Fc gamma receptor II binding and glycan content

Abstract

Provided herein is a method for determining product quality of an antibody composition, the product quality being based on the Fcγ receptor II (FcγII) binding level of the antibody composition. In an exemplary embodiment, the method includes: (a) determining the non-fucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition; (b) optionally calculating a predicted FcγRII binding level based on the non-fucosylated glycan content and / or β-galactosylated glycan content determined in (a); and (c) determining that the product quality of the antibody composition is acceptable if (i) the non-fucosylated glycan content and / or β-galactosylated glycan content is within a target range and / or (ii) the predicted FcγRII binding level is within a target range. Related methods of monitoring product quality and methods of manufacturing the antibody composition are further provided herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS The benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 252,245, filed October 5, 2021, and U.S. Provisional Patent Application No. 63 / 299,104, filed January 13, 2022, is claimed herein, the disclosures of which are incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer readable nucleotide / amino acid sequence listing submitted contemporaneously herewith is incorporated by reference in its entirety and is identified as follows: 15.4 byte XML file entitled "A-2755-WO01-SEC_Sequence_Listing.XML", created on September 22, 2022. [Background technology]

[0003] Glycosylation is one of the most common yet still prominent post-translational modifications, as it is involved in multiple cellular functions, such as protein folding, quality control, molecular trafficking and sorting, and cell surface receptor interactions. Glycosylation influences the therapeutic efficacy of recombinant protein drugs, as it affects the bioactivity, pharmacokinetics, immunogenicity, solubility, and in vivo clearance of therapeutic glycoproteins. The Fc glycoform profile is a product quality attribute, especially for recombinant antibodies, as it directly affects the clinical efficacy and pharmacokinetics of the antibody.

[0004] Specific glycan structures associated with the conserved biantennary glycan in the Fc-CH2 domain can strongly influence the interaction of the Fc domain with Fc gamma receptors (FcγRs) that mediate antibody effector functions (e.g., antibody-dependent cellular cytotoxicity (ADCC)) (see Reusch D, Tejada ML. Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 2015;25:1325-34). For example, core fucose has been shown to have a profound effect on FcγRIIIa binding affinity, resulting in substantial changes in ADCC activity (see Okazaki A, et al. Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcgammaRIIIa. Journal of molecular biology 2004;336:1239-49; Ferrara C, et al. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose. Proceedings of the National Academy of Sciences of the United States of America 2011;108:12669-74). High mannose levels have also been shown to play a role in modulating ADCC activity, albeit to a much more modest and less predictable extent than core fucose (Thomann M, et al. Fc-galactosylation modulates antibody-dependent cellular cytotoxicity of therapeutic antibodies. Molecular immunology 2016;73:69-75).Since core fucose has been reported to sterically hinder the Fc domain from interacting with FcγR, many studies have focused on glycan groups lacking core fucose, including nonfucosylated glycans and high mannose glycans. In addition to this glycan group lacking core fucose, terminal galactose has been suggested to affect ADCC levels. In particular, the presence of terminal galactose enhances ADCC activity. Thomann et al.,Molec Immunol 73:69-75(2016).

[0005] The structure of glycans present in the Fc domain of an antibody can also affect Fc binding to the complement protein C1q, and thus ultimately affect the complement-dependent cytotoxicity (CDC) effector function of the antibody. For example, antibodies with higher β-galactosylation bind C1q with higher affinity and induce higher levels of CDC activity. Similarly, reduced β-galactosylation of the anti-TNF antibody adalimumab is associated with reduced ADCC and CDC activity. Reduced β-galactosylation of adalimumab is also associated with reduced FcγRIIIa binding and binding affinity to the C1q protein. Burzawa et al., “Relationship between structure and function: Influence of galactosylation on Fc-mediated binding and functional properties of adalimumab” Bioprocess Online (2018), available at: https: / / www.bioprocessonline.com / doc / influence-of-galactosylation-on-fc-mediated-binding-and-functional-properties-of-adalimumab-0001. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Reusch D,Tejada ML.Fc glycans of therapeutic antibodies as critical quality attributes.Glycobiology 2015;25:1325-34 [Non-Patent Document 2] Okazaki A,et al.Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcgammaRIIIa.Journal of molecular biology 2004;336:1239-49 [Non-Patent Document 3] Ferrara C, et al.Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.Proceedings of the National Academy of Sciences of the United States of America 2011;108:12669-74 [Non-Patent Document 4] Thomann M,et al.Fc-galactosylation modulates antibody-dependent cellular cytotoxicity of therapeutic antibodies.Molecular immunology 2016;73:69-75 [Non-Patent Document 5] Thomann et al., Molec Immunol 73:69-75 (2016) [Non-Patent Document 6] Burzawa et al., “Relationship between structure and function: Influence of galactosylation on Fc-mediated binding and functional properties of adalimumab” Bioprocess Online (2018) Available at: https: / / www.bioprocessonline.com / doc / influence-of-galactosylation-on-fc-mediated-binding-and-functional-properties-of-adalimumab-0001 Summary of the Invention [Problem to be solved by the invention]

[0007] Various factors affect the glycan structure and therefore the final glycosylated form (glycoform) of a protein (glycoprotein). For example, the cell line expressing the antibody, the cell culture medium, the feed medium composition, and the timing of feeding during cell culture can affect the production of glycoforms of a protein. Although research groups have suggested many ways to affect the level of a specific glycoform of an antibody, there is still a need in the biopharmaceutical industry for a simple and efficient method to predict the level of effector function or binding to FcγR that a specific antibody composition will exhibit based on a given glycoform profile of that antibody composition. In addition, there is a need in the art for a method to determine the level of a specific glycan that will achieve a desired level of effector function or FcγR binding level. [Means for solving the problem]

[0008] Provided herein for the first time are data showing a statistically significant association between the FcγRII binding level of an antibody composition and the level of β-galactosylated glycans and / or the level of non-fucosylated glycans of the antibody composition. As further described herein, representations including but not limited to Equations A-D and Equations 1-10 statistically significantly correlate the FcγRII binding of an antibody composition with the % β-galactosylated glycan content and / or the % non-fucosylated glycan content of the antibody composition. Such representations are useful in methods of predicting the level of FcγRII binding of an antibody composition based on the levels of these glycans. In various aspects, the predicted FcγRII binding level serves as a marker that identifies antibody compositions as acceptable in terms of meeting a therapeutic threshold, and thus identifying those that may be used in one or more downstream manufacturing processes, or those that are unacceptable and should not proceed in the manufacturing process. The correlations of the present disclosure are further useful in identifying the glycoprofile of a desired antibody composition. The correlations shown herein, and a given target FcγRII binding level, identify the glycoprofile (e.g., β-galactosylated glycan, nonfucosylated glycan profile) of an antibody composition having a target FcγRII binding level. With the identified β-galactosylated glycan and nonfucosylated glycan profile of an antibody composition having a target FcγRII binding level, a manufacturing process (e.g., cell culture) can be performed to target the identified profile.

[0009] Thus, the present disclosure provides a method for determining the product quality of an antibody composition. In various embodiments, the product quality is based on the FcγRII binding level of the antibody composition. In an exemplary embodiment, the method includes: (a) determining the non-fucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition; (b) optionally calculating a predicted RcγRII binding level based on the non-fucosylated glycan content and / or β-galactosylated glycan content determined in (a); and (c) determining that the product quality of the antibody composition is acceptable if (i) the non-fucosylated glycan content and / or β-galactosylated glycan content is within a target range and / or (ii) the predicted FcγRII binding level is within a target range.

[0010] The present disclosure also provides a method for monitoring the product quality of an antibody composition. In an exemplary embodiment, the method includes determining the product quality of a first sample of the antibody composition obtained according to the method of the present disclosure at a first time point, and determining the product quality of a second sample of the antibody composition obtained according to the method of the present disclosure at a second time point, the second time point being different from the first time point. In various aspects, the difference in the level of non-fucosylated glycans and / or β-galactosylated glycans of the antibody composition between the first time point and the second time point provides information of the difference in the level of FcRII binding of the antibody composition.

[0011] The present disclosure additionally provides a method of producing an antibody composition. In an exemplary embodiment, the method includes determining the product quality of the antibody composition, the product quality of the antibody composition being determined according to the method of the present disclosure, the sample being a sample of in-process material, the non-fucosylated glycan content and / or the β-galactosylated glycan content determined in (a) is not within the target range, the method further includes (d) modifying one or more conditions of the cell culture to obtain an modified cell culture, and (e) determining the non-fucosylated glycan content and / or the β-galactosylated glycan content of a sample of the antibody composition obtained from the modified cell culture, optionally repeating (d) and (e) until the non-fucosylated glycan content and / or the β-galactosylated glycan content is within the target range. In an alternative or additional exemplary embodiment, the method includes: (a) determining the nonfucosylated glycan content and / or β-galactosylated glycan content of a sample of an antibody composition; (b) determining an FcγRII binding level of the antibody composition based on the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (a); and (c) selecting the antibody composition for downstream processing based on the level of FcγRII binding determined in (b).

[0012] Also provided is a method of modifying the level of FcγRII binding of an antibody composition, in an exemplary embodiment, the method comprises (a) specifying the level of FcγRII, and (b) modifying the level of nonfucosylated glycans and / or β-galactosylated glycans of the antibody composition to achieve the specified level of FcγRII.

[0013] The present disclosure provides a method for determining the level of FcγRII binding of an antibody composition. In an exemplary embodiment, the method comprises determining the level of nonfucosylated glycan and / or β-galactosylated glycan of the antibody composition. The present disclosure also provides a method for predicting the level of FcγRII binding of an antibody composition. In an exemplary embodiment, the method comprises determining the level of nonfucosylated glycan and / or β-galactosylated glycan of the antibody composition. In various aspects, the level of nonfucosylated glycan and / or β-galactosylated glycan of the antibody composition provides information of FcγRII binding of the antibody composition according to the relationships presented herein.

[0014] The present disclosure provides a method for predicting the in vivo efficacy and / or side effects of an antibody composition. In an exemplary embodiment, the method includes: (a) determining the nonfucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition; and (b) predicting the antibody composition as causing an in vivo side effect based on the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (a). [Brief description of the drawings]

[0015] [Figure 1A] 1A and 1B are diagrams of exemplary glycan structures. [Figure 1B] 1A and 1B are diagrams of exemplary glycan structures. [Figure 2A] Figure 2A is a representative glycan map chromatogram (full-scale view) and Figure 2B is a representative glycan map chromatogram (enlarged-scale view). [Figure 2B] Figure 2A is a representative glycan map chromatogram (full-scale view) and Figure 2B is a representative glycan map chromatogram (enlarged-scale view). [Diagram 3] FIG. 3 is a general schematic diagram of part of the binding assay described in Examples 2 and 4. [Figure 4A]FIG. 4A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 4B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 4C is an FcγRIIa binding leverage plot for HM glycans. FIG. 4D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 4E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 4B] FIG. 4A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 4B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 4C is an FcγRIIa binding leverage plot for HM glycans. FIG. 4D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 4E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 4C]FIG. 4A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 4B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 4C is an FcγRIIa binding leverage plot for HM glycans. FIG. 4D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 4E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 4D] FIG. 4A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 4B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 4C is an FcγRIIa binding leverage plot for HM glycans. FIG. 4D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 4E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 4E]FIG. 4A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 4B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 4C is an FcγRIIa binding leverage plot for HM glycans. FIG. 4D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 4E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 4F] FIG. 4A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 4B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 4C is an FcγRIIa binding leverage plot for HM glycans. FIG. 4D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 4E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 4G]FIG. 4A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 4B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 4C is an FcγRIIa binding leverage plot for HM glycans. FIG. 4D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 4E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 4G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 5A] FIG. 5A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 5B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 5C is an FcγRIIb binding leverage plot for HM glycans. FIG. 5D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 5E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 5B]FIG. 5A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 5B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 5C is an FcγRIIb binding leverage plot for HM glycans. FIG. 5D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 5E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 5C] FIG. 5A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 5B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 5C is an FcγRIIb binding leverage plot for HM glycans. FIG. 5D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 5E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 5D]FIG. 5A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 5B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 5C is an FcγRIIb binding leverage plot for HM glycans. FIG. 5D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 5E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 5E] FIG. 5A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 5B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 5C is an FcγRIIb binding leverage plot for HM glycans. FIG. 5D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 5E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 5F]FIG. 5A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 5B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 5C is an FcγRIIb binding leverage plot for HM glycans. FIG. 5D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 5E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 5G] FIG. 5A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 5B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 5C is an FcγRIIb binding leverage plot for HM glycans. FIG. 5D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 5E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 5G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 6] 6 is a diagram of the presently disclosed correlation and its exemplary application for drug substance manufacturing and drug product release assays. Instead of testing an in-process sample or a lot sample for FcγRII binding or effector function, the β-galactosylated glycan % and non-fucosylated glycan % of the sample are measured to determine whether the antibody composition should be selected for continued manufacturing or downstream processing, or whether the lot should be sold. [Figure 7A]FIG. 7A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 7B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 7C is an FcγRIIa binding leverage plot for HM glycans. FIG. 7D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 7E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 7B] FIG. 7A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 7B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 7C is an FcγRIIa binding leverage plot for HM glycans. FIG. 7D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 7E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 7C]FIG. 7A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 7B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 7C is an FcγRIIa binding leverage plot for HM glycans. FIG. 7D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 7E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 7D] FIG. 7A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 7B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 7C is an FcγRIIa binding leverage plot for HM glycans. FIG. 7D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 7E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 7E]FIG. 7A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 7B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 7C is an FcγRIIa binding leverage plot for HM glycans. FIG. 7D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 7E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 7F] FIG. 7A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 7B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 7C is an FcγRIIa binding leverage plot for HM glycans. FIG. 7D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 7E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 7G]FIG. 7A is an FcγRIIa binding leverage plot for β-galactosylated glycans. FIG. 7B is an FcγRIIa binding leverage plot for non-fucosylated glycans. FIG. 7C is an FcγRIIa binding leverage plot for HM glycans. FIG. 7D is a graph plotting actual FcγRIIa binding as a function of predicted FcγIIa binding. FIG. 7E is a graph plotting FcγIIa binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7F is a graph plotting FcγIIa binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown in the shaded region. FIG. 7G is a graph plotting FcγIIa binding as a function of high mannose glycans (%). The 95% confidence space is shown in the shaded region. [Figure 8A] FIG. 8A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 8B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 8C is an FcγRIIb binding leverage plot for HM glycans. FIG. 8D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 8E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 8B]FIG. 8A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 8B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 8C is an FcγRIIb binding leverage plot for HM glycans. FIG. 8D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 8E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 8C] FIG. 8A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 8B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 8C is an FcγRIIb binding leverage plot for HM glycans. FIG. 8D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 8E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 8D]FIG. 8A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 8B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 8C is an FcγRIIb binding leverage plot for HM glycans. FIG. 8D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 8E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 8E] FIG. 8A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 8B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 8C is an FcγRIIb binding leverage plot for HM glycans. FIG. 8D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 8E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 8F]FIG. 8A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 8B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 8C is an FcγRIIb binding leverage plot for HM glycans. FIG. 8D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 8E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. [Figure 8G] FIG. 8A is an FcγRIIb binding leverage plot for β-galactosylated glycans. FIG. 8B is an FcγRIIb binding leverage plot for non-fucosylated glycans. FIG. 8C is an FcγRIIb binding leverage plot for HM glycans. FIG. 8D is a graph plotting actual FcγRIIb binding as a function of predicted FcγRIIb binding. FIG. 8E is a graph plotting FcγIIb binding as a function of β-galactosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8F is a graph plotting FcγIIb binding as a function of non-fucosylated glycans (%). The 95% confidence space is shown as the shaded region. FIG. 8G is a graph plotting FcγIIb binding as a function of high mannose glycans (%). The 95% confidence space is shown as the shaded region. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Glycosylation, glycans, and methods for measuring glycans Many secreted proteins are post-translationally glycosylated, a process by which sugar moieties (e.g., glycans, sugars) are covalently attached to specific amino acids of proteins. In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which the glycan is attached to an asparagine at the recognition sequence Asn-X-Thr / Ser (where "X" is any amino acid except proline), and (2) O-linked glycosylation, in which the glycan is attached to a serine or threonine. Regardless of the type of glycosylation (N-linked or O-linked), there is a wide range of glycan structures attached to each site (O or N), resulting in microheterogeneity of protein glycoforms.

