Methods of determining glycan pairing content

Abstract

Methods of determining glycan pairing content of a protein composition are provided. In exemplary embodiments, the method comprises (a) treating a sample of the protein composition with (i) a mannosidase and (ii) a β1-4 endoglycosidase, to produce a mixture of components of the protein, wherein at least some of the components comprises one or more digested glycan structures; and (b) separating the components of the mixture based on molecular weight; and (c) quantifying the abundance of glycan pairs, optionally, quantifying (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans, of the mixture. In exemplary aspects, the method comprises quantifying the relative unpaired afucosylated (AF) glycan content and / or relative unpaired high mannose (HM) glycan content of the mixture.

Description

METHODS OF DETERMINING GLYCAN PAIRING CONTENTCROSS-REFERENCE TO RELATED APPLICATION

[0001] The benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 711,032 filed on October 23, 2024, is hereby claimed, and the disclosure thereof is hereby incorporated by reference herein.BACKGROUND

[0002] Glycosylation plays a role in multiple cellular functions, including, for example, protein folding, quality control, molecular trafficking and sorting, and cell surface receptor interaction. The glycan structure of a protein drug affects its therapeutic efficacy, impacting its bioactivity, pharmacokinetics, immunogenicity, solubility, and in vivo clearance. Fc glycoform profiles are product quality attributes for recombinant antibodies, since they directly impact the clinical efficacy and pharmacokinetics of the antibodies. See, e.g., Reusch and Tejada, Glycobiology 25(12): 1325-1334 (2015); and Boune et al., Antibodies (Basel) 9(2): 22 (2020).

[0003] Given their importance, the glycan structures attached to recombinant protein drugs are often monitored during development and manufacturing (Li et al., Front Immunol 8: 1554 (2017). Traditionally, glycans are monitored using a released glycan assay, in which glycans are cleaved from the protein backbone, chromatographically separated, and identified. Techniques used for the analysis of immunoglobulin glycosylation are reviewed in de Haan et al., Glycobiology 30(4): 226-240 (2020). The glycan population of IgG therapeutics is innately heterogenous and complex, as glycosylation requires the coordinated functions of multiple metabolic enzymes and is complicated by the linkage of sugar isomers, the glycosylation site occupancy, as well as the outer arm sugar addition, which can include one or more of fucose, galactose, bisecting GIcNAc, and sialic acid. Further diversification of the antibody glycan population is due to the asymmetry of the two N-glycans from every heavy chain and the involvement of random pairing of two different heavy chain glycans.

[0004] Even when successful, the current methods used for glycan analysis ignore the fact that many therapeutic protein molecules, including IgG and fusion proteins with IgG Fc region, contain two glycosylated Fc chains. Because the current commonly used methods include cleavage of the glycans from the protein backbone, such methods fail to impart knowledge on the pairing status of the glycan species within individual protein molecules. For example, while current methods may be used to determine the overall abundance of afucosylated glycans in a given antibody composition sample, it does not inform the level of antibodies comprising a fully afucosylated pair of heavy chains vs. comprising only a hemi-afucosylated pair of heavy chains. Such information on pairingstatus of the protein is suggested to be an important feature of an antibody composition that influences its biological activity level (see, e.g., International Patent Application Publication No. WO 2022 / 081824).

[0005] Glycan pairing analysis is challenging due to the size of the molecules and the highly heterogeneous glycan population. The analysis of specifically afucosylated glycan pairs is further complicated by the low abundance of afucosylated and high mannose glycans relative to other glycans. Accordingly, determining the abundance of afucosylated glycan pairs and teasing out the relative abundance of fully afucosylated pairs or hemi-afucosylated pairs is complex and timeconsuming.

[0006] The same challenges complicating the glycan analysis of fully afucosylated vs hemi-afucosylated glycan pairs also apply to high mannose glycan pairs, as high mannose glycan pairs are often in low abundance, relative to other glycans. The current methods fail to inform the level of antibodies comprising a pair of heavy chains comprising high mannose glycans on one or both chains, and such information on high mannose pairing status may influence the antibodies' biological activity. Thus, there is a need in the biopharmaceutical industry for simple, efficient and reliable methods of determining glycan pairing content of an IgG antibody composition.SUMMARY

[0007] Presented herein are data demonstrating the feasibility and advantages of an efficient method of determining the glycan pairing content, e.g., relative unpaired afucosylated glycan content, relative paired afucosylated glycan content, relative paired high mannose glycan content, and / or relative unpaired high mannose glycan content, of a protein composition. The method comprises preparing a simplified sample using a mannosidase which reduces the heterogeneity of low abundance high mannose glycans. The method further comprises analyzing the simplified sample through separation-based techniques, such as mass spectrometry. This method provides for an easier, faster, and more accurate way of determining the glycan pairing content of a protein composition.

[0008] Accordingly, the present disclosure provides methods of determining the glycan pairing content of a protein composition comprising a protein comprising an Fc region comprising a glycan pair. In exemplary embodiments, the method comprises (a) treating a sample of the protein composition with (i) a mannosidase and (ii) a pi-4 endoglycosidase, to produce a mixture of components of the protein, wherein at least some of the components comprises one or more digested glycan structures; and (b) separating the components of the mixture based on molecular weight; and (c) quantifying the abundance of glycan pairs of the mixture to determine the glycanpairing content of the protein composition. In exemplary aspects, the mannosidase cleaves an al-2 linkage, al-3 linkage, and / or al-6 linkage of a high mannose glycan. In exemplary aspects, the mannosidase is an al-2, 3, 6 mannosidase. In exemplary instances, the mannosidase is an al-6 mannosidase. In various aspects, the mannosidase is an al-2 mannosidase. In various instances, the mannosidase is an al-2, 3 mannosidase. In exemplary aspects, the method comprises treating the sample with two mannosidases. In some aspects, the method comprises treating the sample with an al-2, 3, 6 mannosidase and an al-6 mannosidase. In exemplary instances, the method comprises treating the sample with each mannosidase for less than 4 hours, optionally, less than 3 hours and / or more than 1 hour. The method comprises treating the sample with each mannosidase for about 2 hours in some aspects. In various aspects, the method comprises treating the sample with a single mannosidase, e.g., an al-2 mannosidase or an al-2, 3 mannosidase. In various instances, the method comprises treating the sample with the mannosidase for less than 30 hours, optionally, about 24 hours. In exemplary aspects, none of the digested glycan structures of the components in the mixture produced in (a) comprises six or more mannosyl residues. In various aspects, the digested glycan structures of the components in the mixture produced in (a) comprise less than 6 mannosyl residues or 5 or fewer mannosyl residues. In various instances, the digested glycan structures of the components in the mixture produced in (a) comprise zero, one, three or five mannosyl residues. In various aspects, the digested glycan structures of the components in the mixture produced in (a) comprises zero or one mannosyl residues. In various aspects, the digested glycan structures of the components in the mixture produced in (a) comprises zero or three mannosyl residues. In various aspects, the digested glycan structures of the components in the mixture produced in (a) comprises zero or five mannosyl residues. In exemplary instances, when a digested glycan structure comprises one, three, or five mannosyl residues, the digested glycan structure further comprises two core GIcNac residues. In exemplary instances, when a digested glycan structure comprises zero mannosyl residues, the digested glycan structure further comprises only one core GIcNac residue. The sample is treated with one or more mannosidases after the sample is treated with the pi-4 endoglycosidase in various aspects. In exemplary aspects, the pi-4 endoglycosidase is an Endo S endoglycosidase. In various aspects, the method comprises treating the sample with the endoglycosidase for less than 1 hour, optionally, about 30 minutes. In exemplary instances, the method comprises separating the components of the mixture based on molecular weight which comprises performing mass spectrometry to obtain one or more mass spectral peaks. In various aspects, the method comprises deconvoluting the mass spectral peaks to obtain deconvoluted mass spectral peaks. The method comprises matching the molecular weight of each deconvoluted mass spectral peak against a database of glycan pairs and the associatedmolecular weight to identify the glycan pair in various instances. In exemplary aspects, the separating of (b) and the quantifying of (c) occur in less than 2 hours, optionally, less than 75 minutes, e.g., less than 60 minutes. In various aspects, the protein of the protein composition comprises an antibody comprising the Fc region or is an antibody comprising the Fc region. In various instances, the method further comprises treating the sample with (iii) an enzyme that cleaves each antibody into a Fab fragment and Fc fragment, optionally, a cysteine protease. In various aspects, the cysteine protease cleaves at a site between Thr and His or between Lys and Thr of the sequence KTHTCPP (SEQ ID NO: 1) of an IgGl antibody heavy chain. In various instances, the method comprises treating the sample with the cysteine protease for at least 8 hours or at least 12 hours. In exemplary instances, the method comprises chromatographically separating components of the mixture. In various instances, the method comprises chromatographically separating Fab fragments from Fc fragments. In some aspects, chromatographically separating comprises a reversed phase liquid chromatography (RP-LC). The method, in some aspects, comprises performing mass spectrometry on the Fc fragment-containing chromatographic fraction to obtain one or more mass spectral peaks. In various aspects, the method comprises deconvoluting the mass spectral peaks to obtain deconvoluted mass spectral peaks. In exemplary instances, the method comprises matching the molecular weight of each deconvoluted mass spectral peak against a database of glycan pairs and the associated molecular weight to identify the glycan pair.

[0009] The present disclosure also provides methods of determining the glycan pairing content of a protein composition comprising a protein comprising an antibody. In exemplary embodiments, the method comprises (a) treating a sample of the protein composition with (i) an enzyme that cleaves an antibody heavy chain at a site N-terminal to the hinge region disulfide linkage, (ii) a mannosidase, and (iii) a pi-4 endoglycosidase, to produce a mixture of Fab fragments and Fc fragments of the protein, wherein at least some of the Fc fragments comprise one or more digested glycan structures; (b) chromatographically separating the Fab fragments from the Fc fragments, (c) separating Fc fragments based on molecular weight, (d) quantifying the abundance of glycan pairs to determine the glycan pairing content of the protein composition.

[0010] In exemplary aspects of the presently disclosed methods, quantifying the abundance of glycan pairs of the mixture comprises quantifying the (i) paired afucosylated glycan content, (ii) unpaired afucosylated glycan content, (iii) paired high mannose glycan content, and / or (iv) unpaired high mannose glycan content. In exemplary aspects of the presently disclosed methods, quantifying the abundance of glycan pairs of the mixture comprises quantifying the (i) relative unpaired afucosylated glycan content and / or (ii) relative unpaired high mannose glycan content. In various aspects, the method is, in various aspects, a part of a manufacturing process for the proteincomposition. In various instances, the method is performed in real time during manufacture of the protein composition. In exemplary aspects, the sample is a sample of in-process material. In exemplary instances, the glycan pairing content is determined pre-harvest or post-harvest.Optionally, the glycan pairing content is determined post-harvest. In various instances, the sample is obtained from a manufacturing lot.

[0011] Further provided here are methods of analyzing a protein composition. In exemplary embodiments, the method comprises determining the glycan pairing content of a sample of the protein composition in accordance with any of the presently disclosed methods of determining glycan pairing content, and, optionally, comparing the glycan pairing content of the protein composition to the glycan pairing content of a reference product.

[0012] Additionally provided herein are methods of monitoring production of a protein composition. In exemplary embodiments, the method comprises determining the glycan pairing content of a protein composition in accordance with a presently disclosed method of determining glycan pairing content for a first sample obtained at a first timepoint and for a second sample taken at a second timepoint, which is different from the first timepoint, and, optionally, comparing the glycan pairing content of the first sample to that of the second sample. In various aspects, the method comprises quantifying the relative unpaired AF glycan content, the relative paired AF glycan content, the relative paired HM glycan content, and / or relative unpaired HM glycan content for each of the first sample and of the second sample. In exemplary aspects, each of the first sample and second sample is a sample of in-process material. In exemplary instances, the first sample is a sample of in-process material and the second sample is a sample of a manufacturing lot. In various aspects, the first sample is a sample obtained before one or more conditions of the cell culture are modified and the second sample is a sample obtained after the one or more conditions of the cell culture are modified.

[0013] Further provided herein are methods of producing a protein composition. In exemplary embodiments, the method comprises (A) determining the glycan pairing content of a sample of the protein composition in accordance with a presently disclosed method of determining glycan pairing content, wherein the sample is a sample of in-process material, wherein, when the glycan pairing content is determined as outside a target range, the method further comprises (B) modifying one or more conditions of the cell culture to obtain a modified cell culture and determining the glycan pairing content, and optionally, repeating (A) and (B) until the glycan pairing content is within the target range. In exemplary aspects, one or more conditions of the cell culture are modified to primarily change the relative unpaired HM glycan content and / or the relative paired HM glycan content to achieve the target range of glycan pairing content. In exemplary instances, one or moreconditions of the cell culture are modified to primarily change the relative unpaired AF glycan content and / or the relative paired AF glycan content to achieve the target range of glycan pairing content.

[0014] The present disclosure provides methods of selecting a cell line or cell clone for a protein composition manufacture process. In exemplary embodiments, the method comprises determining the glycan pairing content of a protein composition in accordance with any one of presently disclosed methods of determining glycan pairing content, for each cell line or cell clone producing the protein of the protein composition, and identifying the cell lines or cell clones that have a glycan pairing content within a preselected target range, wherein the identified cell lines or cell clones are selected for producing the protein in the protein composition manufacture process. In various aspects, the preselected target range is based on the range of relative unpaired afucosylated glycan content, relative paired afucosylated glycan content, relative paired high mannose glycan content, and / or the range of relative unpaired high mannose glycan content of a reference cell line or cell clone.

[0015] Additional exemplary embodiments and aspects of the present disclosure are provided below.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A is an illustration of exemplary glycan structures.

[0017] Figure IB is an illustration of exemplary glycan groups.

[0018] Figure 2 is a diagram of the salvage pathway and the de novo pathway of fucose metabolism.

[0019] Figure 3A is a representative glycan map chromatogram (full scale view; y-axis max ~440.00 EU)) obtained through HILIC. Figure 3B is a representative glycan map chromatogram (expanded scale view; y-axis max ~44.00 EU) obtained through HILIC.

[0020] Figure 4A is a series of drawings exemplifying antibodies with unpaired or paired afucosylated glycans and unpaired or paired high mannose glycans. Figure 4B provides exemplary flow charts useful for assigning nomenclature to a glycan pair comprising canonical glycans. As shown in the exemplary flow chart of Box 1, if the glycan pair comprises zero afucosylated glycans (e.g., both glycans are fucosylated), then the glycan pair is "paired fucosylated". If both glycans of the glycan pair are afucosylated, then the glycan pair is "paired afucosylated". If only one glycan is afucosylated, then the glycan pair is "unpaired afucosylated". As shown in the exemplary flow chart of Box 2, if the glycan pair comprises zero high mannose glycans, then Box 1 is used for determiningglycan pair classification. If both glycans comprise a high mannose and are afucosylated, then the glycan pair is "paired high mannose". If only one glycan of the glycan pair comprises a high mannose and is afucosylated, and the other glycan lacks a high mannose and is fucosylated, then the glycan pair is "unpaired high mannose". If only one glycan of the glycan pair comprises a high mannose and the other chain is afucosylated, then the glycan pair is "paired afucosylated". From the perspective of high mannose, this glycan pair is "unpaired high mannose". Additional information useful for assigning nomenclature to a glycan pair is provided herein. See, e.g., Example 1.

[0021] Figure 5 is an illustration of a digestion scheme with three classes of enzymes.

[0022] Figure 6A shows exemplary chromatographic peaks obtained from a 95 min HILIC-MS separation, wherein Fab fragments are separated from glycosylated Fc fragments. The inset shows a zoomed view of the glycosylated Fc fragment peaks. Figure 6B shows a further zoomed view of the glycosylated Fc fragment peaks. The glycopair identity for each peak is noted. Those labeled in bold indicate a relevant glycopair.

[0023] Figure 7 shows examples of an unpaired afucoslyated Fc fragment, paired afucosylated Fc fragment, unpaired high mannose Fc fragment, and paired high mannose Fc fragment, following a two-enzyme digestion.

[0024] Figure 8A is an illustration of the simplified chromatographic separation of Fab fragments from Fc fragments, the extraction of the one Fc fragment chromatographic peak for mass spectrometry, and mass spectra of the Fc fragment chromatographic peak. Figure 8B shows deconvoluted mass spectra (left) and a zoomed view thereof (right). Deconvoluted mass spectra peaks are matched to a database for peak identification.

[0025] Each of Figures 9A-9C is a deconvoluted spectrum of samples of the panel described in Example 2. Peaks for relevant glycoform species are shown.

[0026] Figure 10 is a schematic of a cell-based ADCC assay.

[0027] Figure 11A is leverage plot of relative ADCC activity level (%) as measured by the cell-based ADCC assay plotted as a function of measured released afucosylated glycans (%). The best fit line is the solid diagonal line in the middle of the shaded area. p<0.0001.

[0028] Figure 11B is leverage plot of relative ADCC activity level (%) as measured by the cell-based ADCC assay plotted as a function of measured released high mannose glycans (%). The best fit line is the solid diagonal line in the middle of the shaded area. p=0.2786.

[0029] Figure 11C is graph of the Actual ADCC activity level (%) as measured by the cell-based ADCC assay plotted against the Predicted ADCC activity level (%) as calculated using Equation 8.

[0030] Figure 12A is leverage plot of relative ADCC activity level (%) as measured by the cell-based ADCC assay plotted as a function of measured unpaired afucosylated glycans (%). The best fit line is the solid diagonal line in the middle of the shaded area. pcO.OOOl.

[0031] Figure 12B is leverage plot of relative ADCC activity level (%) as measured by the cell-based ADCC assay plotted as a function of measured unpaired high mannose glycans (%). The best fit line is the solid diagonal line in the middle of the shaded area. p=0.0012.

[0032] Figure 12C is graph of the Actual ADCC activity level (%) as measured by the cell-based ADCC assay plotted against the Predicted ADCC activity level (%) as calculated using Equation 5.

[0033] Figure 13 is an illustration of exemplary species of unpaired afucosylated glycan pairs, paired afucosylated glycan pairs, unpaired high mannose glycan pairs, and paired high mannose glycan pairs.

[0034] Figure 14 is an illustration of high mannose glycans with labeled al-2 linkages, al-3 linkages, and al-6 linkages between mannosyl residues. For Man7 and Man8, the additional one or two mannosyl residues may be attached to any one or two of the three terminal mannosyl residues present in Man6.

[0035] Figure 15 is an illustration of a multi-enzyme digestion and exemplary digested glycan structures (shown in the dotted line box) including e.g., paired afucosylated glycan pairs, unpaired afucosylated glycan pairs, paired high mannose glycan pairs, unpaired high mannose glycan pairs. The digested glycan structures are simplified, comprising fewer monosaccharides compared to the pre-digested glycan structure shown on the left.

[0036] Figure 16A is an illustration of exemplary digested glycan structures and their categorization as paired afucosylated glycan pair, unpaired afucosylated glycan pair, paired high mannose glycan pair, or unpaired high mannose glycan pair.

[0037] Figure 16B is an illustration of exemplary digested glycan structures produced upon treatment with the noted mannosidase.

[0038] Each of Figures 17-19 is a pair of deconvoluted mass spectra for multi-enzyme treated (digested) samples (right) and for untreated samples (undigested) samples (left). In Figures 17 and 18, multi-enzyme treatment included treatment with al-2, 3, 6 mannosidase and al-6 mannosidase. In Figure 19, multi-enzyme treatment included treatment with al-2 mannosidase. As shown in these figures, the peaks for high mannose glycopairs are well-resolved.

[0039] Figure 20 is an illustration of an exemplary multi-enzyme digestion, wherein EndoS and one or more mannosidases are used. Exemplary digested glycan structures (e.g., paired afucosylatedglycan pairs, unpaired afucosylated glycan pairs, paired high mannose glycan pairs, unpaired high mannose glycan pairs) following digestion are shown. Unlike the digested glycan structures of Figure 15, each of the digested glycan structures in this figure comprises a Fab fragment covalently linked to a Fc region comprising a glycopair.DETAILED DESCRIPTION

[0040] The present disclosure provides methods of determining the glycan pairing content of a protein composition, e.g., an antibody composition. Paired or unpaired high mannose glycan content and paired or unpaired afuscosylated glycan content can be informative of the ADCC activity and pharmacokinetics of antibodies. However, due to the number of different species within each of these categories, each with a slightly different mass, resolving the mass spectrum of typical glycosylation pair proteins, such as IgG antibody Fc glycans, can be laborious, and the resolution of individual glycan pair species can be challenging. It is appreciated herein that digestion of paired protein glycans with pi-4 endoglycosidases in combination with mannosidases can simplify the many complex (and diverse) glycans into just a few digested glycan structures. These digested glycan structures are directly informative of whether the undigested glycan pair comprised paired or unpaired high mannose species and / or paired or unpaired afucosylated species. The methods herein greatly simplify the analysis of the glycan pair content of each glycosylated protein in a composition by mass spectrometry by reducing numerous, and often overlapping, mass spectrum peaks into just a few clearly delineated peaks that identify the relevant information about the high mannose and afucosylation status of each glycan pair.

[0041] Accordingly, the present disclosure provides methods of determining the glycan pairing content of a protein composition comprising a protein comprising an Fc region comprising a glycan pair. In exemplary embodiments, the method comprises (a) treating a sample of the protein composition with (i) a mannosidase and (ii) a pi-4 endoglycosidase, to produce a mixture of components of the protein, wherein at least some of the components comprises one or more digested glycan structures; and (b) separating the components of the mixture based on molecular weight; and (c) quantifying the abundance of glycan pairs of the mixture to determine the glycan pairing content of the protein composition. As discussed further below, the present inventive methods of determining the glycan pairing content of a protein composition may be incorporated into methods of analyzing a protein composition to, for instance, determine the Fc glycoform profile of the composition. The presently disclosed methods of determining the glycan pairing content or a protein composition can also be incorporated into methods of monitoring production of a protein composition, methods for producing a protein composition, and methods of selecting a cell line or cell clone for a protein composition manufacture process.

[0042] Glycosylation, Glycans, and Methods ofGlycan Measurement

[0043] Many secreted proteins undergo post-translational glycosylation, a process by which sugar moieties (e.g., glycans, saccharides) are covalently attached to specific amino acids of a protein. In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which glycans are attached to the asparagine of the recognition sequence Asn-X-Thr / Ser, where "X" is any amino acid except proline, and (2) O-linked glycosylation in which glycans are attached to serine or threonine. Regardless of the glycosylation type (N-linked or O-linked), microheterogeneity of protein glycoforms exists due to the large range of glycan structures associated with each site (O or N).

[0044] All N-glycans have a common core sugar sequence: Manal-6(Manal-3)Manpi-4GlcNAcpi-4GlcNAcpi-Asn-X-Ser / Thr (Man3GlcNAc2Asn) and are categorized into one of three types: (A) a high mannose (HM) or oligomannose (OM) type, which consists of two N-acetylglucosamine (GIcNAc or GIcNac) moieties and at least 5 (e.g., 5, 6, 7, 8 or 9) mannose (mannosyl, Man) residues, (B) a complex type, which comprises more than two GIcNAc moieties and any number of other sugar types, or (C) a hybrid type, which comprises a Man residue on one side of the branch and GIcNAc at the base of a complex branch. Figure 1A (adapted from Stanley et al., Chapter 8: N-Glycans, Essentials of Glycobiology, 2nded., Cold Spring Harbor Laboratory Press; 2009) shows the three types of N-glycans.

[0045] N-linked glycans found in IgG molecules typically comprise one or more monosaccharides of galactose (Gal), N- glucose (Glc), N-acetylglucoasamine (GIcNAc), glucoasamine (GlcN), mannose (Man), fucose (Fuc). Exemplary glycans, their identity and group classifications are shown in Figure IB.

[0046] N-linked glycosylation begins in the endoplasmic reticulum (ER), where a complex set of reactions result in the attachment of a core glycan structure made essentially of two GIcNAc residues and three Man residues. The glycan complex formed in the ER is modified by action of enzymes in the Golgi apparatus. If the saccharide is relatively inaccessible to the enzymes, it typically stays in the original HM form. If enzymes can access the saccharide, then many of the Man residues are cleaved off and the saccharide is further modified, resulting in the complex type N-glycans structure. For example, mannosidase-1 located in the cis-Golgi, can cleave or hydrolyze a HM glycan, while fucosyltransferase FUT-8, located in the medial-Golgi, fucosylates the glycan (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84). Fucose metabolism is accomplished through the salvage pathway or the de novo pathway as shown in the diagram of Figure 2. In the salvage pathway, free L-fucose is converted to GDP-fucose, while in the de novo pathway, GDP-fucose is synthesized viathree reactions catalyzed by GMD and FX. GDP-fucose is then transported from the cytosol to the Golgi lumen by GDP-Fuc Transferase and transferred to acceptor oligosaccharides and proteins. The other reaction product, GDP, is converted by a luminal nucleotide diphosphatase to guanosine 5 -monophosphate (GMP) and inorganic phosphate (Pi). The former is exported to the cytosol (via an antiport system that is coupled with the transport of GDP-fucose), whereas the latter is postulated to leave the Golgi lumen via the Golgi anion channel, GOLAC. See, e.g., Nordeen et al. 2000;Hirschberg et al. 2001.

[0047] Accordingly, the sugar composition and the structural configuration of a glycan structure varies, depending on the glycosylation machinery in the ER and the Golgi apparatus, the accessibility of the machinery enzymes to the glycan structure, the order of action of each enzyme and the stage at which the protein is released from the glycosylation machinery, among other factors.

[0048] Various methods may be used for assessing glycans present in a glycoprotein-containing composition or for determining, detecting or measuring a glycoform profile (e.g., a glycoprofile) of a particular sample comprising glycoproteins. Suitable methods include, but are not limited to, positive ion MALDI-TOF analysis, negative ion 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 n.m.r. 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 L.R., Anal Bioanal Chem, 2010, Vol. 397:3457-3481 and Geoffrey, R. G. et. al. Analytical Biochemistry 1996, Vol. 240, pages 210-226. Also, a suitable method of assessing glycans present in a glycoprotein-containing composition may comprise enzymatic cleavage of glycans attached to the glycoprotein. The cleaved or released glycans are subsequently separated by Hydrophilic Interaction Liquid Chromatography (HILIC) and a chromatogram with several peaks is produced. Each peak of the chromatogram represents a distribution (amount or abundance) of a different glycan. Two views of a representative HILIC chromatogram comprising peaks for different glycans are provided in Figures 3A and 3B. For these purposes, % Peak Area = Peak Area / Total Peak Area x 100%. Accordingly, the level of a particular glycan (or groups of glycans) is reported as a %. For example, if an antibody composition is characterized as having a Man6 level of 30%, it is meant that 30% of all glycans cleaved from the antibodies of the composition are Man6. As described in more detail herein, it is noted that such methods that remove glycans from the glycoproteins identify a distribution of glycan content for a glycoprotein, but do not provide information relating to paired glycans and / or unpaired glycans.

[0049] The present disclosure references high mannose glycans and afucosylated glycans of a protein composition or an antibody composition (see Figure IB for examples). As used herein, the term "high mannose glycans" or "HM glycans" encompasses glycans comprising 5, 6, 7, 8, or 9 mannose residues, abbreviated as Man5 or M5, Man6 or M6, Man7 or M7, Man8 or M8, and Man9 or M9, respectively. A level of HM glycans, in various aspects, is obtained by summing the % Man5, the % Man6, the % Man7, the % Man8, and the % Man9. As used herein, the term "afucosylated glycan" or "AF glycan" refers to glycans which lack a core fucose, e.g., an al,6-linked fucose on the GIcNAc residue involved in the amide bond with the Asn of the N-glycosylation site. Afucosylated glycans include, but are not limited to, A1G0, A1G1, A2G0, A2G1 (a and b), A2G2, A1G1M4 and A1G1M5. It is noted that high mannose glycans also lack core fucose (and thus represent a subset of afucosylated glycans), but high mannose glycans have certain characteristics and may be referred to as a separate glycan group. Accordingly, unless explicitly stated otherwise, high mannose is understood to represent a separate characteristic and may be classified separately from, or as an additional characteristic of afucosylated glycans. See, e.g., Reusch and Tejada, Glycobiology 25(12): 1325-1334 (2015). A level of afucosylated glycans, in various aspects, is obtained by summing the % A1G0, the % A2G0, the % A2Gla, the % A2Glb, the % A2G2, the % A1G1M5, the % AlGla.

[0050] The level (e.g., amount, abundance) of glycans (e.g., % HM glycans, % AF glycans) may be determined (e.g., measured) by any of the various methods known in the art for assessing glycans present in a glycoprotein-containing composition or for determining, detecting or measuring a glycoform profile (e.g., a glycoprofile) of a particular sample comprising glycoproteins. The level (e.g., amount, abundance) of glycans (e.g., % HM glycans, % AF glycans) of a protein composition or an antibody composition may be determined by measuring the level (e.g., amount, abundance) of such glycans in a sample of the protein composition or antibody composition though a chromatography-based method, e.g., HILIC, and the level (e.g., amount, abundance) of glycans is expressed as a %, as described herein. The level of glycans of a protein composition or an antibody composition may be expressed as a % of all glycans cleaved from the proteins or antibodies of the composition. The level (e.g., amount, abundance) of glycans (e.g., % HM glycans, % AF glycans) may be determined (e.g., measured) by measuring the level of such glycans in a sample of the protein composition or antibody composition. Samples of a protein composition or an antibody composition may be taken and the level (e.g., amount, abundance) of glycans (e.g., % HM glycans, % AF glycans) for each sample may be determined (e.g., measured). The % HM glycans and / or % AF glycans may be determined.

