapse rate (ARR) in a subject having relapsing multiple sclerosis (MS), comprising the step of administering to the subject an effective amount of the composition according to any one of claims 1 to 23 or the pharmaceutical preparation according to any one of claims 24 to 28, comprising the step of performing intravenous infusion of the composition or the pharmaceutical preparation in a multiple-injection dosing regimen, wherein the dosing regimen a) a first injection containing 150 mg of the anti-CD20 antibody protein on day 1; b) a second injection containing 450 mg of the anti-CD20 antibody protein approximately 2 weeks after the first injection; c) a first subsequent injection containing 450 mg of the anti-CD20 antibody protein at approximately 24 weeks or approximately 6 months from the first injection, and d) one or more subsequent injections containing 450 mg of the anti-CD20 antibody protein at approximately 24 weeks or approximately 6 months from the previous injection is included.

47. The method according to claim 46, wherein the effective amount of the composition or the pharmaceutical preparation is sufficient to result in an ARR of 0.091 or an ARR of 0.

076.

48. The method according to claim 46 or 47, wherein the duration of the second injection, the first subsequent injection, and the one or more subsequent injections of the anti-CD20 antibody protein is approximately 1 hour.

49. A method of treating relapsing multiple sclerosis (MS) in a subject in need thereof, comprising the step of administering to the subject an effective amount of the composition according to any one of claims 1 to 23 or the pharmaceutical preparation according to any one of claims 24 to 28, comprising the step of performing intravenous infusion of the composition or the pharmaceutical preparation in a multiple-injection dosing regimen, wherein the dosing regimen e) a first infusion containing 150 mg of the anti-CD20 antibody protein on day 1; f) a second infusion containing 450 mg of the anti-CD20 antibody protein approximately 2 weeks after the first infusion; g) a first subsequent infusion containing 450 mg of the anti-CD20 antibody protein at approximately 24 weeks or approximately 6 months from the first infusion, and h) one or more subsequent infusions containing 450 mg of the anti-CD20 antibody protein at approximately 24 weeks or approximately 6 months from the previous infusion comprising, wherein the duration of the second infusion, the first subsequent infusion, and the one or more subsequent infusions of the anti-CD20 antibody protein is approximately 1 hour, a method. **Claim 50** The method according to any one of claims 41 to 49, further comprising the step of pre-dosing the subject with a corticosteroid and an antihistamine 30 to 60 minutes before administration of the composition or the pharmaceutical formulation. **Claim 51** The method according to claim 50, wherein the corticosteroid is methylprednisolone or dexamethasone, optionally the methylprednisolone is administered at a dose of approximately 100 mg, and / or the dexamethasone is administered at a dose of approximately 10 - 20 mg. **Claim 52** The method according to any one of claims 46 to 51, wherein the intravenous infusion of the composition or the pharmaceutical formulation is prepared in 250 mL of 0.9% sodium chloride for injection. **Claim 53** The method according to any one of claims 46 to 52, wherein the first subsequent infusion is at approximately 24 weeks from the first infusion. **Claim 54** The method according to any one of claims 46 to 53, wherein the one or more subsequent infusions are at approximately 24 weeks from the previous infusion. **Claim 55** The method according to any one of claims 46 to 54, wherein the first subsequent infusion is at approximately 6 months from the first infusion. **Claim 56** The method according to any one of claims 46 to 55, wherein the one or more subsequent infusions are at approximately 6 months from the previous infusion. **Claim 57** The duration of the first infusion of the anti-CD20 antibody protein is approximately 4 hours, The method according to any one of claims 46 to 56, wherein the first injection of the anti-CD20 antibody protein is optionally performed at a rate of 10 mL per hour for the first 30 minutes, 20 mL per hour for the next 30 minutes, 35 mL per hour for the next 1 hour, and 100 mL per hour for the remaining 2 hours.

58. The method according to any one of claims 48 to 57, wherein the second injection of the anti-CD20 antibody protein, the subsequent injection following the first injection, and the one or more subsequent injections are performed at a rate of 100 mL per hour for the first 30 minutes and 400 mL per hour for the remaining 30 minutes.

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

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

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

62. The method according to any one of claims 46 to 61, wherein the subject administered the multiple injection dosing regimen of the anti-CD20 antibody protein achieves an increase in the no evidence of disease activity (NEDA) state compared to a subject receiving daily oral administration of 14 mg of teriflunomide during the same treatment period.

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

64. The method according to any one of claims 46 to 63, wherein the subject who has undergone the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves an increase in the Multiple Sclerosis Functional Composite (MSFC) score as compared to a subject who has received daily oral administration of 14 mg of teriflunomide during the same treatment period.

65. The method according to any one of claims 46 to 64, wherein the subject who has undergone the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves an improvement in the Timed 25-Foot Walk (T25FW) score as compared to a subject who has received daily oral administration of 14 mg of teriflunomide during the same treatment period.

66. The method according to any one of claims 46 to 65, wherein the subject who has undergone the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves an improvement in the 9-Hole Peg Test (9-HPT) score as compared to a subject who has received daily oral administration of 14 mg of teriflunomide during the same treatment period.

67. The method according to any one of claims 46 to 66, wherein the subject who has undergone the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves a significant reduction in the volume and number of new T1 hypointense lesions by MRI scan as compared to a subject who has received daily oral administration of 14 mg of teriflunomide during the same treatment period.

68. The method according to 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 (CV = 28%) per day and an average maximum concentration of 139 mcg / mL (CV = 15%).

69. The method according to any one of claims 45 to 68, wherein the RMS includes Clinically Isolated Syndrome ("CIS"), Relapsing-Remitting MS ("RRMS"), or Active Secondary Progressive MS ("SPMS").

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

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

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

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

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