Automated high-throughput buffer exchange platform enhances rapid flow analysis of antibody drug conjugates by high resolution mass spectrometry.

Antibody drug conjugates (ADCs) are an increasingly important therapeutic class of molecules for the treatment of cancer. Average drug-to-antibody ratio (DAR) and drug-load distribution are critical quality attributes of ADCs with the potential to impact efficacy and toxicity of the molecule and need to be analytically characterized and understood. Several platform methods including hydrophobic interaction chromatography (HIC) and native size-exclusion chromatography-mass spectrometry (nSEC-MS) have been developed for that purpose; however, each presents some limitations. In this work, we assessed a new sample preparation and buffer exchange platform coupled with high-resolution mass spectrometry for characterizing the drug-load and distribution of several cysteine-linked ADCs conjugated with a variety of chemotypes. Several criteria were evaluated during the optimization of the buffer exchange-mass spectrometry system performance and the data generated with the system were compared with results from nSEC-MS and HIC. The results indicated that the platform enables automated and high throughput quantitative DAR characterization for antibody-drug conjugates with high reproducibility and offers several key advantages over existing approaches that are used for chemotype-agnostic ADC characterization.

Structural characterization of a monomethylauristatin-E based ADC that contains 8 drugs conjugated at interchain cysteine residues.

Antibody-drug conjugates (ADCs) with a drug-to-antibody ratio (DAR) of 8 are attractive as therapeutic anti-cancer agents due to the higher levels of cytotoxic payload delivered to tumors. Biophysical characterization of a DAR 8 ADC fully conjugated at all interchain cysteine residues was carried out to determine if IgG1 interchain disulfide reduction and conjugation led to structural perturbations that impacted product stability. Comparisons between the DAR 8 ADC and the unconjugated parent antibody identified minor tertiary and quaternary structural changes localized to the CL, CH1, and CH2 domains and CH2-CH3 domain interface. Stability studies of the DAR 8 ADC indicated that the structural changes had minimal impacts to product stability as demonstrated by low levels of fragmentation and aggregation under nominal storage and temperature stress stability conditions. Additionally, no detectable higher order structural changes were observed by CD or DSC in the DAR 8 ADC after 3 months at (25 °C) stability conditions. The structural and stability results support the developability of DAR 8 ADCs fully conjugated to interchain cysteines residues with an optimized and clinically relevant second generation monomethylauristatin-E (MMAE) drug-linker.

Scientific Best Practices for Primary Sequence Confirmation and Sequence Variant Analysis in the Development of Therapeutic Proteins.

In this commentary, we will provide a high-level introduction into LC-MS product characterization methodologies deployed throughout biopharmaceutical development. The ICH guidelines for early and late phase filings is broad so that it is applicable to diverse biotherapeutic products in the clinic and industry pipelines. This commentary is meant to address areas of protein primary sequence confirmation and sequence variant analysis where ambiguity exists in industry on the specific scope of work that is needed to fulfill the general guidance that is given in sections Q5b and Q6b. This commentary highlights the discussion and outcomes of two recent workshops centering on the application of LC-MS to primary structure confirmation and sequence variant analysis (SVA) that were held at the 2018 and 2019 CASSS Practical Applications of Mass Spectrometry in the Biotechnology Industry Symposia in San Francisco, CA and Chicago, IL, respectively. Recommendations from the conferences fall into two distinct but related areas; 1) consolidation of opinions amongst industry stakeholders on the specific definitions of peptide mapping and peptide sequencing for primary structure confirmation and the technologies used for both, as they relate to regulatory expectations and submissions and 2) development of fit-for-purpose strategy to define appropriate assay controls in SVA experiments.

Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines.