[0051] Glycan Pairing and Methods of Measuring Glycan Pairing Content

[0052] In exemplary instances of the present disclosure, the protein composition comprises a protein comprising an antibody Fc region comprising two covalently paired polypeptides or protein fragments, wherein each polypeptide or protein fragment comprises an N-linked glycan that together form a glycan pair (also referred to herein as "glycopair") of the Fc region. In exemplary aspects, the protein comprises an antibody comprising an Fc region comprising two covalently linked polypeptides or protein fragments, each of which comprises N-linked glycans that together form a glycan pair or glycopair. In various instances, the protein of the protein composition is an antibody, and the protein composition is an antibody composition. In exemplary instances of the present disclosure, the antibody composition comprises antibodies, each comprising two heavy chains and two light chains. In exemplary aspects, each antibody of the antibody composition comprises a fragment crystallizable (Fc) region and two Fab regions. The Fc region of the antibody or a protein comprises two identical protein fragments derived from constant domains of the antibody heavy chains, and each protein fragment is glycosylated, e.g., comprises a glycan. Accordingly, in exemplary aspects, the Fc region of the antibody or protein comprises a pair of glycans, or a glycan pair or glycopair. In various aspects, the two glycans of the glycan pair are structurally different from each other. Alternatively, the glycans of the glycan pair are structurally identical. The glycan pair may be classified based on the presence or absence of glycans of the glycan pair comprising a core fucose, as well as the presence or absence of glycans of the glycan pair comprising high mannose. Figure 4A is a series of drawings exemplifying an unpaired afucosylated antibody, a paired afucosylated antibody, an unpaired high mannose antibody and a paired high mannose antibody. Figure 4B provides exemplary flow charts useful for assigning nomenclature to a glycan pair comprising canonical glycans. Exemplary paired high mannose glycans include glycans having (a) two identical high mannose glycans (e.g., high mannose of identical structure), such as Man5, Man6, Man7, Man8 or Man9, or (b) two non-identical high mannose glycans (e.g., high mannose glycans having different structures) but each high mannose glycan comprises Man5, Man6, Man7, Man8 or Man9 (e.g., Man5 as one glycan and Man6, Man7, Man8, or Man9 as the other glycan, or Man6 as one glycan and Man7, Man8, or Man9 as the other glycan, or Man7 as one glycan and Man8 or Man9 as the other glycan, or Man8 as one glycan and Man9 as the other glycan). Exemplary unpaired high mannose glycans include glycans having Man5, Man6, Man7, Man8 or Man9 as one glycan and a fucosylated or afucosylated glycan as the other glycan. Exemplary unpaired afucosylated glycan pairs include, for instance A1G0, A2G0, A2Gla, A2Glb, or A2G2 as one glycan and a fucosylated glycan as the other glycan. Exemplary paired afucosylated glycans include, for instance, two identical afucosylated glycans (e.g., A1G0 / A1G0) or two non-identical afucosylatedglycans (e.g., A1G0 / A2G2). Paired afucosylated glycans include, for example, (i) A1G0 as one glycan and A2G0, A2Gla, A2Glb, or A2G2 or (ii) A2G0 on one chain and A2Gla, A2Glb, or A2G2 on the other Fc chain or (iii) A2Gla on one Fc chain and A2Glb or A2G2 on the other Fc chain or (iv) A2Glb on one Fc chain and A2G2 on the other chain. Paired afucosylated glycans can include a high mannose as one glycan and any of A1G0, A2G0, A2Gla, A2Glb, or A2G2 as the other, e.g., M5 / A1G0. From the high mannose perspective, if only one glycan of the pair comprises a high mannose, the paired afucosylated glycan may be considered as an "unpaired high mannose".

[0053] In exemplary instances, the glycan pair is given a designation based on the fucosylation / afucosylation status of each glycan of the glycan pair. In exemplary aspects, the glycan pair is given a designation based on the high mannose status of each glycan of the glycan pair.Suitable designations will be appreciated in the context of the glycan structures of interest in the particular scenario. In various aspects, the glycan pair is given two designations, wherein a first designation is based on the fucosylation / afucosylation status of each glycan of the glycan pair and a second designation is based on the high mannose status of each glycan of the glycan pair, and, optionally, a summary designation, which is used for quantifying the abundance of Fc fragments comprising (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans, is assigned. Table A below provides an exemplary way of assigning these designations. In various instances, a Summary Designation is assigned by applying three rules relating to the First Designation (Designation 1) and Second Designation (Designation 2), as follows: (1) when Designation 1 is paired fucosylated, the Summary Designation is paired fucosylated (regardless of Designation 2); (2) when Designation 2 is no HM, the Summary Designation is the same as Designation 1; and (3) when Designation 2 is paired HM or unpaired HM and Designation 1 is not paired fucosylated, the Summary Designation is the same as Designation 2 (e.g., Scenarios 4 and 10) or is a combination of Designation 1 and Designation 2 (e.g., Scenarios 5, 8, and 9).TABLE AScenario Fc HM Fuc / AF Designation 1 Designation 2 Summary Chain Designation 1 1 negative Fuc paired fucosylated no HM paired fucosylated 2 negative Fuc2 1 negative AF paired AF no HM paired AF 2 negative AF3 1 negative Fuc unpaired AF no HM unpaired AF2 negative AF4 1 positive AF paired AF paired HM paired HM 2 positive AF5* 1 positive AF unpaired AF paired HM paired HM, unpaired AF 2 positive Fuc6 1 positive Fuc paired fucosylated paired HM paired fucosylated 2 positive Fuc7 1 positive Fuc paired fucosylated unpaired HM paired fucosylated 2 negative Fuc8 1 positive AF paired AF unpaired HM unpaired HM, paired AF 2 negative AF9* 1 positive Fuc unpaired AF unpaired HM unpaired HM, unpaired AF 2 negative AF10 1 positive AF unpaired AF unpaired HM unpaired HM 2 negative FucFuc, fucosylated; AF, afucosylated; HM, high mannose. Designation 1 is based on fucosylated / afucosylated status of each glycan on each Fc chain. Designation 2 is based on the high mannose status of each glycan on each Fc chain. Summary designation is based on 3 rules: (1) when Designation 1 is paired fucosylated, the Summary Designation is paired fucosylated (regardless of Designation 2); (2) when Designation 2 is no HM, the Summary Designation is the same as Designation 1; and (3) when Designation 2 is paired HM or unpaired HM and Designation 1 is not paired fucosylated, the Summary Designation is the same as Designation 2 (e.g., Scenarios 4 and 10) or is a combination of Designations 1 and 2 (e.g., Scenarios 5, 8 and 9). *Scenarios 5, 6, 7 and 9, involving a fucosylated glycan comprising a high mannose, are uncommon occurrences.

[0054] In various instances, the antibody, or an Fc region of a protein, comprising the glycan pair may be described in terms of its glycan pair classification. Thus, an antibody or Fc region of a protein may be described as "paired afucosylated", "unpaired afucosylated", "paired fucosylated", "paired high mannose" or "unpaired high mannose" by virtue of its glycan pair classification. Similarly, the antibody or an Fc region of a protein may be described as comprising paired afucosylated glycans (or paired afucosylated glycan pairs), unpaired afucosylated glycans (or unpaired afucosylated glycan pairs), paired high mannose glycans (paired high mannose glycan pairs) and / or unpaired high mannose glycans (unpaired high mannose glycan pairs) by virtue of its glycan pair classification. As discussed below, antibodies may be cleaved into fragments. Fc fragments comprise the Fc region of an antibody and may be described as "paired afucosylated", "unpaired afucosylated", "paired fucosylated", "paired high mannose" or "unpaired high mannose" by virtue of the glycan pair classification of the Fc region.

[0055] A protein composition or an antibody composition may be characterized in terms of its glycan pairing content. The glycan pairing content of a protein composition or antibody compositioninforms the composition's paired glycan content and / or unpaired glycan content. For instance, the glycan pairing content of a protein composition or an antibody composition may characterize the composition in terms of its paired afucosylated glycan content and / or unpaired afucosylated glycan content and / or its paired high mannose content and / or unpaired high mannose content. The abundance of paired afucosylated glycan pairs (e.g., paired afucosylated glycan content) and / or the abundance of unpaired afucosylated glycan pairs (e.g., unpaired afucosylated glycan content) and / or the abundance of paired high mannose glycan pairs (e.g., paired high mannose content) and / or the abundance of unpaired high mannose glycan pairs (e.g., unpaired high mannose content) as described herein may be referred to as relative abundance or absolute abundance. In exemplary instances, the absolute abundance of glycans may be expressed in units measuring levels of the glycans themselves, for example in terms of mass, moles, mass or molar units per volume unit, arbitrary units, area under the curve, or intensities of mass spectral peaks, e.g., deconvoluted mass spectral peaks (e.g., as may be determined from a chromatograph). In exemplary instances, a protein composition or an antibody composition is characterized in terms of its relative abundance of unpaired glycans, meaning that the amount of unpaired glycans is expressed as an amount relative to the sum of paired glycans and unpaired glycans of the protein composition or antibody composition. In exemplary aspects, the protein composition or antibody composition is characterized in terms of its relative abundance of unpaired afucosylated glycans. In exemplary aspects, the protein composition or antibody composition is characterized in terms of its relative abundance of unpaired high mannose glycans. Likewise, a protein composition or an antibody composition in various aspects is characterized in terms of its relative abundance of paired glycans, meaning that the amount of paired glycans is expressed as an amount relative to the sum of paired glycans and unpaired glycans of the protein composition or antibody composition. In exemplary aspects, the protein composition or antibody composition is characterized in terms of its relative abundance of paired afucosylated glycans. In exemplary aspects, the protein composition or antibody composition is characterized in terms of its relative abundance of paired high mannose glycans.

[0056] The term "relative abundance of unpaired afucosylated glycans" which is synonymous with "relative unpaired afucosylated glycan content", "relative abundance of unpaired afucosylated glycan pairs" and "relative % unpaired afucosylated glycans" is calculated as dividing the percentage of unpaired afucosylated glycans by the sum of the percentage of unpaired afucosylated glycans and the percentage of paired afucosylated glycans) and multiplying by 100%.

[0057] The term "relative abundance of unpaired high mannose glycans" which is synonymous with "relative unpaired high mannose glycan content", "relative abundance of unpaired highmannose glycan pairs" and "relative % unpaired high mannose glycans" is calculated as the percentage of unpaired high mannose glycans divided by the sum of the percentage of unpaired high mannose glycans and the percentage of paired high mannose glycans) multiplied by 100%.

[0058] The term "relative abundance of paired afucosylated glycans" is synonymous with "relative paired afucosylated glycan content" "relative abundance of paired afucosylated glycan pairs" and "relative % paired afucosylated glycans" is calculated as dividing the percentage of paired afucosylated glycans by the sum of the percentage of unpaired afucosylated glycans and the percentage of paired afucosylated glycans) and multiplying by 100%.

[0059] The term "relative abundance of paired high mannose glycans" is synonymous with "relative paired high mannose glycan content", "relative abundance of paired high mannose glycan pairs" and "relative % paired high mannose glycans" is calculated as dividing the percentage of paired high mannose glycans by the sum of the percentage of unpaired high mannose glycans and the percentage of paired high mannose glycans) and multiplying by 100%.

[0060] In various aspects of the present disclosure, the sum of the relative % unpaired afucosylated glycans and the relative % paired afucosylated glycans equals 100%. Accordingly, in various aspects, if the relative % paired afucosylated glycans is known, the relative % unpaired afucosylated glycans may be determined (e.g., calculated) by subtracting the relative % paired afucosylated glycans from 100%. As such, the skilled person in view of this disclosure, upon knowing the unpaired status would also immediately and instantly know the paired status and vice versa. Accordingly, methods of determining unpaired glycan status as described herein are also instantly informative of paired glycan status and vice versa. Also, in various instances, if the relative % unpaired afucosylated glycans is known, the relative % paired afucosylated glycans may be determined (e.g., calculated) by subtracting the relative % unpaired afucosylated glycans from 100%. In various aspects of the present disclosure, the sum of the relative % unpaired high mannose glycans and the relative % paired high mannose glycans equals 100%. Accordingly, in various aspects, if the relative % paired high mannose glycans is known, the relative % unpaired high mannose glycans may be determined (e.g., calculated) by subtracting the relative % paired high mannose glycans from 100%. Also, in various instances, if the relative % unpaired high mannose glycans is known, the relative % paired high mannose glycans may be determined (e.g., calculated) by subtracting the relative % unpaired high mannose glycans from 100%.

[0061] Methods of Determining Glycan Pairing Content

[0062] The present disclosure provides methods of determining the glycan pairing content of a protein composition, wherein the heterogeneity of high mannose glycans is reduced by simplifyingthe structure of high mannose glycans and / or the heterogeneity of afucosylated glycans is reduced by simplifying the structure of afucosylated glycans. The simplified structures of the high mannose glycans and / or afucosylated glycans are in various instances referred to as "digested glycan structures", because in various instances the glycan structures are simplified upon digestion with one or more enzymes that effectively remove one or more sugar moieties (e.g., monosaccharide residues) from the pre-digested glycan structure (glycan structure prior to digestion). As used herein, "digested glycan structure" refers to the structure of a glycan following digestion comprising at least one core GIcNAc, which may be either fucosylated or afucosylated, and further comprises a residual product of a mannose digest that is informative of the high mannose status (or absence thereof) of the glycan prior to digestion and / or a residual product of an endoglycosidase digest that is informative of the fucosylated status (or afucosylated status) of the glycan pair prior to digestion. In exemplary aspects, digested glycan structures are simplified and / or comprise fewer monosaccharides, relative to their pre-digestion glycan structure. Exemplary digested glycan structures following EndoS digestion are illustrated in Figure 13. Exemplary digested glycan structures following digestion with one or more mannosidases are illustrated in Figure 16B. By way of example, a digested glycan structure may comprise the proximal GIcNac residue(s) (exemplified as a product of pi-4 endoglycosidase digest) from which a fucose is present or absent, and the residual 0, 1, 3, or 5 mannose residues (exemplified as a product of a mannosidase digest). In exemplary embodiments, the method comprises preparing a simplified sample using a mannosidase which reduces the heterogeneity of low abundance high mannose glycans. In exemplary embodiments, the method comprises preparing a simplified sample using a pi-4 endoglycosidase which reduces the heterogeneity of low abundance afucosylated glycans. Accordingly, in exemplary embodiments, the presently disclosed methods of determining the glycan pairing content is a method of determining the glycan pairing content of a protein composition comprising a protein comprising an Fc region comprising a glycan pair. In exemplary embodiments, the method comprises (a) treating a sample of the protein composition with (i) a mannosidase and (ii) a pi-4 endoglycosidase, to produce a mixture of components of the protein, wherein at least some of the components comprises one or more digested glycan structures; and (b) separating the components of the mixture based on molecular weight; and (c) quantifying the abundance of glycan pairs. In exemplary aspects, the method comprises quantifying (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans, of the mixture. In exemplary instances, the method comprises quantifying the relative unpaired afucosylated (AF) glycan content and / or relative unpaired high mannose (HM) glycan content of the protein composition. In various aspects, the glycan pairing content is quantified based onquantification of the glycan pairs which in turn is based quantification of the digested glycan structures.

[0063] Mannosyl residues (or mannose residues) are linked to the other moieties of a glycan structure through one or more of al-2 linkages, al-3 linkages and / or al-6 linkages. Mannosidases are a class of exoglycosidases that can hydrolyze the al-2, al-3 and / or al-6 linked mannosyl residues from the distal terminal of the glycan structure of a glycoprotein. Mannosidases are known in the art (see, e.g., Rovira et al., Current Opin Struct Biol 62: 79-92 (2020)) and are commercially available from companies such as New England BioLabs (Ipswich, MA) and Agilent Technologies (Santa Clara, CA). The al-2, 3, 6 mannosidase, has a broad substrate specificity cleaving al-2, al-3, and al-6 linkages, while the al-2, 3 mannosidase demonstrates specificity at al-2 and al-3 linkages, and the al-2 mannosidase, and al-6 mannosidase demonstrate specificity towards the al-2 linkage and al-6 linkage, respectively. In exemplary aspects of the presently disclosed methods of determining glycan pairing content, the sample of the protein composition is treated with a mannosidase. In various aspects, the mannosidase cleaves an al-2 linkage, al-3 linkage, and / or al-6 linkage of a high mannose glycan. In exemplary aspects, the mannosidase is an al-2, 3, 6 mannosidase and cleaves al-2 linkages, al-3 linkages, and al-6 linkages of a high mannose glycan. In exemplary instances, the mannosidase is an al-6 mannosidase and cleaves al-6 linkages of a high mannose glycan. In various aspects, the mannosidase is an al-2 mannosidase and cleaves al-2 linkages of a high mannose glycan. In various instances, the mannosidase is an al-2, 3 mannosidase and cleaves al-2 linkages and al-3 linkages of a high mannose glycan. In exemplary aspects, the method comprises treating the sample with more than one mannosidases, optionally, two or three mannosidases. In various aspects, two mannosidases are used to treat the sample. In some aspects, the method comprises treating the sample with an al-2, 3, 6 mannosidase and an al-6 mannosidase. In exemplary instances, the method comprises treating the sample with each mannosidase for less than 4 hours, optionally, less than 3 hours and / or more than 1 hour. The method comprises treating the sample with each mannosidase for about 2 hours in some aspects. In various aspects, the method comprises treating the sample with a single mannosidase, e.g., an al-2 mannosidase or an al-2, 3 mannosidase. In various instances, the method comprises treating the sample with the mannosidase for less than 30 hours, optionally, about 24 hours. In exemplary aspects, the mannosidase digestion(s) are carried out to nearly 100% completion (e.g., greater than 99% completion, greater than 98% completion, greater than 95% completion), while in other aspects, the mannosidase digestions are carried out to 50% completion, 60% completion, 70% completion, 80% completion, 90% completion, or more than 90% completion. Percent completion in variousinstances may be determined by measuring the amount of linkages before and after digestion with the endoglycosidase and calculating the percent completion according toPercent completion = [(B - A) / B] *100%,wherein B is the number of linkages before digestion with the mannosidase, A is the number of linkages after digestion with the mannosidase.

[0064] In exemplary aspects of the presently disclosed method, the heterogeneity of high mannose glycans is reduced by simplifying the structure of high mannose glycans to a digested glycan structure. The simplifying in various aspects is achieved through one or more digestions with one or more mannosidases to reduce the total number of sugar moieties (e.g., monosaccharides) of the N-linked glycan. In exemplary aspects, the methods comprise treating with one or more mannosidases, and after the mannosidase treatment, each digested glycan structure has fewer mannosyl residues compared to the pre-digested glycan structure. In various aspects, none of the digested glycan structures of the components in the mixture produced in (a) (e.g., post-digestion, post-treatment) comprises six or more mannosyl residues. In various aspects, the digested glycan structures of the components in the mixture produced in (a) comprise less than 6 mannosyl residues or 5 or fewer mannosyl residues. In various instances, the digested glycan structures of the components in the mixture produced in (a) comprise zero, one, three or five mannosyl residues. In various aspects, the digested glycan structures of the components in the mixture produced in (a) comprises zero or one mannosyl residues. In various aspects, the digested glycan structures of the components in the mixture produced in (a) comprises zero or three mannosyl residues. In various aspects, the digested glycan structures of the components in the mixture produced in (a) comprises zero or five mannosyl residues. In exemplary instances, when a digested glycan structure of the pair comprises one, three, or five mannosyl residues, the digested glycan structure further comprises two core GIcNac residues. In exemplary instances, when a digested glycan structure of the pair comprises zero mannosyl residues, the digested glycan structure further comprises only one core GIcNac residue. The core GIcNac residue in various aspects comprises a fucose, while in other aspects, the core GIcNac residue does not comprise a fucose (e.g., is afucosylated).

[0065] In exemplary aspects of the presently disclosed methods of determining glycan pairing content, the sample of the protein composition is treated with an endoglycosidase, such as a pi-4 endoglycosidase. Endoglycosidases are enzymes that cleave internal linkages of unexposed sugar residues of N-glycans (Lehrman, M.A., Lipids Carbohydrates Membranes and Membrane Proteins: Glycoproteins, N-Linked, Encyclopedia of Biological Chemistry (2nded.), Elsevier Inc., pages 457-464 (2013). Endoglycosidases are commercially available from companies such as Genovis Inc. (Lund,Sweden) and New England Biolabs (Ipswich, MA). In various aspects, the endoglycosidase is a pi-4 endoglycosidase which cleaves the pi-4 linkage between core GIcNAc residues. In exemplary instances, the pi-4 endoglycosidase is an IgG-specific enzyme. In exemplary aspects, the pi-4 endoglycosidase or IgG-specific enzyme is an Endoglycosidase D, an Endoglycosidase F, an Endoglycosidase Fl, an Endoglycosidase F2, an Endoglycosidase H, or an Endo S or EndoS2 endoglycosidase. In exemplary instances, the pi-4 endoglycosidase is an Endoglycosidase S (Endo S). In exemplary aspects, the method comprises treating the sample of the protein composition with the endoglycosidase (e.g., pi-4 endoglycosidase) for less than 1 hour, e.g., less than 55 minutes, less than 50 minutes, less than 45 minutes, less than 40 minutes, less than 35 minutes, less than 30 minutes. In various aspects, the method comprises treating the protein composition with the endoglycosidase (e.g., pi-4 endoglycosidase( for about 30 minutes. In exemplary aspects, the endoglycosidase digestion(s) are carried out to nearly 100% completion (e.g., greater than 99% completion, greater than 98% completion, greater than 95% completion), while in other aspects, the endoglycosidase digestions are carried out to 50% completion, 60% completion, 70% completion, 80% completion, 90% completion, or more than 90% completion. Percent completion in various instances may be determined by measuring the amount of linkages before and after digestion with the endoglycosidase and calculating the percent completion according toPercent completion = [(B - A) / B] *100%,wherein B is the number of linkages before digestion with the endoglycosidase and A is the number of linkages after digestion with the endoglycosidase.

[0066] In various aspects, the sample is treated with one or more mannosidases after the sample is treated with the pi-4 endoglycosidase. In various aspects, the sample is treated with one or more mannosidases before the sample is treated with the pi-4 endoglycosidase. In various aspects, the sample is treated with one or more mannosidases at the same time the sample is treated with the pi-4 endoglycosidase.

[0067] In exemplary instances, the method comprises separating the components of the mixture based on molecular weight. In various instances, the method comprises separating the components of the mixture based on molecular weight which comprises performing mass spectrometry to obtain one or more mass spectral peaks. Mass spectrometry is known in the art. See, e.g., Habbab et al., Chapter 7: Mass Spectrometry, Analytical Techniques in Biosciences: From Basics to Applications, Academic Press, pages 115-124 (2022). In various aspects, the mass spectrometry is carried out with liquid chromatography (e.g., LC-MS) and in various instances, the mass spectrometry is carried out with a reversed phase liquid chromatography (RPLC-MS). In various aspects, the method comprisesdeconvoluting the mass spectral peaks to obtain deconvoluted mass spectral peaks. Suitable methods of deconvoluting mass spectral peaks are known in the art. See, e.g., Gadgil et al., J. Am. Soc. Mass Spectrom. 17, 867 (2006); Murray et al., Pure and Applied Chemistry, vol. 85, no. 7, 2013, pp. 1515-1609; Marchetti and Mignerey, Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 324(1-2): 288-296 (1993); Xu et al., Rapid Commun Mass Spectrom. 2018 May 30;32(10):763-774. In exemplary instances, the method comprises matching the molecular weight of each deconvoluted mass spectral peak against a database of glycan pairs and the associated molecular weight to identify the glycan pair. In exemplary aspects, the matching is carried out through an automated, computer assisted process. The database in exemplary aspects comprises the glycan pairs and their associated theoretical molecular weights of Tables 19, 22, and 25 of Examples 13, 14, and 15, respectively. In exemplary instances, the method comprises quantifying the abundance of (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans and in exemplary aspect, the method comprises quantifying the relative unpaired afucosylated (AF) glycan content and relative unpaired high mannose (HM) glycan content. In various instances, the abundance of (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans is based on the quantification of digested glycan structures of a glycan pair of the components of the mixture. The digested glycan structures are, in exemplary aspects, categorized as (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, or (iv) unpaired high mannose glycans according to Figure 16A. In various aspects, the abundance of paired afucosylated glycans and the abundance of unpaired afucosylated glycans are summed so that relative unpaired afucosylated glycans and / or relative paired afucosylated glycans may be determined. In various aspects, the abundance of paired high mannose glycans and the abundance of unpaired high mannose glycans are summed so that relative unpaired high mannose glycans and / or relative paired high mannose glycans may be determined. Suitable methods for carrying out the method of the present disclosure is described in detail in Examples 13-19. In various aspects, the separating and quantifying of the presently disclosed methods occur in less than 2 hours, optionally, less than 95 minutes, less than 85 minutes, less than 75 minutes, less than 60 minutes, less than 45 minutes, less than 30 minutes or less than 20 minutes (optionally, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes). The presently disclosed methods in exemplary aspects comprise separating and quantifying in less than 30 minutes or less than 20 minutes. In various aspects, theseparating and quantifying of the presently disclosed methods occur in about 5 minutes to about 25 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 15 minutes, about 5 minutes to about 10 minutes, about 10 minutes to about 25 minutes, about 15 minutes to about 25 minutes, about 20 minutes to about 25 minutes). The presently disclosed methods of determining the glycan pairing content advantageously require less time than prior methods.

[0068] In various aspects, the protein of the protein composition comprises an antibody comprising the Fc region or is an antibody comprising the Fc region. In various instances, the method further comprises treating the sample with (iii) an enzyme that cleaves each antibody into a Fab fragment and Fc fragment. In various aspects, each Fc fragment of the antibodies of the composition comprises a pair of polypeptides each polypeptide of which comprises an N-linked glycan that together form a glycopair. In various instances, the enzyme is a cysteine protease. In various instances, the enzyme is a member of the IgdE protease family, optionally, an IgdE expressed by a Streptococcus. In various instances, the enzyme is structurally identical or highly similar to an IgdE protease expressed by Streptococcus agalactiae. In various aspects, the enzyme is structurally identical or highly similar to an enzyme expressed by a Porphyromonas anaerobe. In various instances, the enzyme is structurally identical or highly similar to an enzyme expressed by a Porphyromonas gingivalis. In various instances, the enzyme (e.g., cysteine protease) cleaves at a single site between Thr and His or between Lys and Thr of the sequence KTHTCPP (SEQ ID NO: 1) of an IgGl antibody heavy chain. In exemplary aspects, the method comprises treating the sample of the protein composition with the cysteine protease for at least 8 hours or at least 12 hours, e.g., at least 9 hours, at least 10 hours, at least 11 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours. In exemplary aspects, the digestion with the cysteine protease is carried out to nearly 100% completion (e.g., greater than 99% completion, greater than 98% completion, greater than 95% completion), while in other aspects, the digestions are carried out to greater than 50% completion, greater than 60% completion, greater than 70% completion, greater than 80% completion, greater than 90% completion, or more than 90% completion.

[0069] In various instances, the method comprises treating the sample of the protein composition with the cysteine protease, followed by treating the sample with the pi-4 endoglycosidase, and then treating the sample with one or more mannosidases. In various aspects, the method comprises treating the sample of the protein composition according to the multi-enzyme digestion scheme of Figure 15 and Digestion Scheme 1 is carried out with the enzyme that cleaves each antibody into a Fab fragment and Fc fragment, Digestion Scheme 2 is carried out with an endoglycosidase andDigestion Scheme 3 is carried out with one or more mannosidases. Examples 13-17 describe suitable methods comprising multi-enzyme digestion schemes. In exemplary instances, the method comprises chromatographically separating components of the mixture comprising Fab fragments and Fc fragments. In various instances, the method comprises chromatographically separating Fab fragments from Fc fragments. In various aspects, the chromatographically separating comprises a reversed phase liquid chromatography (RP-LC), affinity chromatography (e.g., Protein A chromatography), anion or cation exchange chromatography, liquid chromatography (LC, e.g., reversed phase LC, HILIC), and the like. Optionally, the chromatography is a reversed phase liquid chromatography (RP-LC). In various aspects, spectral peaks of the Fc fragments elute as one chromatographic peak which is selected for analysis by mass spectrometry. The method, in some aspects, comprises performing mass spectrometry on the Fc fragment-containing chromatographic fraction to obtain one or more mass spectral peaks. In various aspects, the method comprises deconvoluting the mass spectral peaks to obtain deconvoluted mass spectral peaks, as described herein. In exemplary instances, the method comprises matching the molecular weight of each deconvoluted mass spectral peak against a database of glycan pairs and the associated molecular weight to identify the glycan pair, exemplary aspects, the matching is carried out through an automated, computer assisted process, as described herein (see Examples 13-15, for instance). The database in exemplary aspects comprises the glycan pairs and their associated theoretical molecular weights of Tables 19, 22, and 25 of Examples 13, 14, and 15, respectively. In exemplary instances, the method comprises quantifying the abundance of (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans and in exemplary aspect, the method comprises quantifying the relative unpaired afucosylated (AF) glycan content and relative unpaired high mannose (HM) glycan content. In various instances, the abundance of (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans is based on the quantification of digested glycan structures of a glycan pair of the components of the mixture. The digested glycan structures are, in exemplary aspects, categorized as (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, or (iv) unpaired high mannose glycans according to Figure 16A. In various aspects, the abundance of paired afucosylated glycans and the abundance of unpaired afucosylated glycans are summed so that relative unpaired afucosylated glycans and / or relative paired afucosylated glycans may be determined. In various aspects, the abundance of paired high mannose glycans and the abundance of unpaired high mannose glycans are summed so that relative unpaired high mannose glycans and / or relative paired high mannoseglycans may be determined. Suitable methods for carrying out the method of the present disclosure is described in detail in Examples 13-19.