[0017] All N-glycans have a common core sugar sequence: Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr(Man3GlcNAc2Asn) and are classified into one of three types: (A) high mannose (HM) or oligomannose (OM) types, consisting of two N-acetylglucosamine (GluNAc) moieties and multiple (e.g., 5, 6, 7, 8, or 9) mannose (Man) residues; (B) complex types, containing more than two GlcNAc moieties and any number of other sugar types; or (C) hybrid types, containing a Man residue on one of the branches and a GlcNAc at the base of the complex branch.

[0018] N-linked glycans typically include one or more monosaccharides of galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (GLc), N-acetylglucosamine (GlcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxynonanoic acid (Kdn), fucose (Fuc), glucuronic acid (GLcA), iduronic acid (IdoA), galacturonic acid (GalA), and mannuronic acid (ManA). Exemplary glycan structures, indicated by commonly used saccharide symbols, and their identities are shown in Figures 1A and 1B.

[0019] N-linked glycosylation is initiated in the endoplasmic reticulum (ER) and results in a complex series of reactions that result in the attachment of a core glycan structure that is essentially made up of two GlcNAc and three Man residues. The glycan complex formed in the ER is modified by the action of enzymes in the Golgi apparatus. If the sugar is relatively inaccessible to the enzymes, it typically remains in its original HM form. If the sugar is accessible to the enzymes, many of the Man residues are cleaved off and the sugar is further modified, resulting in complex N-glycan structures. For example, mannosidase-1 located in the cis-Golgi can cleave or hydrolyze HM glycans, while fucosyltransferase FUT-8 located in the medial-Golgi fucosylates the glycans (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84).

[0020] Thus, the sugar composition and structural configuration of a glycan structure varies depending, inter alia, on the glycosylation machinery in the ER and Golgi apparatus, the accessibility of the glycan structure to the enzymes of that machinery, the order of action of each enzyme, and the step at which the protein is released from the glycosylation machinery.

[0021] A variety of methods are known in the art for assessing the glycans present in a glycoprotein-containing composition or for determining, detecting, or measuring the glycoform profile (e.g., glycoprofile) of a particular sample containing a glycoprotein. Suitable methods include, but are not limited to, cationic MALDI-TOF analysis, anionic MALDI-TOF analysis, weak anion exchange (WAX) chromatography, normal phase chromatography (NP-HPLC), exoglycosidase digestion, Bio-Gel P-4 chromatography, anion exchange chromatography, and one-dimensional nmr spectroscopy, and combinations thereof. See, e.g., Mattu et al., JBC 273:2260-2272 (1998); Field et al., Biochem J 299(Pt 1):261-275 (1994); Yoo et al., MAbs 2(3):320-334 (2010) Wuhrer M. et al., Journal of Chromatography B, 2005, Vol. 825, Issue 2, pages 124-133; Ruhaak LR, Anal Bioanal Chem, 2010, Vol. 397:3457-3481 and Geoffrey, RGet. al. Analytical Biochemistry 1996, Vol. 240, pages 210-226. Also provided herein in Example 1 is a method suitable for assessing the glycans present in a glycoprotein-containing composition (e.g., an antibody composition). The method of Example 1 describes an assay in which glycans attached to a glycosylated protein of a composition (e.g., an antibody of an antibody composition) are enzymatically cleaved from the protein (e.g., an antibody). The glycans are then separated by hydrophilic interaction liquid chromatography (HILIC) to produce a chromatogram with several peaks. Each peak in the chromatogram represents the average distribution (amount) of a different glycan. Two views of an exemplary HILIC chromatogram with peaks of different glycans are shown in Figures 2A and 2B.For these purposes, peak area %=peak area / total peak area×100%, and total peak area %=total area of ​​sample / total area of ​​standard×100%. Thus, the level of a particular glycan (or group of glycans) is reported as a percentage. For example, if an antibody composition is characterized as having a Man6 level of 30%, this means that 30% of all glycans cleaved from the antibodies of this composition are Man6.

[0022] The present disclosure (including the correlations, relationships, and equations set forth herein) relates to non-fucosylated glycans, and / or β-galactosylated glycans, and / or high mannose glycans of antibody compositions. As used herein, the term "non-fucosylated glycan" or "AF glycan" refers to a glycan lacking a core fucose, e.g., α1,6-linked fucose on the GlcNAc residue involved in the amide bond with the Asn of the N-glycosylation site. Non-fucosylated glycans include, but are not limited to, A1G0, A2G0, A2G1a, A2G1b, A2G2, and A1G1M5. Additional non-fucosylated glycans include, for example, A1G1a, G0[H3N4], G0[H4N4], G0[H5N4], FO-N[H3N3]. See, e.g., Reusch and Tejada, Glycobiology 25(12):1325-1334 (2015). The level of nonfucosylated glycans is, in various embodiments, obtained by summing the % of each nonfucosylated glycan type, e.g., by summing A1G0%, A2G0%, A2G1a%, A2G1b%, A2G2%, A1G1M5%, A1G1a%, G0[H3N4]%, G0[H4N4]%, G0[H5N4]%, and FO-N[H3N3]%. As used herein, the term "β-galactosylated glycan" is synonymous with "terminal galactose glycan" and refers to any glycan that contains one or two galactose molecules. Glycans containing one galactose molecule are designated "G1" (e.g., "G1a" or "G1b") in the glycan name, and glycans containing two galactose molecules are designated "G2" in the glycan name. Thus, a β-galactosylated glycan is, in various embodiments, a G1-galactosylated glycan, a G1a-galactosylated glycan, a G1b-galactosylated glycan, or a G2-galactosylated glycan. In various embodiments, a β-galactosylated glycan includes a core fucose (e.g., A2G1F, A2G2F). Alternatively, a β-galactosylated glycan lacks a core fucose (e.g., A2G1 (e.g., A2G1a and A2G1b) and A2G2 (or G1 and G2)).In some embodiments, the galactosylated glycans are hybrid glycans containing high mannose arms and galactose-containing arms, as exemplified by A1G1M5 and A1G1, respectively, and single-arm glycans. It should be noted that although β-galactosylated glycans may lack core fucose (and thus represent a subset of non-fucosylated glycans), they have certain characteristics and may be referred to as a separate glycan group. Thus, unless otherwise specified, β-galactosylated glycans are understood to represent a separate characteristic and may be classified separately from non-fucosylated glycans or as an additional property of non-fucosylated glycans. The level of β-galactosylated glycans is, in various embodiments, obtained by summing the percentages of each β-galactosylated glycan species, for example, by summing the percentages of each G1-galactosylated glycan species, each G1a-galactosylated glycan species, each G1b-galactosylated glycan species, and each G2-galactosylated glycan species. As used herein, the term "high mannose glycan" or "HM glycan" encompasses glycans containing 5, 6, 7, 8, or 9 mannose residues, abbreviated as Man5, Man6, Man7, Man8, and Man9, respectively. The level of HM glycans is, in various embodiments, obtained by summing Man5%, Man6%, Man7%, Man8%, and Man9%.

[0023] In exemplary embodiments, the glycan level (e.g., glycan content, optionally expressed as a percentage, e.g., AF glycan%, β-galactosylated glycan%, HM glycan%) is determined (e.g., measured) by any of a variety of methods known in the art for assessing the glycans present in a glycoprotein-containing composition or for determining, detecting, or measuring the glycoform profile (e.g., glycoprofile) of a particular sample containing a glycoprotein. In exemplary cases, the glycan level (e.g., AF glycan%, β-galactosylated glycan%, HM glycan%) of an antibody composition is determined by measuring the level of such glycans in a sample of the antibody composition by a chromatography-based method (e.g., HILIC), and the glycan level is expressed as a percentage as described herein. See, e.g., Example 1. In exemplary cases, the glycan level of an antibody composition is expressed as a percentage of all glycans cleaved from the antibodies of the composition. In various embodiments, glycan levels (e.g., AF glycan%, β-galactosylated glycan%, HM glycan%) are determined (e.g., measured) by measuring the levels of such glycans in samples of the antibody composition. In exemplary cases, at least 5, at least 6, at least 7, at least 8, or at least 9 samples of the antibody composition are taken and the glycan levels (e.g., AF glycan%, β-galactosylated glycan%, HM glycan%) are determined (e.g., measured) for each sample. In various embodiments, the mean or average of AF glycan%, and / or β-galactosylated glycan%, and / or HM glycan% is determined.

[0024] FcγRII binding Fc receptors are receptors on the surface of B lymphocytes, follicular dendritic cells, natural killer (NK) cells, macrophages, neutrophils, eosinophils, basophils, platelets, and mast cells that bind to the Fc region of antibodies. Fc receptors are classified into different classes based on the type of antibody they bind to. For example, Fc gamma receptors are receptors for the Fc region of IgG antibodies, Fc alpha receptors are receptors for the Fc region of IgA antibodies, and Fc epsilon receptors are receptors for the Fc region of IgE antibodies.

[0025] The term "FcγR" or "Fc-gamma receptor" refers to a protein belonging to the IgG superfamily that is involved in inducing phagocytosis of opsonized cells or microorganisms. See, e.g., Fridman WH. Fc receptors and immunoglobulin binding factors. FASEB Journal. 5(12):2684-90 (1991). Members of the Fc gamma receptor family include FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). The sequences of FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and FcγRIIIB can be found in many sequence databases, for example in the Uniprot database (www.uniprot.org) under accession numbers P12314 (FCGR1_HUMAN), P12318 (FCG2A_HUMAN), P31994 (FCG2B_HUMAN), P08637 (FCG3A_HUMAN), and P08637 (FCG3A_HUMAN), respectively.

[0026] The FcγRII family of human integral membrane receptor glycoproteins includes FcγRIIa, FcγRIIc, and FcγRIIb. FcγRIIa and FcγRIIc have cellular functions opposite to those of FcγRIIb. FcγRIIa protein is an activating Fc receptor, whereas FcγRIIb is inhibitory and is considered an immune checkpoint that regulates the action of activating Fc receptors and antigen receptors on B cells. FcγRIIc is similar to FcγRIIa and is considered an activating Fc receptor. FcγRIIa is expressed on granulocytes, monocytes, and monocyte-derived cells (e.g., macrophages and dendritic cells (DCs)). Engagement of FcγRIIa by IgG cross-linking can initiate a variety of effector functions, such as phagocytosis, activation of neutrophils and other myeloid effector cells to kill IgG-ozonated target cells, activation of granulocytes to release inflammatory mediators, T cell proliferation, and T cell-mediated cytokine secretion, as well as platelet activation, adhesion, and aggregation after vascular injury. The structure and function of FcγRII proteins are reviewed in Anania et al., Front. Immunol. 10:464 (2019); available on the World Wide Web at doi.org / 10.3389 / fimmu.2019.00464.

[0027] The present disclosure (including the correlations, associations, and equations presented herein) relates to the level of FcγRII binding of an antibody composition. Methods for measuring the level of FcγRII binding of an antibody composition are known in the art, exemplary methods of which are described herein (see, e.g., Examples 2 and 4), and the data presented herein support that the level of FcγRII binding of an antibody composition can be predicted by the glycoprofile of the antibody composition. In exemplary cases, the non-fucosylated glycan %, and / or β-galactosylated glycan %, and / or HM glycan % of the antibody composition can be used to calculate or predict the level of FcγRII binding of the antibody composition. Similarly, given that antibody effector function is induced upon binding of the antibody Fc domain to FcγRII, the level of FcγRII binding of an antibody composition may in various cases serve as a proxy for effector function, such that the non-fucosylated glycan %, and / or β-galactosylated glycan %, and / or HM glycan % of an antibody composition may be used to calculate or predict the level of effector function of the antibody composition, which effector function is activated upon FcγRII binding. In an exemplary embodiment, the present disclosure relates the non-fucosylated glycan %, and / or β-galactosylated glycan %, and / or HM glycan % of an antibody composition to the level of FcγRIIa binding. In an alternative or additional embodiment, the present disclosure relates the non-fucosylated glycan %, and / or β-galactosylated glycan %, and / or HM glycan % of an antibody composition to the level of FcγRIIb binding.

[0028] The disclosed relationship linking the non-fucosylated glycan %, and / or β-galactosylated glycan %, and / or HM glycan % of an antibody composition with the level of FcγRII binding is useful in various cases for the design of process control measures to ensure that the desired FcγRII binding activity can be delivered at consistent and intended levels. This correlation can be used to ensure consistent clinical outcomes, achieve functional similarity of biosimilar candidates, and predict potential in vivo efficacy of therapeutic antibody treatments.

[0029] In various embodiments, the FcγRII binding level may be calculated based on the non-fucosylated glycan %, and / or the β-galactosylated glycan %, and / or the HM glycan % of the antibody composition based on the present disclosure. In various embodiments, the non-fucosylated glycan %, and / or the β-galactosylated glycan %, and / or the HM glycan % of the antibody composition are amounts measured based on a sample of the antibody composition. In various cases, the measured non-fucosylated glycan %, and / or the measured β-galactosylated glycan %, and / or the measured HM glycan % are measured by a method including, but not limited to, HILIC. In various cases, the measured non-fucosylated glycan %, and / or the measured β-galactosylated glycan %, and / or the measured HM glycan % are measured by a method including, but not limited to, the method described in Example 1.