With the advent of highly sensitive technologies such as tandem mass spectrometry and next-generation sequencing, recombinant antibodies are now routinely analyzed for the presence of low-level sequence variants including amino acid misincorporations. During mAb cell culture process development, we found that proline was replaced with the non-canonical amino acid, hydroxyproline, in the protein sequence. We investigated the relationship between proline content in the cell culture media and proline sequence variants and found that the proline concentration was inversely correlated with the amount of sequence variants detected in the protein sequence. Hydroxyproline incorporation has been previously reported in recombinant proteins produced in mammalian expression systems as a post-translational modification. Given the dependency on proline levels, the mechanism was then investigated. To address the possibility of co-translational misincorporation of hydroxyproline, we used tandem mass spectrometry to measure incorporation of stable-isotope labelled hydroxyproline added to the feed of a production bioreactor. We discovered co-translational misincorporation of labelled hydroxyproline in the recombinant antibody. These findings are significant, since they underscore the need to track non-canonical amino acid incorporation as a co-translational event in CHO cells. Understanding the mechanism of hydroxyproline incorporation is crucial in developing an appropriate control strategy during biologics production.

Native size-exclusion chromatography-mass spectrometry: suitability for antibody-drug conjugate drug-to-antibody ratio quantitation across a range of chemotypes and drug-loading levels.

Native size-exclusion chromatography-mass spectrometry (nSEC-MS) is an analytical methodology that is appropriate for accurately quantitating the drug-to-antibody ratio (DAR) on a wide variety of interchain cysteine-linked antibody-drug conjugates (ADCs), irrespective of chemotype. In the current preclinical environment, novel ADCs conjugated with unique drug-linkers need to progress toward the clinic as quickly as possible. Platform analytical approaches can reduce time-to-clinic because key process development and optimization activities can be decoupled from the development of bespoke, molecule-specific analytical methods. In this work, we assessed the potential of nSEC-MS as a platformable, quantitative DAR method. The nSEC-MS method was evaluated according to performance characteristics and parameters described in the ICH guideline Validation of Analytical Procedures: Text and Methodology Q2(R1). In order to comprehensively assess the accuracy and bias of nSEC-MS DAR quantitation, ADCs were generated using three different drug-linker chemotypes with DARs ranging from 2 to 8. These molecules were tested by hydrophobic interaction chromatography (HIC) and nSEC-MS, and DARs obtained from both methods were compared to assess the degree to which nSEC-MS quantitation aligned with the HIC release assay. Our results indicated that there is no bias introduced by nSEC-MS quantitation of DAR and that SEC-MS data can be bridged to HIC data without the need for a correction factor or offset. nSEC-MS was also found to be suitable for unbiased DAR quantitation in the other ADC chemotypes that were evaluated. Based on the totality of our work, we conclude that, used as intended, nSEC-MS is well suited for quantitating DAR on a variety of interchain cysteine-linked ADCs in an accurate, unbiased manner.

Control of Antibody Impurities Induced by Riboflavin in Culture Media During Production.

During the manufacturing of protein biologics, product variability during cell culture production and harvest needs to be actively controlled and monitored to maintain acceptable product quality. To a large degree, variants that have previously been described are covalent in nature and are easily analyzed by a variety of techniques. Here, we describe a noncovalent post translational modification of recombinantly expressed antibodies, containing variable domain tryptophans, that are exposed to culture media components and ambient laboratory light. The modified species, designated as conformer, can be monitored by hydrophobic interaction chromatography and often exhibits reduced potency. We studied conformer formation and identified key elements driving its accelerated growth using an IgG2 monoclonal antibody. Conformer is a result of a noncovalent interaction of the antibody with riboflavin, an essential vitamin added to many production cell culture formulations. Chemical and physical factors that influence the impact of riboflavin are identified, and methods for process control of this product quality attribute are addressed in order to prevent loss of antibody potency and potential safety issues. Identifying therapeutic antibody drug candidates with the potential to form conformers can be performed early in development to avoid this undesirable product quality propensity.

Biopharmaceutical Industry Practices for Sequence Variant Analyses of Recombinant Protein Therapeutics.