[0070] In various aspects, the protein composition comprises an antibody. In various aspects, the protein of the protein composition is an antibody. The present disclosure also provides methods of determining the relative unpaired glycan content of a protein composition comprising a protein comprising an antibody. In exemplary embodiments, the method comprises (a) treating a sample of the protein composition with (i) an enzyme that cleaves an antibody heavy chain at a site N-terminal to the hinge region disulfide linkage to produce Fab fragments and Fc fragments, (ii) a mannosidase, and (iii) a pi-4 endoglycosidase, to produce a mixture of Fab fragments and Fc fragments of the protein, wherein at least some of the Fc fragments comprise digested glycan structures; (b) chromatographically separating the Fab fragments from the Fc fragments, (c) separating Fc fragments based on molecular weight, (d) quantifying the abundance of glycan pairs, optionally, (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans, of the mixture. In various aspects, the method comprises quantifying the relative unpaired afucosylated (AF) glycan content and / or relative unpaired high mannose (HM) glycan content of the protein composition.

[0071] With regard to any one of the presently disclosed methods, the method is, in various aspects, a part of a manufacturing process for the protein composition or the protein of the protein composition. In various instances, the method is performed in real time during manufacture of the protein composition. In exemplary aspects, the sample is a sample of in-process material. In exemplary instances, the glycan pairing content is determined pre-harvest or post-harvest.Optionally, the glycan pairing content is determined post-harvest. In various instances, the sample is obtained from a manufacturing lot.

[0072] Applications of Methods of Determining Glycan Pairing Content

[0073] The present inventive methods of determining glycan pairing content are useful during the development and / or production of a protein composition or an antibody composition. For example, the method of determining relative unpaired glycans may be used for analyzing an IgG antibody composition or a protein composition comprising a fusion protein comprising a glycosylated antibody Fc region or comprising a glycosylated antibody. Accordingly, methods of analyzing a protein composition or an IgG antibody composition are provided herein. In exemplary embodiments, the method comprises determining the glycan pairing content of a sample of the protein composition or IgG antibody composition in accordance with any one of the presently disclosed methods of determining the glycan pairing content. In various aspects, the methodcomprises comparing the glycan pairing content (e.g., relative unpaired AF glycan content and / or relative unpaired HM glycan content) of the protein composition or IgG antibody composition to the glycan pairing content (e.g., relative unpaired AF glycan content and / or relative unpaired HM glycan content) of a reference product. The method may be performed in manufacturing, for example to determine if a lot of the protein composition or IgG antibody composition meets a specification.

[0074] Additionally provided are methods of monitoring production of a protein composition or an IgG antibody composition. In exemplary embodiments, the method comprises determining the glycan pairing content of a protein composition or an IgG antibody composition in accordance with any one of the presently disclosed methods of determining the glycan pairing content, for a first sample obtained at a first timepoint and for a second sample taken at a second timepoint which is different from the first timepoint. In exemplary aspects, the method comprises comparing the glycan pairing content (e.g., relative unpaired AF glycan content and / or relative unpaired HM glycan content) of the first sample to the glycan pairing content (e.g., the relative unpaired AF glycan content and / or relative unpaired HM glycan content) of the second sample.

[0075] Additionally provided herein are methods of monitoring production of a protein composition. In exemplary embodiments, the method comprises determining the glycan pairing content of a protein composition in accordance with a presently disclosed method for a first sample obtained at a first timepoint and for a second sample taken at a second timepoint, which is different from the first timepoint, and, optionally, comparing the glycan pairing content (e.g., relative unpaired AF glycan content and / or relative unpaired HM glycan content) of the first sample to the glycan pairing content (e.g., relative unpaired AF glycan content and / or relative unpaired HM glycan content) of the second sample.

[0076] In various aspects, each of the first sample and second sample is a sample of in-process material. In various instances, 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 one or more conditions of the cell culture are modified and the second sample is a sample obtained after the one or more conditions of the cell culture are modified.

[0077] In various aspects, the presently disclosed methods are useful for determining whether a protein composition or an IgG antibody composition meets the statistical criteria which includes appropriate acceptance levels and / or appropriate rejection levels.

[0078] In various aspects, the methods of analyzing a protein composition or an IgG antibody composition and / or methods of monitoring production of a protein composition or an IgG antibody composition occur during the development or production of a protein composition or an antibodycomposition. Accordingly, methods of producing a protein composition or an antibody composition are also provided by the present disclosure. In exemplary embodiments, the method comprises (A) determining the glycan pairing content (e.g., relative unpaired glycan content) of a sample of a protein composition or an IgG antibody composition, wherein the glycan pairing content (e.g., relative unpaired glycan content) of the sample of the protein composition or antibody composition is determined in accordance with any one of the presently disclosed methods of determining the glycan pairing content, wherein the sample is a sample of in-process material, wherein, when the glycan pairing content (e.g., relative unpaired glycan content) is determined as outside a predetermined target range, the method further comprises (B) modifying one or more conditions of the cell culture to obtain a modified cell culture and determining the glycan pairing content (e.g., relative unpaired glycan content), and optionally, repeating (A) and (B) until the glycan pairing content (e.g., relative unpaired glycan content) is within the predetermined target range. In various instances, one or more conditions of the cell culture are modified to primarily change the relative unpaired HM glycan content to achieve the target range of glycan pairing content (e.g., relative unpaired glycan content). In various aspects, one or more conditions of the cell culture are modified to primarily change the relative unpaired AF glycan content to achieve the target range of glycan pairing content (e.g., relative unpaired glycan content). Additional embodiments of methods of producing antibody compositions are further provided below. See, e.g., Methods of Producing Antibody Compositions.

[0079] In various aspects, the presently disclosed methods are useful for selecting a cell line or cell clone for producing a protein in a protein composition manufacture process. In various aspects, the method comprises determining the glycan pairing content of a protein composition in accordance with any one of the presently disclosed methods of determining glycan pairing content for each cell line or cell clone within a series of cell lines or cell clones producing the protein of the protein composition, and identifying the cell line(s) or cell clone(s) that has / have a glycan pairing content within a preselected target range, wherein the identified cell line or cell clone is selected for producing the protein in the manufacture process. In various aspects, the preselected target range is based on the range of glycan pairing content, e.g., relative unpaired afucosylated glycan content and / or the range of relative unpaired high mannose glycan content, of a reference cell line or cell clone. In various aspects, the method comprises determining for each protein composition produced by each cell line or cell clone of the series, the glycan pairing content, the released glycan content and the ADCC levels. In various aspects, the method comprises the steps outlined herein at Examples 5 and 19.

[0080] ADCC and Methods of Modifying ADCC Activity Levels

[0081] The data presented herein support that the glycan pairing content (e.g., relative unpaired glycan content) of an antibody composition is related to the ADCC activity level for the antibody composition and that the ADCC activity level of the antibody composition may be modified by modifying the glycan pairing content (e.g., relative unpaired glycan content) of the antibody composition. Without being bound to a particular theory, the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content) of an antibody composition is related to the ADCC activity level of the antibody composition, and, changing the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content) of the antibody composition leads to changing the ADCC activity level of the antibody composition. It is further contemplated that relative unpaired glycan content (e.g., relative unpaired afucosylated glycan content and / or relative unpaired high mannose glycan content)) has greater leverage on ADCC than the relative paired glycan content (e.g., relative paired afucosylated glycan content and / or relative paired high mannose glycan content), so that a percent change in the relative unpaired glycan content will have a greater effect on ADCC than the same percent change in relative paired glycan content. Accordingly, provided herein are methods of modifying the ADCC level of an antibody composition. In exemplary embodiments, the method comprises modifying the relative unpaired afucosylated glycan content of an antibody composition and / or the relative unpaired high mannose glycan content of an antibody composition. Provided herein are methods of modifying the ADCC level of a protein composition. In exemplary embodiments, the method comprises modifying the relative unpaired afucosylated glycan content of protein composition and / or the relative unpaired high mannose glycan content of a protein composition.

[0082] The term "ADCC" or "antibody-dependent cell-mediated cytotoxicity" or "antibodydependent cellular cytotoxicity" refers to the mechanism by which an effector cell of the immune system (e.g., natural killer cells (NK cells), macrophages, neutrophils, eosinophils) actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies. ADCC is a part of the adaptive immune response and occurs when antigen-specific antibodies bind to (1) the membrane-surface antigens on a target cell through its antigen-binding regions and (2) to Fc receptors on the surface of the effector cells through its Fc region. Binding of the Fc region of the antibody to the Fc receptor causes the effector cells to release cytotoxic factors that lead to death of the target cell (e.g., through cell lysis or cellular degranulation).

[0083] Fc receptors are receptors on the surfaces of B lymphocytes, follicular dendritic cells, NK cells, macrophages, neutrophils, eosinophils, basophils, platelets and mast cells that bind to the Fcregion of an antibody. Fc receptors are grouped into different classes based on the type of antibody that they bind. For example, an Fey receptor is a receptor for the Fc region of an IgG antibody, an Fc-alpha receptor is a receptor for the Fc region of an IgA antibody, and an Fc-epsilon receptor is a receptor for the Fc region of an IgE antibody.

[0084] The term "FcyR" or "Fc-gamma receptor" is a protein belonging to the IgG superfamily involved in inducing phagocytosis of opsonized cells or microbes. 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: FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRI 11 A (CD16a), and FcyRI I IB (CD16b). The sequences of FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and FcyRIIIB can be found in many sequence databases, for example, at 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.

[0085] The term "ADCC activity" or "ADCC level" refers to the extent to which ADCC is activated or stimulated. Methods of measuring or determining the ADCC level of an antibody composition, including commercially available assays and kits for measuring or determining the ADCC level, are well-known in the art, as described, Yamashita et al., Scientific Reports 6: article number 19772 (2016), doi:10.1038 / srepl9772); Kantakamalakul et al., "A novel EGFP-CEM-NKr flow cytometric method for measuring antibody dependent cell mediated-cytotoxicity (ADCC) activity in HIV-l infected individuals", J Immunol Methods 315 (Issues 1-2): 1-10; (2006); Gomez-Roman et al., "A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity", J Immunol Methods 308 (Issues 1-2): 53-67 (2006); Schnueriger et al., Development of a quantitative, cell-line based assay to measure ADCC activity mediated by therapeutic antibodies", Molec Immunology 38 (Issues 12-13): 1512-1517 (2011); and Mata et al., "Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in51Cr-release and CD107a assays", J Immunol Methods 406: 1-9 (2014); all herein incorporated by reference for all purposes. The term "ADCC Assay" or "FcyR reporter gene assay" refers to an assay, kit or method useful to determine the ADCC activity of an antibody. Exemplary methods of measuring or determining the ADCC activity of an antibody in the methods described herein include the ADCC assay described in the Example 3 or the ADCC Reporter Assay commercially available from Promega (Catalog No. G7010 and G7018). In some embodiments, ADCC activity is measured or determined using a calcein release assay containing one or more of the following: a FcyRllla (158\Z)-expressing NK92(M1) cells as effector cells and HCC2218 cells or MT-3 cells as target cells labeled with calcein-AM. An illustration of an exemplary calcein release assay isprovided as Figure 10. In exemplary aspects of the calcein release assay, a standard curve is created using various concentrations of a reference antibody (Figure 10B).

[0086] In exemplary aspects, the level of ADCC of an antibody composition is determined by a quantitative cell-based assay which measures the ability of the antibodies of the antibody composition to mediate cell cytotoxicity in a dose-dependent manner in cells expressing the antigen of the antibodies and engaging FcyRIIIA receptors on effector cells through the Fc domain of the antibodies. In various embodiments, the method comprises the use of target cells harboring detectable labels that are released when the target cells are lysed by the effector cells. The amount of detectable label released from the target cells is a measure of the ADCC activity of the antibody composition. The amount of detectable label released from the target cells, in some aspects, is compared to a baseline. Also, the ADCC level may be reported as a % ADCC relative to a control % ADCC. In various aspects, the % ADCC is a relative % ADCC, which optionally, is relative to a control % ADCC. In various aspects, the control % ADCC is the % ADCC of a reference antibody. In exemplary instances, the control % ADCC is within a range of about 60% to about 130%. Optionally, the % ADCC is determined by the assay described in Example 3.

[0087] In exemplary embodiments, the method of modifying (increasing or decreasing) the ADCC level of an antibody composition comprises modifying (increasing or decreasing) the glycan pairing content (e.g., relative unpaired afucosylated glycan content of an antibody composition and / or the relative unpaired high mannose glycan content) of an antibody composition. In exemplary aspects, the presently disclosed method of modifying the ADCC level of an antibody composition comprises increasing the glycan pairing content (e.g., relative unpaired afucosylated glycan content) to increase the level of ADCC activity. In exemplary instances, the method of modifying the ADCC level of an antibody composition comprises increasing the glycan pairing content (e.g., relative unpaired high mannose glycan content) to increase the level of ADCC activity. In various aspects, the increase in ADCC activity level provided by the methods of the disclosure is at least or about a 1% to about a 20% increase (e.g., at least or about a 1% increase, at least or about a 2% increase, at least or about a 3% increase, at least or about a 4% increase, at least or about a 5% increase, at least or about a 6% increase, at least or about a 7% increase, at least or about a 8% increase, at least or about a 9% increase, at least or about a 10% increase, at least or about a 11% increase, at least or about a 12% increase, at least or about a 13% increase, at least or about a 14% increase, at least or about a 15% increase, at least or about a 16% increase, at least or about a 17% increase, at least or about a 18% increase, at least or about a 19% increase, at least or about a 20% increase) relative to a control. A suitable control may be the same protein or antibody composition without the increase in the glycan pairing content (e.g., relative unpaired glycan content). In exemplary embodiments, the increase inADCC activity level provided by the methods of the disclosure is about 10% to about 100%, optionally, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 70%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 10% to about 15%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, or about 95% to about 100%. The increase can be relative to the control. In exemplary embodiments, the increase in ADCC activity level provided by the methods of the disclosure is over 100%, e.g., 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or even 1000% relative a control. In exemplary embodiments, the level of ADCC activity increases by at least about 1.5-fold, relative a control. A suitable control may be an ADCC activity level of the same protein or antibody composition without the change in the glycan pairing content (e.g., relative unpaired glycan content). In exemplary embodiments, the level of ADCC activity increases by at least about 2-fold, relative a control. In exemplary embodiments, the level of ADCC activity increases by at least about 3-fold, relative a control. In exemplary embodiments, the level of ADCC activity increases by at least about 4-fold or about 5-fold, relative to a control. In various aspects, the increase in the level of ADCC activity of the antibody composition is related to the increase in glycan pairing content (e.g., relative unpaired glycan content). For instance, the increase in the level of ADCC activity of the antibody composition is at least or about X% per ~1% increase in glycan pairing content (e.g., relative unpaired glycan content), wherein X% is at least or about a 1% to about a 20% increase (e.g., at least or about a 1% increase, at least or about a 2% increase, at least or about a 3% increase, at least or about a 4% increase, at least or about a 5% increase, at least or about a 6% increase, at least or about a 7% increase, at least or about a 8% increase, at least or about a 9% increase, at least or about a 10% increase, at least or about a 11% increase, at least or about a 12% increase, at least or about a 13% increase, at least or about a 14% increase, at least or about a 15% increase, at least or about a 16% increase, at least or about a 17% increase, at least or about a 18% increase, at least or about a 19% increase, at least or about a 20% increase). Also, for example, X% may be about 10% to about 100%, optionally, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 10% to about 15%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, or about 95% to about 100%.

[0088] In various aspects, the method of modifying the ADCC level of an antibody composition comprises decreasing the glycan pairing content (e.g., relative unpaired afucosylated glycan content)to decrease the level of ADCC activity. In various instances, the method of modifying the ADCC level of an antibody composition comprises decreasing the glycan pairing content (e.g., relative unpaired high mannose glycan content) to decrease the level of ADCC activity. In various aspects, the decrease in the ADCC activity level provided by the methods of the disclosure is at least or about a 1% to about a 20% decrease (e.g., at least or about a 1% decrease, at least or about a 2% decrease, at least or about a 3% decrease, at least or about a 4% decrease, at least or about a 5% decrease, at least or about a 6% decrease, at least or about a 7% decrease, at least or about a 8% increase, at least or about a 9% increase, at least or about a 10% increase, at least or about a 11% increase, at least or about a 12% increase, at least or about a 13% increase, at least or about a 14% increase, at least or about a 15% increase, at least or about a 16% increase, at least or about a 17% increase, at least or about a 18% increase, at least or about a 19% increase, at least or about a 20% increase) relative a control. A suitable control may be the same protein or antibody composition without the change in the glycan pairing content (e.g., relative unpaired glycan) and overall glycan composition content. In exemplary embodiments, the decrease in the ADCC activity level provided by the methods of the disclosure is about 10% to about 100%, optionally, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 10% to about 15%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, or about 95% to about 100%. The decrease can be relative to a control. In exemplary embodiments, the decrease in the ADCC activity level provided by the methods of the disclosure is over 100%, e.g., 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or even 1000% relative a control. In exemplary embodiments, the level of ADCC activity decreases by at least about 1.5-fold, relative a control. A suitable control may be the ADCC activity level of the same protein or antibody composition without the change in the glycan content. In exemplary embodiments, the level of ADCC activity decreases by at least about 2-fold, relative a control. In exemplary embodiments, the level of ADCC activity decreases by at least about 3-fold, relative a control. In exemplary embodiments, the level of ADCC activity decreases by at least about 4-fold or about 5-fold, relative to a control. In various aspects, the decrease in the level of ADCC activity of the antibody composition is related to the decrease in glycan pairing content (e.g., relative unpaired glycan content). For instance, the decrease in the level of ADCC activity of the antibody composition is at least or about X% per ~1% decrease in glycan pairing content (e.g., relative unpaired glycan content), wherein X% is at least or about a 1% to about a 20% decrease (e.g., at least or about a 1% decrease, at least or about a 2% decrease, at least or about a 3% decrease, at least or about a 4%decrease, at least or about a 5% decrease, at least or about a 6% decrease, at least or about a 7% decrease, at least or about a 8% increase, at least or about a 9% increase, at least or about a 10% increase, at least or about a 11% increase, at least or about a 12% increase, at least or about a 13% increase, at least or about a 14% increase, at least or about a 15% increase, at least or about a 16% increase, at least or about a 17% increase, at least or about a 18% increase, at least or about a 19% increase, at least or about a 20% increase). Also, for example, X% may be about 10% to about 100%, optionally, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 10% to about 15%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, or about 95% to about 100%.

[0089] In exemplary aspects, the modification (increase or decrease) effected by the presently disclosed methods are relative to a "control". In exemplary aspects, the control is the level of ADCC activity when the steps of the method are not carried out. In exemplary aspects, the control is the level of ADCC activity when the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content) is not modified (increased or decreased). For example, a suitable control may be the ADCC activity level of the same protein or antibody composition but without the increase in the glycan pairing content (e.g., relative unpaired glycan content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content)), or a suitable control may be the ADCC activity level of the same protein or antibody composition but without the decrease in the glycan pairing content (e.g., relative unpaired glycan content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content)). In exemplary instances, the control may be the ADCC activity level of the same protein or antibody composition produced under the same cell culture conditions with exception of those conditions that were modified to cause a change in the glycan pairing content (e.g., relative unpaired glycan content). In exemplary aspects, the control may be the ADCC activity level of the same protein or antibody composition produced under a first set of cell culture conditions which lead to an ADCC activity level which is outside of a target range of ADCC activity level. In various aspects, the control may be the ADCC activity level of the same protein or antibody composition produced under a first set of cell culture conditions which lead to a glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content) which is / are outside of a target range.

[0090] The term "target range" refers to a range of values based on a reference standard or reference product. For instance, the target range of ADCC activity level may be the range of ADCCactivity level exhibited by a reference product. Also, for example, the target range of glycan pairing content (e.g., relative unpaired glycan content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content)) may be the range of glycan pairing content (e.g., relative unpaired glycan content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content)) for a reference product. In various aspects, the target range is a predetermined target range, meaning that the target range was determined or ascertained at a prior timepoint.

[0091] Methods of Modifying Unpaired Glycan Content

[0092] In exemplary embodiments, the method of modifying (increasing or decreasing) the ADCC level of an antibody composition comprises modifying (increasing or decreasing) the glycan pairing content (e.g., unpaired afucosylated glycan content of an antibody composition and / or the unpaired high mannose glycan content) of an antibody composition. In exemplary aspects, the presently disclosed method of modifying the ADCC level of an antibody composition comprises increasing the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose content) to increase the level of ADCC activity. In various aspects, the method of modifying the ADCC level of an antibody composition comprises increasing the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose content) by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, or more. In various aspects, the method of modifying the ADCC level of an antibody composition comprises increasing the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose content) by more than 5% or more than 10%, e.g., by about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In various aspects, the method comprises increasing the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose content) by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more.

[0093] In exemplary aspects, the presently disclosed method of modifying the ADCC level of an antibody composition comprises decreasing the glycan pairing content (e.g., unpaired afucosylated glycan content) to decrease the level of ADCC activity. In various aspects, the method of modifying the ADCC level of an antibody composition comprises decreasing the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose content) by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, or more. In various aspects, the method of modifying the ADCC level of an antibody composition comprises decreasingthe glycan pairing content (e.g., the unpaired afucosylated glycan content and / or the unpaired high mannose content) by more than 5% or more than 10%, e.g., by about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In various aspects, the method comprises decreasing the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose content) by at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more.

[0094] In exemplary aspects, the increase or decrease in the glycan pairing content (e.g., unpaired afucosylated glycan content and / or unpaired high mannose glycan content) is / are relative to a "control". In exemplary aspects, the control is the glycan pairing content (e.g., unpaired glycan content) of a control protein or antibody composition produced under the same cell culture conditions with exception of those conditions that lead to increased or decreased glycan pairing content (e.g., unpaired glycan content). In exemplary aspects, the control may be the glycan pairing content (e.g., unpaired glycan content) of the same protein or antibody composition produced under a first set of cell culture conditions which lead to an ADCC activity level which is outside of a target range of ADCC activity level. In various aspects, the control may be the glycan pairing content (e.g., unpaired glycan content) of the same protein or antibody composition produced under a first set of cell culture conditions which lead to a glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose glycan content) which is / are outside of a target range.

[0095] Without being bound to a particular theory, conditions which lead to a modified (increased or decreased) afucosylated glycan content and / or high mannose content may lead to increased or decreased unpaired afucosylated glycan content and / or unpaired high mannose content of an antibody composition. In various instances, the unpaired afucosylated glycan content and / or unpaired high mannose content is increased or decreased by following the teachings of any one of International Patent Application Publication Nos. WO2013 / 114164, WO2013 / 114245, WO2013 / 114167, WO2015128793, or WO2016 / 089919, W02018 / 170099, W02019 / 191150, each of which is incorporated herein by reference. In various instances, the unpaired afucosylated glycan content and / or unpaired high mannose content is increased or decreased by selecting a clone that produces antibody or antibody protein product comprising a level of unpaired afucosylated glycan content and / or unpaired high mannose content within a target range. See, e.g., Examples 5 and 19.

[0096] Methods of Producing Antibody Compositions

[0097] Simple and efficient methods to predict the level of effector function (e.g., ADCC) a particular antibody composition will exhibit based on a given glycoform profile for that antibody composition are described herein. The data provided herein support that the ADCC activity level for the antibody composition may be predicted based on the glycan pairing content (e.g., the relative unpaired glycan content) of an antibody composition. Without being bound to a particular theory, the unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content of an antibody composition is predictive of the ADCC activity level of the antibody composition. Such predicted ADCC levels are useful during antibody production, when, it is necessary for the antibody to have an ADCC activity level within a target range. For instance, by monitoring the glycan pairing content (e.g., the unpaired afucosylated glycan content and / or the unpaired high mannose glycan content) of an antibody composition, it may be predicted whether the antibody composition will exhibit an ADCC activity level within a target range. If a target range of ADCC activity levels for an antibody composition is known, the target range of the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content) may be determined. Selection of the antibody composition for continued processing, e.g., downstream processing, may occur when the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose glycan, or the ADCC activity level, as calculated based on the unpaired afucosylated glycan content and / or the unpaired high mannose glycan content), is / are within a target range. In exemplary aspects, the target range is based on a target range of ADCC activity levels for a reference antibody and a model which correlates ADCC activity level of the antibody composition to afucosylated glycan content and / or high mannose glycan content of the antibody composition, optionally, a model which correlates ADCC activity level of the antibody composition to glycan pairing content (e.g., unpaired afucosylated glycan content and / or unpaired high mannose glycan content) of the antibody composition.

[0098] Accordingly, the present disclosure provides methods of producing an antibody composition. In exemplary embodiments, the method comprises (i) determining the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content) of a sample of the antibody composition; and (ii) selecting the antibody composition for downstream processing based on the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or relative unpaired high mannose glycan content) determined in (i). In exemplary embodiments, the method of producing an antibody composition comprises (i) determining the glycan pairing content (e.g., unpaired afucosylated glycan content of an antibody composition and / or the unpaired high mannose glycan content) of an antibody composition; (ii)determining the ADCC level of the antibody composition based on the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose glycan content) determined in (i); and (iii) selecting the antibody composition for downstream processing when the ADCC level of the antibody composition determined in (ii) is within a target ADCC range. In exemplary embodiments, the method of producing an antibody composition comprises (i) determining the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose glycan content) of a sample of the antibody composition taken from a cell culture comprising glycosylation-competent cells expressing an antibody of the antibody composition; (ii) optionally, modifying the cell culture to modulate the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose glycan content) and determining the glycan pairing content (e.g., unpaired afucosylated glycan content and / or the unpaired high mannose glycan content) of a sample of the antibody composition taken from the modified cell culture; and (iii) selecting the antibody composition for downstream processing based on the glycan pairing content (e.g., unpaired afucosylated glycan content and / or unpaired high mannose glycan content).

[0099] Also provided herein are methods of preparing an IgG antibody composition having an ADCC activity level within a target range. In exemplary embodiments, the method comprises determining the glycan pairing content (e.g., relative unpaired glycan content) of a sample of the IgG antibody composition in accordance with any one of the presently disclosed methods of determining the glycan pairing content (e.g., relative unpaired glycan content). The glycan pairing content (e.g., relative unpaired AF glycan content and / or relative unpaired HM glycan content) of the IgG antibody composition may be compared to a target range of glycan pairing content (e.g., unpaired AF glycan content and / or relative unpaired HM glycan content). For example, the target range may be a reference level of a specification, or of a reference IgG antibody composition. Also, for instance, the target range of glycan pairing content (e.g., unpaired AF glycan content and / or relative unpaired HM glycan content) may be a predetermined target range of glycan pairing content (e.g., unpaired AF glycan content and / or relative unpaired HM glycan content) which statistically correlates with a predetermined target range of ADCC activity levels of a reference IgG antibody composition.

[0100] Methods of producing an antibody composition are also provided by the present disclosure. In exemplary embodiments, the method comprises determining glycan pairing content (e.g., relative unpaired glycan content) of the antibody composition in accordance with any one of the presently disclosed methods of determining the glycan pairing content (e.g., relative unpaired glycan content), wherein, when the glycan pairing content (e.g., relative unpaired glycan content) is determined as outside a predetermined target range, the method further comprises modifying oneor more conditions of the cell culture to obtain a modified cell culture and determining the glycan pairing content (e.g., relative unpaired glycan content). The method in various instance comprises repeating steps of the method until the glycan pairing content (e.g., relative unpaired glycan content) is within the predetermined target range.

[0101] In various aspects, the sample is taken from a cell culture comprising glycosylation-competent cells expressing an antibody of the antibody composition. Optionally, the method further comprises modifying one or more conditions of the cell culture to modify the glycan pairing content (e.g, relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content) of the antibody composition and determining the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content) of a sample of the antibody composition taken from the modified cell culture. In exemplary aspects, the method comprises repeating the modifying until the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or relative unpaired high mannose glycan content) is within a target range. In various aspects, the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or the relative unpaired high mannose glycan content) is / are determined in real time with respect to production of the antibody composition. As used herein "real time" refers to determinations that are made while a production process is ongoing, without interruption to the process. It will be appreciated that production of therapeutic proteins involves living cells and sensitive materials that cannot be put on hold indefinitely while assays and determinations are performed. If numerical examples of interest, a "real time" determination can be a determination that is made within 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minute, 10 minutes, 5 minutes, 1 minute, 30 seconds, 20 seconds, 10 seconds, 5 seconds, 1 seconds, or 0.1 seconds from the time a measurement is made (while the production process is ongoing). In various instances, the method comprises selecting the antibody composition for downstream processing when the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or relative unpaired high mannose glycan content) is / are in a target range. In various aspects, the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or relative unpaired high mannose glycan content) correlate with the ADCC activity level of the antibody composition. Optionally, the method further comprises determining the ADCC activity level of the antibody composition based on the glycan pairing content (e.g., relative unpaired afucosylated glycan content and / or relative unpaired high mannose glycan content) determined in (i). Optionally, the method comprises selecting the antibody composition for downstream processing when the ADCC activity level is in a target range.

[0102] In various aspects, the method of producing an antibody composition comprises modifying the ADCC level of an antibody composition according to a method of modifying the ADCC level of the present disclosure.