[0030] In various aspects, based on the present disclosure, the non-fucosylated glycan %, and / or the β-galactosylated glycan %, and / or the HM glycan % can be calculated based on a known, or predetermined, or preselected, or target FcγRII binding level. In various cases, assuming that a particular antibody of the antibody composition is produced, the target FcγRII binding level or the target range of FcγRII binding levels is known. For example, the antibody can include an amino acid sequence identical to a reference antibody (or an amino acid sequence at least 95%, 97%, or 99% identical to the amino acid sequence of the reference antibody), and the target FcγRII binding level or its range is known for the reference antibody. In exemplary embodiments, the target nonfucosylated glycan %, and / or the target β-galactosylated glycan %, and / or the target HM glycan %, are calculated based on a first model correlating the nonfucosylated glycan %, and / or the β-galactosylated glycan %, and / or the HM glycan %, with the FcγRII binding level. In various cases, the first model is a linear regression model. In various embodiments, the first model correlating the FcγRII binding level with the nonfucosylated glycan %, and / or the β-galactosylated glycan %, and / or the HM glycan %, is statistically significant as demonstrated by its low p-value. In various embodiments, the p-value is less than 0.05. In various cases, the p-value is less than 0.01 or less than 0.001. In various cases, the p-value is less than 0.0001.

[0031] In an exemplary embodiment, the β-galactosylated glycan content of the antibody composition positively correlates with the FcγRII binding level. In various embodiments, a higher level of β-galactosylated glycan content correlates with a higher level of FcγRII binding, and a lower level of β-galactosylated glycan content correlates with a lower level of FcγRII binding. In an exemplary embodiment, the non-fucosylated glycan content of the antibody composition negatively correlates with the FcγRII binding level. In various embodiments, a higher level of non-fucosylated glycan content correlates with a lower level of FcγRII binding, and a lower level of non-fucosylated glycan content correlates with a higher level of FcγRII binding. In an exemplary embodiment, the high mannose glycan content of the antibody composition correlates with the FcγRII binding level. In an exemplary case, this correlation is a negative correlation. In various embodiments, higher levels of HM glycan content correlate with lower levels of FcγRII binding and lower levels of HM glycan content correlate with higher levels of FcγRII binding.

[0032] In an exemplary embodiment, the FcγRII binding level is a level of FcγRIIa binding. In an exemplary case, the FcγRIIa binding level is calculated based on a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan). In various embodiments, the FcγRII binding level is calculated according to Equation A: FcγRII binding level = m*BG%+y [Equation A] (wherein m is from about 0.535 to about 1.091, y is from about 72.58 to about 85.78, and BG% is the β-galactosylated glycan content % determined in (a). It is calculated according to:

[0033] In an exemplary case, m of Equation A is 0.813 and / or y of Equation A is 79.18. In an alternative exemplary case, m of Equation A is 0.778 and / or y of Equation A is 81.76.

[0034] In exemplary cases, the FcγRII binding level is calculated based on the determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan). The FcγRII binding level is calculated, in various cases, according to Equation B: FcγRII binding level = m*AF%+y [Equation B] (wherein m is about -13.73 to about -7.54, y is about 108.8 to about 119.1, and AF% is the non-fucosylated glycan content%) It is calculated according to:

[0035] In various embodiments, m of Equation B is −10.63 and / or y of Equation B is 114. In alternative exemplary cases, m of Equation B is −9.53 and / or y of Equation B is 114.

[0036] In an exemplary embodiment, the FcγRII binding level is based on a determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan) and a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan).

[0037] In an exemplary case, the FcγRII binding level is calculated according to Equation 3: FcγRII binding=0.576*BG%+(-4.978)*AF%+98.877 [Equation 3] (wherein BG% is the β-galactosylated glycan content % and AF% is the non-fucosylated glycan content %) This is the level within the 95% confidence interval of the line.

[0038] In an exemplary embodiment, the FcγRII binding level is a level of FcγRIIb binding. In an exemplary case, the FcγRIIb binding level is calculated based on a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan). In various embodiments, the FcγRII binding level is calculated according to Equation C: FcγRII binding level = m*BG%+y [Equation C] (In the formula, m is about 0.3260 to about 0.9697, y is about 77.72 to about 92.99, and BG% is the β-galactosylated glycan content%.) It is calculated according to:

[0039] In various cases, m of equation C is 0.648 and / or y of equation C is 85.36. In alternative exemplary cases, m of equation C is 0.644 and / or y of equation C is 86.34.

[0040] In exemplary cases, the FcγRIIb binding level is calculated based on the determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan). The FcγRII binding level is, in various cases, calculated according to Equation D: FcγRII binding level = m*AF%+y [Equation D] (wherein m is about -12.02 to about -6.247, y is about 109.3 to about 118.9, and AF% is the non-fucosylated glycan content%) It is calculated according to:

[0041] In various embodiments, m of Equation D is about −9.132 and / or y of Equation D is about 114. In alternative exemplary cases, m of Equation D is about −7.102 and / or y of Equation D is about 111.9.

[0042] In various aspects, the FcγRIIb binding level is calculated based on a determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan) and a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan). In an exemplary embodiment, the FcγRII binding level is calculated based on Equation 4: FcγRII binding=0.461*BG%+(-4.429)*AF%+105.731 [Equation 4] (wherein BG% is the β-galactosylated glycan content % and AF% is the non-fucosylated glycan content %) This is the level within the 95% confidence interval of the line.

[0043] In exemplary cases, the FcγRII binding level is calculated based on a determined or measured high mannose (HM) glycan content (e.g., HM glycan %). In various aspects, the FcγRII binding level is calculated based on a determined or measured non-fucosylated glycan content (e.g., non-fucosylated glycan %), a determined or measured β-galactosylated glycan content (e.g., β-galactosylated glycan %), and a determined or measured HM glycan content (HM glycan %). In exemplary aspects, the FcγRIIa binding level of an antibody composition is calculated based on Equation 5: FcγRII binding=0.576*BG%+(-4.978)*AF%+98.877+(-1.343)*HM% [Equation 5] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0044] In an exemplary embodiment, the FcγRIIa binding level of an antibody composition is calculated according to Equation 9: FcγRII binding=0.545*BG%+(-4.466)*AF%+102.7+(-2.036)*HM% [Equation 9] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0045] In an exemplary embodiment, the FcγRIIb binding level of an antibody composition is calculated according to Equation 6: FcγRII binding=0.461*BG%+(-4.429)*AF%+105.731+(-1.883)*HM% [Equation 6] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0046] In an exemplary embodiment, the FcγRIIb binding level of an antibody composition is calculated according to Equation 10: FcγRII binding=0.590*BG%+(-2.04)*AF%+99.2+(-1.91)*HM% [Equation 10] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0047] Methods for determining and / or monitoring product quality Based on the present disclosure, the product quality of an antibody composition can be determined and / or monitored. Thus, the present disclosure provides a method for determining the product quality of an antibody composition, the product quality of the antibody composition being based on the FcγII binding level of the antibody composition. In an exemplary embodiment, the method includes: (a) determining the non-fucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition; (b) optionally calculating the FcγRII binding level based on the non-fucosylated glycan content and / or β-galactosylated glycan content determined in (a); and (c) determining the product quality of the antibody composition as acceptable if (i) the non-fucosylated glycan content and / or β-galactosylated glycan content is within a target range and / or (ii) the FcγRII binding level is within a target range.

[0048] In various aspects, the target range of FcγRII binding level, the target range of nonfucosylated glycan content, and / or the target range of β-glycosylated glycan content are based on the FcγRII binding level, the nonfucosylated glycan content, and / or the β-galactosylated glycan content of a reference antibody. In various cases, the reference antibody comprises a chimeric constant region. In an exemplary case, the chimeric constant region of the reference antibody comprises a portion of an IgG2 constant region and a portion of an IgG4 constant region. In various aspects, the chimeric constant region comprises a CH1 and / or a hinge of an IgG2 and / or a CH2-CH3 of an IgG4. In an exemplary case, the chimeric constant region comprises a chimeric constant region of SEQ ID NO: 15. Optionally, the reference antibody is eculizumab.

[0049] In an exemplary embodiment, the FcγRII binding level is a level of FcγRIIa binding. In an exemplary case, the FcγRIIa binding level is calculated based on a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan). In various embodiments, the FcγRII binding level is calculated according to Equation A: FcγRII binding level = m*BG%+y [Equation A] (wherein m is from about 0.535 to about 1.091, y is from about 72.58 to about 85.78, and BG% is the β-galactosylated glycan content % determined in (a). It is calculated according to:

[0050] In an exemplary case, m of Equation A is 0.813 and / or y of Equation A is 79.18. In an alternative exemplary case, m of Equation A is 0.778 and / or y of Equation A is 81.76.

[0051] In exemplary cases, the FcγRII binding level is calculated based on the determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan). The FcγRII binding level is calculated, in various cases, according to Equation B: FcγRII binding level = m*AF%+y [Equation B] (wherein m is about -13.73 to about -7.54, y is about 108.8 to about 119.1, and AF% is the non-fucosylated glycan content%) It is calculated according to:

[0052] In various embodiments, m of Equation B is −10.63 and / or y of Equation B is 114. In alternative exemplary cases, m of Equation B is −9.53 and / or y of Equation B is 114.

[0053] In an exemplary embodiment, the FcγRII binding level is based on a determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan) and a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan).

[0054] In an exemplary case, the FcγRII binding level is calculated according to Equation 3: FcγRII binding=0.576*BG%+(-4.978)*AF%+98.877 [Equation 3] (wherein BG% is the β-galactosylated glycan content % and AF% is the non-fucosylated glycan content %) This is the level within the 95% confidence interval of the line.

[0055] In an exemplary embodiment, the FcγRII binding level is a level of FcγRIIb binding. In an exemplary case, the FcγRIIb binding level is calculated based on a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan). In various embodiments, the FcγRII binding level is calculated according to Equation C: FcγRII binding level = m*BG%+y [Equation C] (In the formula, m is about 0.3260 to about 0.9697, y is about 77.72 to about 92.99, and BG% is the β-galactosylated glycan content%.) It is calculated according to:

[0056] In various cases, m of equation C is 0.648 and / or y of equation C is 85.36. In alternative exemplary cases, m of equation C is 0.644 and / or y of equation C is 86.34.

[0057] In exemplary cases, the FcγRIIb binding level is calculated based on the determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan). The FcγRII binding level is, in various cases, calculated according to Equation D: FcγRII binding level = m*AF%+y [Equation D] (wherein m is about -12.02 to about -6.247, y is about 109.3 to about 118.9, and AF% is the non-fucosylated glycan content%) It is calculated according to:

[0058] In various embodiments, m of Equation D is about −9.132 and / or y of Equation D is about 114. In alternative exemplary cases, m of Equation D is about −7.102 and / or y of Equation D is about 111.9.

[0059] In various aspects, the FcγRIIb binding level is calculated based on a determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan) and a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan). In an exemplary embodiment, the FcγRII binding level is calculated based on Equation 4: FcγRII binding=0.461*BG%+(-4.429)*AF%+105.731 [Equation 4] (wherein BG% is the β-galactosylated glycan content % and AF% is the non-fucosylated glycan content %) This is the level within the 95% confidence interval of the line.

[0060] In exemplary cases, the FcγRII binding level is calculated based on a determined or measured high mannose (HM) glycan content (e.g., HM glycan %). In various aspects, the FcγRII binding level is calculated based on a determined or measured non-fucosylated glycan content (e.g., non-fucosylated glycan %), a determined or measured β-galactosylated glycan content (e.g., β-galactosylated glycan %), and a determined or measured HM glycan content (HM glycan %). In exemplary aspects, the FcγRIIa binding level of an antibody composition is calculated based on Equation 5: FcγRII binding=0.576*BG%+(-4.978)*AF%+98.877+(-1.343)*HM% [Equation 5] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0061] In an exemplary embodiment, the FcγRIIa binding level of an antibody composition is calculated according to Equation 9: FcγRII binding=0.545*BG%+(-4.466)*AF%+102.7+(-2.036)*HM% [Equation 9] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0062] In an exemplary embodiment, the FcγRIIb binding level of an antibody composition is calculated according to Equation 6: FcγRII binding=0.461*BG%+(-4.429)*AF%+105.731+(-1.883)*HM% [Equation 6] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0063] In an exemplary embodiment, the FcγRIIb binding level of an antibody composition is calculated according to Equation 10: FcγRII binding=0.590*BG%+(-2.04)*AF%+99.2+(-1.91)*HM% [Equation 10] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0064] In an exemplary embodiment, the method is a quality control (QC) assay. In an exemplary embodiment, the method is an in-process QC assay. In various embodiments, the sample is a sample of an in-process material. In various cases, the AF glycan content and / or the β-galactosylated glycan content is determined before harvesting or after harvesting. In exemplary cases, the AF glycan content and / or the β-galactosylated glycan content is determined after chromatography. Optionally, the chromatography includes capture chromatography, intermediate chromatography, and / or polish chromatography. In some embodiments, the AF glycan content and / or the β-galactosylated glycan content is determined after viral inactivation and neutralization, viral filtration, or buffer exchange. In various instances, the method is a lot release assay. The sample is, in some embodiments, a sample of a manufacturing lot.

[0065] In various aspects, the method further comprises (i) selecting the antibody composition for downstream processing if the non-fucosylated glycan content and / or β-galactosylated glycan content is within the target range and / or if the FcγRII binding level is within the target range. If the AF glycan content and / or β-galactosylated glycan content determined in (a) is not within the target range, in various aspects, modifying one or more conditions of the cell culture to obtain an engineered cell culture. In some aspects, the method further comprises determining the non-fucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition obtained after modifying one or more conditions of the cell culture, e.g., determining the non-fucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition of the engineered cell culture. In various aspects, if the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (a) is not within the target range, the method further comprises (d) modifying one or more conditions of the cell culture to obtain an altered cell culture, and (e) determining the nonfucosylated glycan content and / or β-galactosylated glycan content of a sample of antibody composition obtained from the altered cell culture. In an exemplary aspect, if the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (a) is not within the target range, the method further comprises (d) and (d) until the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (d) is within the target range.

[0066] In an exemplary case, an assay that directly measures FcγRII binding of the antibody composition is performed on the antibody composition only if the non-fucosylated glycan content and / or β-galactosylated glycan content determined in (a) is not within the target range (e.g., outside the target range). An assay that directly measures FcγRII binding activity includes, for example, an assay described in Example 2 or Example 4. In an exemplary case, an assay that directly measures FcγRII binding of the antibody composition is not performed on the antibody composition. In various embodiments, determining the non-fucosylated glycan content and / or β-galactosylated glycan content is the only step required to determine the product quality of the antibody composition. Without being bound by theory, the statistically significant correlations described herein allow the non-fucosylated glycan content and / or β-galactosylated glycan content to be indicative of FcγRII binding levels, and therefore an assay that directly measures FcγRII binding levels is not required. Thus, direct measurement of FcγRII binding levels of an antibody composition is not necessary and therefore is not performed in various embodiments of the disclosed methods.