The application of advanced methodologies such as next-generation sequencing (NGS) and mass spectrometry (MS) to the characterization of cell lines and recombinant proteins has enabled the highly sensitive detection of sequence variants (SVs). However, although these approaches can be leveraged to provide deep insight into product microheterogeneity caused by SVs, they are not used in a standardized manner across the industry. Currently, there is little clarity and consensus on the utilization, timing, and significance of SV findings. This white paper addresses the current practices, logistics, and strategies for the analysis of SVs using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences including approaches for detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback. Although SVs are a potential issue for all recombinant protein therapeutics, the scope of this discussion will be limited to SVs produced in mammalian cells. Ultimately, it is our hope that the findings from the survey and deliberations of the committee are useful to decision makers in industry and positions them to respond to findings of SVs in recombinant proteins that are destined for clinical or commercial use in a strategic manner.LAY ABSTRACT: This white paper addresses the current practices, logistics, and strategies for the analysis of amino acid sequence variants using a benchmarking survey coordinated by the International Consortium for Innovation & Quality in Pharmaceutical Development (IQ) as well as a series of deliberations among a panel of experts assembled from across the biopharmaceutical industry. Discussion includes current industry experiences regarding detection and quantitation of SVs during cell-line and process development, risk assessments, and regulatory feedback.

Characterization of SGN-CD123A, A Potent CD123-Directed Antibody-Drug Conjugate for Acute Myeloid Leukemia.

Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody-drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro, SGN-CD123A-mediated potent cytotoxicity of 11/12 CD123+ AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo, SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554-64. ©2017 AACR.

ELISA reagent coverage evaluation by affinity purification tandem mass spectrometry.

Host cell proteins (HCPs) must be adequately removed from recombinant therapeutics by downstream processing to ensure patient safety, product quality, and regulatory compliance. HCP process clearance is typically monitored by enzyme-linked immunosorbent assay (ELISA) using a polyclonal reagent. Recently, mass spectrometry (MS) has been used to identify specific HCP process impurities and monitor their clearance. Despite this capability, ELISA remains the preferred analytical approach due to its simplicity and throughput. There are, however, inherent difficulties reconciling the protein-centric results of MS characterization with ELISA, or providing assurance that ELISA has acceptable coverage against all process-specific HCP impurities that could pose safety or efficacy risks. Here, we describe efficient determination of ELISA reagent coverage by proteomic analysis following affinity purification with a polyclonal anti-HCP reagent (AP-MS). The resulting HCP identifications can be compared with the actual downstream process impurities for a given process to enable a highly focused assessment of ELISA reagent suitability. We illustrate the utility of this approach by performing coverage evaluation of an anti-HCP polyclonal against both an HCP immunogen and the downstream HCP impurities identified in a therapeutic monoclonal antibody after Protein A purification. The overall goal is to strategically implement affinity-based mass spectrometry as part of a holistic framework for evaluating HCP process clearance, ELISA reagent coverage, and process clearance risks. We envision coverage analysis by AP-MS will further enable a framework for HCP impurity analysis driven by characterization of actual product-specific process impurities, complimenting analytical methods centered on consideration of the total host cell proteome.

Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting.

Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains. Our results for two case studies are shown here. The first study was aimed at defining the conformational changes of mAbs induced by deglycosylation. We found that two residues in CH2 domain (D268 and E297) show significantly enhanced side chain accessibility upon deglycosylation. This site-specific result highlighted the advantage of monitoring the labeling kinetics at the amino acid level as opposed to the peptide level, which would result in averaging out of highly localized conformational differences. The second study was designed to assess conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules.

Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation.

Characterization of both the acidic and basic regions of imaged capillary isoelectric focusing (icIEF) profile of an IgG1 antibody was achieved through preparative immobilized pH gradient isoelectric focusing (IPG-IEF) fractionation. Recent attempts at using this method to fractionate charge variants of monoclonal antibodies (mAbs) have shown promising results, but identification of the chemical modifications in the variants was limited to the basic species. We have optimized the method to achieve enrichment of each variant across the icIEF profile of an IgG1 mAb. The fractionation was followed by extended characterization to elucidate the composition of the acidic, main, and basic species observed in the icIEF profile. Deamidation, sialylation, glycation, and fragmentation were identified as the main modifications contributing to acidic variants of the mAb while C-terminal lysine, C-terminal proline amidation, and uncyclized N-terminal glutamine were the major species contributing to the basic variants. This characterization allows a better understanding of the modifications that contribute to the charge variants observed by icIEF, facilitating the evaluation of impacts on product safety and efficacy.