[0103] In various instances, the method of producing an antibody composition comprises determining the glycan pairing content (e.g., relative unpaired glycan content) of an antibody composition according to any of the presently disclosed methods of determining the glycan pairing content (e.g., relative unpaired glycan content) of an antibody composition.

[0104] Downstream Processing

[0105] The glycan pairing content (e.g., relative % unpaired high mannose glycans and / or relative % unpaired afucosylated glycans) are determined (e.g., measured) to better inform as to the % antibody-dependent cell-mediated cytotoxicity (ADCC) of the antibody composition. The determining (e.g., measuring) may occur at any stage of manufacture. In particular, measurements may be taken pre- or post-harvest, preceding or during any stage of downstream processing.Example downstream processing includes any chromatography unit operation, including capture chromatography, intermediate chromatography, and / or polish chromatography unit operations; virus inactivation and neutralization; virus filtration; and / or final formulation. The glycan pairing content (e.g., relative % unpaired high mannose glycans, and / or relative % unpaired afucosylated glycans) in various aspects is determined (e.g., measured) in real-time, near real-time, and / or after the fact. Monitoring and measurements can be done using known techniques and commercially available equipment.

[0106] In various aspects of the present disclosure, the determining (e.g., measuring) of the glycan pairing content (e.g., % unpaired high mannose glycans and / or % unpaired afucosylated glycans) is carried out before a harvest. As used herein the term "harvest" refers to cell culture media containing the recombinant protein of interest being collected and separated at least from the cells of the cell culture. The harvest can be performed continuously. The harvest in some aspects is performed using centrifugation and can further comprise precipitation, filtration, and the like. In various aspects, the determining is carried out before harvest. In various aspects, the determining is carried out before chromatography, optionally, Protein A chromatography. In some aspects, the determining (e.g., measuring) of the glycan pairing content (e.g., relative % unpaired high mannose glycans and / or relative % unpaired afucosylated glycans) is carried out at least 3 days, at least 4 days, or at least 5 days before harvest. Optionally, determining (e.g., measuring) the glycan pairing content (e.g., relative % unpaired high mannose glycans and / or relative % unpaired afucosylated glycans) is carried out in real-time with regard to antibody production.

[0107] In various aspects of the present disclosure, determining (e.g., measuring) the glycan pairing content (e.g., relative % unpaired high mannose glycans, and / or relative % unpaired afucosylated glycans) is carried out after a harvest. In various aspects, the determining is carried out after chromatography, optionally, Protein A chromatography. In various aspects, the determining is carried out after harvest and after chromatography, e.g., a Protein A chromatography.

[0108] With regard to the presently disclosed methods, the antibody composition in various aspects is selected or chosen for further processing, e.g., for downstream processing, and the selection is based on a particular parameter, e.g., % ADCC, glycan pairing content (e.g., relative % unpaired high mannose glycans, and / or relative % unpaired afucosylated glycans). In various instances, the presently disclosed methods comprise using the antibody composition in further processing, e.g., downstream processing, based on a particular parameter, e.g., based on the % ADCC, glycan pairing content (e.g., relative % unpaired high mannose glycans, and / or relative % unpaired afucosylated glycans). In various instances, the presently disclosed methods comprise carrying out further processing, e.g., downstream processing, with the antibody composition, based on a particular parameter, e.g., based on the % ADCC, glycan pairing content (e.g., relative % unpaired high mannose glycans, and / or relative % unpaired afucosylated glycans).

[0109] In exemplary instances the downstream processing comprises or consists of any processing which occurs after (or downstream of) the processing at which the glycan pairing content (e.g., relative % unpaired high mannose glycans, and / or relative % unpaired afucosylated glycans) are determined (e.g., measured). For example, if the glycan pairing content (e.g., relative % unpaired high mannose glycans, and / or relative % unpaired afucosylated glycans) were determined (e.g., measured) at harvest, then the downstream processing is any processing which occurs after (or downstream of) the harvest, which in various aspects comprise(s): dilution, filling, filtration, formulation, chromatography, viral filtration, viral inactivation, or a combination thereof. Also, for example, if the glycan pairing content (e.g., relative % unpaired high mannose glycans, and / or relative % unpaired afucosylated glycans) were determined (e.g., measured) after chromatography, e.g., Protein A chromatography, then the downstream processing comprises or consists of any processing which occurs after (or downstream of) the chromatography, and the downstream processing in various aspects comprise(s): a dilution, a filling, a filtration, a formulation, further chromatography, a viral filtration, a viral inactivation, or a combination thereof. In exemplary instances the further chromatography is an ion exchange chromatography (e.g., cation exchange chromatography or anion exchange chromatography).

[0110] Stages / types of chromatography used during downstream processing include capture or affinity chromatography which is used to separate the recombinant product from other proteins,aggregates, DNA, viruses and other such impurities. In exemplary instances, initial chromatography is carried out with Protein A (e.g., Protein A attached to a resin). Intermediate and polish chromatography in various aspects further purify the recombinant protein, removing bulk contaminants, adventitious viruses, trace impurities, aggregates, isoforms, etc. The chromatography can either be performed in bind and elute mode, where the recombinant protein of interest is bound to the chromatography medium and the impurities flow through, or in flow-through mode, where the impurities are bound and the recombinant protein flows through. Examples of such chromatography methods include ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed modal or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reverse phase chromatography and gel filtration.

[0111] In various aspects, the downstream processing comprises viral inactivation. Enveloped viruses have a capsid enclosed by a lipoprotein membrane or "envelope" and are therefore susceptible to inactivation. The virus inactivation in various instances includes heat inactivation / pasteurization, pH inactivation, UV and gamma ray irradiation, use of high intensity broad spectrum white light, addition of chemical inactivating agents, surfactants, and solvent / detergent treatments.

[0112] In various aspects, the downstream processing comprises virus filtration. In various aspects, the virus filtration comprises removing non-enveloped viruses. In various aspects, the virus filtration comprises the use of micro- or nano-filters.

[0113] In various aspects, the downstream processing comprises formulation, which may be performed in one or more steps. Following completion of the chromatography, the purified recombinant proteins are in various aspects buffer exchanged into a formulation buffer. In exemplary aspects, the buffer exchange is performed using ultrafiltration and diafiltration (UF / DF). In exemplary aspects, the recombinant protein is buffer exchanged into a desired formulation buffer using diafiltration and concentrated to a desired final formulation concentration using ultrafiltration. Additional stability-enhancing excipients in various aspects are added following a UF / DF formulation.

[0114] Additional Steps

[0115] The methods disclosed herein, in various aspects, comprise additional steps. For example, in some aspects, the methods comprise one or more upstream steps or downstream steps involved in producing, purifying, and formulating an antibody composition. Optionally, the downstream steps are any one of those downstream processing steps described herein or known in the art. See, e.g., Downstream Processing. In exemplary embodiments, the method comprises stepsfor generating host cells that express a recombinant glycosylated protein (e.g., antibody). The host cells, in some aspects, are prokaryotic host cells, e.g., E. coli or Bacillus subtilis, or the host cells, in some aspects, are eukaryotic host cells, e.g., yeast cells, filamentous fungi cells, protozoa cells, insect cells, or mammalian cells (e.g., CHO cells). Such host cells are described in the art. See, e.g., Kunert et al., Appl. Microbiol Biotechnol. 100: 3451-61 (2016) and herein under "Cells." For example, the methods comprise, in some instances, introducing into host cells a vector comprising a nucleic acid comprising a nucleotide sequence encoding the recombinant glycosylated protein, or a polypeptide chain thereof.

[0116] In exemplary aspects, the methods comprise maintaining cells, e.g., glycosylation-competent cells in a cell culture. Accordingly, the methods may comprise carrying out any one or more steps described herein in Maintaining Cells In A Cell Culture.

[0117] In exemplary embodiments, the methods disclosed herein comprise steps for isolating and / or purifying the antibodies from the culture. In exemplary aspects, the method comprises one or more chromatography steps including, but not limited to, e.g., affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, and / or hydrophobic interaction chromatography. In exemplary aspects, the method comprises steps for producing crystalline biomolecules from a solution comprising the recombinant glycosylated proteins.

[0118] The methods of the disclosure, in various aspects, comprise one or more steps for preparing a composition, including, in some aspects, a pharmaceutical composition, comprising the purified recombinant glycosylated protein. Such compositions are discussed herein.

[0119] Maintaining Cells In A Cell Culture

[0120] With regard to the methods of producing an antibody composition of the present disclosure, the antibody composition may be produced by maintaining cells in a cell culture. The cell culture may be maintained according to any set of conditions suitable for production of a recombinant glycosylated protein. For example, in some aspects, the cell culture is maintained at a particular pH, temperature, cell density, culture volume, dissolved oxygen level, pressure, osmolality, and the like. In exemplary aspects, the cell culture prior to inoculation is shaken (e.g., at 70 rpm) at 5% CO2under standard humidified conditions in a CO2incubator. In exemplary aspects, the cell culture is inoculated with a seeding density of about 106cells / mL in 1.5 L medium.

[0121] In exemplary aspects, the methods of the disclosure comprise maintaining the glycosylation-competent cells in a cell culture medium at a pH of about 6.85 to about 7.05, e.g., in various aspects, 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.

[0122] In exemplary aspects, the methods comprise maintaining the cell culture at a temperature between 305C and 405C. In exemplary embodiments, the temperature is between about 32^C to about 38^C or between about 35^C to about 385C.

[0123] In exemplary aspects, the methods comprise maintaining the osmolality between about 200 mOsm / kg to about 500 mOsm / kg. In exemplary aspects, the method comprises maintaining the osmolality between about 225 mOsm / kg to about 400 mOsm / kg or about 225 mOsm / kg to about 375 mOsm / kg. In exemplary aspects, the method comprises maintaining the osmolality between about 225 mOsm / kg to about 350 mOsm / kg. In various aspects, 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.

[0124] In exemplary aspects, the methods comprise maintaining dissolved the oxygen (DO) level of the cell culture at about 20% to about 60% oxygen saturation during the initial cell culture period. In exemplary instances, the method comprises maintaining 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 exemplary instances, the method comprises maintaining 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 exemplary aspects, the DO level is about 35 mm Hg to about 85 mmHg or about 40 mm Hg to about 80 mmHg or about 45 mm Hg to about 75 mm Hg.

[0125] The cell culture is maintained in any one or more culture medium. In exemplary aspects, the cell culture is maintained in a medium suitable for cell growth and / or is provided with one or more feeding media according to any suitable feeding schedule. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprising glucose, fucose, lactate, ammonia, glutamine, and / or glutamate. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprising manganese at a concentration less than or about 1 pM during the initial cell culture period. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprising about 0.25 pM to about 1 pM manganese. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprising negligible amounts of manganese. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprising copper at a concentration less than or about 50 ppb during the initial cell culture period. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprisingcopper at a concentration less than or about 40 ppb during the initial cell culture period. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprising copper at a concentration less than or about 30 ppb during the initial cell culture period. In exemplary aspects, the method comprises maintaining the cell culture in a medium comprising copper at a concentration less than or about 20 ppb during the initial cell culture period. In exemplary aspects, the medium comprises copper at a concentration greater than or about 5 ppb or greater than or about 10 ppb. In exemplary aspects, the cell culture medium comprises mannose. In exemplary aspects, the cell culture medium does not comprise mannose.

[0126] In exemplary embodiments, the type of cell culture is a fed-batch culture or a continuous perfusion culture. However, the methods of the disclosure are advantageously not limited to any particular type of cell culture.

[0127] The cells maintained in cell culture may be glycosylation-competent cells. In exemplary aspects, the glycosylation-competent cells are eukaryotic cells, including, but not limited to, yeast cells, filamentous fungi cells, protozoa cells, algae cells, insect cells, or mammalian cells. Such host cells are described in the art. See, e.g., Kunert et al., Appl. Microbiol Biotechnol. 100: 3451-61 (2016). In exemplary aspects, the eukaryotic cells are mammalian cells. In exemplary aspects, the mammalian cells are non-human mammalian cells. In some aspects, the cells are Chinese Hamster Ovary (CHO) cells and derivatives thereof (e.g., CHO-K1, CHO pro-3), mouse myeloma cells (e.g., NSO, GS-NSO, Sp2 / 0), cells engineered to be deficient in dihydrofolatereductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or derivatives thereof (e.g., HEK293T, HEK293-EBNA), green African monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human bone osteosarcoma epithelial cells U2-OS, adenocarcinomic human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, mouse embryo fibroblast cells NIH 3T3, mouse fibroblast cells L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer 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)).

[0128] Cells that are not glycosylation-competent can also be transformed into glycosylation-competent cells, e.g. by transfecting them with genes encoding relevant enzymes necessary for glycosylation. Exemplary enzymes include but are not limited to oligosaccharyltransferases, glycosidases, glucosidase I, glucosidease II, calnexin / calreticulin, glycosyltransferases, mannosidases, GIcNAc transferases, galactosyltransferases, and sialyltransferases.

[0129] In exemplary embodiments, the glycosylation-competent cells are not genetically modified to alter the activity of an enzyme of the de novo pathway or the salvage pathway. These two pathways of fucose metabolism are shown in Figure 2. In exemplary embodiments, the glycosylation-competent cells are not genetically modified to alter the activity of any one or more of: a fucosyl-transferase (FUT, e.g.,FUTl, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9), a fucose kinase, a GDP-fucose pyrophosphorylase, GDP-D-mannose-4,6-dehydratase (GMD), and GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase (FX). In exemplary embodiments, the glycosylation-competent cells are not genetically modified to knock-out a gene encoding FX.

[0130] In exemplary embodiments, the glycosylation-competent cells are not genetically modified to alter the activity P(l,4)- / V-acetylglucosaminyltransferase III (GNTIII) or GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD). In exemplary aspects, the glycosylation-competent cells are not genetically modified to overexpress GNTIII or RMD.

[0131] Proteins of the Protein Compositions

[0132] In exemplary embodiments, the presently disclosed methods of determining glycan pairing content is carried out for determining glycan pairing content of a protein composition. In various aspects, the protein of the protein composition comprises an antibody comprising an Fc region or is an antibody comprising an Fc region. In the latter instance, the protein composition is an antibody composition. In various aspects, the protein of the protein composition comprises a fusion protein comprising an Fc region. In various aspects, one or more of the polypeptides of the Fc region is fused to a polypeptide. The polypeptide may be for instance an interleukin, cytokine, hormone, growth factor, cell-surface receptor or any ligand thereof, or other protein. The polypeptide may be any of the antigens listed herein under "Antibodies and Fragments Thereof. In various instances, the fusion protein comprising an Fc region comprises a pair of polypeptide wherein each polypeptide comprises an N-linked glycan that together form a glycopair. In various aspects, the fusion protein comprising an Fc region further comprises an antigen binding fragment, e.g., a Fab. In various aspects, the fusion protein comprises an antibody fused to a polypeptide an the C-terminus of one or both of the polypeptides of the Fc region.

[0133] Antibodies and Fragments Thereof

[0134] As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, and comprising variable and constant regions. For example, an antibody may be an IgG which is a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa).. Anantibody has a variable region and a constant region. In IgG formats, the variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and substantially varies among other antibodies that bind to different antigens. See, e.g., Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4th ed.Elsevier Science Ltd. / Garland Publishing, (1999).

[0135] Briefly, in an antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition. A variable region comprises at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342: 877-883), within a framework region (designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0136] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgGl, lgG2, lgG3, and lgG4. IgM has subclasses, including, but not limited to, IgMl and lgM2. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In various aspects, the antibody is an IgG, including any one of IgGl, lgG2, lgG3 or lgG4. In various instances, the antibody is an IgGl.

[0137] In various aspects, the antibody can be a monoclonal antibody or a polyclonal antibody. In exemplary instances, the antibody is a mammalian antibody, e.g., a mouse antibody, rat antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, pig antibody, human antibody, and the like. In certain aspects, the recombinant glycosylated protein is a monoclonal human antibody.

[0138] In various aspects, the antibody is a chimeric antibody or a humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody containing constant domains from one species and the variable domains from a second, or more generally, containing stretches of amino acid sequence from at least two species. The term "humanized" when used in relation to antibodies refers to antibodies having at least CDR regions from a non-human source which areengineered to have a structure and immunological function more similar to true human antibodies than the original source antibodies. For example, humanizing can involve grafting CDR from a nonhuman antibody, such as a mouse antibody, into a human antibody. Humanizing also can involve select amino acid substitutions to make a non-human sequence look more like a human sequence.

[0139] An antibody, in various aspects, is cleaved into fragments by enzymes, such as, e.g., papain and pepsin. Papain cleaves an antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves an antibody to produce a F(ab')2 fragment and a pFc' fragment. As described herein, the methods of determining unpaired glycan content comprises a two-enzyme digestion, one of which is used to produce a mixture of Fab fragments and Fc fragments.

[0140] Advantageously, the methods are not limited to the antigen-specificity of the antibody. Accordingly, the antibody has any binding specificity for virtually any antigen. In exemplary aspects, the antibody binds to a hormone, growth factor, cytokine, a cell-surface receptor, or any ligand thereof. In exemplary aspects, the antibody binds to a protein expressed on the cell surface of an immune cell. In exemplary aspects, the antibody binds to a cluster of differentiation molecule selected from the group consisting of: CDla, CDlb, CDlc, CDld, CD2, CDS, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11A, CD11B, CD11C, CDwl2, CD13, CD14, CD15, CD15s, CD16, CDwl7, 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, CD79a, CD79P, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDwlO8, CD109, CD114, CD 115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CDwl21b, CD122, CD123, CD124, CD125, CD126, CD127, CDwl28, CD129, CD130, CDwl31, CD132, CD134, CD135, CDwl36, CDwl37, 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.

[0141] In exemplary aspects, the antibody is one of those described in U.S. Patent No.7947809 and U.S. Patent Application Publication No. 20090041784 (glucagon receptor), U.S. Patent No. 7939070, U.S. Patent No. 7833527, U.S. Patent No. 7767206, and U.S. Patent No. 7786284 (IL-17 receptor A), U.S. Patent No. 7872106 and U.S. Patent No. 7592429 (Sclerostin), U.S. 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. Patent No. 7728110 (SARS coronavirus), U.S. Patent No. 7718776 and U.S. Patent Application Publication No. 20100209435 (OPGL), U.S. Patent No. 7658924 and U.S. Patent No. 7521053 (Angiopoietin-2), U.S. Patent No. 7601818, U.S. Patent No. 7795413, U.S. Patent Application Publication No.20090155274, U.S. Patent Application Publication No. 20110040076 (NGF), U.S. Patent No. 7579186 (TGF-P type II receptor), U.S. Patent No. 7541438 (connective tissue growth factor), U.S. Patent No.7438910 (IL1-R1), U.S. Patent No. 7423128 (properdin), U.S. Patent No. 7411057, U.S. Patent No. 7824679, U.S. Patent No. 7109003, U.S. Patent No. 6682736, U.S. Patent No. 7132281, and U.S. Patent No. 7807797 (CTLA-4), U.S. Patent No. 7084257, U.S. Patent No. 7790859, U.S. Patent No. 7335743, U.S. Patent No. 7084257, and U.S. Patent Application Publication No. 20110045537 (interferon-gamma), U.S. Patent No. 7932372 (MAdCAM), U.S. Patent No. 7906625, U.S. Patent Application Publication No. 20080292639, and U.S. Patent Application Publicaiton No. 20110044986 (amyloid), U.S. Patent No. 7815907 and U.S. Patent No. 7700742 (insulin-like growth factor I), U.S. Patent No. 7566772 and U.S. Patent No. 7964193 (interleukin-lp), U.S. Patent No. 7563442, U.S. Patent No. 7288251, U.S. Patent No. 7338660, U.S. Patent No. 7626012, U.S. Patent No. 7618633, and U.S. Patent Application Publication No. 20100098694 (CD40), U.S. Patent No. 7498420 (c-Met), U.S. Patent No. 7326414, U.S. Patent No. 7592430, and U.S. Patent No. 7728113 (M-CSF), U.S. Patent No. 6924360, U.S. Patent No. 7067131, and U.S. Patent No. 7090844 (MUC18), U.S. Patent No. 6235883, U.S. Patent No. 7807798, and U.S. Patent Application Publication No. 20100305307 (epidermal growth factor receptor), U.S. Patent No. 6716587, U.S. Patent No. 7872113, U.S. Patent No. 7465450, U.S. Patent No. 7186809, U.S. Patent No. 7317090, and U.S. Patent No. 7638606 (interleukin-4 receptor), U.S. Patent Application Publication No. 20110135657 (BETA-KLOTHO), U.S. Patent No. 7887799 and U.S. Patent No. 7879323 (fibroblast growth factor-like polypeptides), U.S. Patent No. 7867494 (IgE), U.S. Patent Application Publication No. 20100254975 (ALPHA-4 BETA-7), U.S. Patent Application Publication No. 20100197005 and U.S. Patent No. 7537762 (ACTIVIN RECEPTOR-LIKE KINASE-1), U.S. Patent No. 7585500 and U.S. Patent Application Publication No. 20100047253 (IL-13), U.S. Patent Application Publication No. 20090263383 and U.S. Patent No. 7449555 (CD148), U.S. Patent Application Publication No. 20090234106 (ACTIVIN A), U.S. Patent Application Publication No. 20090226447 (angiopoietin-1 and angiopoietin-2), U.S. Patent Application Publication No. 20090191212 (Angiopoietin-2), U.S. Patent Application Publication No.20090155164 (C-FMS), U.S. Patent No. 7537762 (activin receptor-like kinase-1), U.S. Patent No.7371381 (galanin), U.S. Patent Application Publication No. 20070196376 (INSULIN-LIKE GROWTH FACTORS), U.S. Patent No. 7267960 and U.S. Patent No. 7741115 (LDCAM), US7265212 (CD45RB), U.S. Patent No. 7709611, U.S. Patent Application Publication No. 20060127393 and U.S. Patent Application Publication No. 20100040619 (DKK1), U.S. Patent No. 7807795, U.S. Patent Application Publication No. 20030103978 and U.S. Patent No. 7923008 (osteoprotegerin), U.S. Patent Application Publication No. 20090208489 (OV064), U.S. Patent Application Publication No.20080286284 (PSMA), U.S. Patent No. 7888482, U.S. Patent Application Publication No.20110165171, and U.S. Patent Application Publication No. 20110059063 (PAR2), U.S. Patent Application Publication No. 20110150888 (HEPCIDIN), U.S. Patent No. 7939640 (B7L-1), U.S. Patent No. 7915391 (c-Kit), U.S. Patent No. 7807796, U.S. Patent No. 7193058, and U.S. Patent No. 7427669 (ULBP), U.S. Patent No. 7786271, U.S. Patent No. 7304144, and U.S. Patent Application Publication No. 20090238823 (TSLP), U.S. Patent No. 7767793 (SIGIRR), U.S. Patent No. 7705130 (HER-3), U.S. Patent No. 7704501 (ataxin-l-like polypeptide), U.S. Patent No. 7695948 and U.S. Patent No.7199224 (TNF-a converting enzyme), U.S. Patent Application Publication No. 20090234106 (ACTIVIN A), U.S. Patent Application Publication No. 20090214559 and U.S. Patent No. 7438910 (IL1-R1), U.S. Patent No. 7579186 (TGF-P type II receptor), U.S. Patent No. 7569387 (TNF receptor-like molecules), U.S. Patent No. 7541438, (connective tissue growth factor), U.S. Patent No. 7521048 (TRAIL receptor-2), U.S. Patent No. 6319499, U.S. Patent No. 7081523, and U.S. Patent Application Publication No. 20080182976 (erythropoietin receptor), U.S. Patent Application Publication No. 20080166352 and U.S. Patent No. 7435796 (B7RP1), U.S. Patent No. 7423128 (properdin), U.S. Patent No. 7422742 and U.S. Patent No. 7141653 (interleukin-5), U.S. Patent No. 6740522 and U.S. Patent No. 7411050 (RANKL), U.S. Patent No. 7378091 (carbonic anhydrase IX (CA IX) tumor antigen), U.S. Patent No. 7318925and U.S. Patent No. 7288253 (parathyroid hormone), U.S. Patent No. 7285269 (TNF), U.S. Patent No. 6692740 and U.S. Patent No. 7270817 (ACPL), U.S. Patent No.7202343 (monocyte chemo-attractant protein-1), U.S. Patent No. 7144731 (SCF), U.S. Patent No. 6355779 and U.S. Patent No. 7138500 (4-1BB), U.S. Patent No. 7135174 (PDGFD), U.S. Patent No.6630143 and U.S. Patent No. 7045128 (Flt-3 ligand), U.S. Patent No. 6849450 (metalloproteinase inhibitor), U.S. Patent No. 6596852 (LERK-5), U.S. Patent No. 6232447 (LERK-6), U.S. Patent No. 6500429 (brain-derived neurotrophic factor), U.S. Patent No. 6184359 (epithelium-derived T-cell factor), U.S. Patent No. 6143874 (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 purposes of their disclosure of variable domainpolypeptides, variable domain encoding nucleic acids, host cells, vectors, methods of making polypeptides encoding said variable domains, pharmaceutical compositions, and methods of treating diseases associated with the respective target of the variable domain-containing antigen binding protein or antibody.

[0142] In exemplary embodiments, the antibody is one of Muromonab-CD3 (product marketed with the brand name Orthoclone Okt3®), Abciximab (product marketed with the brand name Reopro®), Rituximab (product marketed with the brand name MabThera®, Rituxan®), Basiliximab (product marketed with the brand name Simulect®), Daclizumab (product marketed with the brand name Zenapax®), Palivizumab (product marketed with the brand name Synagis®), Infliximab (product marketed with the brand name Remicade®), Trastuzumab (product marketed with the brand name Herceptin®), Alemtuzumab (product marketed with the brand name MabCampath®, Campath-1H®), Adalimumab (product marketed with the brand name Humira®), Tositumomab-1131 (product marketed with the brand name Bexxar®), Efalizumab (product marketed with the brand name Raptiva®), Cetuximab (product marketed with the brand name Erbitux®), Ibritumomab tiuxetan (product marketed with the brand name Zevalin®), Omalizumab (product marketed with the brand name Xolair®), ocrelizumab (product marketed with the brand name Ocrevus®), Bevacizumab (product marketed with the brand name Avastin®), Bemarituzumab; Natalizumab (product marketed with the brand name Tysabri®), Ranibizumab (product marketed with the brand name Lucentis®), Panitumumab (product marketed with the brand name Vectibix®), Eculizumab (product marketed with the brand name Soliris®), Certolizumab pegol (product marketed with the brand name Cimzia®), Golimumab (product marketed with the brand name Simponi®), Canakinumab (product marketed with the brand name Haris®), Catumaxomab (product marketed with the brand name Removab®), Ustekinumab (product marketed with the brand name Stelara®), Tocilizumab (product marketed with the brand name RoActemra®, Actemra®), Ofatumumab (product marketed with the brand name Arzerra®), Denosumab (product marketed with the brand name Prolia®), Belimumab (product marketed with the brand name Benlysta®), Raxibacumab, Rocatinlimab, Ipilimumab (product marketed with the brand name Yervoy®), and Pertuzumab (product marketed with the brand name Perjeta antibodies, such as daclizumab.

[0143] In exemplary aspects, the antibody binds to a tumor associated antigen and is an anticancer antibody. Examples of suitable anti-cancer antibodies include, but are not limited to, anti-BAFF antibodies such as belimumab; anti-CD20 antibodies such as rituximab; anti-CD22 antibodies such as epratuzumab; anti-CD25 antibodies such as daclizumab; anti-CD30 antibodies such as iratumumab, anti-CD33 antibodies such as gemtuzumab, anti-CD52 antibodies such as alemtuzumab; anti-CD152 antibodies such as ipilimumab; anti-EGFR antibodies such as cetuximab;and FGFR2b antibodies such as bemarituzuman; anti-HER2 antibodies such as trastuzumab and pertuzumab; anti-l L6 antibodies, such as siltuximab; anti 0X40 antibodies such as rocatinlimab; and anti-VEGF antibodies such as bevacizumab; anti-l L6 receptor antibodies such as tocilizumab.

[0144] In various aspects, the antibody comprises high mannose glycans. In various instances, the antibody comprises high mannose glycans and the level of Mannose 3 (Man3) glycans are undetectable, as determined by, e.g., HILIC.

[0145] Protein Compositions and Antibody Compositions

[0146] In various aspects, the presently disclosed methods relate to protein compositions. In various aspects, the protein composition comprises only one type of protein. In various instances, the protein composition comprises proteins wherein each protein of the protein composition comprises the same or essentially the amino acid sequence. In various aspects, the protein composition comprises proteins wherein each protein of the protein composition comprises an amino acid sequence which is at least 90% identical to the amino acid sequences of all other proteins of the composition. In various aspects, the composition comprises proteins wherein each protein of the protein composition comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences of all other proteins of the composition. In various aspects, the composition comprises proteins wherein each protein of the composition comprises an amino acid sequence which is the same or essentially the same (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 sequences of all other proteins of the composition) but the glycan pair classifications of the proteins of the composition may differ from each other. In exemplary aspects, the protein composition comprises a heterogeneous mixture of proteins having different glycan pair classifications. In various instances, the protein composition may be characterized in terms of its glycan pairing content (e.g., unpaired AF glycan content, unpaired HM glycan content, paired AF glycan content, and / or its paired HM glycan content). In various instances, the protein composition may be characterized in terms of its relative abundance of glycan pairs (e.g., relative abundance of unpaired AF glycans and / or relative abundance of unpaired HM glycans).