[0067] In various embodiments, the method determines product quality with respect to an FcγRII binding level criterion. In various embodiments, the FcγRII binding level criterion is one of the antibody composition's acceptance criteria. The method of the present disclosure, in various embodiments, is aimed at ensuring that a batch of a pharmaceutical product meets the appropriate specifications and appropriate statistical quality control criteria, respectively, as a condition of its approval and sale (e.g., approval and sale in the United States pursuant to 21 CFR 211.165). In various embodiments, the method of the present disclosure for determining product quality meets the statistical quality control criteria, including the appropriate acceptance level and / or the appropriate rejection level. Terms including, but not limited to, "acceptance criteria," "lot," and "in-process" are as defined by their meanings in Title 21 of the Code of Federal Regulations (CFR), Section 210.3.

[0068] The present disclosure also provides a method for monitoring the product quality of an antibody composition, wherein the FcγRII binding level of the antibody composition is a criterion on which the product quality of the antibody composition is based. In an exemplary embodiment, the method includes determining the product quality of the antibody composition according to the method of the present disclosure using a first sample obtained at a first time point and a second sample taken at a second time point different from the first time point. In various cases, each of the first sample and the second sample is a sample of in-process material. In various aspects, the first sample is a sample of in-process material and the second sample is a sample of a manufacturing lot. Optionally, the first sample is a sample obtained before modifying one or more conditions of the cell culture, and the second sample is a sample obtained after modifying one or more conditions of the cell culture. In an exemplary case, a non-fucosylated glycan content and / or a β-galactosylated glycan content is determined for each of the first sample and the second sample. Additional samples can be obtained to determine the product quality of the antibody composition and to determine the non-fucosylated glycan content and / or the β-galactosylated glycan content. The product quality of the antibody composition depends on whether the non-fucosylated glycan content and / or the β-galactosylated glycan content is within a target range. In an exemplary embodiment, the target range of the non-fucosylated glycan content and / or the β-galactosylated glycan content is based on a reference antibody. In various embodiments, the target range of the FcγRII binding level, the target range of the non-fucosylated glycan content, and / or the target range of the β-galactosylated glycan content is based on the FcγRII binding level, the non-fucosylated glycan content, and / or the β-galactosylated glycan content of the reference antibody. In various cases, the reference antibody comprises a chimeric constant region. In an exemplary embodiment, the chimeric constant region of the reference antibody comprises a portion of an IgG2 constant region and a portion of an IgG4 constant region. In various aspects, the chimeric constant region comprises a CH1 and / or a hinge of an IgG2 and / or a CH2-CH3 of an IgG4. In an exemplary case, the chimeric constant region comprises the chimeric constant region of SEQ ID NO: 15. Optionally, the reference antibody is eculizumab.

[0069] Methods for Producing Antibody Compositions The present disclosure provides a method of producing an antibody composition. In an exemplary embodiment, the method includes determining the product quality of the antibody composition, where the product quality of the antibody composition is determined according to the method of the present disclosure. Optionally, the method includes determining the non-fucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition, where the sample is a sample of in-process material. In various cases, the method includes determining that the product quality of the antibody composition is acceptable and / or that the FcγRII binding level criterion is achieved if the non-fucosylated glycan content and / or β-galactosylated glycan content determined in (a) is within a target range as defined herein. In an exemplary embodiment, the target range of the non-fucosylated glycan content and / or β-galactosylated glycan content is based on the target range of the FcγrII binding level of a reference antibody. In various aspects, if the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (a) is not within the target range, the method further comprises (iii) modifying one or more conditions of the cell culture to obtain an altered cell culture, and (d) determining the nonfucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition obtained from the altered cell culture, optionally repeating (iii) and (d) until the nonfucosylated glycan content and / or β-galactosylated glycan content is within the target range. In various cases, the sample is a sample of a cell culture comprising cells expressing an antibody of the antibody composition. In various cases, one or more conditions of the cell culture are modified to modify the nonfucosylated glycan content and / or β-galactosylated glycan content. In various cases, the host cells or clones are selected to obtain modified non-fucosylated glycan content and / or β-galactosylated glycan content. In various aspects, the method includes modifying the AF glycan content. In an exemplary aspect, one or more conditions of the cell culture are modified to modify the AF glycan content of the antibody composition. In an exemplary aspect, the one or more conditions modify primarily the AF glycan content.In various cases, the one or more conditions modify the AF glycan content and do not modify the β-galactosylated glycan content. In exemplary embodiments, the method includes modifying the β-galactosylated glycan content. Optionally, the one or more conditions of the cell culture are modified to modify the β-galactosylated glycan content of the antibody composition. In some cases, the one or more conditions modify primarily the β-galactosylated glycan content. In some embodiments, the one or more conditions modify the β-galactosylated glycan content and do not modify the AF glycan content. In various cases, the method includes repeating the modification of the nonfucosylated (AF) glycan content and / or repeating the modification of the β-galactosylated glycan content until both the nonfucosylated glycan content and the β-galactosylated glycan content are within a target range. Finally, the method includes modifying the nonfucosylated (AF) glycan content and / or modifying the β-galactosylated glycan content until FcγRII binding (calculated or predicted) is within the target range. In various embodiments, one or more conditions of the cell culture are modified to primarily alter the HM glycan content to achieve the target range of FcγRII binding and / or one or more conditions of the cell culture are modified to primarily alter the β-galactosylated glycan content to achieve the target range of FcγRII binding.

[0070] In an exemplary embodiment, the target range is the target range of the reference antibody. For example, if the target range of the FcγRII binding level of the reference antibody is known, the target level of the non-fucosylated glycan content and / or the β-galactosylated glycan content can be calculated according to the correlation described herein. Alternatively, if the target range of the non-fucosylated glycan content of the reference antibody is known and / or the target range of the β-galactosylated glycan content of the reference antibody is known, the target range of the FcγRII binding level of the reference antibody can be calculated.

[0071] In an exemplary embodiment, the FcγRII binding level is a level of FcγRIIa binding. In an exemplary case, the FcγRIIa binding level is calculated based on a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan). In various embodiments, the FcγRII binding level is calculated according to Equation A: FcγRII binding level = m*BG%+y [Equation A] (wherein m is from about 0.535 to about 1.091, y is from about 72.58 to about 85.78, and BG% is the β-galactosylated glycan content % determined in (a). It is calculated according to:

[0072] In an exemplary case, m of Equation A is 0.813 and / or y of Equation A is 79.18. In an alternative exemplary case, m of Equation A is 0.778 and / or y of Equation A is 81.76.

[0073] In exemplary cases, the FcγRII binding level is calculated based on the determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan). The FcγRII binding level is calculated, in various cases, according to Equation B: FcγRII binding level = m*AF%+y [Equation B] (wherein m is about -13.73 to about -7.54, y is about 108.8 to about 119.1, and AF% is the non-fucosylated glycan content%) It is calculated according to:

[0074] In various embodiments, m of Equation B is −10.63 and / or y of Equation B is 114. In alternative exemplary cases, m of Equation B is −9.53 and / or y of Equation B is 114.

[0075] In an exemplary embodiment, the FcγRII binding level is based on a determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan) and a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan).

[0076] In an exemplary case, the FcγRII binding level is calculated according to Equation 3: FcγRII binding=0.576*BG%+(-4.978)*AF%+98.877 [Equation 3] (wherein BG% is the β-galactosylated glycan content % and AF% is the non-fucosylated glycan content %) This is the level within the 95% confidence interval of the line.

[0077] In an exemplary embodiment, the FcγRII binding level is a level of FcγRIIb binding. In an exemplary case, the FcγRIIb binding level is calculated based on a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan). In various embodiments, the FcγRII binding level is calculated according to Equation C: FcγRII binding level = m*BG%+y [Equation C] (In the formula, m is about 0.3260 to about 0.9697, y is about 77.72 to about 92.99, and BG% is the β-galactosylated glycan content%.) It is calculated according to:

[0078] In various cases, m of equation C is 0.648 and / or y of equation C is 85.36. In alternative exemplary cases, m of equation C is 0.644 and / or y of equation C is 86.34.

[0079] In exemplary cases, the FcγRIIb binding level is calculated based on the determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan). The FcγRII binding level is, in various cases, calculated according to Equation D: FcγRII binding level = m*AF%+y [Equation D] (wherein m is about -12.02 to about -6.247, y is about 109.3 to about 118.9, and AF% is the non-fucosylated glycan content%) It is calculated according to:

[0080] In various embodiments, m of Equation D is about −9.132 and / or y of Equation D is about 114. In alternative exemplary cases, m of Equation D is about −7.102 and / or y of Equation D is about 111.9.

[0081] In various aspects, the FcγRIIb binding level is calculated based on a determined or measured non-fucosylated glycan content (e.g., % non-fucosylated glycan) and a determined or measured β-galactosylated glycan content (e.g., % β-galactosylated glycan). In an exemplary embodiment, the FcγRII binding level is calculated based on Equation 4: FcγRII binding=0.461*BG%+(-4.429)*AF%+105.731 [Equation 4] (wherein BG% is the β-galactosylated glycan content % and AF% is the non-fucosylated glycan content %) This is the level within the 95% confidence interval of the line.

[0082] In exemplary cases, the FcγRII binding level is calculated based on a determined or measured high mannose (HM) glycan content (e.g., HM glycan %). In various aspects, the FcγRII binding level is calculated based on a determined or measured non-fucosylated glycan content (e.g., non-fucosylated glycan %), a determined or measured β-galactosylated glycan content (e.g., β-galactosylated glycan %), and a determined or measured HM glycan content (HM glycan %). In exemplary aspects, the FcγRIIa binding level of an antibody composition is calculated based on Equation 5: FcγRII binding=0.576*BG%+(-4.978)*AF%+98.877+(-1.343)*HM% [Equation 5] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0083] In an exemplary embodiment, the FcγRIIa binding level of an antibody composition is calculated according to Equation 9: FcγRII binding=0.545*BG%+(-4.466)*AF%+102.7+(-2.036)*HM% [Equation 9] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0084] In an exemplary embodiment, the FcγRIIb binding level of an antibody composition is calculated according to Equation 6: FcγRII binding=0.461*BG%+(-4.429)*AF%+105.731+(-1.883)*HM% [Equation 6] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0085] In an exemplary embodiment, the FcγRIIb binding level of an antibody composition is calculated according to Equation 10: FcγRII binding=0.590*BG%+(-2.04)*AF%+99.2+(-1.91)*HM% [Equation 10] (wherein BG% is the % β-galactosylated glycan content, AF% is the % non-fucosylated glycan content, and HM% is the % high mannose glycan content). This is the level within the 95% confidence interval of the line.

[0086] In an exemplary embodiment, a method of producing an antibody composition of the present disclosure includes: (a) determining the nonfucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition; (b) determining the FcγRII binding level of the antibody composition based on the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (a); and (c) selecting the antibody composition for downstream processing based on the level of FcγRII binding determined in (b). In various cases, an antibody of the antibody composition comprises a chimeric constant region. In an exemplary case, the chimeric constant region of an antibody of the antibody composition comprises a portion of an IgG2 constant region and a portion of an IgG4 constant region. In various aspects, the chimeric constant region comprises a CH1 and / or a hinge of an IgG2 and / or a CH2-CH3 of an IgG4. In an exemplary case, the chimeric constant region comprises a chimeric constant region of SEQ ID NO: 15.

[0087] In various cases, the antibody composition comprises an anti-C5 antibody comprising the heavy and light chains of eculizumab. Optionally, the sample is derived from a cell culture comprising glycosylation-competent cells expressing the antibodies of the antibody composition. In an exemplary embodiment, the method further comprises modifying one or more conditions of the cell culture to modify the non-fucosylated glycan content and / or the β-galactosylated glycan content of the antibody composition, and determining the non-fucosylated glycan content and / or the β-galactosylated glycan content of a sample of the antibody composition taken from the modified cell culture. In an exemplary embodiment, the method further comprises modifying one or more conditions of the cell culture to increase the level of non-fucosylated glycans of the antibody composition to decrease the level of FcγRII binding of the antibody composition, and / or modifying one or more conditions of the cell culture to decrease the level of β-galactosylated glycans of the antibody composition to decrease the level of FcγRII binding of the antibody composition. Optionally, the method further comprises modifying one or more conditions of the cell culture to reduce the level of nonfucosylated glycans of the antibody composition to increase the level of FcγRII binding of the antibody composition, and / or modifying one or more conditions of the cell culture to increase the level of β-galactosylated glycans of the antibody composition to increase the level of FcγRII binding of the antibody composition. In an exemplary embodiment, the method further comprises repeating the modifying until the nonfucosylated glycan content and / or the β-galactosylated glycan content is within a target range. In an exemplary case, the nonfucosylated glycan content and / or the β-galactosylated glycan content is determined in real time with respect to the production of the antibody composition. In an exemplary embodiment, the method comprises selecting the antibody composition for downstream processing if the nonfucosylated glycan content and / or the β-galactosylated glycan content is within a target range. Optionally, the method includes selecting the antibody composition for downstream processing if the level of FcγRII binding is within a target range, hi various cases, determining the level of FcγRII binding includes determining the level of ADCC, ADCP, and / or CDC.In various cases, the method further includes designating a level of ADCC, ADCP, and / or CDC of the antibody composition, where the selected antibody composition comprises the designated level of ADCC, ADCP, and / or CDC.

[0088] Treatment process The non-fucosylated glycan % and / or β-galactosylated glycan content % are determined (e.g., measured) to provide better information regarding the FcγRII binding level of the antibody composition. The determining (e.g., measuring) can be performed at any point in the production. In particular, the measuring can be performed before or after harvesting, for example, at any stage during downstream processing following any chromatographic unit operation, such as capture chromatography, intermediate chromatography, and / or polishing chromatography unit operations; viral inactivation and neutralization, viral filtration; and / or final formulation. The non-fucosylated glycan % and / or β-galactosylated glycan content % in various embodiments is determined (e.g., measured) in real time, near real time, and / or after the fact. The monitoring and measuring can be performed using known techniques and commercially available equipment.

[0089] In various aspects of the disclosure, the determining (e.g., measuring) of the % nonfucosylated glycan and / or % β-galactosylated glycan content is performed prior to harvesting. As used herein, the term "harvest" refers to a process in which the cell culture medium containing the recombinant protein of interest is collected and separated from at least the cells of the cell culture. Harvesting may be performed continuously. In some aspects, harvesting is performed using centrifugation and may further include precipitation, filtration, etc. In various aspects, the determining is performed after chromatography (optionally, Protein A chromatography). In various aspects, the determining is performed after harvesting and after chromatography (optionally, Protein A chromatography).