Antibody structural integrity of site-specific antibody-drug conjugates investigated by hydrogen/deuterium exchange mass spectrometry.

We present the results of a hydrogen/deuterium exchange mass spectrometric (HDX-MS) investigation of an antibody-drug conjugate (ADC) comprised of drug-linkers conjugated to cysteine residues that have been engineered into heavy chain (HC) fragment crystallizable (Fc) domain at position 239. A side-by-side comparison of the HC Ser239 wild type (wt) monoclonal antibody (mAb) and the engineered Cys239 mAb indicates that site directed mutagenesis of Ser239 to cysteine has no impact on the HDX kinetics of the mAb. According to the crystal structure of a homologous immunoglobulin G1 (IgG1) antibody (PDB: 1HZH ), the backbone amide of Ser239 is hydrogen-bonded to Val264 backbone amide in the wt-mAb studied here. Replacing Ser239 with a Cys residue does not alter the exchange kinetics of the backbone amide of Val264 suggesting that either Ser or Cys at position 239 has similar amide-hydrogen bonding with Val264. However, a small segment in CH2 domain of the ADC ((264)VDVS) was found to have a slightly increased HDX rate compared to the wt- and C239-mAb constructs. The slightly increased HDX rate of the segment (264)VDVS in ADCs indicates that the further modification of Cys239 with drug-linkers only attenuates the local backbone amide hydrogen-bonding network between Cys239 and Val264. All other regions which are proximal to the site of drug conjugation are unaffected. The results demonstrate that the site-specific drug conjugation at the engineered Cys residue at the position 239 of HC does not impact the structural integrity of antibodies. The results also highlight the utility of applying HDX-MS to ADCs to gain a molecular level insight into the impact of site-specific conjugation technologies on the higher-order structure (HOS) of mAbs. The methodology can be applied generally to site-specific ADC modalities to understand the individual contributions of site-mutagenesis and drug-linker conjugation on the HOS of therapeutic candidate ADCs.

Solid-state mAbs and ADCs subjected to heat-stress stability conditions can be covalently modified with buffer and excipient molecules.

We report that a unique type of chemical modification occurs on lyophilized proteins. Freeze-dried mAbs and antibody-drug conjugates (ADCs) can be covalently modified with buffer and excipient molecules on the side chains of Glu, Asp, Thr, and Ser amino acids when subjected to temperature stress. The reaction occurs primarily via condensation of common buffers and excipients such as histidine, tris, trehalose and sucrose, with Glu and Asp carboxylates in the primary sequence of proteins. The reaction was also found to proceed through condensation of carboxylate containing buffers such as citrate, with Thr and Ser hydroxyls in the primary sequence of proteins. Based on the mass of the covalent adducts observed on mAbs and ADCs, it is apparent that the reaction produces water as a product and is thus favored in a low moisture environments such as a lyophilized protein cake. Herein, we present the evidence for the covalent modification of proteins drawn from case studies of in-depth characterization of heat-stressed mAbs and ADCs in the solid state. We also demonstrate how common charge variant assays such as imaged capillary isoelectric focusing and mass spectrometry can be used to monitor this specific class of protein modification.

Approaches to Interchain Cysteine-Linked ADC Characterization by Mass Spectrometry.