[0147] The presently disclosed methods in various aspects relate to antibody compositions. In various aspects, the composition comprises only one type of antibody. In various instances, the composition comprises antibodies wherein each antibody of the composition comprises the same or essentially the amino acid sequence. In various aspects, the composition comprises antibodies wherein each antibody of the composition comprises an amino acid sequence which is at least 90% identical to the amino acid sequences of all other antibodies of the composition. In various aspects,the composition comprises antibodies wherein each antibody of the composition comprises an amino acid sequence which is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences of all other antibodies of the composition. In various aspects, the composition comprises antibodies wherein each antibody of the composition comprises an amino acid sequence which is the same or essentially the same (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 sequences of all other antibodies of the composition) but the glycan pair classifications of the antibodies of the composition may differ from each other. In exemplary aspects, the antibody composition comprises a heterogeneous mixture of antibodies having different glycan pair classifications. In various instances, the antibody composition may be characterized in terms of its glycan pairing content (e.g., unpaired AF glycan content, unpaired HM glycan content, paired AF glycan content, and / or its paired HM glycan content). In various instances, the protein composition may be characterized in terms of its relative abundance of glycan pairs (e.g., relative abundance of unpaired AF glycans and / or relative abundance of unpaired HM glycans).

[0148] In various aspects, the protein composition comprises or is an antibody comprising an Fc region. In various aspects, the protein composition or antibody composition comprises antibodies, wherein each antibody comprises the same heavy and light chain amino acid sequences as another antibody of the antibody composition. The antibody is in various aspects any of those described in the art or described herein, e.g., at "Antibodies and Fragments Thereof". It is noted that endogenous mannose 3 species may have the same mass as the digested glycan structure comprising 3 mannose residues produced by digesting a high mannose species with a mannosidase such as 1-2,3 mannosidase. However, it is appreciated that Mannose 3 (Man 3) is typically a low abundance species, often near or below the limit of detection. Moreover, to the extent that endogenous Man 3 is present, it is expected to be distributed among both paired and unpaired glycans. As such, it is appreciated herein that the possible presence of low levels of Man3 species will not significantly impact the determination of glycan pairing using methods comprising 1-2,3 mannosidase as described herein. In various aspects, at least some of the antibodies of the antibody composition comprise high mannose glycans. In various instances, the antibodies of the antibody composition comprise high mannose glycans and the level of mannose 3 glycans is undetectable, as determined by, e.g., HILIC. It is noted that al-2 mannosidase can convert high mannose glycans into a mannose 5 species (Man 5), which has a distinct mass from any Man 3 species. In various instances, the antibodies of the antibody composition comprise high mannose glycans and the level of Mannose 3 (Man3) glycans is detectable, as determined by, e.g., HILIC, and the method employs al-2 mannosidase.

[0149] Exemplary Embodiments

[0150] The following is a listing of exemplary embodiments provided merely to illustrate the present invention and not in any way to limit its scope.1. A method of determining the glycan pairing content of a protein composition comprising a protein comprising an Fc region comprising a glycan pair, the method comprising:a. treating a sample of the protein composition with (i) a mannosidase and (ii) a pi-4 endoglycosidase, to produce a mixture of components of the protein, wherein at least some of the components comprises one or more digested glycan structures; andb. separating the components of the mixture based on molecular weight; and c. quantifying the abundance of glycan pairs of the mixture to determine the glycan pairing content of the protein composition.2. The method of embodiment 1, wherein the mannosidase cleaves an al-2 linkage, al-3 linkage, and / or al-6 linkage of a high mannose glycan.3. The method of any one of the preceding embodiments, wherein the mannosidase is an al-2, 3, 6 mannosidase.4. The method of any one of the preceding embodiments, wherein the mannosidase is an al-6 mannosidase.5. The method of any one of the preceding embodiments, wherein the mannosidase is an al-2 mannosidase.6. The method of any one of the preceding embodiments, wherein the mannosidase is an al-2, 3 mannosidase.7. The method of any one of the preceding embodiments, comprising treating the sample with two mannosidases.8. The method of embodiment 7, comprising treating the sample with an al-2, 3, 6 mannosidase and an al-6 mannosidase.9. The method of embodiment 7 or 8, comprising treating the sample with each mannosidase for less than 4 hours, optionally, less than 3 hours.10. The method of embodiment 9, comprising treating the sample with each mannosidase for more than 1 hour.11. The method of any one of embodiments 7-9, comprising treating the sample with each mannosidase for about 2 hours.12. The method of any one of embodiments 1-6, comprising treating the sample with a single mannosidase.13. The method of embodiment 12, wherein the mannosidase is an al-2 mannosidase.14. The method of embodiment 12, wherein the mannosidase is an al-2, 3 mannosidase.15. The method of any one of embodiments 12-14, comprising treating the sample with the mannosidase for less than 30 hours, optionally, about 24 hours.16. The method of any one of the preceding embodiments, wherein none of the digested glycan structures of the components in the mixture produced in (a) comprises six or more mannosyl residues.17. The method of any one of the preceding embodiments, wherein the digested glycan structures of the components in the mixture produced in (a) comprise less than 6 mannosyl residues.18. The method of embodiment 17, wherein the digested glycan structures of the components in the mixture produced in (a) comprise 5 or fewer mannosyl residues.19. The method of embodiment 18, wherein the digested glycan structures of the components in the mixture produced in (a) comprise zero, one , three or five mannosyl residues.20. The method of embodiment 19, wherein the digested glycan structures of the components in the mixture produced in (a) comprises zero or one mannosyl residues.21. The method of embodiment 19, wherein the digested glycan structures of the components in the mixture produced in (a) comprises zero or three mannosyl residues.22. The method of embodiment 19, wherein the digested glycan structures of the components in the mixture produced in (a) comprises zero or five mannosyl residues.23. The method of any one of embodiments 16 to 22, wherein, when a digested glycan structure comprises one, three, or five mannosyl residues, the digested glycan structure further comprises two core N-acetylglucosamine (GalNac) residues.24. The method of any one of embodiments 16 to 22, wherein, when a digested glycan structure comprises zero mannosyl residues, the digested glycan structure further comprises only one core GIcNac residue.25. The method of any one of the preceding embodiments, wherein the sample is treated with one or more mannosidases after the sample is treated with the pi-4 endoglycosidase.26. The method of any one of the preceding embodiments, wherein the pi-4 endoglycosidase is an Endo S endoglycosidase.27. The method of any one of embodiments 24-26, comprising treating the sample with the endoglycosidase for less than 1 hour.28. The method of embodiment 27, comprising treating the sample with the endoglycosidase for about 30 minutes.29. The method of any one of the preceding embodiments, wherein separating the components of the mixture based on molecular weight comprises performing mass spectrometry to obtain one or more mass spectral peaks.30. The method of embodiment 29, comprising deconvoluting the mass spectral peaks to obtain deconvoluted mass spectral peaks.31. The method of embodiment 30, comprising matching the molecular weight of each deconvoluted mass spectral peak against a database of glycan pairs and the associated molecular weight to identify the glycan pair.32. The method of any one of the preceding embodiments, wherein the separating of (b) and the quantifying of (c) occurs in less than 2 hours.33. The method of embodiment 32, wherein (b) and (c) occur in less than 75 minutes.34. The method of embodiment 33, wherein (b) and (c) occur in less than 60 minutes.35. The method of any one of the preceding embodiments, wherein the protein of the protein composition comprises an antibody comprising the Fc region or is an antibody comprising the Fc region.36. The method of embodiment 35, further comprising treating the sample with (iii) an enzyme that cleaves each antibody into a Fab fragment and Fc fragment.37. The method of embodiment 36, wherein the enzyme is a cysteine protease.38. The method of embodiment 37, wherein the cysteine protease cleaves at a site between Thr and His or between Lys and Thr of the sequence KTHTCPP (SEQ ID NO: 1) of an IgGl antibody heavy chain.39. The method of embodiment 37 or 38, comprising treating the sample with the cysteine protease for at least 8 hours or at least 12 hours.40. The method of any one of the preceding embodiments 36-39, comprising chromatographically separating components of the mixture.41. The method of any one of embodiments 36-40, comprising chromatographically separating Fab fragments from Fc fragments.42. The method of embodiment 40 or 41, wherein chromatographically separating comprises a reversed phase liquid chromatography (RP-LC).43. The method of any one of embodiments 40-42, comprising performing mass spectrometry on the Fc fragment-containing chromatographic fraction to obtain one or more mass spectral peaks.44. The method of embodiment 43, comprising deconvoluting the mass spectral peaks to obtain deconvoluted mass spectral peaks.45. The method of embodiment 44, comprising matching the molecular weight of each deconvoluted mass spectral peak against a database of glycan pairs and the associated molecular weight to identify the glycan pair.46. A method of determining the glycan pairing content of a protein composition comprising a protein comprising an antibody, the method comprisinga. treating a sample of the protein composition with (i) an enzyme that cleaves an antibody heavy chain at a site N-terminal to the hinge region disulfide linkage, (ii) a mannosidase, and (iii) a pi-4 endoglycosidase, to produce a mixture of Fab fragments and Fc fragments of the protein, wherein at least some of the Fc fragments comprise one or more digested glycan structures;b. chromatographically separating the Fab fragments from the Fc fragments c. separating Fc fragments based on molecular weight, andd. quantifying the abundance of glycan pairs to determine the glycan pairing content of the protein composition.47. The method of any one of the preceding embodiments, comprising quantifying the i. paired afucosylated glycan content,ii. unpaired afucosylated glycan content,iii. paired high mannose glycan content, and / oriv. unpaired high mannose glycan content.48. The method of any one of the preceding embodiments, wherein the method is a part of a manufacturing process for the protein composition.49. The method of any one of the preceding embodiments, wherein the method is performed in real time during manufacture of the protein composition.50. The method of any one of the preceding embodiments, wherein the sample is a sample of in-process material.51. The method of any one of the preceding embodiments, wherein the glycan pairing content is determined pre-harvest or post-harvest.52. The method of embodiment 51, wherein the glycan pairing content is determined post-harvest.53. The method of any one of the preceding embodiments, wherein the sample is obtained from a manufacturing lot.54. The method of any one of the preceding embodiments, wherein the protein of the protein composition is an antibody comprising the Fc region.55. A method of analyzing a protein composition, comprising determining the glycan pairing content of a sample of the protein composition in accordance with a method of any one of embodiments 1 to 54, and, optionally, comparing the glycan pairing content of the protein composition to the glycan pairing content of a reference product.56. A method of monitoring production of a protein composition, comprising determining the glycan pairing content of a protein composition in accordance with a method of any one of embodiments 1 to 54, for a first sample obtained at a first timepoint and for a second sample taken at a second timepoint which is different from the first timepoint, and, optionally, comparing the glycan pairing content of the first sample to glycan pairing content of the second sample.57. The method of embodiment 56, wherein each of the first sample and second sample is a sample of in-process material.58. The method of embodiment 57, wherein the first sample is a sample of in-process material and the second sample is a sample of a manufacturing lot.59. The method of embodiment 57, wherein the first sample is a sample obtained before one or more conditions of the cell culture are modified and the second sample is a sample obtained after the one or more conditions of the cell culture are modified.60. A method of producing a protein composition, comprising (A) determining glycan pairing content of a sample of the protein composition in accordance with a method of any one of embodiments 1-54, wherein the sample is a sample of in-process material, wherein, when the glycan pairing content is determined as outside a target range, the method further comprises (B) modifying one or more conditions of the cell culture to obtain a modified cell culture and determining the glycan pairing content, and optionally, repeating (A) and (B) until the glycan pairing content is within the target range.61. The method of embodiment 60, wherein one or more conditions of the cell culture are modified to primarily change the relative unpaired HM glycan content to achieve the target range of glycan pairing content.62. The method of embodiment 61, wherein one or more conditions of the cell culture are modified to primarily change the relative unpaired AF glycan content to achieve the target range of glycan pairing content.63. A method of selecting a cell line or cell clone for a protein composition manufacture process, comprising determining the glycan pairing content of a protein composition in accordance with a method of any one of embodiments 1 to 54, for cell lines or cell clones producing the protein of the protein composition, and identifying a cell line or cell clone that have a glycan pairing content within a preselected target range, wherein the identified cell line or cell clone is selected for the protein composition manufacture process.64. The method of embodiment 63, wherein the preselected target range is based on the range of relative unpaired afucosylated glycan content and / or the range of relative unpaired high mannose glycan content of a reference cell line or cell clone.65. A method of determining the glycan pairing content of an IgG antibody composition, comprisinga. treating the IgG antibody composition with enzymes to form a mixture of Fab fragments and Fc fragments, wherein a first enzyme cleaves an antibody heavy chain at a site N-terminal to the hinge region disulfide linkages, a second enzyme cleaves the pi-4 linkage between core GIcNAc residues, and a third enzyme cleaves the al-2, al-3, and / or al-6 linkages to remove one or more mannosyl residues, to form Fc fragments each comprising one or more digested glycan structures,b. separating the Fab fragments from the Fc fragments, andc. quantifying the abundance of glycan pairs to determine the glycan pairing content of the IgG antibody composition.66. The method of embodiment 65, wherein the first enzyme is a cysteine protease. 67. The method of embodiment 66, wherein the cysteine protease cleaves at a site between Thr and His or between Lys and Thr of the sequence KTHTCPP (SEQ ID NO: 1) of an IgGl antibody heavy chain.68. The method of embodiment 66 or 67, comprising treating the IgG antibody composition with the cysteine protease for at least 8 hours or at least 12 hours.69. The method of any one of embodiments 65-68, wherein the second enzyme is an IgG-specific enzyme.70. The method of embodiment 69, wherein the IgG-specific enzyme is an endoglycosidase.71. The method of embodiment 70, wherein the endoglycosidase is an Endo S endoglycosidase.72. The method of any one of embodiments 69-71, comprising treating the mixture with the IgG-specific enzyme for less than 1 hour.73. The method of embodiment 72, comprising treating the IgG antibody composition with the IgG-specific enzyme for about 30 minutes.74. The method of any one of embodiments 65-73, wherein the Fab fragments are separated from the Fc fragments by a chromatography.75. The method of embodiment 74, wherein the chromatography is a reversed phase liquid chromatography.76. The method of embodiment 74 or 75, comprising performing mass spectrometry on a chromatographic fraction comprising the Fc fragments to obtain one or more mass spectral peaks.77. The method of embodiment 76, comprising deconvoluting the mass spectral peaks to obtain deconvoluted mass spectral peaks.78. The method of embodiment 77, comprising matching the molecular weight of each deconvoluted mass spectral peak against a database of glycan pairs and the associated molecular weight to identify the glycan pair.79. The method of any one of embodiments 65-78, wherein (b) and (c) occurs in less than 2 hours.80. The method of embodiment 79, wherein (b) and (c) occurs in less than 75 minutes.81. The method of embodiment 80, wherein (b) and (c) occurs in less than 60 minutes.82. A method of preparing an IgG antibody composition having an ADCC activity within a target range, comprising determining the glycan pairing content of a sample of the IgG antibody composition in accordance with a method of any one of embodiments 65 to 81, and comparing the glycan pairing content of the IgG antibody composition to a target range of glycan pairing content.83. A method of analyzing an IgG antibody composition, comprising determining the glycan pairing content of a sample of the IgG antibody composition in accordance with a method of any one of embodiments 65 to 81, and, optionally, comparing the glycan pairing content to that of a reference product.84. The method of embodiment 82 or 83, wherein the method is a part of a manufacturing process for the IgG antibody composition.85. The method of any one of embodiments 82 to 84, wherein the method is performed in real time during manufacturing of the IgG antibody composition.86. The method of any one of embodiments 82 to 85, wherein the sample is a sample of in-process material.87. The method of any one of embodiments 82 to 86, wherein the glycan pairing content is determined pre-harvest or post-harvest.88. The method of embodiment 87 , wherein the glycan pairing content is determined post-harvest.89. The method of any one of embodiments 82 to 88, wherein the sample is obtained from a manufacturing lot.90. A method of monitoring production of an IgG antibody composition, comprising determining the glycan pairing content of an IgG antibody composition in accordance with a method of any one of embodiments 65 to 81, for a first sample obtained at a first timepoint and for a second sample taken at a second timepoint which is different from the first timepoint, and, optionally, comparing the glycan pairing content of the first sample to the glycan pairing content of the second sample.91. The method of embodiment 90, wherein each of the first sample and second sample is a sample of in-process material.92. The method of embodiment 90, wherein the first sample is a sample of in-process material and the second sample is a sample of a manufacturing lot.93. The method of embodiment 90, wherein the first sample is a sample obtained before one or more conditions of the cell culture are modified and the second sample is a sample obtained after the one or more conditions of the cell culture are modified.94. A method of producing an IgG antibody composition, comprising (A) determining glycan pairing content of a sample of the IgG antibody composition in accordance with a method of any one of embodiments 65-81, wherein the sample is a sample of in-process material, wherein, when the glycan pairing content is determined as outside a target range, the method further comprises (B) modifying one or more conditions of the cell culture to obtain a modified cell culture and determining the glycan pairing content, and optionally, repeating (A) and (B) until the glycan pairing content is within the target range.95. The method of embodiment 94, wherein one or more conditions of the cell culture are modified to primarily change the relative unpaired HM glycan content to achieve the target range of glycan pairing content.96. The method of embodiment 94, wherein one or more conditions of the cell culture are modified to primarily change the relative unpaired AF glycan content to achieve the target range of glycan pairing content.97. The method of any one of embodiments 65-96, comprising quantifying thei. paired afucosylated Fc fragments,ii. unpaired afucosylated Fc fragments,iii. paired high mannose Fc fragments, and / oriv. unpaired high mannose Fc fragments.98. The method of any one of embodiments 65-97, comprising quantifying the relative unpaired afucosylated (AF) glycan content and relative unpaired high mannose (HM) glycan content.99. The method of any one of embodiments 65-98, the third enzyme is an al-2 mannosidase, al-3 mannosidase, al-6 mannosidase, al-2, 3, 6 mannosidase, or al-2, 3 mannosidase.

[0151] The following examples are given merely to illustrate the present invention and not in any way to limit its scope.EXAMPLES EXAMPLE 1

[0152] This example describes an exemplary simplified method of determining paired glycan content and unpaired glycan content for an antibody composition.

[0153] In a previous study, the paired afucosylated and high mannose glycan content and unpaired afucosylated and high mannose glycan content were determined for two different antibody compositions, and statistical relationships correlating unpaired glycan content of the antibody composition with ADCC activity and FcyRllla binding were established. In this previous study, the method used to measure the levels of unpaired or paired glycopairs comprised three steps: an antibody cleavage step, a chromatographic separation step, and a mass spectrometry (MS)-based detection step. Briefly, samples of an antibody composition comprising Antibody A or Antibody B were treated with the IgdE (FabALACTICA® or FL from Genovis Inc., Cambridge, MA) enzyme which cleaves the heavy chain of antibodies above the hinge to create Fab and Fc antibody fragments. The FL enzyme cleaves between the second and third amino acid of the IgGl heavy chain sequence KTHTCPP (SEQ ID NO: 1). The resulting Fab fragments and glycosylated Fc fragments were subsequently separated and characterized by hydrophilic interaction liquid chromatography (HILIC) with water / organic with ion-pairing reagent as solvents and MS-based detection. The HILIC separation was carried out on a Waters Acquity UPLC Glycoprotein Amide column over a course of 95 min with mobile phase composition 20:80 (v / v) of water and acetonitrile with 0.1% TFA. At 1 min, a linear gradient of 0.44 ml / min was applied for the next 74 min for separating glycopairs based on their interaction with the stationary phase. MS detection was carried out using an Agilent 6545XT QToF MS with an Agilent Jet Stream (AJS) electrospray ionization source (ESI) with settings of: capillary voltage - 4500 V; drying gas - 11 mL / min; nebulizer pressure - 25 psi and gas temperature -340 °C using a mass range of 1000-3000 m / z. Deconvolution was performed using Bioconfirm B.09.00 (Agilent) using S / N of 30 and output mass range set to 40-60 kDa.

[0154] Though the above method successfully measured the content of unpaired and paired afucosylated and high mannose glycans to establish statistically significant correlations with biological functions, a subsequent study was conducted to improve the efficiency of the method for determining the levels of unpaired and paired afucosylated and high mannose glycans. Glycan pairing analysis is complicated due to the high degree of heterogeneity of the glycopair population, and the HILIC separation aims to reduce the complexity through the introduction of a high-resolution separation prior to MS analysis. The glycopairs are separated into a glycopairing map of approximately 15 chromatographic peaks (Figure 6A). However, the HILIC-MS glycan pairing analysis remains complicated for several reasons. First, the paired fucosylated (Fc, A2G0F / A2G0F, Fc, A2G0F / A2G1F and Fc, A2G1F / A2G1F) glycopairs, which do not significantly impact ADCC, are the dominant species in the analysis, eluting across the entire chromatographic region of the glycosylated Fc fragment chromatogram (Figure 6A-6B). Second, many of the important chromatographic peaks are poorly resolved: the key afucosylated and high mannose glycopairs coelute with and / or poorly separate from the dominant paired fucoslyated glycopairs. As a result, it is difficult to establish the correct window to extract the mass spectra for each chromatographic peak, and a flawed extraction window can artificially inflate or deflate the peak areas of the key afucosylated and high mannose glycopairs reducing the accuracy of the analysis. Third, structural isomers and glycopairs with partially reduced disulfide bonds (Rd, +2Da) lead to multiple chromatographic peaks for species categorized as the same glycopair. Finally, key afucosylated and high mannose glycopairs (shown in bold in Figure 6B) have extremely low abundances, and often represent less than 5% of the population. Taken together, these challenges complicate the data analysis and quantitation and require the careful consideration of a highly-skilled analyst, and ultimately increase the time required for these overall method.

[0155] A new method was designed to include the preparation, separation, measurement, and analysis of a simplified sample comprising the components essential for quantitation of afucosylated and high mannose glycans (paired or unpaired) without an abundance of non-essential components. For example, in the previous method, every type of glycopair including the high mannose, and all fucosylated and afucosylated glycopairs were identified and quantified, and this degree of resolution was unnecessary for the purposes of determining levels of unpaired and paired afucosylated and high mannose glycopairs. Further, the separation prior to the MS-based detection was designed so the glycosylated Fc fragments eluted under one chromatographic peak in the total ion chromatogram which was chromatographically separated from the Fab region. In the previous HILIC-MS method, poor chromatographic resolution of key afucosylated and high mannose glycopairs from the abundant fucosylated glycopairs lead to difficulties determining the correct mass spectral extraction windows for accurate quantitation. To simplify analysis in the new method all glycosylated Fc fragments intentionally co-elute under one chromatographic peak in the total ion chromatogram. The mass spectra are extracted from the entire chromatographic peak of the Fc fragment and deconvoluted. The deconvoluted mass spectral peaks in the one deconvoluted mass spectrum are leveraged for identification and quantitation of the glycopairs. Using this approach, the Fc fragments are separated by their differences (delta) in molecular weights (MW). By intentionally removing the chromatographic separation of the glycosylated Fc fragments the data analysis is simplified, and subsequently quantitative accuracy and sample-to-sample comparability is improved. Additionally, the sample through-put is dramatically increased. An automated identification system for faster identification and quantitation of the paired and unpaired afucosylated and high mannose glycopairs was additionally included in the new method.

[0156] The new method comprised a two-enzyme digestion of the antibody composition before chromatographic separation and a MS-based detection. This two-enzyme digestion is depicted in Figure 5. Briefly, samples of the antibody composition were treated with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc., Cambridge, MA) which cleaves the heavy chain of IgGl antibodies above the hinge to create Fab and Fc antibody fragments. The FL enzyme cleaves between the second and third amino acid of the IgGl heavy chain sequence KTHTCPP (SEQ ID NO: 1). The digestion was carried out overnight at 37 °C. A second digestion with EndoS (IgGZERO® from Genovis Inc.,), an IgG specific endoglycosidase S enzyme, was carried out for 30 min at 37°C. EndoS is an IgG-specific endoglycosidase acting on complex type N-glycans at the Fc-glycosylation site of IgGs. The enzyme hydrolyzes the pi-4 linkage between the core GIcNAc residues in the N-glycans, leaving the innermost GIcNAc intact on the Fc with or without a fucose. EndoS has limited activity on high mannose and hybrid glycans, this is leveraged to identify and quantify the high mannose glycopairs and subsequently the % relative abundance of paired and unpaired high mannose.

[0157] This two-enzyme digestion yielded a simplified population of glycopairs which could be categorized into one of two afucosylated glycopair classes: (1) paired afucosylated (both heavy chains of the Fc fragment were afucosylated) and (2) unpaired afucosylated (only one heavy chain of the Fc fragment was afucosylated, while the other heavy chain was fucosylated) (Figure 7). Based on the assumption that both afucosylated and high mannose glycans are highly potent, pairs containing a combination of high mannose and afucosylated glycans were classed as afucosylated paired, unpaired high mannose, as appropriate and according to Table A.

[0158] EndoS has limited activity on high mannose and hybrid glycans, this was leveraged to identify high mannose glycopairs and quantify the high mannose glycan pairing. The population of individual Fc high mannose glycopairs could be categorized into one of two high mannose classes: (1) paired high mannose (both heavy chains of the Fc fragment contained high mannose glycans) and (2) unpaired high mannose (only one heavy chain of the Fc fragment contained high mannose, while the other heavy chain was fucosylated or afucosylated) (Figure 7). A summary designation was given according to Table 1 and following the three rules described herein.

[0159] Following the two-enzyme digestion, resulting Fc and Fab fragments were chromatographically separated by using a rapid reversed phase liquid chromatography (RP-LC) separation with MS detection. This separation yielded one chromatographic peak comprising the Fc fragments that was well separated from the Fab fragments and treatment enzymes. A diagram of the chromatographic separation and MS analysis steps are shown in Figures 8A-8B.

[0160] The equipment used to perform steps of the method has the following capabilities:Equipment capabilities:UPLC system with column heating capabilitiesMS system with an ESI source and high-resolution mass analyzer (time-of- flight or orbitrap)Temperature controlled autosamplerReversed phase column

[0161] The autosampler temperature was held at 4°C and an aliquot was injected and the separation was carried out using a 2.1 ID x 50 mm RP analytical column at 60 °C. Mobile Phase A was 0.1% (v / v) trifluoroacetic acid in water while Mobile Phase B was 0.1% (v / v) trifluoroacetic acid in acetonitrile. The mobile phase gradient example is provided below:Time Flow Rate Mobile Phase A Mobile Phase B (minutes) (mL / minute) (%) (%)0.0 0.25 70 3010 0.25 55 4510.3 0.25 10 9011.3 0.25 10 9012 0.25 70 3015 0.25 70 3017 0.25 70 30

[0162] MS acquisition was performed using an Agilent 6545 XT Q-TOF with an AJS ESI source in positive ion mode. The source conditions were: gas temperature - 325 °C, drying and sheath gas flow - 12 L / min, sheath gas temperature - 300 °C, nebulizer - 40 psi, capillary voltage - 5000 M, fragmentor voltage - 280 M, and skimmer - 120 V. Spectra were acquired with a m / z range of 1200-3200.

[0163] The chromatographic peak that contained the Fc fragments, was then selected for data analysis, the mass spectra of the one chromatographic peak that contained the Fc fragments was extracted and deconvoluted (Figures 8A-8B). The deconvolution was performed using Byos (Protein Metrics Inc, Cupertino, CA) the input m / z range was 1300-3200 m / z while the output range was 30-70 kDa. The intensity of each deconvoluted mass spectral peak of the antibody composition was employed for quantitation.

[0164] The EndoS treated glycopairs were identified by matching the molecular weights (MW) of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs (Table 1). The glycan MWs used in this approach is the glycan MW associated with the N-linkage (native glycan MW - H2O). The database comprised of the theoretical MWs of two EndoS treated glycans (glycan pairs) whereby complex glycans were hydrolyzed to either GIcNac or GlucNac + Fuc while high mannose and hybrid glycans remained intact. The database comprised of the theoretical MWs of approximately 40 individual EndoS glycan pairs classed as (1) afucosylated paired and (2) afucosylated unpaired and (3) high mannose paired and (4) high mannose unpaired (Table 1). Glycan pairs that have been hydrolyzed by EndoS are italicized in the subsequent text.