[0090] With respect to the methods of the present disclosure, the antibody composition is in various aspects selected for further processing steps (e.g., one or more downstream processing steps), which selection is based on a particular parameter (e.g., % FcγRII binding, % nonfucosylated glycan, and / or % β-galactosylated glycan content). In various cases, the methods of the present disclosure include using the antibody composition in further processing steps (e.g., one or more downstream processing steps) based on a particular parameter (e.g., % FcγRII binding, % nonfucosylated glycan, and / or % β-galactosylated glycan content). In various cases, the methods of the present disclosure include carrying out further processing steps (e.g., one or more downstream processing steps) with the antibody composition based on a particular parameter (e.g., % FcγRII binding, % nonfucosylated glycan, and / or % β-galactosylated glycan content). Optionally, these processing steps may be performed sequentially, simultaneously, and / or overlapping with each other.

[0091] In exemplary cases, the one or more downstream processing steps are any processing steps that occur after (or downstream of) the processing step in which the % nonfucosylated glycan and / or % β-galactosylated glycan content is determined (e.g., measured). For example, if the % nonfucosylated glycan and / or % β-galactosylated glycan content is determined (e.g., measured) at the time of harvesting, the one or more downstream processing steps are any processing steps that occur after (or downstream of) the harvesting step, and in various embodiments include the following: a dilution step, a loading step, a filtration step, a formulation step, a chromatography step, a viral filtration step, a viral inactivation step, or a combination thereof. Also, for example, if the % nonfucosylated glycan and / or % β-galactosylated glycan content is determined (e.g., measured) after chromatography (e.g., Protein A chromatography), the one or more downstream processing steps are any processing steps that are performed after (or downstream of) this chromatography, and in various embodiments include: a dilution step, a loading step, a filtration step, a formulation step, a further chromatography step, a viral filtration step, a viral inactivation step, or a combination thereof. In an exemplary case, the further chromatography is an ion exchange chromatography (e.g., a cation exchange chromatography or an anion exchange chromatography). Optionally, these downstream processing steps may be performed sequentially, simultaneously, and / or overlapping with each other.

[0092] Chromatographic steps / types used during downstream processing include capture or affinity chromatography, which are used to separate the recombinant product from other proteins, aggregates, DNA, viruses, and other such impurities. In an exemplary case, the initial chromatography is performed with Protein A (e.g., Protein A bound to a resin). In various embodiments, intermediate and polish chromatography further purifies the recombinant protein to remove bulk contaminants, adventitious viruses, trace impurities, aggregates, isoforms, etc. This chromatography can be performed in a bind and elute mode, where the recombinant protein of interest binds to the chromatography medium and the impurities flow through, or in a flow-through mode, where the impurities bind and the recombinant protein flows through. Examples of such chromatographic methods include: ion exchange chromatography (IEX), such as anion exchange chromatography (AEX), and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography, and gel filtration.

[0093] In various embodiments, the downstream process is a virus inactivation process. Enveloped viruses have a capsid surrounded by a lipoprotein membrane or "envelope" and are therefore susceptible to inactivation. In various instances, virus inactivation processes include heat inactivation / pasteurization, pH inactivation, UV and gamma irradiation, use of high intensity broad spectrum white light, addition of chemical inactivating agents, surfactants, and solvent / detergent treatments.

[0094] In various aspects, the downstream process is a virus filtration process. In various aspects, the virus filtration process comprises removing non-enveloped viruses. In various aspects, the virus filtration process comprises the use of a microfilter or a nanofilter.

[0095] In various embodiments, downstream processing steps include one or more formulation steps. In various embodiments, after completion of the chromatography steps, the purified recombinant protein is buffer exchanged into a formulation buffer. In an exemplary embodiment, the buffer exchange is performed using ultrafiltration and diafiltration (UF / DF). In an exemplary embodiment, the recombinant protein is buffer exchanged into a desired formulation buffer using diafiltration and concentrated to a desired final formulation concentration using ultrafiltration. In various embodiments, following the UF / DF formulation step, additional stability enhancing excipients are added.

[0096] Recombinant Glycosylated Proteins The methods of the disclosure relate to compositions comprising a recombinant glycosylated protein. In various aspects, the recombinant glycosylated protein has the formula: Asn-Xaa1-Xaa2 (wherein Xaa1 is any amino acid except Pro, and Xaa2 is Ser or Thr). The amino acid sequence includes one or more N-glycosylation consensus sequences.

[0097] In an exemplary embodiment, the recombinant glycosylated protein comprises a crystallizable fragment (Fc) polypeptide. The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides containing the hinge region that promotes dimerization. Fusion proteins that include an Fc portion (and oligomers formed therefrom) offer the advantage of easy purification by affinity chromatography using Protein A or Protein G columns. In an exemplary embodiment, the recombinant glycosylated protein comprises an Fc of an IgG (e.g., human IgG). In an exemplary aspect, the recombinant glycosylated protein comprises an Fc of an IgG1 or IgG2. In an exemplary aspect, the recombinant glycosylated protein is an antibody, an antibody protein product, a peptibody, or an Fc-fusion protein.

[0098] In an exemplary embodiment, the recombinant glycosylated protein is an antibody. As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin type, including heavy and light chains, and including a variable region and a constant region. For example, an antibody can be an IgG, which is a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having one "light" chain (typically with a molecular weight of about 25 kDa) and one "heavy" chain (typically with a molecular weight of about 50-70 kDa). An antibody has a variable region and a constant region. In the IgG type, the variable region is generally about 100-110 or more amino acids, includes three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and is substantially different from other antibodies that bind to different antigens. See, for example, Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4 th ed. Elsevier Science Ltd. / Garland Publishing, (1999).

[0099] Briefly, in the framework of an antibody, the CDRs are embedded within the framework in the variable regions of the heavy and light chains, where they constitute the regions that play a major role in antigen binding and recognition. The variable regions contain at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), which are located within framework regions (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0100] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. The embodiments of the present disclosure include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa-type or lambda-type light chain constant region, for example, a human kappa-type or human lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-type, delta-type, epsilon-type, gamma-type, or mu-type heavy chain constant region, for example, a human alpha-type, human delta-type, human epsilon-type, human gamma-type, or human mu-type heavy chain constant region. Thus, in exemplary embodiments, the antibody is of the isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4.

[0101] In an exemplary embodiment, the recombinant glycosylated protein (e.g., an antibody) comprises a chimeric constant region. In an exemplary case, the chimeric constant region of the recombinant glycosylated protein comprises a portion of an IgG2 constant region and a portion of an IgG4 constant region. In various embodiments, the chimeric constant region comprises a CH1 and / or a hinge of an IgG2 and / or a CH2-CH3 of an IgG4. In an exemplary case, the chimeric constant region comprises a chimeric constant region of SEQ ID NO: 15. The recombinant glycosylated protein can be an antibody of the antibody composition described herein.

[0102] In various aspects, the antibody can be a monoclonal antibody or a polyclonal antibody. In exemplary cases, the antibody is a mammalian antibody, such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a pig antibody, a human antibody, etc. In certain aspects, the recombinant glycosylated protein is a monoclonal human antibody.

[0103] In various embodiments, the antibody is cleaved into fragments by enzymes such as, for example, papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and one Fc fragment. Pepsin cleaves the antibody to generate a F(ab')2 fragment and a pFc' fragment. In exemplary embodiments, the recombinant glycosylated protein is an antibody fragment (e.g., Fab, Fc, F(ab')2, or pFc') that retains at least one glycosylation site. In the context of the methods of the present disclosure, the antibody may lack certain portions of the antibody and may be an antibody fragment. In various embodiments, the antibody fragment includes a glycosylation site. In some embodiments, the fragment is a "glycosylated Fc fragment" that includes at least a portion of the Fc region of the antibody that is glycosylated by post-translational modification in eukaryotic cells. In various cases, the recombinant glycosylated protein is a glycosylated Fc fragment.

[0104] The structure of antibodies has been utilized to expand the range of alternative antibody formats spanning the molecular weight range of at least or about 12-150 kDa and valency (n) range from monomers (n=1), dimers (n=2), and trimers (n=3) to tetramers (n=4) and potentially higher; such alternative antibody formats are referred to herein as "antibody protein products" or "antibody binding proteins."

[0105] The antibody protein product can be an antigen-binding format based on antibody fragments (e.g., scFv, Fab, and VHH / VH) that retain complete antigen-binding ability. The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists entirely of the variable (V) region. A soluble and flexible amino acid peptide linker is used to link the V region to a scFv (single-chain fragment variable) fragment to stabilize the molecule, or a constant (C) domain is added to the V region to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab are widely used fragments that can be easily produced in prokaryotic hosts. Other antibody protein products include disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and dimeric and multimeric antibody types, such as diabodies, triabodies, and tetrabodies, which include different types consisting of scFv linked to oligomerization domains, or minibodies (miniAbs). The smallest fragments are the VHH / VH of camelid heavy chain Abs and single domain Abs (sdAbs). The building blocks most frequently used to generate new antibody types are single chain variable (V)-domain antibody fragments (scFv), which contain V domains (VH and VL domains) from heavy and light chains linked by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted onto the Fc domain. Peptibodies are well described in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0106] Other antibody protein products include single chain antibodies (SCAs); diabodies; triabodies; tetrabodies; bispecific or trispecific antibodies, etc. Bispecific antibodies can be classified into five major classes: BsIgG, adduct IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2)Part A:97-106(2015).

[0107] In exemplary aspects, the recombinant glycosylated protein comprises any one of these antibody protein products (e.g., scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camelid heavy chain antibody, sdAb, diabody; triabody; tetrabody; bi- or triabody, BsIgG, adjunct IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate) and comprises one or more N-glycosylation consensus sequences (optionally one or more Fc polypeptides). In various aspects, the antibody protein product comprises a glycosylation site. In exemplary aspects, the antibody protein product can be a glycosylated Fc fragment conjugated to an antibody binding fragment ("glycosylated Fc fragment antibody product").

[0108] The recombinant glycosylated protein may be an antibody protein product in monomeric, or polymeric, oligomeric, or multimeric form. In certain embodiments, where an antibody comprises two or more distinct antigen-binding region fragments, the antibody is considered to be bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of distinct epitopes recognized and bound by the antibody.

[0109] In various embodiments, the recombinant glycosylated protein is a chimeric or humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody that contains a constant domain from one species and a variable domain from a second species, or more commonly, contains stretches of amino acid sequences from at least two species. The term "humanized" when used in reference to an antibody refers to an antibody with at least the CDR regions from a non-human source that have been engineered to have a structure and immunological function that is more similar to a true human antibody than the original source antibody. For example, humanization can include grafting CDRs from a non-human antibody (e.g., a murine antibody) onto a human antibody. Humanization can also include the selection of amino acid substitutions to make the non-human sequence appear more human.

[0110] Advantageously, the method is not limited to the antigen specificity of the antibody, glycosylated Fc fragment, antibody protein product, chimeric antibody, or humanized antibody. Thus, the antibody, glycosylated Fc fragment, antibody protein product, chimeric antibody, or humanized antibody can have virtually any binding specificity for any antigen. In an exemplary embodiment, the antibody binds to a hormone, growth factor, cytokine, cell surface receptor, or any ligand thereof. In an exemplary embodiment, the antibody binds to a protein expressed on the cell surface of an immune cell. In exemplary embodiments, the antibody is selected from the group consisting of CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11A, CD11B, CD11C, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31,CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61 , CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD79α, CD79β, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD 98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CD109, CD114, CD115, CD116, CD117, CD118, CD1 19, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CD125, CD126, CD127, CDw128, CD129, CD130, CDw131, CD132, CD134, CD135 , CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166 and CD182.

[0111] In exemplary embodiments, the antibody, glycosylated Fc fragment, antibody protein product, chimeric antibody, or humanized antibody is one of those described in U.S. Pat. No. 7,947,809 and U.S. Patent Publication No. 20090041784 (glucagon receptor), U.S. Pat. No. 7,939,070, U.S. Pat. No. 7,833,527, U.S. Pat. No. 7,767,206, and U.S. Pat. No. 7,786,284 (IL-17 receptor A), U.S. Pat. No. 7,872,106, and U.S. Pat. No. 7,592,429 (sclerostin), U.S. Pat. Japanese Patent No. 7871611, U.S. Patent No. 7815907, U.S. Patent No. 7037498, U.S. Patent No. 7700742 and U.S. Patent Application Publication No. 20100255538 (IGF-1 receptor), U.S. Patent No. 7868140 (B7RP1), U.S. Patent No. 7807159 and U.S. Patent Application Publication No. 20110091455 (myostatin), U.S. Patent No. 7736644, U.S. Patent No. 7628986, U.S. Patent No. 7524496 and U.S. Patent Application Publication No. 20100111979 (deletion mutants of epidermal growth factor receptor), U.S. Pat. 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Nos. 7,318,925 and 7,288,253 (parathyroid hormone), 7,285,269 (TNF), 6,692,740 and 7,270,817 (ACPL), 7,202,343 (monocyte chemotactic protein 1), 7,144,731 (SCF), 6,355,779 and 7,138,500 (4-1BB), 7,135,174 (PDGFD), 6,630,143 and 7,045,128 (Flt -3 ligand), U.S. Patent No. 6,849,450 (metalloprotease inhibitor), U.S. Patent No. 6,596,852 (LERK-5), U.S. Patent No. 6,232,447 (LERK-6), U.S. Patent No. 6,500,429 (brain-derived neurotrophic factor), U.S. Patent No. 6,184,359 (epithelial-derived T-cell factor), U.S. Patent No. 6,143,874 (neurotrophic factor NNT-1), U.S. Patent Application Publication No. 20110027287 (proprotein convertase subtilisin kexin type 9 (PCSK9)), U.S. Patent Application Publication No. 20110014201 (IL-18 RECEPTOR),and U.S. Patent Application Publication No. 20090155164 (C-FMS). The above patents and published patent applications are incorporated herein by reference in their entirety for the purposes of their disclosure of variable domain polypeptides, nucleic acids encoding variable domains, host cells, vectors, methods of making polypeptides encoding said variable domains, pharmaceutical compositions, and methods of treating diseases associated with the respective targets of the variable domain-containing antigen binding proteins or antibodies.

[0112] In exemplary embodiments, the glycosylated Fc fragment, antibody protein product, chimeric antibody, or humanized antibody is one of the following: muromonab-CD3 (product commercially available under the trade name Orthoclone Okt3®), abciximab (product commercially available under the trade name Reopro®), rituximab (product commercially available under the trade name MabThera®, Rituxan®), basiliximab (product commercially available under the trade name Simulect®), daclizumab (product commercially available under the trade name Zenapax®), palivizumab (product commercially available under the trade name Synagis®), infliximab (product commercially available under the trade name Remicade®), trachomatis (product commercially available under the trade name Telomerase®), telomerase ... rastuzumab (product marketed under the trade name Herceptin®), alemtuzumab (product marketed under the trade name MabCampath®, Campath-1H®), adalimumab (product marketed under the trade name Humira®), tositumomab-I131 (product marketed under the trade name Bexxar®), efalizumab (product marketed under the trade name Raptiva®), cetuximab (product marketed under the trade name Erbitux®), ibritumomab tiuxeta (product marketed under the trade name Zevalin®), omalizumab (product marketed under the trade name Xolair®), bevacizumab (product marketed under the trade name Avastin®), natalizumab (product marketed under the trade name Tysabri®), ranibizumab (product marketed under the trade name Lucentis®), panitumumab (product marketed under the trade name Vectibix®), eculizumab (product marketed under the trade name Soliris®), certolizumab pegol (product product marketed under the trade name Cimzia®), golimumab (product marketed under the trade name Simponi®), canakinumab (product marketed under the trade name Ilaris®), catumaxomab (product marketed under the trade name Removab®), ustekinumab (product marketed under the trade name Stelara®), tocilizumab (product marketed under the trade name RoActemra®, Actemra®), ofatumumab (product marketed under the trade name Arzerra®),denosumab (product marketed under the trade name Prolia®), belimumab (product marketed under the trade name Benlysta®), raxibacumab, ipilimumab (product marketed under the trade name Yervoy®), and pertuzumab (product marketed under the trade name Perjeta®). In exemplary embodiments, the antibody is one of the following: an anti-TNF alpha antibody, such as adalimumab, infliximab, etanercept, golimumab, and certolizumab pegol; an anti-IL1.beta antibody, such as canakinumab; an anti-IL12 / 23 (p40) antibody, such as ustekinumab and briakinumab; and an anti-IL2R antibody, such as daclizumab.

[0113] In an exemplary embodiment, the antigen of the antibody is complement protein C5 (e.g., human complement C5) and the antibody is an anti-C5 antibody (e.g., an anti-human C5 monoclonal antibody). C5 is a component of the complement system, which is part of the innate immune system. The C5 preproprotein is proteolytically processed to generate multiple protein products, including the C5 alpha chain, the C5 beta chain, the C5a anaphylatoxin, and C5b. The C5 protein is composed of the C5 alpha chain and the C5 beta chain, which are linked by disulfide bridges. The amino acid sequence of the preproprotein is set forth herein as SEQ ID NO:2, where residues 19-673 represent the sequence of the complement C5 beta chain, residues 752-1676 represent the sequence of the complement C5 alpha chain, and residues 678-751 represent the sequence of the C5a anaphylatoxin. SEQ ID NO:3 is the sequence of the mRNA sequence of transcript variant 1 encoded by the human C5 gene. In various embodiments, the antibody is eculizumab or a biosimilar thereof. The term eculizumab refers to a chimeric monoclonal antibody comprising the hinge and CH1 domains of IgG2 and the CH2 and CH3 domains of IgG4, which binds to complement protein C5 (see CAS Number: 219685-50, DrugBank Accession No. DB01257). In an exemplary embodiment, the antibody comprises a light chain comprising the CDR1, CDR2, and CDR3 of the variable region of eculizumab light chain set forth in Table A. In an exemplary embodiment, the antibody comprises a heavy chain comprising the CDR1, CDR2, and CDR3 of the variable region of eculizumab heavy chain set forth in Table A. In various cases, the antibody comprises the VH and VL of eculizumab, or comprises the VH-IgG1 kappa sequence and the VL-IgG kappa sequence.

[0114] [Table 1]

[0115] [Table 2]

[0116] In various embodiments, the antibody comprises: i. a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:4, or a variant amino acid sequence of SEQ ID NO:4 having one or two amino acid substitutions; ii. a LC CDR2 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:5, or a variant amino acid sequence of SEQ ID NO:5 having one or two amino acid substitutions; iii. a LC CDR3 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:6, or a variant amino acid sequence of SEQ ID NO:6 having one or two amino acid substitutions; iv. a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:7, or a variant amino acid sequence of SEQ ID NO:7 having one or two amino acid substitutions; v. HC CDR2 comprising the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:8, or a variant amino acid sequence of SEQ ID NO:8 having one or two amino acid substitutions; vi. A HC CDR3 comprising the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:9, or a variant amino acid sequence of SEQ ID NO:9 having one or two amino acid substitutions.

[0117] In various cases, the antibody comprises an LC variable region comprising the amino acid sequence of SEQ ID NO:10, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:10, or a variant amino acid sequence of SEQ ID NO:10 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0118] In exemplary aspects, the antibody comprises a HC variable region comprising the amino acid sequence of SEQ ID NO:11, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:11, or a variant amino acid sequence of SEQ ID NO:11 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0119] In exemplary cases, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 12, or a variant amino acid sequence of SEQ ID NO: 12 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0120] In various aspects, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:13, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:13, or a variant amino acid sequence of SEQ ID NO:13 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0121] In exemplary cases, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 14, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 14, or a variant amino acid sequence of SEQ ID NO: 14 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0122] In various aspects, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:15, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:15, or a variant amino acid sequence of SEQ ID NO:15 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0123] composition The disclosed methods relate to compositions comprising recombinant glycosylated proteins. In various embodiments, the composition comprises only one type of recombinant glycosylated protein. In various cases, the composition comprises recombinant glycosylated proteins, where each recombinant glycosylated protein of the composition comprises the same or essentially the same amino acid sequence. In various embodiments, the composition comprises recombinant glycosylated proteins, where each recombinant glycosylated protein of the composition comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of all other recombinant glycosylated proteins of the composition. In various embodiments, the composition comprises recombinant glycosylated proteins, where each recombinant glycosylated protein of the composition comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of all other recombinant glycosylated proteins of the composition. In various aspects, the composition comprises recombinant glycosylated proteins, each of which comprises an amino acid sequence that is identical or essentially identical (e.g., at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of every other recombinant glycosylated protein of the composition), although the glycoprofiles of the recombinant glycosylated proteins of the composition can differ from one another.

[0124] In an exemplary embodiment, the recombinant glycosylated protein is an antibody fragment, and thus the composition can be an antibody fragment composition.

[0125] In an exemplary embodiment, the recombinant glycosylated protein is an antibody protein product, and thus the composition can be an antibody protein product composition.

[0126] In an exemplary embodiment, the recombinant glycosylated protein is a glycosylated Fc fragment, and thus the composition may be a glycosylated Fc fragment composition.

[0127] In an exemplary embodiment, the recombinant glycosylated protein is a glycosylated Fc fragment antibody product, and thus the composition may be a glycosylated Fc fragment antibody product composition.

[0128] In an exemplary embodiment, the recombinant glycosylated protein is a chimeric antibody, and thus the composition may be a chimeric antibody composition.

[0129] In an exemplary embodiment, the recombinant glycosylated protein is a humanized antibody, and thus the composition may be a humanized antibody composition.

[0130] In an exemplary embodiment, the recombinant glycosylated protein is an antibody and the composition is an antibody composition. In various embodiments, the composition comprises only one type of antibody. In various cases, the composition comprises an antibody, and each antibody of the antibody composition comprises the same or essentially the same amino acid sequence. In various embodiments, the antibody composition comprises an antibody, and each antibody of the antibody composition comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of every other antibody of the antibody composition. In various embodiments, the antibody composition comprises an antibody, and each antibody of the antibody composition comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of every other antibody of the antibody composition. In various aspects, the antibody composition comprises antibodies, each of which comprises an amino acid sequence that is identical or essentially identical (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the protein of every other antibody of the antibody composition), but the glycoprofiles of the antibodies of the antibody composition may differ from one another. In exemplary aspects, the antibody composition comprises a known heterogeneous mixture of various glycoforms. In various cases, the antibody composition may be characterized in terms of its AF glycan content and / or its β-galactosylated glycan content. In various aspects, the antibody composition is described in terms of its % AF glycan content and / or its % β-galactosylated glycan content. Optionally, the antibody composition may be characterized in terms of the content of other types of glycans (e.g., high mannose glycoforms, fucosylated glycoforms, etc.).

[0131] In various aspects, each antibody of the antibody composition is an IgG, optionally an IgG comprising an IgG2 hinge and CH1 domain and an IgG4 CH2 and CH3 domain. In various cases, each antibody of the antibody composition binds to complement protein C5. In an exemplary aspect, each antibody of the antibody composition is an anti-C5 antibody. In various aspects, each antibody of the antibody composition comprises: i. a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:4, or a variant amino acid sequence of SEQ ID NO:4 having one or two amino acid substitutions; ii. a LC CDR2 comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:5, or a variant amino acid sequence of SEQ ID NO:5 having one or two amino acid substitutions; iii. a LC CDR3 comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:6, or a variant amino acid sequence of SEQ ID NO:6 having one or two amino acid substitutions; iv. a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:7, or a variant amino acid sequence of SEQ ID NO:7 having one or two amino acid substitutions; v. a HC CDR2 comprising the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:8, or a variant amino acid sequence of SEQ ID NO:8 having one or two amino acid substitutions; and / or vi. A HC CDR3 comprising the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:9, or a variant amino acid sequence of SEQ ID NO:9 having one or two amino acid substitutions.

[0132] In various cases, each antibody of the antibody composition comprises an LC variable region comprising the amino acid sequence of SEQ ID NO:10, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:10, or a variant amino acid sequence of SEQ ID NO:10 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0133] In an exemplary embodiment, each antibody of the antibody composition comprises an HC variable region comprising the amino acid sequence of SEQ ID NO:11, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:11, or a variant amino acid sequence of SEQ ID NO:11 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0134] In exemplary cases, each antibody of the antibody composition comprises a light chain comprising the amino acid sequence of SEQ ID NO:12, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:12, or a variant amino acid sequence of SEQ ID NO:12 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0135] In various aspects, each antibody of the antibody composition comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:13, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:13, or a variant amino acid sequence of SEQ ID NO:13 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0136] In exemplary cases, each antibody of the antibody composition comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 14, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 14, or a variant amino acid sequence of SEQ ID NO: 14 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0137] In various aspects, each antibody of the antibody composition comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:15, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:15, or a variant amino acid sequence of SEQ ID NO:15 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0138] In exemplary embodiments, the antibody composition comprises a heterogeneous mixture of antibodies of various glycoforms. In various cases, the antibody composition may be characterized in terms of its AF glycan content and / or its β-galactosylated glycan content. In various embodiments, the antibody composition is described in terms of its % AF glycan content and / or its % β-galactosylated glycan content. Optionally, the antibody composition may be characterized in terms of the content of other types of glycans (e.g., high mannose glycoforms, fucosylated glycoforms, etc.).

[0139] In an exemplary embodiment, the composition is combined with a pharma- ceutically acceptable carrier, diluent, or excipient. Thus, provided herein is a pharmaceutical composition comprising a recombinant glycosylated protein composition described herein (e.g., an antibody composition, or an antibody binding protein composition) and a pharma- ceutically acceptable carrier, diluent, or excipient. As used herein, the term "pharma- ceutically acceptable carrier" includes any of the standard pharmaceutical carriers, including, for example, phosphate buffered saline, water, emulsions (e.g., oil / water emulsions, or water / oil emulsions), and various types of wetting agents.

[0140] In an exemplary embodiment, the antibody compositions are produced by glycosylation competent cells in cell culture as described herein.

[0141] Additional Process The methods disclosed herein include additional steps in various aspects. For example, in some aspects, the methods include one or more upstream or downstream steps involved in the production, purification, and formulation of recombinant glycosylated proteins (e.g., antibodies). Optionally, the downstream steps are any one of the downstream processing steps described herein or known in the art. See, e.g., processing steps. In exemplary embodiments, the methods include steps for producing host cells that express recombinant glycosylated proteins (e.g., antibodies). The host cell, in some embodiments, is a prokaryotic host cell (e.g., E. coli or Bacillus subtilis), or the host cell, in some embodiments, is a eukaryotic host cell (e.g., a yeast cell, a filamentous fungal cell, a protozoan cell, an insect cell, or a mammalian cell (e.g., a CHO cell). Such host cells are described in the art, see, e.g., Frenzel, et al., Front Immunol 4:217 (2013) and the "Cells" section herein. For example, the method, in some cases, includes introducing into the host cell a vector that includes a nucleic acid that includes a nucleotide sequence encoding the recombinant glycosylated protein or a polypeptide chain thereof.

[0142] In an exemplary embodiment, the method includes maintaining the cells (e.g., glycosylation-competent cells) in cell culture. Thus, the method can include performing any one or more steps described herein for maintaining the cells in cell culture.

[0143] In exemplary embodiments, the methods disclosed herein include isolating and / or purifying a recombinant glycosylated protein (e.g., a recombinant antibody) from a culture. In exemplary aspects, the methods include one or more chromatography steps (such as, but not limited to, affinity chromatography (e.g., Protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography). In exemplary aspects, the methods include producing a crystalline biomolecule from a solution containing the recombinant glycosylated protein.

[0144] The methods of the disclosure, in various aspects, include one or more steps for preparing a composition (e.g., in some aspects, a pharmaceutical composition) that includes the purified recombinant glycosylated protein. Such compositions are discussed herein.

[0145] Maintaining cells in cell culture With regard to the method of producing the antibody composition of the present disclosure, the antibody composition may be produced by maintaining cells in cell culture. The cell culture may be maintained according to any set of conditions suitable for the production of recombinant glycosylated proteins. For example, in some embodiments, the cell culture is maintained at a particular pH, temperature, cell density, culture volume, dissolved oxygen level, pressure, osmolality, etc. In an exemplary embodiment, the pre-seeded cell culture is shaken (e.g., 70 rpm) in a CO2 incubator under standard humidified conditions with 5% CO2. In an exemplary embodiment, about 10 cells are cultured in 1.5 L of medium. 6 Seed the cell cultures at a seeding density of 10 cells / mL.

[0146] In exemplary embodiments, the methods of the disclosure include maintaining glycosylation competent cells in a cell culture medium at a pH of about 6.85 to about 7.05, e.g., in various embodiments, at a pH of about 6.85, about 6.86, about 6.87, about 6.88, about 6.89, about 6.90, about 6.91, about 6.92, about 6.93, about 6.94, about 6.95, about 6.96, about 6.97, about 6.98, about 6.99, about 7.00, about 7.01, about 7.02, about 7.03, about 7.04, or about 7.05.

[0147] In an exemplary aspect, the method includes maintaining the cell culture at a temperature between 30° C. and 40° C. In an exemplary embodiment, the temperature is between about 32° C. and about 38° C. or between about 35° C. and about 38° C.

[0148] In an exemplary embodiment, the method includes maintaining an osmolality of about 200 mOsm / kg to about 500 mOsm / kg. In an exemplary embodiment, the method includes maintaining an osmolality of about 225 mOsm / kg to about 400 mOsm / kg or about 225 mOsm / kg to about 375 mOsm / kg. In an exemplary embodiment, the method includes maintaining an osmolality of about 225 mOsm / kg to about 350 mOsm / kg. In various embodiments, the osmolality (mOsm / kg) is maintained at about 200, 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500.