[0165] To facilitate the automated identification of the EndoS treated glycopairs (defined as the intact Fc and two glycan moieties) the amino acid sequence of the FL treated Fc was first loaded into the protein analysis software to generate a theoretical MW of the Fc, additionally the software was instructed to consider that all possible disulfide bonds remain intact and both C-terminal lysine's were clipped. Second, the database of the theoretical MWs of EndoS glycan pairs was loaded into the protein analysis software as a list of custom modifications. Following this user input, the protein analysis software then automatically determined a list of theoretical MWs for the EndoS glycopairs (seen in Table 1). The software automatically identified the deconvoluted mass spectral peaks if the MW matched with a theoretical MW in the custom-built database of EndoS glycopairs.TABLE 1EndoS GlycanDominant Paired Fucosylated EndoS Glycan Pair EndoS Glycopair MW (Da)Pair MW (Da)GlcNac+ Fuc / GIcNac + Fuc 698.6 50901.2EndoS GlycanPaired Afucosylated EndoS Glycan Pairs EndoS Glycopair MW (Da)Pair MW (Da)GIcNac / GIcNac 406.4 50608.9 M5 / GlcNac 1420.3 51622.8 M6 / GlcNac 1582.4 51785 M7 / GlcNac 1744.6 51947.1 M8 / GlcNac 1906.7 52109.2 AlG0M5 / G / cA / ac 1623.5 51826 A1G0M5 / A1G0M5 2840.6 53043.1 AlGlM5 / G / cA / ac 1785.6 51988.2 A1G1M5 / A1G1M5 3164.8 53367.4 M5 / A1G0M5 2637.4 52839.9 M6 / A1G0M5 or M5 / A1G1M5 2799.5 53002 M7 / A1G0M5 or M6 / A1G1M5 2961.6 53164.2 M8 / A1G0M5 or M7 / A1G1M5 3123.8 53326.3 M8 / A1G1M5 3285.9 53488.5EndoS GlycanUnpaired Afucosylated EndoS Glycan Pairs EndoS Glycopair MW (Da)Pair MW (Da)GIcNac / GIcNac + Fuc 552.5 50755.1 GIcNac 203.2 50405.7 AlG0M5 / G / cA / ac or G / cA / ac / AlG0M5F 1769.6 51972.2 AlGlM5 / G / cA / ac + Fuc or G / cA / ac / AlGlM5F 1931.8 52134.3 G / cA / ac / AlG0M4F 1753.6 51956.2 G / cA / ac / AlSlM4F 2060.9 52263.4 GIcNac / A1G1S1M5F 2223 52425.5EndoS GlycanPaired High Mannose EndoS Glycan Pairs EndoS Glycopair MW (Da)Pair MW (Da)M5 / M5 2434.2 52636.7 M5 / M6 2596.3 52798.9 M5 / M7 or FC, M6 / M6 2758.5 52961 M5 / M8 or FC, M6 / M7 2920.6 53123.1 M6 / M8 or FC, M7 / M7 3082.7 53285.3 M7 / M8 3244.9 53447.4 M8 / M8 3407 53609.6EndoS GlycanUnpaired High Mannose EndoS Glycan Pairs EndoS Glycopair MW (Da)Pair MW (Da)M5 / GlcNac + Fuc 1566.4 51769 M6 / GlcNac + Fuc 1728.6 51931.1M7 / GlcNac + Fuc 1890.7 52093.2 M8 / GlcNac + Fuc 2052.8 52255.4 M5 / A1G0M4F 2621.4 52823.9 M6 / A1G0M4F or FC, M5 / A1G0M5F 2783.5 52986.1 M7 / A1G0M4F or FC, M6 / A1G0M5F or FC + M5 / A1G1M5F 2945.7 53148.2 M8 / A1G0M4F or FC, M7 / A1G0M5F or FC + M6 / A1G1M5F 3107.8 53310.3 M8 / A1G0M5F or FC + M7 / A1G1M5F 3269.9 53472.5 M8 / A1G1M5F 3432.1 53634.6 M5 / A1S1M4F 3074.8 53277.3 M6 / A1S1M4F or FC + M5 / A1G1S1M5F 3236.9 53439.4 M7 / A1S1M4F or FC + M6 / A1G1S1M5F 3399 53601.6 M8 / A1S1M4F or FC + M7 / A1G1S1M5F 3561.2 53763.7 M8 / A1G1S1M5F 3723.3 53925.9Note: Additional MWs for EndoS treated glycopairs which have only a single glycoslyation or a bisecting GIcNac are included in the database but not listed above.

[0166] The most abundant EndoS glycopair is Fc, GlucNac + Fucose / GlucNac + Fucose which contains a fucosylated glycan on each heavy chain and is therefore not applicable to the determination of afucoslyated or high mannose glycan pairing. In the previous study using HILIC-MS, the highly abundant and not applicable paired fucosylated glycopairs elute across the entire region of the chromatogram where the glycosylated Fc elutes often masking the presence of the species of interest (Figure 6B). In the current study, the EndoS treatment simplifies the multitude of highly abundant paired fucoslyated glycopairs into a single species, Fc, GlucNac + Fucose / GlucNac + Fucose. This single deconvoluted mass peak is well separated, by the delta molecular weight, from the species of interest which facilitates streamlined data interpretation (Figure 8B).

[0167] The abundance of the individual EndoS glycopairs was determined from the relative intensities or areas of the identified deconvoluted mass spectral peaks as follows:% Glycopair Intensity = Deconvoluted Peak Intensity / Total Deconvoluted Peak Intensity x 100[Equation 1]

[0168] The abundance of paired afucosyated glycopairs was determined by summing the individual glycopairs categorized as paired afucosylated glycopairs, similarly the abundance of unpaired afucosyated glycopairs was determined by summing the individual glycopairs categorized as unpaired afucosylated glycopairs. To calculate the % relative abundance of the Unpaired Afucosylated Glycan Content of the antibody composition Equation 2 was employed:% Unpaired Afucosylated Glycan Content = (% Unpaired Afucoslyated Glycopairs / % Total Afucoslyated Glycopairs (Unpaired+Paired)) x 100[Equation 2]

[0169] The abundance of paired high mannose glycopairs was determined by summing the individual glycopairs categorized as paired high mannose glycopairs, similarly the abundance of unpaired high mannose glycopairs was determined by summing the individual glycopairs categorized as unpaired high mannose glycopairs. To calculate the % relative abundance of the Unpaired High Mannose Glycan content of the antibody composition Equation 3 was employed:% Unpaired High Mannose Glycan Content = (% Unpaired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 3]

[0170] In the previous study, a 95 min HILIC-MS analysis was employed to separate a highly complex population of glycopairs for the determination of the levels of afucoslyated and high mannose glycan pairing. This approach remains challenging as the highly abundant and not applicable paired fucosylated glycopairs elute across the entire glycoslyated Fc chromatographic region often masking the presence of the glycopairs of interest. Key high mannose and afucosylated glycopairs co-elute with and / or elute as shoulders of the highly abundant paired fucosylated glycopairs making accurate quantitation challenging. This example demonstrates the feasibility of a streamlined method where the glycopair population is simplified prior to analysis with EndoS. A rapid 17-min chromatographic separation prior to MS-detection simplifies analysis by yielding one, well resolved, chromatographic peak of Fc fragments for further quantitation. The quantitation of the EndoS glycopairs is achieved using the deconvoluted mass spectral peaks where the highly abundant paired fucosylated species is a single deconvoluted mass spectral peak well separated, by MW, from the key afucosylated and high mannose glycopairs. The custom built EndoS glycan pair MW database facilitates automated identification and quantitation of the critical afucosylated and high mannose glycopairs. This new method affords a rapid analysis and increased quantitative accuracy when determining % relative abundance of the unpaired and paired afucosylated glycan content and % relative abundance of the unpaired and paired high mannose glycan content of an antibody composition.EXAMPLE 2

[0171] This example describes an exemplary application of the simplified method of determining the % relative paired and unpaired afucosylated and high mannose glycan content for an antibody composition.

[0172] The new method described in Example 1 was carried out on a panel of antibody composition samples, wherein individual samples of the panel comprised an IgGl monoclonalantibody produced by a particular clone or a sample of a reference product (RP) lot. Deconvoluted mass spectra for selected samples are shown in Figures 9A-9C. Data for paired afucosylated glycopairs and unpaired afucosylated glycopairs are provided in Tables 2A and 2B, respectively. Data for paired high mannose glycopairs and unpaired high mannose glycopairs are provided in Tables 2C and 2D, respectively.TABLE 2A% Paired Afucosylated Sample % FC , GIcNac / GIcNac % FC , N\5 / GlcNacGlycopairs Clone 113 0.59 0.36 0.95 Clone 116 1.37 0.08 1.45 Clone 126 0.73 0.42 1.15 Clone 203 1.3 0.57 1.87 Clone 236 0.17 0.36 0.53 Clone 85 1.43 0.57 2.00 Clone 93 1.65 0.76 2.41 Clone 98 1.72 0.77 2.49 Clone 160 0.71 ND 0.71 Clone 160.2 0.07 ND 0.07 Clone 182 0.72 ND 0.72 Clone 260 1.47 ND 1.47RP15 1.21 ND 1.21RP16 1.23 ND 1.23RP44 1.07 ND 1.07RP51 0.94 ND 0.94RP54 1.34 ND 1.34 *Note: Only the paired afucosylated glycopairs detected in 1 or more samples are listed. ND = Not detected for sample. Italicized glycans have been hydrolyzed by EndoSTABLE 2B% Fc, % Fc, % Paired Samples % Fc, GIcNac / GIcNac + Fuc A1G1M4 / GIcNac + AlGlM5 / G / c / Vac + Afucosylated Fuc Fuc Glycopairs Clone 113 9.75 ND ND 9.75 Clone 116 13.28 ND ND 13.28 Clone 126 10.26 ND ND 10.26 Clone 203 12.51 ND ND 12.51 Clone 236 8.34 ND ND 8.34 Clone 85 13.72 ND ND 13.72 Clone 93 14.58 ND ND 14.58Clone 98 14.71 ND ND 14.71Clone 160 6.19 0.4 ND 6.59 Clone 160.2 3.78 0.74 ND 4.52 Clone 182 5.09 0.05 ND 5.14 Clone 260 9.9 1.86 0.23 11.99 RP15 7.67 ND ND 7.67 RP16 8.11 ND ND 8.11 RP44 7.08 ND ND 7.08 RP51 7.15 ND ND 7.15RP54 7.86 ND ND 7.86 *Note: Only the paired afucosylated glycopairs detected in 1 or more samples are listed. ND = Not detected for sample. Italicized glycans have been hydrolyzed by EndoSTABLE 2C% Paired % Fc, % Fc,High Sample Name % Fc, M5 / M5 % Fc, M5 / M6 M6 / M6 or M5 / M8or % Fc, M8 / M8Mannose Fc, M5 / M7 Fc, M6 / M7Glyco pairs Clone 113 0.02 ND 0.08 ND ND 0.10 Clone 116 0.13 ND 0.02 ND ND 0.15 Clone 126 ND ND 0.02 0.02 ND 0.04 Clone 203 0.2 0.01 0.01 ND ND 0.22 Clone 236 ND ND 0.02 ND ND 0.02 Clone 85 0.21 0.01 0.01 ND ND 0.23 Clone 93 0.35 ND 0.02 ND ND 0.37 Clone 98 0.37 0.01 ND ND ND 0.38 Clone 160 ND ND 0.08 ND ND 0.08 Clone 160.2 ND ND 0.07 ND 0.02 0.09 Clone 182 ND ND 0.12 ND 0.01 0.13 Clone 260 ND ND 0.04 ND ND 0.04 RP15 0.38 ND 0 ND ND 0.38 RP16 0.58 ND ND ND ND 0.58 RP44 0.19 ND ND ND ND 0.19 RP51 0.35 ND ND ND ND 0.35RP54 0.24 ND 0.01 ND ND 0.25 *Note: Only the paired afucosylated glycopairs detected in 1 or more samples are listed. ND = Not detected for sample. Italicized glycans have been hydrolyzed by EndoSTABLE 2D% Unpaired % Fc, % Fc,% Fc, M6 / GlcNac High Sample % Fc, M5 / GIcNac + Fuc M7 / GlcNac M8 / GlcNac ++ Fuc Mannose + Fuc FucGlycopairsClone 113 0.35 ND ND 0.22 0.57 Clone 116 0.67 ND ND 0.07 0.74 Clone 126 ND ND ND 0.2 0.20 Clone 203 0.62 ND ND 0.15 0.77 Clone 236 ND ND ND 0.42 0.42 Clone 85 0.41 ND ND 0.15 0.56 Clone 93 1.04 ND ND 0.04 1.08 Clone 98 1.04 ND ND ND 1.04 Clone 160 ND 0.07 ND ND 0.07 Clone 160.2 ND 0.6 ND 0.36 0.96 Clone 182 0.24 0.01 ND 0.16 0.41 Clone 260 ND 1.1 0.45 ND 1.55 RP15 1.09 ND ND ND 1.09 RP16 1.22 ND ND ND 1.22 RP44 0.82 ND ND ND 0.82 RP51 0.96 ND ND ND 0.96RP54 0.92 ND ND 0.02 0.94 *Note: Only the paired afucosylated glycopairs detected in 1 or more samples are listed. ND = Not detected for sample. Italicized glycans have been hydrolyzed by EndoS

[0173] The relative unpaired and paired afucosylated glycan content and the relative unpaired and paired high mannose glycan content were calculated as described in Example 1 and the data is provided below in Table 3.TABLE 3% Unpaired % Paired % Unpaired High% Paired High Mannose Sample Afucosylated Afucosylated Mannose GlycanGlycan Content Glycan Content Glycan Content ContentClone 113 91.1 8.9 85.1 14.9 Clone 116 90.2 9.8 83.1 16.9 Clone 126 89.9 10.1 83.3 16.7 Clone 203 87.0 13.0 77.8 22.2 Clone 236 94.0 6.0 95.5 4.5 Clone 85 87.3 12.7 70.9 29.1 Clone 93 85.8 14.2 74.5 25.5 Clone 98 85.5 14.5 73.2 26.8 Clone 160 90.3 9.7 46.7 53.3 Clone 160.2 98.4 1.6 92 8.0 Clone 182 87.7 12.3 75.9 24.1 Clone 260 89.1 10.9 97.5 2.5RP 15 86.4 13.6 74.1 25.9RP 16 86.8 13.2 67.8 32.2RP 44 86.9 13.1 81.2 18.8RP 51 88.4 11.6 73.3 26.7RP 54 85.4 14.6 79.0 21.0

[0174] Based on these data, the RP range of unpaired afucosylated glycan content was 85.4 -88.4 and the range of unpaired high mannose glycan content was 67.8 - 81.2. The clones having the best unpaired glycan profiles are Clones 203, 116, 113, 126 and 236, with clones 116 and 126 having an unpaired afucosylated glycan content within the RP range and clone 203 having both unpaired afucosylated glycan content and unpaired high mannose glycan within the RP range.EXAMPLE 3

[0175] This example describes an exemplary method for measuring ADCC activity levels.

[0176] ADCC activity levels (expressed as a % relative value) for a panel of samples of an IgGl monoclonal antibody composition are determined using a quantitative cell-based assay that measures the ability of the antibody to mediate cell cytotoxicity in a dose-dependent manner of target cells stably expressing antigens while engaging FcyRIIIA (158V) receptors on NK92-M1 effector cells via the antibody Fc domain. These events lead to the activation of the effector cells and destruction of the target cells via exocytosis of the cytolytic granule complex perforin / granzyme. A schematic of the ADCC assay is provided in Figure 10. Briefly, target cells are labeled with calcein-acetoxymethyl (calcein-AM), which readily enters the cells and is subsequently cleaved by intercellular esterases and trapped within the cells. When target cells are lysed, fluorescent calcein is released into the medium. The level of calcein released from lysed target cells is determined by measuring the fluorescence of the reaction supernatant in an Envision (Perkin Elmer) fluorescence plate reader. Each assay is performed in triplicate and the mean and standard deviation are recorded. The data are fitted to the mean fluorescence values using a constrained 4 parameter fit using SoftMaxPro software and reported as percentage ADCC activity relative to a reference standard as calculated by the EC50 standard / EC50 sample ratio.EXAMPLE 4

[0177] HILIC is a quantitative analysis of the N-linked glycan distribution of the antibody and comprises three steps: (1) release and label N-linked glycans from reference and test samples using PNGase F and a fluorophore that can specifically derivatize free glycan, (2) load samples within the validated linear range onto a HILIC column, the labeled N-linked glycans are separated using a gradient of decreasing organic solvent, and (3) monitor elution of glycan species with fluorescence detector.

[0178] The standard and test samples are prepared by carrying out the following steps: (1) dilute samples and controls with water, (2) add PNGase F and incubate the samples and controls to release N-linked glycans, (3) mix with fluorophore labeling solution using a fluorophore such as 2-aminobenzoic acid. Vortex and incubate the samples and controls, (4) centrifuge down to pellet protein and remove supernatant, and (5) dry and reconstitute labeled glycans in the injection solution.

[0179] The solutions used in this assay are a Mobile Phase A (100 mM ammonium formate, target pH 3.0) and a Mobile Phase B (acetonitrile). The equipment used to perform steps of the method has the following capabilities:Equipment capabilities:UPLC systemFluorescence detector set to appropriate excitation / emission wavelength optimized to labeling fluorophoreData collection systemTemperature controlled autosamplerHydrophilic interaction column

[0180] The instrument settings for HPLC using a hydrophilic interaction analytical BEH Glycan 1.7 pm column (2.1 mm ID X 150 mm) and 2-aminobenzoic acid fluorophore labeling method are provided below:Target sample load 2 pLColumn heater set point 35°CAuto-sampler set point 10°CDetection Excitation 360 nmEmission 425 nm

[0181] The mobile phase gradient example is provided below:Time Flow Rate Mobile Phase A Mobile Phase B (minutes) (mL / minute) (%) (%)0.0 0.25 22.0 78.0111.2 0.25 40.1 59.9117.9 0.20 90.0 10.0124.5 0.20 90.0 10.0129.1 0.25 22.0 78.0 155 0.25 22.0 78.0

[0182] Reports of the results comprise the following format:Report area % for high mannose glycans and afucosylated glycans*% High mannose glycans = % M5 + % M6 + % M7 + % M8% Afucosylated glycans = %A1GO + %A2G0 + %AlGla +%AlGlb + %A2Gla + %A2Glb + %A1G1M4 +%A2G2 + %A1G1M5 + %A1G1S1M5*Calculation formulas depend on presence of individual high mannose and afucosylated glycans EXAMPLE 5

[0183] This example describes the relationships of ADCC activity to % released high mannose glycans, % released afucosylated glycans, % unpaired high mannose glycans and % unpaired afucosylated glycans for an antibody composition.

[0184] The ADCC activity level of a panel of antibody compositions was determined by a cellbased ADCC assay. The % released high mannose glycans and % released afucosylated glycans were determined by the method described in Example 4. The ADCC activity levels, % released high mannose glycans, and % released afucosylated glycans of each antibody composition of the panel are provided in Table 4.TABLE 4%Released Afucosylated %Released High %Relative ADCC Glycans Mannose Glycans Response Clone 203 7.7 2.6 181 Clone 116 7.5 2.5 181 Clone 113 5.7 2.8 150 Clone 126 5.4 2.7 128 Clone 236 4.5 1.8 117 Clone 85 8.8 2.2 195 Clone 93 8.9 3 197 Clone 98 9.7 3.6 201 Clone 160 5.9 0.6 114 Clone 160.2 2.9 1.4 61 Clone 182 4.7 1.2 97 Clone 260 7.9 0.8 177 RP15 4.3 2.7 95 RP16 4.6 2.8 102 RP44 4.0 1.9 83 RP51 3.8 2.1 85RP54 4.6 1.9 85

[0185] These data were analyzed using the JMP suite of computer programs for statistical analysis (SAS Institute, Cary, NC). Figure 11A is an ADCC leverage plot for % released afucosylated glycans and Figure 11B is a leverage plot for % released high mannose glycans. The best fit line is the solid diagonal line in the middle of the shaded area. As shown in Figures 11A and 11B, released afucosylated glycans demonstrated a statistically significant relationship with the ADCC activity level of the antibody composition (pcO.OOOl), whereas released high mannose glycans did not (p=0.2786).

[0186] The relationship between % ADCC and the % released afucosylated glycans and % released high mannose glycans may be described by Equation 4:Predicted % ADCC = -5.8 + (21.7* % Released Afucosylated Glycans) + (4.3 * % Released High Mannose Glycans)[Equation 4]

[0187] Plugging the measured values for % released afucosylated glycans and % released high mannose glycans into Equation 4, a predicted % ADCC value was calculated for each sample. The actual % ADCC (as measured in the cell-based assay) was plotted against the predicted % ADCC (as calculated by Equation 4) and the plot is provided as Figure 11C. Statistical parameters, including Root Mean Square Error (RMSE), r2, and p-value, are shown in Figure 11C. These results suggested that Equation 4 predicted the actual (measured) ADCC with accuracy and underlines the statistically significant correlation between % released afucosylated glycans and ADCC (pcO.OOOl). Higher levels of afucosylated glycans result in higher ADCC activity. The effect of high mannose on ADCC was weak and not statistically significant.

[0188] As the glycans are released from the Fc of the antibody composition in the approach outlined in Example 4 no glycan pairing information is captured and the leverage of both the unpaired and paired afucosylated glycans and the unpaired and paired high mannose glycans on ADCC activity cannot be determined. In the previous HILIC-MS method outlined in Example 1, an increased level of unpaired afucosylated glycans was shown to have an increased leverage on ADCC, likely because the percentage of afucosylated glycans is spread over a wider distribution of molecules of the antibody composition. Paired high mannose glycans has been shown to have a very low leverage on ADCC activity, however the structural reason for this is currently unknown.

[0189] The RP-LC MS approach outlined in Example 1 to measure the levels of paired and unpaired afucosylated and high mannose glycan content offers a powerful, but simplified approach to determine the relationship between ADCC activity and glycan pairing. The overall distribution of total afucosylated and total high mannose glycans determined by the approaches described inExample 1 and Example 4 should provide similar results. However, the RP-LC MS method was developed as a targeted workflow focused on the analysis of the unpaired afucosylated and high mannose glycan content, the full composition data are likely less accurate compared to the well-established released glycan assay described in Example 4. The optimal use of the glycan pairing data on the afucosylated and high mannose subgroups is in application in a form of a correction factor to the released afucosylated and high mannose glycan content determined using Example 4 to account for the contribution of glycan pairing. This correction can be used to determine the relationship between ADCC activity and unpaired afucosylated glycans and unpaired high mannose glycans for an antibody composition.

[0190] The paired and unpaired afucosylated glycan content and paired and unpaired high mannose glycan content of the same antibody composition were determined following the procedure outlined in Example 1. The results are provided in Table 3.

[0191] The fraction of paired afucosylated glycans was used to apply a correction to the released afucosylated glycan data in Table 4 to account for the portion of the antibody composition where the afucosylated glycans are paired (paired afucosylated fraction). Using Equation 5, the % Unpaired Afucosylated Glycans of the entire antibody composition can be determined.% Unpaired Afucosylated Glycans = % released afucosylated glycans (1- paired afucosylated fraction) / (l+ paired afucosylated fraction) x 100[Equation 5]

[0192] The relative paired high mannose glycan content was used to apply a correction to the released high mannose glycan data in Table 4 to account for the portion of the antibody composition where the high mannose glycans are paired (paired high mannose fraction). Using Equation 6, the % Unpaired High Mannose Glycans of the entire antibody composition can be determined.% Unpaired High Mannose Glycans = % released high mannose glycans (1- paired high mannose fraction) / (l+ paired high mannose fraction) x 100[Equation 6]

[0193] The ADCC activity levels, the % Unpaired Afucosylated Glycans and the % Unpaired High Mannose Glycans were calculated as described in Equation 5 and Equation 6 and the data is provided below in Table 5.TABLE 5% Unpaired % Unpaired High %Relative ADCC SampleAfucosylated Glycans Mannose Glycans ResponseClone 203 5.9 1.7 181Clone 116 6.2 1.8 181 Clone 113 4.8 2.1 150 Clone 126 4.4 1.9 128 Clone 236 4 1.6 117 Clone 85 6.8 1.2 195 Clone 93 6.7 1.8 197 Clone 98 7.2 2.1 201 Clone 160 4.9 0.2 114 Clone 160.2 2.8 1.2 61 Clone 182 3.9 0.7 97 Clone 260 6.8 0.8 177 RP15 3.3 1.6 95 RP16 3.5 1.4 102 RP44 3.1 1.3 83 RP51 3 1.2 85RP54 3.4 1.3 85

[0194] The data in Table 5 was then analyzed using the JMP suite of computer programs for statistical analysis. Figure 12A is an ADCC leverage plot for % unpaired afucosylated glycans and Figure 12B is a leverage plot for % unpaired high mannose glycans. The best fit line is the solid diagonal line in the middle of the shaded area. As shown in Figures 12A and 12B, both % unpaired afucosylated glycans and % unpaired high mannose glycans demonstrated a statistically significant relationship with ADCC activity levels (pcO.OOOl for % unpaired afucosylated glycans and p=0.0012 for % unpaired high mannose glycans). These results support that the % unpaired high mannose glycans do significantly contribute to ADCC activity.

[0195] Taken together, these data support that the pairing status matters. The overall % released high mannose glycan content did not appear to have a statistically significant relationship with the ADCC activity level, whereas the % unpaired high mannose glycan content was demonstrated to correlate with ADCC activity statistically significantly.

[0196] The relationship between % ADCC and the % unpaired afucosylated glycans and % unpaired high mannose glycans may be described by Equation 7:Predicted % ADCC = -27.5 + (29.6* % Unpaired Afucosylated Glycans) + (14.5 * % Unpaired High Mannose Glycans)[Equation 7]

[0197] Plugging the measured values for % unpaired high mannose glycans and % unpaired afucosylated glycans into Equation 7, a predicted % ADCC value was calculated for each sample. The actual % ADCC (as measured in the cell-based assay) was plotted against the predicted % ADCC (ascalculated by Equation 7) and the plot is provided as Figure 12C. Statistical parameters, including Root Mean Square Error (RMSE), r2, and p-value, are shown in Figure 12C.

[0198] These results suggested that Equation 7 predicted the actual (measured) ADCC with accuracy and underlines the statistically significant direct correlation between % unpaired afucosylated glycans, % unpaired high mannose glycans, and ADCC activity level (pcO.OOOl). Higher levels of unpaired afucosylated glycan content and unpaired high mannoses content result in higher ADCC activity. The leverage of unpaired afucosylated glycans was stronger than the leverage of unpaired high mannose glycans on ADCC activity (29.6 and 14.5, respectively). Determining unpaired afucosylated glycans and unpaired high mannose glycans can predict ADCC levels of an antibody composition and thus predict product quality, e.g., quality of the antibody composition.

[0199] A similar approach can be used to correct the released afucosylated and high mannose glycan content to determine the paired afucosylated and paired high mannose glycan content respectively. For the current antibody composition, as the abundance of the paired glycan species are much lower, as seen in Table 3, a lower impact to ADCC activity is expected. Nonetheless, this approach can be employed to further understand the relationship between the paired glycan content and ADCC activity. It is expected that paired afucosylated glycans will have a statistically significant relationship with ADCC. As discussed above it is expected that the paired high mannose glycan content will have a very low leverage on ADCC activity.

[0200] These data support that glycan pairing analysis is important for understanding the structure-function relationship.EXAMPLE 6

[0201] This example describes a method of determining the product quality during manufacture of an antibody composition.

[0202] Samples of a cell culture comprising Clone 203 producing an antibody are collected at different pre-harvest times post-inoculation and post-harvest prior to Protein A chromatography. The new method described in Example 1 is carried out on the obtained samples. Data for paired high mannose glycan content unpaired high mannose glycan pair content, paired afucosylated glycan content and unpaired afucosylated glycan pair content for each sample are recorded. The range of unpaired high mannose glycan content and unpaired afucosylated glycan content is predetermined. The samples that are deemed to be outside this range are marked, and troubleshooting on the corresponding manufacture days are monitored. It is discovered that a cell culture parameter shifts on this day and efforts to prevent the shift are taken. The process of collecting samples at different times pre-harvest and post-harvest as well as determining the unpaired highmannose glycan content and unpaired afucosylated glycan content of the samples are repeated. The samples are deemed as within the pre-determined range.EXAMPLE 7

[0203] This example describes a method of determining the product quality of different manufacture lots of an antibody composition.

[0204] Samples of Lots 1-10 of an IgGl monoclonal antibody are obtained following storage for 12, 24 and 36 months at 4 °C. The new method described in Example 1 is carried out on the obtained samples. Data for paired high mannose glycan species, unpaired high mannose glycan pair content, paired afucosylated glycan content and unpaired afucosylated glycan pair content for each sample are recorded. Using Equation 7 and the data for % unpaired high mannose glycan molecules and % unpaired afucosylated glycan molecule, predicted ADCC activity levels for each lot are determined. Lots that are within the pre-determined ADCC range are determined as acceptable for administration to a patient, whereas lots that are outside the range are discarded.EXAMPLE 8

[0205] This example describes a simplified method of determining paired glycan content and unpaired glycan content for a large scale-preparation of the IgG described in previous Examples 1-7.

[0206] Three batches of an IgG were made in large-scale bioreactors and samples from each batch were obtained and used in the two-enzyme digestion as essentially described in Example 1. Briefly, the samples were treated overnight with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc., Cambridge, MA), and a second digestion was carried out with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. Following the two-enzyme digestion, resulting Fc and Fab fragments were chromatographically separated by using a rapid reversed phase liquid chromatography (RP-LC) separation with MS detection, as essentially described in Example 1. This separation yielded one chromatographic peak comprising the Fc fragments that was well seperated from the Fab fragments and treatment enzymes. The chromatographic peak that contained the Fc fragments, was then selected for data analysis, the mass spectra of the one chromatographic peak that contained the Fc fragments was extracted and deconvoluted, as essentially described in Example 1. The EndoS treated glycopairs were identified by matching the molecular weights (MW) of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs (Table 1), as essentially described in Example 1. The data for unpaired afucosylated glycopairs, paired afucosylated glycopairs, unpaired high mannose glycopairs and paired afucosylated glycopairs are provided in Table 6.TABLE 6Detected EndoS Glycopairs 500L Batchl 500L Batch2 500L Batch3 FC , M5 / M5 0.53 0.73 0.76 % Paired High Mannose Glycopairs 0.53 0.73 0.76 FC , M5 / GlcNac + Fuc 1.35 1.35 0.97 FC , M8 / GlcNac + Fuc 0.72 0.60 0.44 % Unpaired High Mannose Glycopairs 2.07 1.95 1.41 FC , GIcNac / GIcNac + Fuc 8.71 8.66 8.2 % Unpaired Afucosylated Glycopairs 8.71 8.66 8.20 FC , GIcNac / GIcNac 0.61 0.67 0.58 FC , M5 / GlcNac + Fuc 0.46 0.43 0.19% Paired Afucosylated Glycopairs 1.07 1.10 0.77

[0207] The relative unpaired and paired afucosylated glycan content and the relative unpaired and paired high mannose glycan content were calculated as described in Example 1 and the data is provided below in Table 7.TABLE 7Relative Unpaired and Paired Glycan Content 500L Batchl 500L Batch2 500L Batch3 % Unpaired Afucosylated 89.1 88.7 91.4 % Unpaired High Mannose 79.6 72.8 65.0 % Paired Afucosylated 10.9 11.3 8.6% Paired High Mannose 20.4 27.2 35.0

[0208] This example demonstrated that the new, simplified method of determining the relative unpaired glycan content can be carried out with a large-scale preparation of an IgG antibody composition.EXAMPLE 9

[0209] This example describes a simplified method of determining paired glycan content and unpaired glycan content for an antibody composition comprising an IgG different from the IgG described in Examples 1-8. The IgG of this example targeted an antigen different from the target of the IgG of Examples 1-8.

[0210] Samples of an antibody composition comprising an IgG different from the IgG described in Examples 1-8 were used in a two-enzyme digestion as essentially described in Example 1. Briefly, the samples were treated overnight with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc., Cambridge, MA), and a second digestion was carried out with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. Following the two-enzyme digestion, resulting Fc and Fab fragments were chromatographically separated by using a rapid reversed phase liquid chromatography (RP-LC)separation with MS detection, as essentially described in Example 1. This separation yielded one chromatographic peak comprising the Fc fragments that was well seperated from the Fab fragments and treatment enzymes. The chromatographic peak that contained the Fc fragments, was then selected for data analysis, the mass spectra of the one chromatographic peak that contained the Fc fragments was extracted and deconvoluted, as essentially described in Example 1. The EndoS treated glycopairs were identified by matching the molecular weights (MW) of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs (Table 1), as essentially described in Example 1. The data for unpaired afucosylated glycopairs, paired afucosylated glycopairs, unpaired high mannose glycopairs and paired afucosylated glycopairs are provided in Table 8.TABLE 8Detected EndoS Glycopairs Relative Abundance (%) FC , M5 / M5 0.01% Paired High Mannose Glycopairs 0.01FC , M5 / GlcNac + Fuc 0.53FC , M8 / GlcNac + Fuc 0.07% Unpaired High Mannose Glycopairs 0.60FC , GIcNac / GIcNac + Fuc 6.04% Unpaired Afucosylated Glycopairs 6.04FC , GIcNac / GIcNac 1.19FC , GIcNac 0.22% Paired Afucosylated Glycopairs 1.41

[0211] The relative unpaired and paired afucosylated glycan content and the relative unpaired and paired high mannose glycan content were calculated as described in Example 1 and the data is provided below in Table 9.TABLE 9Relative Unpaired and Paired Glycan Content Relative Abundance (%) % Unpaired Afucosylated Glycan Content 81.1% Unpaired High Mannose Glycan Content 98.4% Paired Afucosylated Glycan Content 18.9% Paired High Mannose Glycan Content 1.6

[0212] This example demonstrated that the new, simplified method of determining the relative unpaired glycan content can be used with IgG antibody compositions other than the ones described in Examples 1-8.EXAMPLE 10

[0213] This example describes a simplified method of determining paired glycan content and unpaired glycan content for an antibody composition comprising an IgG of the same isotype and antigen-specificity as the IgG of Examples 1-7 but different allotype (e.g., comprising a modified Fc region sequence, relative to the IgG of Examples 1-7).

[0214] Samples of an antibody composition comprising an IgG of a different allotype (relative to the IgG described in Examples 1-7) were used in a two-enzyme digestion as essentially described in Example 1. Briefly, the samples were treated overnight with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc., Cambridge, MA), and a second digestion was carried out with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. Following the two-enzyme digestion, resulting Fc and Fab fragments were chromatographically separated by using a rapid reversed phase liquid chromatography (RP-LC) separation with MS detection, as essentially described in Example 1. This separation yielded one chromatographic peak comprising the Fc fragments that was well separated from the Fab fragments and treatment enzymes. The chromatographic peak that contained the Fc fragments, was then selected for data analysis, the mass spectra of the one chromatographic peak that contained the Fc fragments was extracted and deconvoluted, as essentially described in Example 1. The EndoS treated glycopairs were identified by matching the molecular weights (MW) of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs, as essentially described in Example 1, but the custom-built database used in this example is described in Table 10. The data for unpaired afucosylated glycopairs, paired afucosylated glycopairs, unpaired high mannose glycopairs and paired afucosylated glycopairs are provided in Table 11.TABLE 10EndoS GlycanDominant Paired Fucosylated EndoS Glycan Pair EndoS Glycopair MW (Da)Pair MW (Da)GIcNac / GIcNac + Fuc 698.6 50837.1EndoS GlycanPaired Afucosylated EndoS Glycan Pairs EndoS Glycopair MW (Da)Pair MW (Da)GIcNac / GIcNac 406.4 50544.8 M5 / GlcNac 1420.3 51558.7 M6 / GlcNac 1582.4 51720.8 M7 / GlcNac 1744.6 51883.0 M8 / GlcNac 1906.7 52045.1 AlG0M5 / G / cA / ac 1623.5 51761.9 A1G0M5 / A1G0M5 2840.6 52979.0 AlGlM5 / G / cA / ac 1785.6 52062.2 A1G1M5 / A1G1M5 3164.8 53579.6 M5 / A1G0M5 2637.4 52775.8M6 / A1G0M5 or M5 / A1G1M5 2799.5 52937.9 M7 / A1G0M5 or M6 / A1G1M5 2961.6 53100.1 M8 / A1G0M5 or M7 / A1G1M5 3123.8 53262.2 M8 / A1G1M5 3285.9 53562.5EndoS GlycanUnpaired Afucosylated EndoS Glycan Pairs EndoS Glycopair MW (Da)Pair MW (Da)GIcNac / GIcNac + Fuc 552.5 50487.7 GIcNac 203.2 50341.6 AlG0M5 / G / cA / ac or G / cA / ac / AlG0M5F 1769.6 51908.0 AlGlM5 / G / cA / ac + Fuc or G / cA / ac / AlGlM5F 1931.8 52070.2 G / cA / ac / AlG0M4F 1753.6 51892.0 G / cA / ac / AlSlM4F 2060.9 52199.3 GIcNac / A1G1S1M5F 2223 52361.4EndoS GlycanPaired High Mannose EndoS Glycan Pairs EndoS Glycopair MW (Da)Pair MW (Da)M5 / M5 2434.2 52572.6 M5 / M6 2596.3 52734.7 M5 / M7 or FC, M6 / M6 2758.5 52896.9 M5 / M8 or FC, M6 / M7 2920.6 53059.0 M6 / M8 or FC, M7 / M7 3082.7 53221.1 M7 / M8 3244.9 53383.3 M8 / M8 3407 53545.4EndoS GlycanUnpaired High Mannose EndoS Glycan Pairs EndoS Glycopair MW (Da)Pair MW (Da)M5 / GlcNac + Fuc 1566.4 51704.8 M6 / GlcNac + Fuc 1728.6 51867.0 M7 / GlcNac + Fuc 1890.7 52029.1 M8 / GlcNac + Fuc 2052.8 52191.3 M5 / A1G0M4F 2621.4 52759.8 M6 / A1G0M4F or FC, M5 / A1G0M5F 2783.5 52921.9 M7 / A1G0M4F or FC, M6 / A1G0M5F or FC + M5 / A1G1M5F 2945.7 53084.1 M8 / A1G0M4F or FC, M7 / A1G0M5F or FC + M6 / A1G1M5F 3107.8 53246.2 M8 / A1G0M5F or FC + M7 / A1G1M5F 3269.9 53408.3 M8 / A1G1M5F 3432.1 53570.5 M5 / A1S1M4F 3074.8 53213.2 M6 / A1S1M4F or FC + M5 / A1G1S1M5F 3236.9 53375.3 M7 / A1S1M4F or FC + M6 / A1G1S1M5F 3399.0 53537.5 M8 / A1S1M4F or FC + M7 / A1G1S1M5F 3561.2 53699.6 M8 / A1G1S1M5F 3723.3 53861.7TABLE 11Detected EndoS Glycopairs Relative Abundance (%) FC , M5 / M5 0.05FC , M6 / M6 0.08% Paired High Mannose Glycopairs 0.05FC , M5 / GlcNac + Fuc 0.66FC , M8 / GlcNac + Fuc 0.02% Unpaired High Mannose Glycopairs 0.68FC , GIcNac / GIcNac + Fuc 3.15% Unpaired Afucosylated Glycopairs 3.15FC , GIcNac / GIcNac 0.53FC , GIcNac 0.08% Paired Afucosylated Glycopairs 0.61

[0215] The relative unpaired and paired afucosylated glycan content and the relative unpaired and paired high mannose glycan content were calculated as described in Example 1 and the data is provided below in Table 12.TABLE 12Relative Unpaired and Paired Glycan Content Relative Abundance (%) % Unpaired Afucosylated Glycan Content 83.8% Unpaired High Mannose Glycan Content 93.2% Paired Afucosylated Glycan Content 16.2% Paired High Mannose Glycan Content 6.8EXAMPLE 11

[0216] This example describes a simplified method of determining paired glycan content and unpaired glycan content for a panel of antibody composition samples, wherein each individual sample of the panel comprised an IgGl monoclonal antibody produced by a particular clone or was a sample of a reference product (RP) lot.

[0217] Samples of the panel were used in a two-enzyme digestion as essentially described in Example 1. Briefly, the samples were treated overnight with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc., Cambridge, MA), and a second digestion was carried out with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. Following the two-enzyme digestion, resulting Fc and Fab fragments were chromatographically separated by using a rapid reversed phase liquid chromatography (RP-LC) separation with MS detection, as essentially described in Example 1. This separation yielded one chromatographic peak comprising the Fc fragments that was well separated from the Fab fragments and treatment enzymes. The chromatographic peak that contained the Fc fragments, was thenselected for data analysis, the mass spectra of the one chromatographic peak that contained the Fc fragments was extracted and deconvoluted, as essentially described in Example 1. The EndoS treated glycopairs were identified by matching the molecular weights (MW) of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs (Table 10), as essentially described in Example 1. The data for unpaired afucosylated glycopairs, paired afucosylated glycopairs, unpaired high mannose glycopairs and paired afucosylated glycopairs are provided in Table 13.TABLE 13Detected EndoS Glycopairs Lot 1 Lot i Lot 3 Lot 4 Lot 5 Lot 6Relative Abundance (%)FC , M5 / M5 0.07 0.14 0.03 0.09 0.1 0.12 % Paired High Mannose Glycopairs 0.07 0.14 0.03 0.09 0.10 0.12 FC , M5 / GlcNac + Fuc 0.83 2.03 ND 1.47 1.53 1.95 FC , M7 / GlcNac + Fuc ND ND ND ND 0.21 ND % Unpaired High Mannose Glycopairs 0.83 2.03 0.00 1.47 1.74 1.95 FC , GIcNac / GIcNac + Fuc 7.17 1.71 7.16 4.07 4.4 2.03 FC , AlG0M5 / G / cA / ac + Fuc ND ND ND 0.27 0.52 ND FC , AlGlM5 / G / cA / ac + Fuc ND ND ND 0.09 ND ND % Unpaired Afucosylated Glycopairs 7.17 1.71 7.16 4.43 4.92 2.03 FC , GIcNac 0.41 ND ND 0.5 0.58 ND FC , GIcNac / GIcNac 0.94 0.26 1 0.81 0.82 0.21 FC , A1G1M5 / A1G1M5 ND ND 0.01 ND 0.02 ND FC , M5 / GIcNac ND ND ND 0.35 0.41 ND FC , M8 / GIcNac ND ND ND 0.18 ND ND % Paired Afucosylated Glycopairs 1.35 0.26 1.01 1.84 1.83 0.21Relative Unpaired and Paired Glycan Content Lot 1 Lot i Lot 3 Lot 4 Lot 5 Lot 6

[0218] The relative unpaired and paired afucosylated glycan content and the relative unpaired and paired high mannose glycan content were calculated as described in Example 1 and the data is provided below in Table 14.TABLE 14Relative Unpaired and Paired Glycan Content Relative Abundance (%)Lot 1 Lot i Lot 3 Lot 4 Lot 5 Lot 6 % Unpaired Afucosylated Glycan Content 84.2 86.8 87.6 70.7 72.9 90.6 % Unpaired High Mannose Glycan Content 92.2 93.5 0.0 94.2 94.6 94.2 % Paired Afucosylated Glycan Content 15.8 13.2 12.4 29.3 27.1 9.4% Paired High Mannose Glycan Content 7.8 6.5 100.0 5.8 5.4 5.8EXAMPLE 12

[0219] This example describes a simplified method of determining paired high mannose glycan content and unpaired high mannose glycan content for an antibody composition comprising a completely afucosylated IgG.

[0220] Samples of an antibody composition comprising completely afucosylated IgG were used in a two-enzyme digestion as essentially described in Example 1. Briefly, the samples were treated overnight with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc., Cambridge, MA), and a second digestion was carried out with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. Following the two-enzyme digestion, resulting Fc and Fab fragments were chromatographically separated by using a rapid reversed phase liquid chromatography (RP-LC) separation with MS detection, as essentially described in Example 1. This separation yielded one chromatographic peak comprising the Fc fragments that was well separated from the Fab fragments and treatment enzymes. The chromatographic peak that contained the Fc fragments, was then selected for data analysis, the mass spectra of the one chromatographic peak that contained the Fc fragments was extracted and deconvoluted, as essentially described in Example 1. The EndoS treated glycopairs were identified by matching the molecular weights (MW) of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs (Table 15), as essentially described in Example 1. The data for unpaired high mannose glycopairs and paired afucosylated glycopairs are provided in Table 16.TABLE 15EndoS Glycan PairDominant Paired Afucosylated EndoS Glycan Pair EndoS Glycopair MW (Da)MW (Da)GIcNac / GIcNac 406.4 50544.8EndoS Glycan PairPaired Unpaired High Mannose EndoS Glycan Pairs EndoS Glycopair MW (Da)MW (Da)M5 / GlcNac 1420.3 51558.7 M6 / GlcNac 1582.4 51720.8 M7 / GlcNac 1744.6 51883.0 M8 / GlcNac 1906.7 52045.1 M5 / A1G0M5 2637.4 52775.8 M6 / A1G0M5 or M5 / A1G1M5 2799.5 52937.9 M7 / A1G0M5 or M6 / A1G1M5 2961.6 53100.0 M8 / A1G0M5 or M7 / A1G1M5 3123.8 53262.2 M8 / A1G1M5 3285.9 53424.3EndoS Glycan PairPaired High Mannose EndoS Glycan Pairs EndoS Glycopair MW (Da)MW (Da)M5 / M5 2434.2 52572.6 M5 / M6 2596.3 52734.7 M5 / M7 or FC, M6 / M6 2758.5 52896.9 M5 / M8 or FC, M6 / M7 2920.6 53059.0 M6 / M8 or FC, M7 / M7 3082.7 52896.9 M7 / M8 3244.9 53059.0 M8 / M8 3407 53221.1TABLE 16Detected EndoS Glycopairs %Relative Abundance FC , M5 / M5 1.1% Paired High Mannose Glycopairs 1.1FC , M5 / GlcNac 3.9FC , M6 / GlcNac 0.6FC , M8 / GlcNac 0.2% Unpaired High Mannose Glycopairs 4.6

[0221] The relative unpaired and paired high mannose glycan content were calculated as described in Example 1 and the data is provided below in Table 17.TABLE 17Relative Unpaired and Paired Glycan Content %Relative Abundance % Unpaired High Mannose 80.4% Paired High Mannose 19.6EXAMPLE 13

[0222] This example describes an exemplary method of determining glycan pairing content (e.g., paired high mannose glycan content and unpaired high mannose glycan content) for an antibody composition.

[0223] Glycan pairing analysis is challenging due to the size of the molecules and the highly heterogeneous glycan population. The analysis of specifically afucosylated glycan pairs and high mannose glycan pairs is further complicated by the low abundance of these glycans relative to other glycans. Accordingly, determining the relative unpaired afucosylated glycan content (or paired afucosylated glycan content) and relative unpaired high mannose glycan content (or paired high mannose glycan content) is complex and time-consuming.

[0224] To illustrate the low abundances of high mannose glycans, the high mannose released glycan data for an exemplary antibody composition is provided in Table 18. As seen in this table,Man5 is the most abundant of the high mannose glycans, though its relative abundance (relative to all other glycans present in the antibody composition) is very low (<2%). Man6, Man7, and Man8 are also present, though the relative abundance of these high mannose glycans are even lower than that of Man5 and do not even reach 1%. Man9 for this antibody composition was not detectable. These low relative abundances make the quantitation of high mannose glycopairs particularly challenging, as the signal for the high mannose glycopairs is often close to the limit of detection.TABLE 18High mannose Glycan % Relative Abundance Man5 1.9%Man6 0.1%Man7 <0.1%Man8 0.5%Man9 ND

[0225] The high mannose glycopairs retain a relatively high degree of heterogeneity. Up to 25 discrete high mannose glycopairs are possible following EndoS treatment (Figure 13). The high degree of heterogeneity coupled with the low abundances make it very difficult to determine the relative unpaired afucosylated glycan content (or paired afucosylated glycan content) and relative unpaired high mannose glycan content (or paired high mannose glycan content).

[0226] Using endoglycosidases (such as EndoS2 (from Genovis Inc.) or EndoH, EndoHf, EndoD, EndoF2 and EndoS (all from New England Biolabs)), provide an opportunity to simplify high mannose glycopair structure. However, such endoglycosidases fail to inform the specific high mannose glycopair status, as cleavage with these endoglycosidases render high mannose glycopairs indistinguishable from paired afucosylated glycopairs.

[0227] Herein, an exemplary method that simplifies the structures of the glycopairs of the antibody composition prior to the RP-LC-MS analysis for the quantitation of the paired and unpaired afucosylated and high mannose glycan content is described. In this method, a digestion with an exoglycosidase is carried out to simplify the high mannose glycan pair structures prior to quantitation of the glycopairs. Unlike endoglycosidases, exoglycosidases are enzymes that cleave the glycosidic linkages in the terminal residues of oligosaccharides. As seen in Figure 14, high mannose glycans typically, contain between four to eight al-2-, al-3- and / or al-6-linked mannoseresidues attached to the structure of one P-l inked mannose and two GIcNAc residues. Mannosidases are a class of exoglycosidases that can hydrolyze the al-2, al-3 and / or al-6 linked mannose residues from the distal terminal of the glycan structure of a glycoprotein. One mannosidase, the al-2, 3, 6 mannosidase, has a broad substrate specificity cleaving al-2, 3, 6 mannosidase linkages, while the al-2 mannosidase, al-2, 3 mannosidase and al-6 mannosidase demonstrate specificity towards the al-2 linkage, al-2 and al-3 linkages, and al-6 linkage, respectively. It was hypothesized that these enzymes could be used separately or together to simplify the heterogeneity of the high mannose glycopairs, to ultimately compress a heterogenous high mannose population into just two species: paired high mannose and unpaired high mannose.

[0228] In the exemplary method described herein, a multi-enzyme digestion scheme was carried out (Figure 15) with a sample of an antibody composition comprising the same antibody used in Examples 1-8. The sample was first treated overnight with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc.,) which allows for Fab fragments to be chromatographically separated from glycosylated Fc species. A second digestion was carried out with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. EndoS hydrolyzes the pi,4 linkage between the core GIcNAc residues in the N-glycans, leaving the innermost GIcNAc intact on the Fc with or without a fucose. The sample was further digested through a sequential digestion of al-2, 3, 6 mannosidase (from Agilent Technologies) at 37°C for 2 hours followed by a digestion with al-6 mannosidase (from New England Biolabs) at 37°C for 2 hours. The al-6 mannosidase was used to supplement the cleavage activity at the al-6 linkage, since the al-2, 3, 6 mannosidase may demonstrate limited activity on a single al-6 linked mannose from core P-mannose. Following the digestion with al-2, 3, 6 mannosidase and al-6 mannosidase, a single P-linked mannose and two GIcNAc's (2GlcNac+Man) remained (Figure 16B). As depicted in Figure 16A, the highly heterogenous high mannose glycopair population is simplified to just two glycopair classes: unpaired high mannose and paired high mannose.

[0229] Following the multi-enzyme digestion scheme, the resulting sample comprising glycosylated Fc fragments and Fab fragments was chromatographically separated using a RP-LC separation with online MS detection. The chromatographic peak comprising the Fc fragments was then selected for data analysis. The mass spectra of the one chromatographic peak that contained the Fc fragments was extracted and deconvoluted. The deconvoluted spectra is shown on the right side of Figure 17 and, for comparison purposes, the deconvoluted spectra of a sample that was not digested with the enzymes is shown on the left. The deconvoluted spectra on the left side highlights the sample complexity of an untreated sample, and, encompassed in the dashed box is the low abundant and unresolved mass spectral region containing a complex population of glycopairs, including critical unpaired (and paired) afucosylated and high mannose species, that are very difficultto quantitate. In contrast, following the muti-enzyme sample treatment workflow, the heterogenous population of glycopairs is condensed into approximately six well resolved spectral peaks for the target glycopairs permitting the efficient quantitation of critical unpaired (and paired) afucosylated and high mannose species as shown on the right side of Figure 17.

[0230] Glycopairs having simplified structures (due to treatment with EndoS and the mannosidases) were identified by an automated, computer-assisted matching of the molecular weight (MW) of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs (Table 19), like the method of Example 1. The high mannose glycopairs were differentiated from the afucosylated pairs by their unique MWs.TABLE 19Dominant Paired FucosylatedEndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6Glycan Pair MW (Da) Glycopair MW (Da) Mannosidase Glycan PairGlcNac+ Fuc / GlcNac+ Fuc 698.6 50901.2Paired Afucosylated EndoS +EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase al-2,3,6 Mannosidase GlycanGlycan Pair MW (Da) Glycopair MW (Da) PairsGIcNac / GIcNac 406.4 50608.9 2GlcNac + Man / GIcNac 771.8 50974.3 Unpaired Afucosylated EndoSEndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase + al-2,3,6 MannosidaseGlycan Pair MW (Da) Glycopair MW (Da) Glycan PairsGIcNac / GIcNac + Fuc 552.5 50755.1Paired High Mannose EndoS +EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase al-2,3,6 Mannosidase GlycanGlycan Pair MW (Da) Glycopair MW (Da) Pairs2GlcNac + Man / 2GlcNac +1137.2 51339.7ManUnpaired High MannoseEndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6Glycan Pair MW (Da) Glycopair MW (Da) Mannosidase Glycan Pairs2GlcNac + Man / GIcNac + Fuc 917.9 51120.4 2GlcNac+ Man / GIcNac 771.8 50974.3

[0231] The abundance of the individual glycopairs was determined from the relative intensities or areas of the identified deconvoluted mass spectral peaks as follows:% Glycopair Intensity = Deconvoluted Peak Intensity / Total Deconvoluted Peak Intensity x 100[Equation 1]The relative abundances of the simplified glycopairs is shown in Table 20.TABLE 20Detected EndoS + al-2,3,6 Mannosidase Glycopairs %Relative Abundance FC , 2GlcNac + Man / 2GlcNac + Man 0.29% Paired High Mannose Glycopairs 0.29FC , 2GlcNac + Man / GIcNac + Fuc 1.07% Unpaired High Mannose Glycopairs 1.07FC , GIcNac / GIcNac + Fuc 8.08% Unpaired Afucosylated Glycopairs 8.08FC , GIcNac / GIcNac 0.42% Paired Afucosylated Glycopairs 0.42

[0232] The glycan pairing content, in particular, the relative unpaired and paired afucosylated glycan content and the relative unpaired and paired high mannose glycan content, were calculated with Equation 2 and Equation 3 as described in Example 1. Briefly, the abundance of paired afucosyated glycopairs was determined by summing the individual glycopairs categorized as paired afucosylated glycopairs, similarly the abundance of unpaired afucosyated glycopairs was determined by summing the individual glycopairs categorized as unpaired afucosylated glycopairs. To calculate the % relative abundance of the Unpaired Afucosylated Glycan Content of the antibody composition Equation 2 was employed:% Unpaired Afucosylated Glycan Content = (% Unpaired Afucoslyated Glycopairs / % Total Afucoslyated Glycopairs (Unpaired+Paired)) x 100[Equation 2]

[0233] The abundance of paired high mannose glycopairs was determined by summing the individual glycopairs categorized as paired high mannose glycopairs, similarly the abundance of unpaired high mannose glycopairs was determined by summing the individual glycopairs categorized as unpaired high mannose glycopairs. To calculate the % relative abundance of the Unpaired High Mannose Glycan content of the antibody composition Equation 3 was employed:% Unpaired High Mannose Glycan Content = (% Unpaired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 3]

[0234] To calculate the % relative abundance of the Paired Afucosylated Glycan Content of the antibody composition Equation 8 was employed:% Paired Afucosylated Glycan Content = (% Paired Afucoslyated Glycopairs / % Total Afucoslyated Glycopairs (Unpaired+Paired)) x 100[Equation 8]

[0235] To calculate the % relative abundance of the Paired High Mannose Glycan Content of the antibody composition Equation 9 was employed:% Paired High Mannose Glycan Content = (% Paired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 9]

[0236] The data is provided below in Table 21.TABLE 21Relative Unpaired and Paired Glycan Content Relative Abundance (%) % Unpaired Afucosylated Glycan Content 95.1% Unpaired High Mannose Glycan Content 78.7% Paired Afucosylated Glycan Content 4.9% Paired High Mannose Glycan Content 21.3

[0237] This example demonstrated the successful simplification of a heterogeneous mixture of glycopairs through a multi-enzyme digestion scheme and determination of glycan pairing content, e.g., relative unpaired afucosylated glycan content and relative unpaired high mannose glycan content, for an antibody composition.EXAMPLE 14

[0238] This example describes an exemplary method of determining glycan pairing content of an antibody composition comprising a completely afucosylated IgG, wherein the method comprises simplifying the structures of high mannose glycans and quantifying relative unpaired high mannose glycan content (relative paired high mannose glycan content) for the antibody composition.

[0239] Samples of the antibody composition described in Example 12 were used in the method of Example 13 to determine the glycan pairing content of the antibody composition. Briefly, the samples were first treated overnight with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc.,), and then treated with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. The samples were then treated with al-2,3,6 mannosidase (from Agilent Technologies) at 37°C for 2 hours, followed by treatment with al-6 mannosidase (from New England Biolabs) at 37°C for 2 hours.

[0240] After treatment with the enzymes, the samples were applied to an RP-LC column with online MS detection to chromatographically separate Fab fragments from glycosylated Fc fragments. The chromatographic peak comprising the Fc fragments was then selected for data analysis. The mass spectra of the Fc containing chromatographic peak was extracted and deconvoluted, as essentially described in Example 13. The deconvoluted spectra is shown on the right side of Figure18 and, for comparison purposes, the deconvoluted spectra of a sample that was not digested with the enzymes is shown on the left. The deconvoluted spectra on the left side highlights the sample complexity of an untreated sample, and, encompassed in the dashed box is the low abundant and unresolved mass spectral region containing a complex population of glycopairs, including critical unpaired (and paired) high mannose species, that are very difficult to quantitate. In contrast, following the multi-enzyme sample treatment workflow, the heterogenous population of glycopairs is condensed into approximately three well resolved spectral peaks for the target glycopairs, as shown on the right of Figure 18, which well resolved peaks facilitate the efficient quantitation of critical unpaired (and paired) high mannose species.

[0241] Glycopairs are identified by an automated, computer-assisted matching of the MW of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs (Table 22).TABLE 22Dominant Paired Afucosylated EndoS+ EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase al-2,3,6 Mannosidase Glycan Pair Glycan Pair MW (Da) Glycopair MW (Da) GIcNa / GIcNac 406.4 50544.8 Paired High Mannose EndoS+ al- EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase 2,3,6 Mannosidase Glycan Pair Glycan Pair MW (Da) Glycopair MW (Da) 2GlcNac +Man / 2GlcNac + Man 1137.2 51275.6 Unpaired High Mannose EndoS+ al- EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase 2,3,6 Mannosidase Glycan Pair Glycan Pair MW (Da) Glycopair MW (Da) 2GlcNac+Man / GlcNac 771.8 50910.2

[0242] The abundance of the individual glycopairs was determined from the relative intensities or areas of the identified deconvoluted mass spectral peaks as follows:% Glycopair Intensity = Deconvoluted Peak Intensity / Total Deconvoluted Peak Intensity x 100[Equation 1]The relative abundances of the simplified glycopairs is shown in Table 23.TABLE 23Detected EndoS + al-2,3,6 Mannosidase Glycopairs %Relative Abundance FC , 2GlcNac+Man / 2GlcNac + Man 1.38% Paired High Mannose Glycopairs 1.38FC , 2GlcNac + Man / GIcNac 3.89% Unpaired High Mannose Glycopairs 3.89

[0243] The relative unpaired and paired high mannose glycan content were calculated using Equation 3 as described in Example 1 and Equation 9. Briefly, the abundance of paired high mannose glycopairs was determined by summing the individual glycopairs categorized as paired high mannose glycopairs, similarly the abundance of unpaired high mannose glycopairs was determined by summing the individual glycopairs categorized as unpaired high mannose glycopairs. To calculate the % relative abundance of the Unpaired High Mannose Glycan content of the antibody composition Equation 3 was employed:% Unpaired High Mannose Glycan Content = (% Unpaired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 3]To calculate the % relative abundance of the Paired High Mannose Glycan content of the antibody composition Equation 9 was employed:% Paired High Mannose Glycan Content = (% Paired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 9]

[0244] The data is provided below in Table 24.TABLE 24Relative Unpaired and Paired Glycan Content Relative Abundance (%) % Unpaired High Mannose Glycan Content 73.8% Paired High Mannose Glycan Content 26.2

[0245] This example demonstrated the successful simplification of a heterogeneous mixture of glycopairs through a multi-enzyme digestion scheme and determination of glycan pairing content, e.g., relative unpaired high mannose glycan content, for an antibody composition.EXAMPLE 15

[0246] This example describes another exemplary method of determining the glycan pairing content for an antibody composition comprising a completely afucosylated IgG, wherein the method comprises simplifying the structures of high mannose glycans and quantifying relative unpaired high mannose glycan content / relative paired high mannose glycan content for the antibody composition.