[0149] In an exemplary embodiment, the method includes maintaining a dissolved oxygen (DO) level of the cell culture at about 20% to about 60% oxygen saturation during the initial cell culture period. In an exemplary case, the method includes maintaining a DO level of the cell culture at about 30% to about 50% (e.g., about 35% to about 45%) oxygen saturation during the initial cell culture period. In an exemplary case, the method includes maintaining a DO level of the cell culture at about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% oxygen saturation during the initial cell culture period. In an exemplary embodiment, the DO level is about 35 mmHg to about 85 mmHg, or about 40 mmHg to about 80 mmHg, or about 45 mmHg to about 75 mmHg.

[0150] The cell culture is maintained in any one or more culture media. In an exemplary embodiment, the cell culture is maintained in a medium suitable for cell growth and / or is fed with one or more feed media according to any suitable feeding schedule. In an exemplary embodiment, the method includes maintaining the cell culture in a medium comprising glucose, fucose, lactate, ammonia, glutamine, and / or glutamate. In an exemplary embodiment, the method includes maintaining the cell culture in a medium comprising manganese at a concentration of about 1 μM or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium comprising manganese at about 0.25 μM to about 1 μM. In an exemplary embodiment, the method includes maintaining the cell culture in a medium comprising negligible amounts of manganese. In an exemplary embodiment, the method includes maintaining the cell culture in a medium comprising copper at a concentration of about 50 ppb or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium comprising copper at a concentration of about 40 ppb or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 30 ppb or less during the initial cell culture period. In an exemplary embodiment, the method includes maintaining the cell culture in a medium containing copper at a concentration of about 20 ppb or less during the initial cell culture period. In an exemplary embodiment, the medium contains copper at a concentration of about 5 ppb or more or about 10 ppb or more. In an exemplary embodiment, the cell culture medium contains mannose. In an exemplary embodiment, the cell culture medium does not contain mannose.

[0151] In exemplary embodiments, the type of cell culture is fed-batch or continuous perfusion, however, the methods of the present disclosure are advantageously not limited to any particular type of cell culture.

[0152] The cell maintained in the cell culture can be a glycosylation competent cell. In an exemplary embodiment, the glycosylation competent cell is a eukaryotic cell, such as, but not limited to, a yeast cell, a filamentous fungal cell, a protozoan cell, an algae cell, an insect cell, or a mammalian cell. Such host cells are described in the art. See, for example, Frenzel, et al., Front Immunol 4:217 (2013). In an exemplary embodiment, the eukaryotic cell is a mammalian cell. In an exemplary embodiment, the mammalian cell is a non-human mammalian cell. In some embodiments, the cells are selected from the group consisting of Chinese hamster ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryonic fibroblast cells NIH 3T3, mouse fibroblast L929 cells, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human hepatocellular carcinoma cells Hep G2, mouse B myeloma cells J558L, or baby hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)).

[0153] Cells that are not glycosylation competent can also be transformed into glycosylation competent cells, for example, by introducing into them genes encoding the relevant enzymes required for glycosylation. Exemplary enzymes include, but are not limited to, oligosaccharyltransferase, glycosidase, glucosidase I, glucosidase II, calnexin / calreticulin, glycosyltransferase, mannosidase, GlcNAc transferase, galactosyltransferase, and sialyltransferase.

[0154] In an exemplary embodiment, the glycosylation competent cells are not genetically modified to alter the activity of the enzymes of the de novo or salvage pathways. These two pathways of fucose metabolism are shown in FIG. 2. In an exemplary embodiment, the glycosylation competent cells are not genetically modified to alter the activity of any one or more of fucosyl-transferases (FUTs, e.g., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), fucose kinase, GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In an exemplary embodiment, the glycosylation competent cells are not genetically modified to knock out the gene encoding FX.

[0155] In exemplary embodiments, the glycosylation competent cells have not been genetically modified to alter the activity of β(1,4)-N-acetylglucosaminyltransferase III (GNTIII) or GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). In exemplary aspects, the glycosylation competent cells have not been genetically modified to overexpress GNTIII or an RMD. EXAMPLES

[0156] The following examples are presented merely to illustrate embodiments of the present invention and are not intended to limit the scope of the invention in any way.

[0157] Example 1 This example describes an exemplary method for determining the N-linked glycosylation profile (glycan profile) of a monoclonal antibody.

[0158] The objective of this analytical method is to determine the N-linked glycosylation profile of an antibody in an antibody-containing sample by hydrophilic interaction liquid chromatography (HILIC) ultra-high performance liquid chromatography (UHPLC) glycan map analysis, which is a quantitative analysis of the N-linked glycan distribution of an antibody, and involves releasing and labeling N-linked glycans from reference and test samples using PNGase F and a fluorophore that can specifically derivatize the released glycans, loading the samples within the effective linear range onto a HILIC column, separating the labeled N-linked glycans using a decreasing organic solvent gradient, and monitoring the elution of the glycan species using a fluorescence detector.

[0159] Standards and test samples are prepared by: (1) diluting samples and controls with water, (2) adding PNGase F to the samples and controls and incubating to release N-linked glycans, (3) mixing with a fluorophore-labeled solution using a fluorophore such as 2-aminobenzoic acid, vortexing and incubating samples and controls, (4) centrifuging to pellet the protein and removing the supernatant, and (5) drying and reconstituting the labeled glycans in injection solution.

[0160] The solutions used in this assay are Mobile Phase A (100 mM ammonium formate, target pH 3.0) and Mobile Phase B (acetonitrile). The equipment used to carry out the steps of this method has the following capabilities:

[0161] [Table 3]

[0162] Hydrophilic Interaction Analysis The HPLC instrument settings using a BEH Glycan 1.7 μm column (2.1 mm ID×150 mm) and 2-aminobenzoic acid fluorophore labeling method are shown below.

[0163] [Table 4]

[0164] An example of a mobile phase gradient is shown below.

[0165] [Table 5]

[0166] The results report will be structured in the following format:

[0167] [Table 6]

[0168] Examples of representative glycan map chromatograms are shown in Figure 2A (full-scale view) and Figure 2B (enlarged-scale view).

[0169] Example 2 In this example, an exemplary FcγRIIa binding assay is described.

[0170] To measure the FcγRIIa binding activity of antibody-containing samples, an FcγRIIa binding assay was developed using a Biacore T200 (GE Healthcare). A schematic of the assay is shown in FIG. 3. In this assay, serially diluted samples of antibody 1, an antibody against human complement C5 with an IgG2 / IgG4 hybrid Fc domain, were used for capture on a Protein A sensor chip (Series S Sensor Chip Protein A; GE Healthcare). Binding to the isoform of FcγRIIa containing His at amino acid position 131 (hereafter referred to as "FcγRIIa-H") was detected by injecting a constant concentration of FcγRIIa-H over the antibody-containing surface captured with Protein A. Binding data were fitted to a linear model using statistical software PLA3.0, and the relative binding percentage of the samples was calculated compared to the binding level of the antibody 1 reference standard (Ab1 RS).

[0171] This FcγRIIa binding assay was analyzed for method linearity, intermediate precision, and accuracy. For this experiment, five mock activity samples (60%, 80%, 100%, 130%, and 160% levels) were prepared using Ab 1 RS (49.8 mg / mL) or Ab 2 (10.1 mg / mL). A sample with 100% nominal level was also used as an assay control.

[0172] The linearity of the method, i.e., the ability of the method to obtain results that are directly proportional to the concentration of analyte in the sample, was established. Five simulated binding levels (60, 80, 100, 130, and 160%) were evaluated in six independent assays. For samples with binding levels ranging from 60 to 160%, a linear relationship between expected and observed natural log (Ln) binding levels was demonstrated. The slope, Y-intercept, and R 2 The observed values ​​for were 0.9908, 0.0473, and 0.9998, respectively.

[0173] Data obtained from the linearity experiment was used to determine the intermediate precision and accuracy of the binding assay. The intermediate precision of the method was estimated to be 1.1%, and the precision of the method across all binding levels was observed to be 100.5%.

[0174] The reproducibility of the FcγIIa binding assay was determined by testing four independently prepared Ab 1 RS at 100% nominal level. Four independently prepared samples at 1× nominal concentration were tested by two analysts in a total of six assays. The overall CV% for the reproducibility of this assay is 1.1%.

[0175] The specificity of the FcγRIIa binding assay was also assessed as follows: Ab1 (100 nM) was captured on flow cell 2 of a Protein A sensor chip, and 200 nM FcγRIIa-H was injected over flow cell 1 (without Ab1) and flow cell 2 (with Ab1). The sensorgrams showed that FcγRIIa-H specifically bound to Ab1 captured on the Protein A chip, and only background signal was detected for the Protein A chip without Ab1.

[0176] These results confirm that the FcγRIIa binding assay was qualified with respect to method reproducibility, linearity, precision and accuracy over the binding range of 60% to 160%.

[0177] Example 3 In this example, a correlation between FcγRIIa binding and glycan content is demonstrated.

[0178] Antibody 1 is an antibody against human complement C5 with a hybrid IgG2 / IgG4 Fc domain. Antibody 1 has the amino acid sequence of eculizumab, an antibody approved in the United States and Europe for the treatment of paroxysmal nocturnal hemoglobinuria (PIN) and atypical hemolytic uremic syndrome (aHUC). The glycan profile of various samples containing Antibody 1 was determined according to the procedure described in Example 1. These samples were also characterized for FcγRIIa binding activity by carrying out the assay described in Example 2.

[0179] Table 1 lists the measured amounts of high mannose (HM) glycans, β-galactosylated glycans, and non-fucosylated glycans, as well as the measured FcγRIIa binding activity (expressed as % relative binding).

[0180] [Table 7]

[0181] The measured data for FcγRIIa binding, HM content, β-galactosylated content, and nonfucosylated content were analyzed using the JMP suite of computer programs for statistical analysis (SAS Institute, Cary, NC). The results are shown in Figures 4A-4C, where Figure 4A is an FcγRIIa binding leverage plot for β-galactosylated glycans, Figure 4B is a leverage plot for nonfucosylated glycans, and Figure 4C is a leverage plot for HM glycans. The best fit line for each graph is shown as a dark red line. As shown in these figures, β-galactosylation content, nonfucosylated glycan content, and high mannose content were each associated with FcγRIIa binding, and each association was statistically significant (p<0.0001 for β-galactosylated glycan; p=0.0002 for nonfucosylated glycan; and p=0.0142 for high mannose). The relationship between % FcγRIIa binding for Antibody 1 and β-galactosylation content, nonfucosylated glycan content, and high mannose content is calculated using Equation 1: Predicted FcγRIIa binding% = 98.877 + (0.576 * β-galactosylated glycan%) + (-4.978 * non-fucosylated glycan% + (-1.343 high mannose glycan%) [Equation 1] This can be explained as follows.

[0182] The measured values ​​for % β-galactosylated glycan, % nonfucosylated glycan, and % high mannose glycan were substituted into Equation 1 to calculate the predicted % FcγRIIa binding value for each sample, shown in Table 1. The actual % FcγRIIa binding (as measured by the FcγRIIa binding assay) was plotted against the predicted % FcγRIIa binding (as calculated by Equation 1), and this plot is provided as Figure 4D. Figure 4D also shows the root mean square error (RMSE), r 2 Statistical parameters including (RSq) and p-values ​​are also provided. These results suggest that Equation 1 accurately predicts actual (measured) FcγRIIa binding and highlights a statistically significant direct correlation (p<0.0001) between β-galactosylated glycans, nonfucosylated glycans, high mannose glycans, and FcγRIIa binding. Higher levels of β-galactosylated glycans and lower levels of nonfucosylated and high mannose glycans are associated with higher FcγRIIa binding activity. The leverage of β-galactosylated glycans and nonfucosylated glycans were very similar (p<0.0001 and p<0.0002, respectively).

[0183] The measured data of FcγRIIa binding, β-galactosylation content, nonfucosylated glycan content, and HM content were also analyzed using GraphPad Prism software (GraphPad, San Diego, CA) for statistical analysis. The results are shown in Figures 4E-4G, with the equation of the regression line (shown as a dashed line) and the 95% confidence interval (shown as a light blue area) shown in each figure. As shown in these figures, most of the data points fell within the 95% confidence interval of Figures 4E and 4F. As shown in Figure 4G, this confidence interval is much wider than that of Figures 4E and 4F. The ranges of the slopes and y-intercepts of the equations of Figures 4E and 4F are shown in Table 2 below.

[0184] [Table 8]

[0185] Based on the data set used in this study, FcγRIIa binding of antibody compositions can be predicted by measuring β-galactosylation content, non-fucosylated glycan content, and / or HM content. This data supports the strong influence of β-galactosylation content and non-fucosylated glycan content on FcγRIIa binding. Thus, only measuring β-galactosylation content and non-fucosylated glycan content can predict FcγRIIa binding of antibody compositions. The data in Figures 4E-4F support that only measuring β-galactosylation content of non-fucosylated glycan content can be sufficient to reasonably predict FcγRIIa binding. Data points within the 95% confidence interval will reasonably predict FcRIIa binding of antibody compositions.

[0186] Example 4 In this example, an exemplary FcγRIIb binding assay is described.

[0187] To measure FcγRIIb binding of antibody-containing samples, an FcγRIIb binding assay was developed using a Biacore T200 (GE Healthcare). In this assay, samples with various serial dilutions of antibody 1 were used for capture on a protein A sensor chip (Series S Sensor Chip Protein A; GE Healthcare). Binding to human FcγRIIb-GST-H6 recombinant protein (hereafter referred to as "FcγRIIB-GST-H6") was detected by injecting a constant concentration of FcγRIIb-GST-H6 over a surface containing antibody captured with protein A. Binding data were fitted to a linear model using statistical software PLA3.0, and the relative binding percentage of the samples was calculated compared to the binding level of the test antibody 1 reference standard (Ab1 RS).

[0188] Example 5 In this example, a correlation between FcγRIIb binding and glycan content is demonstrated.

[0189] The glycan profile for various samples containing Antibody 1 was determined according to the procedure described in Example 1. These samples were also characterized for FcγRIIb binding activity by performing the assay described in Example 4.

[0190] Table 3 lists the measured amounts of high mannose (HM) glycans, β-galactosylated glycans, and non-fucosylated glycans, as well as the measured FcγRIIb binding activity (expressed as % relative binding).

[0191] [Table 9]

[0192] The measured data on FcγRIIb binding, high mannose content, β-galactosylated content, and nonfucosylated glycan content were analyzed using the JMP suite of computer programs for statistical analysis (SAS Institute, Cary, NC). The results are shown in Figures 5A-5C, where Figure 5A is an FcγRIIb binding leverage plot of β-galactosylated glycans, Figure 5B is a leverage plot of nonfucosylated glycans, and Figure 5C is a leverage plot of HM glycans. The best-fit line for each graph is shown as a dark red line. As shown in these figures, β-galactosylated content and nonfucosylated glycan content were associated with FcγRIIb binding, and each association was statistically significant (p=0.0258 for β-galactosylated glycans; p=0.0600 for nonfucosylated glycans). As shown in Figure 5C, the association between HM content and FcγRIIb binding was not statistically significant (p=0.1533). The relationship between % FcγRIIb binding for Antibody 1 and β-galactosylated content and non-fucosylated glycan content was calculated using Equation 2: Predicted FcγRIIb binding% = 105.731 + (0.461 * β-galactosylated glycan%) + (-4.429 * non-fucosylated glycan% + (-1.883) HM glycan%) [Equation 2] This can be explained as follows.