[0247] Samples of the antibody composition described in Example 12 and 14 comprising a completely afucosylated IgG were used in a multi-enzyme digestion to simplify the heterogenous high mannose glycopair population. In this method, an al-2 mannosidase was used instead of theal-2, 3, 6 mannosidase and al-6 mannosidase used in the method Example 13. Briefly, the samples were first treated overnight with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc.,), and a second digestion was carried out with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. Samples were then treated with al-2 mannosidase (from Agilent Technologies) at 37°C for 24 hours. The al-2 mannosidase is highly specific for cleaving just al-2 mannose linkages of terminal mannose residues and yields Man5 upon cleavage of Man9, Man8, Man7 and Man6. The samples were then chromatographically separated by a RP-LC separation with online MS detection, as described in Example 13. The chromatographic peak comprising the Fc fragments was then selected for data analysis. The mass spectra of the Fc containing chromatographic peak was extracted and deconvoluted, as essentially described in Example 13.

[0248] The deconvoluted spectra is shown on the right side of Figure 19 and, for comparison purposes, the deconvoluted spectra of a sample that was not digested with the enzymes is shown on the left. The deconvoluted spectra on the left side highlights the sample complexity of an untreated sample, and, encompassed in the dashed box is the low abundant and unresolved mass spectral region containing a complex population of glycopairs, including critical unpaired (and paired) high mannose species, that are very difficult to quantitate. In contrast, following the multi-enzyme sample treatment workflow, the heterogenous population of glycopairs is condensed into approximately three well resolved spectral peaks for the target glycopairs, as shown on the right of Figure 19, which well resolved peaks facilitate the efficient quantitation of critical unpaired (and paired) high mannose species.

[0249] Glycopairs were identified by an automated, computer-assisted matching of the MW of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs (Table 25), as essentially described in Examples 1 and 13.TABLE 25Dominant Paired Afucosylated EndoS+ EndoS + al-2 Mannosidase EndoS + al-2 Mannosidase al-2 Mannosidase Glycan Pair Glycan Pair MW (Da) Glycopair MW (Da) GIcNa / GIcNac 406.4 50544.8Paired High Mannose EndoS+ al-2 EndoS + al-2 Mannosidase EndoS + al-2 Mannosidase Mannosidase Glycan Pair Glycan Pair MW (Da) Glycopair MW (Da) Man5 / Man5 2434.2 52572.6 Unpaired High Mannose EndoS+ al-2 EndoS + al-2 Mannosidase EndoS + al-2 Mannosidase Mannosidase Glycan Pair Glycan Pair MW (Da) Glycopair MW (Da) GlcNac / Man5 1420.3 51558.7

[0250] The abundance of the individual glycopairs was determined from the relative intensities or areas of the identified deconvoluted mass spectral peaks as follows:% Glycopair Intensity = Deconvoluted Peak Intensity / Total Deconvoluted Peak Intensity x 100[Equation 1]

[0251] The relative abundances of the simplified glycopairs are shown in Table 26.TABLE 26Detected EndoS+ al-2 Mannosidase %Relative Abundance FC , Man5 / Man5 0.69% Paired High Mannose Glycopairs 0.69FC , GlcNac / Man5 3.96% Unpaired High Mannose Glycopairs 3.96

[0252] The relative unpaired and paired high mannose glycan content were calculated using Equation 3 as described in Example 1 and Equation 9. Briefly, the abundance of paired high mannose glycopairs was determined by summing the individual glycopairs categorized as paired high mannose glycopairs, similarly the abundance of unpaired high mannose glycopairs was determined by summing the individual glycopairs categorized as unpaired high mannose glycopairs. To calculate the % relative abundance of the Unpaired High Mannose Glycan content of the antibody composition Equation 3 was employed:% Unpaired High Mannose Glycan Content = (% Unpaired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 3]To calculate the % relative abundance of the Paired High Mannose Glycan content of the antibody composition Equation 9 was employed:% Paired High Mannose Glycan Content = (% Paired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 9]

[0253] The data is provided below in Table 27.TABLE 27Relative Unpaired and Paired Glycan Content Relative Abundance (%) % Unpaired High Mannose Glycan Content 85.3% Paired High Mannose Glycan Content 14.8

[0254] This example demonstrated the successful simplification of a heterogeneous mixture of glycopairs through a multi-enzyme digestion scheme and determination of glycan pairing content, e.g., relative unpaired high mannose glycan content, for an antibody composition.EXAMPLE 16

[0001] This example describes an exemplary method of determining glycan pairing content for an antibody composition, wherein the method comprises simplifying the structures of high mannose glycans and quantifying the glycan pairing content, e.g., relative unpaired high mannose glycan content / relative paired high mannose glycan content, for the antibody composition.

[0002] A sample of the antibody composition described in Example 12 is used in a multi-enzyme digestion, as essentially described in Example 13, except that an al-2,3 mannosidase is used instead of the al-2,3, 6 mannosidase and al-6 mannosidase used in Example 13. Briefly, the samples are treated overnight with the enzyme IgdE (FabALACTICA® or FL from Genovis Inc.,) followed a treatment with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. The samples are then treated with an al-2,3 mannosidase (from New England Biolabs) at 37°C for approximately 18 hours. The al-2,3 mannosidase is highly specific for cleaving at just al-2 and al-3 mannose linkages of terminal mannose residues and yield two al-6 mannoses and a single P-linked mannose attached to the two core GIcNAc's (2GlcNac+3Man). See Figure 16B.

[0003] Following the multi-enzyme digestion, the sample comprising glycosylated Fc and Fab fragments are chromatographically separated by RP-LC with online MS detection. The chromatographic peak comprising the Fc fragments is selected for data analysis. The mass spectra of the one Fc containing chromatographic peak are extracted and deconvoluted as previously described in Examples 1 and 13.

[0004] Glycopairs are identified by an automated, computer-assisted matching of the MW of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs (Table 28).TABLE 28EndoS + al-2,3 Dominant Paired Afucosylated EndoS + EndoS + al-2,3 MannosidaseMannosidase Glycopair MW al-2,3 Mannosidase Glycan Pair Glycan Pair MW (Da)(Da) GIcNac / GIcNac 406.4 50544.8EndoS + al-2,3 Paired High Mannose EndoS + al-2,3 EndoS + al-2,3 MannosidaseMannosidase Glycopair MW Mannosidase Glycan Pairs Glycan Pair MW (Da)(Da)2GlcNac + 3Man / 2GlcNac + 3Man 1786.0 51924.4EndoS + al-2,3 Unpaired High Mannose EndoS + al-2,3 EndoS + al-2,3 MannosidaseMannosidase Glycopair MW Mannosidase Glycan Pairs Glycan Pair MW (Da)(Da) 2GlcNac + 3Man / GlcNac 1096.2 51234.6

[0005] The abundance of the individual glycopairs is determined from the relative intensities or areas of the identified deconvoluted mass spectral peaks as follows:% Glycopair Intensity = Deconvoluted Peak Intensity / Total Deconvoluted Peak Intensity x 100[Equation 1]

[0006] The relative abundances of the simplified glycopairs are recorded in Table 29.TABLE 29Detected EndoS + al-2,3 Mannosidase Glycopairs %Relative Abundance FC , 2GlcNac + 3Man / 2GlcNac + 3Man% Paired High Mannose GlycopairsFC , 2GlcNac + 3Man / GlcNac% Unpaired High Mannose Glycopairs

[0007] The relative unpaired and paired high mannose glycan content are calculated using Equation 3 as described in Example 1 and Equation 9. Briefly, the abundance of paired high mannose glycopairs was determined by summing the individual glycopairs categorized as paired high mannose glycopairs, similarly the abundance of unpaired high mannose glycopairs was determined by summing the individual glycopairs categorized as unpaired high mannose glycopairs. To calculate the % relative abundance of the Unpaired High Mannose Glycan content of the antibody composition Equation 3 was employed:% Unpaired High Mannose Glycan Content = (% Unpaired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 3]

[0008] To calculate the % relative abundance of the Paired High Mannose Glycan content of the antibody composition Equation 9 was employed:% Paired High Mannose Glycan Content = (% Paired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 9]

[0009] The data is provided below in Table 30.TABLE 30Relative Unpaired and Paired Glycan Content Relative Abundance (%) % Unpaired Afucosylated Glycan Content% Unpaired High Mannose Glycan Content% Paired Afucosylated Glycan Content% Paired High Mannose Glycan Content

[0010] This example demonstrates another exemplary method for determining glycan pairing content for an antibody composition.EXAMPLE 17

[0011] This example describes an exemplary method of simplifying the structures of high mannose glycans and quantifying relative unpaired high mannose glycan content / relative paired high mannose glycan content for an antibody composition comprising an antibody comprising Man3.

[0012] Man3 is a glycan comprising three mannosyl residues: a first mannosyl residue linked to a core GIcNac and two mannosyl residues branching from the first mannosyl residue, one which is linked by an al-3 linkage and the other which is linked by an al-6 linkage. In the previous studies described in Examples 13-16, one or more mannosidases were used to remove one or more mannosyl residues from Man5, Man6, Man7, Man8, and / or Man9 glycans to yield a simplified digested glycan structure comprising one, three, or five mannosyl residues depending on the mannosidase(s) used for digestion. In each of these studies, the antibodies of the antibody compositions were known to have undetectable levels of Man3 by HILIC. In this example, an antibody comprising detectable levels of Man3 (as ascertained by HILIC) is used and glycan pairing content is determined by carrying out the method described in Example 15 without the digestion with the enzyme IgdE. Briefly, samples of the antibody composition comprising an IgG with Man3 are used in a multi-enzyme digestion to simplify the heterogenous high mannose glycopair population. The samples are first treated with EndoS (IgGZERO® from Genovis Inc.,) for 30 min. Samples are then treated with al-2 mannosidase (from Agilent Technologies) at 37°C for 24 hours. The al-2 mannosidase is highly specific for cleaving just al-2 mannose linkages of terminal mannose residues and yields Man5 upon cleavage of Man9, Man8, Man7 and Man6. Man 3 would not be cleaved by al-2 mannosidase given the lack of the al-2 linkages and would remain in its original form.

[0013] Following the multi-enzyme digestion, the sample comprising glycosylated Fc and Fab fragments are chromatographically separated by RP-LC with online MS detection. The chromatographic peak comprising the Fc fragments is selected for data analysis. The mass spectra ofthe one Fc containing chromatographic peak is extracted and deconvoluted as previously described in Examples 1 and 13. Glycopairs are identified by an automated, computer-assisted matching of the MW of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs. As the MW of Man5 differs from the MW of Man3, and thus, glycopairs comprising Man3 glycans are distinguishable from the glycopairs comprising a Man5 digested glycan structure.

[0014] The abundance of the individual glycopairs is determined from the relative intensities or areas of the identified deconvoluted mass spectral peaks as follows:% Glycopair Intensity = Deconvoluted Peak Intensity / Total Deconvoluted Peak Intensity x 100[Equation 1]

[0015] The relative abundances of the simplified glycopairs are recorded in Table 31.TABLE 31Detected EndoS+ al-2 Mannosidase %Relative Abundance FC , Man5 / Man5% Paired High Mannose GlycopairsFC , GlcNac / Man5% Unpaired High Mannose Glycopairs

[0016] The relative unpaired and paired high mannose glycan content are calculated using Equation 3 as described in Example 1 and Equation 9. Briefly, the abundance of paired high mannose glycopairs is determined by summing the individual glycopairs categorized as paired high mannose glycopairs, similarly the abundance of unpaired high mannose glycopairs is determined by summing the individual glycopairs categorized as unpaired high mannose glycopairs. To calculate the % relative abundance of the Unpaired High Mannose Glycan content of the antibody composition Equation 3 is employed:% Unpaired High Mannose Glycan Content = (% Unpaired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 3]

[0017] To calculate the % relative abundance of the Paired High Mannose Glycan Content of the antibody composition Equation 9 is employed:% Paired High Mannose Glycan Content = (% Paired High Mannose Glycopairs / Total High Mannose Glycopairs (Unpaired+Paired)) x 100[Equation 9]

[0018] The data is recorded in Table 32.TABLE 32Relative Unpaired and Paired Glycan Content Relative Abundance (%) % Unpaired High Mannose Glycan Content% Paired High Mannose Glycan ContentEXAMPLE 18

[0019] This example describes another method to simplify the structures of high mannose glycans on an intact antibody composition and quantifying relative unpaired high mannose glycan content / relative paired high mannose glycan content for an antibody composition.

[0020] In the previous studies described in Examples 13-16, IgdE is used to create a mixture of Fab fragments and glycosylated Fc fragments comprising a glycan pair. In this method, the digestions are carried out with the sample of an antibody composition without IgdE as depicted in Figure 20.Briefly, the sample of the antibody composition is treated with EndoS (IgGZERO® from Genovis Inc.,) for 30 min and then one or two mannosidases, as essentially described in Examples 13-16. Following digestion with EndoS and the mannosidase(s), samples are chromatographically separated from digestion enzymes using a rapid RP-LC separation or HILIC with MS detection. The mass spectra is extracted and deconvoluted as previously described in Examples 1 and 13. Glycopairs are identified by an automated, computer-assisted matching of the MW of the deconvoluted mass spectral peaks of the antibody composition against a custom-built database of theoretical MWs as provided in Table 33 (when EndoS and al-2,3,6 Mannosidase is used for digestion), Table 34 (when EndoS and al-2 Mannosidase is used for digestion), and / or Table 35 (when EndoS and al-2,3 Mannosidase is used for digestion).TABLE 33Dominant Paired FucosylatedEndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6Glycan Pair MW (Da) Glycopair MW (Da) Mannosidase Glycan PairGlcNac+ Fuc / GlcNac+ Fuc 698.6 146259.1Paired Afucosylated EndoS +EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase al-2,3,6 Mannosidase GlycanGlycan Pair MW (Da) Glycopair MW (Da) PairsGIcNac / GIcNac 406.4 145966.8 2GlcNac+ Man / GIcNac 771.8 146332.2 Unpaired Afucosylated EndoSEndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase + al-2,3,6 MannosidaseGlycan Pair MW (Da) Glycopair MW (Da)Glycan PairsGIcNac / GIcNac + Fuc 552.5 146112.9 Paired High Mannose EndoS +EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase al-2,3,6 Mannosidase GlycanGlycan Pair MW (Da) Glycopair MW (Da) Pairs2GlcNac + Man / 2GlcNac + Man 1137.2 146697.6 Unpaired High MannoseEndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6 Mannosidase EndoS + al-2,3,6Glycan Pair MW (Da) Glycopair MW (Da) Mannosidase Glycan Pairs2GlcNac + Man / GIcNac + Fuc 917.9 146478.3 2GlcNac+ Man / GIcNac 771.8 146332.2TABLE 34Dominant Paired FucosylatedEndoS + al-2 Mannosidase Glycan EndoS + al-2 Mannosidase EndoS + al-2 MannosidasePair MW (Da) Glycopair MW (Da) Glycan PairGlcNac+ Fuc / GlcNac+ Fuc 698.6 146259.1 Paired Afucosylated EndoS +EndoS + al-2 Mannosidase Glycan EndoS + al-2 Mannosidase al-2 Mannosidase GlycanPair MW (Da) Glycopair MW (Da) PairsGIcNac / GIcNac 406.4 145966.8 Man5 / GlcNac 1420.3 146980.7 Unpaired Afucosylated EndoSEndoS + al-2 Mannosidase Glycan EndoS + al-2 Mannosidase + al-2 Mannosidase GlycanPair MW (Da) Glycopair MW (Da) PairsGIcNac / GIcNac + Fuc 552.5 146112.9 Paired High Mannose EndoS +EndoS + al-2 Mannosidase Glycan EndoS + al-2 Mannosidase al-2 Mannosidase GlycanPair MW (Da) Glycopair MW (Da) PairsMan5 / Man5 2434.2 147994.6 Unpaired High MannoseEndoS + al-2 Mannosidase Glycan EndoS + al-2 Mannosidase EndoS + al-2 MannosidasePair MW (Da) Glycopair MW (Da) Glycan PairsMan5 / GlcNac + Fuc 1566.4 147126.8 Man5 / GlcNac 1420.3 146980.7TABLE 35Dominant Paired FucosylatedEndoS + al-2, 3 Mannosidase Glycan EndoS + al-2, 3 Mannosidase EndoS + al-2, 3 MannosidasePair MW (Da) Glycopair MW (Da) Glycan PairGlcNac+ Fuc / GlcNac+ Fuc 698.6 146259.1 Paired Afucosylated EndoS +EndoS + al-2, 3 Mannosidase Glycan EndoS + al-2, 3 Mannosidase al-2, 3 Mannosidase GlycanPair MW (Da) Glycopair MW (Da)PairsGIcNac / GIcNac 406.4 145966.8 2GlcNac + 3Man / GlcNac 1096.2 146656.6 Unpaired Afucosylated EndoSEndoS + al-2,3 Mannosidase Glycan EndoS + al-2,3 Mannosidase + al-2,3 Mannosidase GlycanPair MW (Da) Glycopair MW (Da) PairsGIcNac / GIcNac + Fuc 552.5 146112.9Paired High Mannose EndoS +EndoS + al-2,3 Mannosidase Glycan EndoS + al-2,3 Mannosidase al-2,3 Mannosidase GlycanPair MW (Da) Glycopair MW (Da) Pairs2GlcNac + 3Man / 2GlcNac +1786.0 147346.43ManUnpaired High MannoseEndoS + al-2,3 Mannosidase Glycan EndoS + al-2,3 Mannosidase EndoS + al-2,3 MannosidasePair MW (Da) Glycopair MW (Da) Glycan Pairs2GlcNac + 3Man / GlcNac + Fuc 1242.3 146802.7 2GlcNac + 3Man / GlcNac 1096.2 146656.6

[0021] The abundance of paired high mannose glycopairs is determined by summing the individual glycopairs categorized as paired and unpaired high mannose glycopairs, as described in Examples 1 and 13.

[0022] This example describes an exemplary method of determining glycan pairing content of a protein composition, wherein an IgdE is not used in the multi-enzyme digestion of the sample of the protein composition prior to separation and quantification.EXAMPLE 19

[0023] This example describes utilizing the methods described in Example 13-17 to analyze the relationships of ADCC activity to % released high mannose glycans, % released afucosylated glycans, % unpaired high mannose glycans and % unpaired afucosylated glycans for an antibody composition to support clone selection.

[0024] Briefly, the % released high mannose glycans and % released afucosylated glycans are determined by the method described in Example 4. The ADCC activity levels are measured as described in Example 3. The % unpaired high mannose glycans and % unpaired afucosylated glycans for an antibody composition is determined using the methods described in Example 13-17. The statistical relationships between the ADCC activity to % released high mannose glycans and % released afucosylated glycans is determined using the JMP suite of computer programs for statistical analysis (SAS Institute, Cary, NC), similar to the statistical analyses described in Example 5. Finally, the % unpaired high mannose glycans and % unpaired afucosylated glycan content are leveraged toapply a correction to the released high mannose glycan data and the released afucosylated glycan data as described in Example 5.

[0025] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0026] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms including the indicated component(s) but not excluding other elements (i.e., meaning "including, but not limited to,") unless otherwise noted.

[0027] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0028] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation 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.

[0029] Preferred embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein.Accordingly, this disclosure includes suitable modifications and equivalents of the subject matter recited in the claims appended hereto, as would be appreciated by those of ordinary skill in the art in the context of this disclosure. Moreover, any combination of the herein-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A method of determining the glycan pairing content of a protein composition comprising a protein comprising an Fc region comprising a glycan pair, the method comprising:a. treating a sample of the protein composition with (i) a mannosidase and (ii) a pi-4 endoglycosidase, to produce a mixture of components of the protein, wherein at least some of the components comprises one or more digested glycan structures; and b. separating the components of the mixture based on molecular weight; andc. quantifying the abundance of glycan pairs of the mixture to determine the glycan pairing content of the protein composition.

2. The method of claim 1, wherein the mannosidase cleaves an al-2 linkage, al-3 linkage, and / or al-6 linkage of a high mannose glycan.

3. The method of any one of the preceding claims, wherein the mannosidase is an al-2, 3, 6 mannosidase.

4. The method of any one of the preceding claims, wherein the mannosidase is an al-6 mannosidase.

5. The method of any one of the preceding claims, wherein the mannosidase is an al-2 mannosidase.

6. The method of any one of the preceding claims, wherein the mannosidase is an al-2, 3 mannosidase.

7. The method of any one of the preceding claims, comprising treating the sample with two mannosidases.

8. The method of claim 7, comprising treating the sample with an al-2, 3, 6 mannosidase and an al-6 mannosidase.

9. The method of claim 7 or 8, comprising treating the sample with each mannosidase for less than 4 hours, optionally, less than 3 hours.

10. The method of claim 9, comprising treating the sample with each mannosidase for more than 1 hour.

11. The method of any one of claims 7-9, comprising treating the sample with each mannosidase for about 2 hours.

12. The method of any one of claims 1-6, comprising treating the sample with a single mannosidase.

13. The method of claim 12, wherein the mannosidase is an al-2 mannosidase.

14. The method of claim 12, wherein the mannosidase is an al-2, 3 mannosidase.

15. The method of any one of claims 12-14, comprising treating the sample with the mannosidase for less than 30 hours, optionally, about 24 hours.

16. The method of any one of the preceding claims, wherein none of the digested glycan structures of the components in the mixture produced in (a) comprises six or more mannosyl residues.

17. The method of any one of the preceding claims, wherein the digested glycan structures of the components in the mixture produced in (a) comprise less than 6 mannosyl residues.

18. The method of claim 17, wherein the digested glycan structures of thecomponents in the mixture produced in (a) comprise 5 or fewer mannosyl residues.

19. The method of claim 18, wherein the digested glycan structures of the components in the mixture produced in (a) comprise zero, one, three or five mannosyl residues.

20. The method of claim 19, wherein the digested glycan structures of the components in the mixture produced in (a) comprises zero or one mannosyl residues.

21. The method of claim 19, wherein the digested glycan structures of the components in the mixture produced in (a) comprises zero or three mannosyl residues.

22. The method of claim 19, wherein the digested glycan structures of the components in the mixture produced in (a) comprises zero or five mannosyl residues.

23. The method of any one of claims 16 to 22, wherein, when a digested glycan structure comprises one, three, or five mannosyl residues, the digested glycan structure further comprises two core N-acetylglucosamine (GalNac) residues.

24. The method of any one of claims 16 to 22, wherein, when a digested glycan structure comprises zero mannosyl residues, the digested glycan structure further comprises only one core GIcNac residue.

25. The method of any one of the preceding claims, wherein the sample is treated with one or more mannosidases after the sample is treated with the pi-4 endoglycosidase.

26. The method of any one of the preceding claims, wherein the pi-4 endoglycosidase is an Endo S endoglycosidase.

27. The method of any one of the preceding claims, comprising treating the sample with the pi- 4 endoglycosidase for less than 1 hour.

28. The method of claim 27, comprising treating the sample with the pi-4 endoglycosidase for about 30 minutes.

29. The method of any one of the preceding claims, wherein separating the components of the mixture based on molecular weight comprises performing mass spectrometry to obtain one or more mass spectral peaks.

30. The method of claim 29, comprising deconvoluting the mass spectral peaks to obtain deconvoluted mass spectral peaks.

31. The method of claim 30, comprising matching the molecular weight of each deconvoluted mass spectral peak against a database of glycan pairs and the associated molecular weight to identify the glycan pair.

32. The method of any one of the preceding claims, wherein the separating of (b) and the quantifying of (c) occurs in less than 2 hours.

33. The method of claim 32, wherein (b) and (c) occur in less than 75 minutes.

34. The method of claim 33, wherein (b) and (c) occur in less than 60 minutes.

35. The method of any one of the preceding claims, wherein the protein of the protein composition comprises an antibody comprising the Fc region or is an antibody comprising the Fc region.

36. The method of claim 35, further comprising treating the sample with (iii) an enzyme that cleaves each antibody into a Fab fragment and Fc fragment.

37. The method of claim 36, wherein the enzyme is a cysteine protease.

38. The method of claim 37, wherein the cysteine protease cleaves at a site between Thr and His or between Lys and Thr of the sequence KTHTCPP (SEQ ID NO: 1) of an IgGl antibody heavy chain.

39. The method of claim 37 or 38, comprising treating the sample with the cysteine protease for at least 8 hours or at least 12 hours.

40. The method of any one of claims 36-40, comprising chromatographically separating Fab fragments from Fc fragments.

41. The method of claim 40, wherein chromatographically separating comprises a reversed phase liquid chromatography (RP-LC).

42. The method of claim 40 or 41, comprising performing mass spectrometry on the Fc fragment-containing chromatographic fraction to obtain one or more mass spectral peaks.

43. The method of claim 42, comprising deconvoluting the mass spectral peaks to obtain deconvoluted mass spectral peaks.

44. The method of claim 43, comprising matching the molecular weight of each deconvoluted mass spectral peak against a database of glycan pairs and the associated molecular weight to identify the glycan pair.

45. A method of determining the glycan pairing content of a protein composition comprising a protein comprising an antibody, the method comprisinga. treating a sample of the protein composition with (i) an enzyme that cleaves an antibody heavy chain at a site N-terminal to the hinge region disulfide linkage, (ii) a mannosidase, and (iii) a pi-4 endoglycosidase, to produce a mixture of Fab fragments and Fc fragments of the protein, wherein at least some of the Fc fragments comprise one or more digested glycan structures;b. chromatographically separating the Fab fragments from the Fc fragmentsc. separating Fc fragments based on molecular weight, andd. quantifying the abundance of glycan pairs to determine the glycan pairing content of the protein composition.

46. The method of any one of the preceding claims, comprising quantifying thei. paired afucosylated glycan content,ii. unpaired afucosylated glycan content,iii. paired high mannose glycan content, and / oriv. unpaired high mannose glycan content.

47. The method of any one of the preceding claims, wherein the method is a part of a manufacturing process for the protein composition.

48. The method of any one of the preceding claims, wherein the method is performed in real time during manufacture of the protein composition.

49. The method of any one of the preceding claims, wherein the sample is a sample of in- process material.

50. The method of any one of the preceding claims, wherein the glycan pairing content is determined pre-harvest or post-harvest.

51. The method of claim 50, wherein the glycan pairing content is determined post-harvest.

52. The method of any one of the preceding claims, wherein the sample is obtained from a manufacturing lot.

53. A method of analyzing a protein composition, comprising determining the glycan pairing content of a sample of the protein composition in accordance with a method of any one of claims 1 to 52, and, optionally, comparing the glycan pairing content to the glycan pairing content of a reference product.

54. A method of monitoring production of a protein composition, comprising determining the glycan pairing content of a protein composition in accordance with a method of any one of claims 1 to 52, for a first sample obtained at a first timepoint and for a second sample taken at a second timepoint which is different from the first timepoint, and, optionally, comparingthe glycan pairing content of the first sample to the glycan pairing content of the second sample.

55. The method of claim 54, wherein each of the first sample and second sample is a sample of in-process material.

56. The method of claim 55, wherein the first sample is a sample of in-process material and the second sample is a sample of a manufacturing lot.

57. The method of claim 55, wherein the first sample is a sample obtained before one or more conditions of the cell culture are modified and the second sample is a sample obtained after the one or more conditions of the cell culture are modified.

58. A method of producing a protein composition, comprising (A) determining glycan pairing content of a sample of the protein composition in accordance with a method of any one of claims 1-52, wherein the sample is a sample of in-process material, wherein, when the glycan pairing content is determined as outside a target range, the method further comprises (B) modifying one or more conditions of the cell culture to obtain a modified cell culture and determining the glycan pairing content, and optionally, repeating (A) and (B) until the glycan pairing content is within the target range.

59. The method of claim 58, wherein one or more conditions of the cell culture are modified to primarily change the relative unpaired HM glycan content to achieve the target range of glycan pairing content.

60. The method of claim 59, wherein one or more conditions of the cell culture are modified to primarily change the relative unpaired AF glycan content to achieve the target range of glycan pairing content.

61. A method of selecting a cell line or cell clone for a protein composition manufacture process, comprising determining the glycan pairing content of a protein composition in accordance with a method of any one of claims 1 to 52, for candidate cell lines or cell clones producing the protein of the protein composition, and identifying a cell line or cell clone that have a glycan pairing content within a preselected target range, wherein the identified cell line or cell clone is selected for producing the protein in the manufacture process.

62. The method of claim 61, wherein the preselected target range is based on the range of relative unpaired afucosylated glycan content and / or the range of relative unpaired high mannose glycan content of a reference cell line or cell clone.

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