[0193] The measured values ​​for % β-galactosylated glycans and % nonfucosylated glycans were substituted into Equation 2 to calculate the predicted % FcγRIIb binding value for each sample, shown in Table 3. The actual % FcγRIIb binding (as measured by the FcγRIIb binding assay) was plotted against the predicted % FcγRIIb binding (as calculated by Equation 2), and this plot is provided as Figure 5D. Figure 5D also shows the root mean square error (RMSE), r 2Statistical parameters including, p-value and p-value are also provided. These results suggest that Equation 2 accurately predicts actual (measured) FcγRIIb binding and highlights a clear correlation between β-galactosylated glycans, nonfucosylated glycans, and FcγRIIb binding. Higher levels of β-galactosylated glycans and lower levels of nonfucosylated glycans are associated with higher FcγRIIb binding activity. The leverage of β-galactosylated glycans and nonfucosylated glycans were similar (p=0.0258 and p=0.0600, respectively).

[0194] The measured data on FcγRIIb binding, HM content, β-galactosylated content, and nonfucosylated glycan content were also analyzed using GraphPad Prism software (GraphPad, San Diego, CA) for statistical analysis. The results are shown in Figures 5E-5G, with the equation of the regression line (shown as a dashed line) and the 95% confidence interval (shown as a light blue area) shown in each figure. As shown in these figures, most of the data points fell within the 95% confidence intervals in Figures 5E and 5F. The slopes and y-intercept ranges of the equations in Figures 5E and 5F are shown in Table 4 below.

[0195] [Table 10]

[0196] Based on the data set used in this study, FcγRIIb binding of antibody compositions can be predicted by measuring β-galactosylation content and non-fucosylated glycan content. The data in Figures 5E-5F confirm that FcγRIIb binding can be predicted with reasonable confidence by simply measuring the β-galactosylation content of non-fucosylated glycan content. Data points within the 95% confidence interval would be reasonably predictive of FcRIIb binding of antibody compositions.

[0197] Example 6 In this example, a correlation between FcγRII binding and glycan content is demonstrated.

[0198] The experiments and analyses of Examples 1-5 were carried out on additional samples containing Antibody 1. Briefly, the glycan profile of samples containing Antibody 1 was determined according to the procedures described in Example 1. These samples were also characterized with respect to FcγRIIa and FcγRIIb binding activity by carrying out the assays described in Examples 2 and 4, respectively.

[0199] Table 5 lists the measured amounts of high mannose (HM) glycans, β-galactosylated (β-gal) glycans, and non-fucosylated (afuco) glycans, as well as the measured FcγRIIa and FcγRIIb binding activities (each expressed as % relative binding) for previously analyzed samples (sample ID numbers 1-11) and additional samples (sample ID numbers 12-19).

[0200] [Table 11]

[0201] The data for measured FcγRIIa binding, measured FcγRIIb binding, high mannose content, β-galactosylated content, and nonfucosylated glycan content in Table 5 were analyzed using the JMP suite of computer programs for statistical analysis (SAS Institute, Cary, NC). The results are shown in Figures 7A-7C and 8A-8C, where Figure 7A is an FcγRIIa binding leverage plot of β-galactosylated glycans, Figure 7B is an FcγRIIa binding leverage plot of nonfucosylated glycans, Figure 7C is an FcγRIIa binding leverage plot of HM glycans, Figure 8A is an FcγRIIb binding leverage plot of β-galactosylated glycans, Figure 8B is an FcγRIIb binding leverage plot of nonfucosylated glycans, and Figure 8C is an FcγRIIb binding leverage plot of HM glycans. The best fit line for each graph is shown as a dark red line.

[0202] As shown in Figures 7A-7B, β-galactosylation content and nonfucosylated glycan content were associated with FcγRIIa binding, and each association was statistically significant (p<0.0001 for β-galactosylated glycans; p=0.0033 for nonfucosylated glycans). As shown in Figure 7C, the association between HM content and FcγRIIa binding was also statistically significant (p=0.0035). The relationship between % FcγRIIa binding for Antibody 1 and β-galactosylation content, nonfucosylated glycan content, and HM content is calculated using Equation 7: Predicted FcγRIIa binding% = 102.704 + (0.545 * β-galactosylated glycan%) + (-4.466 * non-fucosylated glycan% + (-2.0356) HM glycan% [Equation 7] This can be explained as follows.

[0203] The measured values ​​for β-galactosylated glycan %, nonfucosylated glycan %, and HM glycan in Table 5 were substituted into Equation 7 to calculate the predicted % FcγRIIa binding value for each sample. The predicted % FcγRIIa binding values ​​are also presented in Table 5. The actual % FcγRIIa binding (as measured in the FcγRIIa binding assay) was plotted against the predicted % FcγRIIa binding (as calculated by Equation 7), and this plot is provided as Figure 7D. Figure 7D also shows the root mean square error (RMSE), r 2 Statistical parameters including, , and p-values ​​are also provided. These results confirm that Equation 7 accurately predicts actual (measured) FcγRIIa binding and highlights the clear correlation between β-galactosylated glycans, non-fucosylated glycans, HM glycans and FcγRIIa binding. Higher levels of β-galactosylated glycans and lower levels of non-fucosylated and HM glycans are associated with higher FcγRIIa binding activity. The leverage of each glycan group was similar to each other.

[0204] As shown in Figures 8A and 8C, β-galactosylation content and HM glycan content were associated with FcγRIIb binding, and each association was statistically significant (p<0.0001 for β-galactosylated glycans; p=0.0024 for HM glycans). As shown in Figure 8B, nonfucosylated glycan content tended to be associated with FcγRIIb binding (p=0.0947). The relationship between % FcγRIIb binding for Antibody 1 and β-galactosylation content, nonfucosylated glycan content, and HM content is calculated using Equation 8: Predicted FcγRIIb binding% = 99.211 + (0.590 * β-galactosylated glycan%) + (-2.04 * non-fucosylated glycan% + (-1.911) HM glycan% [Equation 8] This can be explained as follows.

[0205] The measured values ​​for β-galactosylated glycan %, nonfucosylated glycan %, and HM glycan in Table 5 were substituted into Equation 8 to calculate the predicted % FcγRIIb binding value for each sample. The predicted % FcγRIIb binding values ​​are also presented in Table 5. The actual % FcγRIIb binding (as measured in the FcγRIIb binding assay) was plotted against the predicted % FcγRIIb binding (as calculated by Equation 8), and this plot is provided as Figure 8D. Figure 8D also shows the root mean square error (RMSE), r 2 Statistical parameters including, , and p-values ​​are also provided. These results confirm that Equation 8 accurately predicts actual (measured) FcγRIIb binding and highlights the clear correlation between β-galactosylated glycans, non-fucosylated glycans, HM glycans and FcγRIIb binding. Higher levels of β-galactosylated glycans and lower levels of non-fucosylated and HM glycans are associated with higher FcγRIIb binding activity. The leverage of each glycan group was similar to each other.

[0206] The data in Table 5 for measured FcγRIIa binding, as well as measured FcγRIIb binding, measured HM content, β-galactosylated content, and non-fucosylated glycan content were further analyzed using GraphPad Prism software (GraphPad, San Diego, Calif.) for statistical analysis. The results are shown in Figures 7E-7G and 8E-8G, where Figure 7E is a graph plotting FcγRIIa binding as a function of β-galactosylation content, Figure 7F is a graph plotting FcγRIIa binding as a function of non-fucosylation content, Figure 7G is a graph plotting FcγRIIa binding as a function of HM content, Figure 8E is a graph plotting FcγRIIb binding as a function of β-galactosylation content, Figure 8F is a graph plotting FcγRIIb binding as a function of non-fucosylation content, and Figure 8G is a graph plotting FcγRIIb binding as a function of HM content. In each figure, the equation of the regression line (shown as a dashed line) and the 95% confidence interval (shown as a light blue region) are shown.

[0207] Based on the data set used in this study, FcγRIIa binding of antibody compositions can be predicted by measuring β-galactosylation content, non-fucosylated glycan content, and HM content. The data in Figures 7E-7G confirm that FcγRIIa binding can be predicted with reasonable confidence by measuring these glycans. Data points within the 95% confidence interval would reasonably predict FcRIIa binding of antibody compositions. Similar observations were made with respect to measured FcγRIIb binding and glycan content. Based on the data set used in this study, FcγRIIb binding of antibody compositions can be predicted by measuring β-galactosylation content, non-fucosylated glycan content, and HM content. The data in Figures 8E-8G confirm that FcγRIIb binding can be predicted with reasonable confidence by measuring these glycans. Data points within the 95% confidence interval would reasonably predict FcRIIb binding of antibody compositions.

[0208] All references cited in this specification, including publications, patent applications, and patents, are incorporated by reference herein to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0209] The use of the terms "a," "an," and "the" and similar referents with respect to the description of this disclosure (and particularly with respect to the claims below) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms including the specified components but not excluding other elements (i.e., meaning "including, but not limited to"), unless otherwise specified.

[0210] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value and each endpoint falling within the range, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0211] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended only to further clarify the disclosure and does not impose limitations on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0212] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the present disclosure. Variations of those preferred embodiments may become apparent to those skilled in the art upon reading the above description. The inventors anticipate that those skilled in the art will adopt such variations as necessary, and the inventors intend for the present disclosure to be practiced in other forms than those specifically described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements is encompassed in the present disclosure in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. A method for determining product quality of an antibody composition, wherein the product quality is based on the Fcγ receptor II (FcγRII) binding level of the antibody composition, the method comprising: a. determining the nonfucosylated glycan content and / or the β-galactosylated glycan content of a sample of the antibody composition; and b. Determining that the product quality of the antibody composition is acceptable if (i) the nonfucosylated glycan content and / or β-galactosylated glycan content is within a target range, and / or (ii) the FcγRII binding level is within a target range. A method comprising:

2. The method of claim 1, wherein the target range of FcγRII binding level, the target range of nonfucosylated glycan content, and / or the target range of β-galactosylated glycan content are based on the FcγRII binding level, nonfucosylated glycan content, and / or β-galactosylated glycan content of a reference antibody.

3. The method described in claim 1 or 2, further comprising calculating an FcγRII binding level based on the non-fucosylated glycan content and / or β-galactosylated glycan content determined in (a).

4. 4. A method for monitoring the product quality of an antibody composition, comprising determining the product quality of the antibody composition according to the method of any one of claims 1 to 3 using a first sample obtained at a first time point and a second sample taken at a second time point different from the first time point.

5. 10. A method for producing an antibody composition, comprising determining a product quality of the antibody composition, wherein the product quality of the antibody composition is determined according to the method of any one of claims 1 to 4, wherein the sample is a sample of in-process material, and if the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (a) is not within the target range, the method further comprises: (d) modifying one or more conditions of the cell culture to obtain an engineered cell culture; and (e) determining the nonfucosylated glycan content and / or β-galactosylated glycan content of the sample of antibody composition obtained from the engineered cell culture, optionally repeating steps (d) and (e) until the nonfucosylated glycan content and / or β-galactosylated glycan content is within the target range.

6. 1. A method for producing an antibody composition, comprising: a. determining the nonfucosylated glycan content and / or the β-galactosylated glycan content of a sample of said antibody composition; b. determining the FcγRII binding level of the antibody composition based on the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (a); and c. selecting the antibody composition for downstream processing based on the level of FcγRII binding determined in (b). A method comprising:

7. The antibody composition comprises an anti-C5 antibody comprising the heavy chain and light chain of eculizumab; b. the sample is derived from a cell culture comprising glycosylation-competent cells expressing the antibodies of the antibody composition; and / or c) the method further comprises modifying one or more conditions of the cell culture to modify the nonfucosylated glycan content and / or β-galactosylated glycan content of the antibody composition, and determining the nonfucosylated glycan content and / or β-galactosylated glycan content of a sample of the antibody composition obtained from the modified cell culture. The method according to any one of claims 1 to 6.

8. (a) modifying one or more conditions of the cell culture to increase the level of nonfucosylated glycans of the antibody composition and decrease the level of FcγRII binding of the antibody composition; b. modifying one or more conditions of the cell culture to reduce the level of β-galactosylated glycans of the antibody composition and thereby reduce the level of FcγRII binding of the antibody composition; c. modifying one or more conditions of the cell culture to reduce the level of nonfucosylated glycans of the antibody composition and increase the level of FcγRII binding of the antibody composition; d. modifying one or more conditions of the cell culture to increase the level of β-galactosylated glycans of the antibody composition to increase the level of FcγRII binding of the antibody composition; and / or e. repeating the modification until the nonfucosylated glycan content and / or the β-galactosylated glycan content is within a target range. The method of claim 7 further comprising:

9. (a) determining the nonfucosylated glycan content and / or the β-galactosylated glycan content in real time with respect to production of the antibody composition; b. the method includes selecting the antibody composition for downstream processing if the nonfucosylated glycan content and / or the β-galactosylated glycan content is within a target range; c. the method comprises selecting the antibody composition for downstream processing if the FcγRII binding level is within a target range; d. determining the level of FcγRII binding comprises determining the level of ADCC, ADCP, and / or CDC; and / or e. the method further comprises assigning a level of ADCC, ADCP, and / or CDC of the antibody composition, wherein the selected antibody composition comprises the assigned level of ADCC, ADCP, and / or CDC. The method according to any one of claims 1 to 8.

10. A method for determining the level of Fcγ receptor II (FcγRII) binding of an antibody composition, comprising determining the level of nonfucosylated glycans and / or β-galactosylated glycans of the antibody composition.

11. A method for predicting the in vivo efficacy and / or side effects of an antibody composition, comprising: a. determining the nonfucosylated glycan content and / or the β-galactosylated glycan content of a sample of said antibody composition; b. predicting the antibody composition as a cause of adverse effects in vivo based on the nonfucosylated glycan content and / or β-galactosylated glycan content determined in (a). A method comprising:

12. The method of any one of claims 1 to 11, wherein the antibody composition comprises a chimeric constant region.

13. the chimeric constant region comprises a portion of an IgG2 constant region and a portion of an IgG4 constant region; and / or the chimeric constant region comprises IgG2 CH1 and / or IgG4 CH2-CH3; The method of claim 12.

14. The method of claim 13 , wherein the antibody composition comprises a chimeric constant region comprising the amino acid sequence of SEQ ID NO:

15.

15. The method of any one of claims 1 to 14, wherein the antibody comprises eculizumab.

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