Therapeutic antibody-drug conjugates (ADCs) harness the cell-killing potential of cytotoxic agents and the tumor targeting specificity of monoclonal antibodies to selectively kill tumor cells. Recent years have witnessed the development of several promising modalities that follow the same basic principles of ADC based therapies but which employ unique cytotoxic agents and conjugation strategies in order to realize therapeutic benefit. The complexity and heterogeneity of ADCs present a challenge to some of the conventional analytical methods that industry has relied upon for biologics characterization. This current review will highlight some of the more recent methodological approaches in mass spectrometry that have bridged the gap that is created when conventional analytical techniques provide an incomplete picture of ADC product quality. Specifically, we will discuss mass spectrometric approaches that preserve and/or capture information about the native structure of ADCs and provide unique insights into the higher order structure (HOS) of these therapeutic molecules.

Measurement of in vivo drug load distribution of cysteine-linked antibody-drug conjugates using microscale liquid chromatography mass spectrometry.

Analysis of samples containing intact antibody-drug conjugates (ADC) using mass spectrometry provides a direct measurement of the drug-load distribution. Once dosed, the drug load distribution changes due to a combination of biological and chemical factors. Liquid chromatography-mass spectrometry (LC-MS) methods to measure the in vivo drug load distribution have been established for ADCs containing native disulfide bonds (lysine-linked or cysteine-linked). However, because of an IgG reduction step in conjugation processes, using LC-MS to analyze intact cysteine-linked ADCs requires native conditions, thus limiting sensitivity. While this limitation has been overcome at the analytical scale, to date, these methods have not been translated to a smaller scale that is required for animal or clinical doses/sampling. In this manuscript, we describe the development of ADC specific affinity capture reagents for processing in vivo samples and optimization of native LC-MS methods at a microscale. These methods are then used to detect the changing drug load distribution over time from a set of in vivo samples, representing to our knowledge the first native mass spectra of cysteine-linked ADCs from an in vivo source.

Conformation and dynamics of interchain cysteine-linked antibody-drug conjugates as revealed by hydrogen/deuterium exchange mass spectrometry.

Antibody-drug conjugates (ADCs) are protein therapeutics in which a target specific monoclonal antibody (mAb) is conjugated with drug molecules. The manufacturing of ADCs involves additional conjugation steps, which are carried out on the parent mAbs, and it is important to evaluate how the drug conjugation process impacts the conformation and dynamics of the mAb. Here, we present a comparative study of interchain cysteine linked IgG1 ADCs and the corresponding mAb by hydrogen/deuterium exchange mass spectrometry (HDX-MS). We found that ∼90% of the primary sequence of the ADC conjugated with either monomethyl auristatin E or F (vcMMAE/mcMMAF) displayed the same HDX kinetics as the mAb, indicating the ADCs and mAbs share very similar conformation and dynamics in solution. Minor increases in HDX kinetic rates were observed in two Fc regions in the ADCs relative to the mAb which indicated that both regions become more structurally dynamic and/or more solvent-accessible in the ADCs. The findings led to a subsequent inquiry into whether the local conformational changes were due to the presence of drugs on the interchain cysteine residues or the absence of intact interchain disulfides or both. To address this question, a side-by-side HDX comparison of ADCs, mAbs, reduced mAbs (containing 8 reduced interchain cysteine thiols), and partially reduced mAbs (conjugation process intermediate) was performed. Our results indicated that the slight increase in conformational dynamics detected at the two regions in the ADCs was due to the absence of intact interchain disulfide bonds and not the presence of vcMMAE or mcMMAF on the alkylated interchain cysteine residues. These results highlight the utility of HDX-MS for interrogating the higher-order structure of ADCs and other protein therapeutics.

Native intact mass determination of antibodies conjugated with monomethyl Auristatin E and F at interchain cysteine residues.

We present here a method for the rapid determination of the intact mass of noncovalently associated antibody heavy chains (HC) and light chains (LC) which result from the attachment of drug conjugates to interchain cysteine residues. By analyzing the antibody-drug conjugate (ADC) using native desalting conditions, we maintain the intact bivalent structure of the ADC, which ordinarily would decompose as a consequence of denaturing chromatographic conditions typically used for liquid chromatographic-mass spectrometric (LC-MS) analysis. The mass of the desalted ADC is subsequently determined using standard desolvation and ionization conditions. Methods presented previously in the literature for analyzing interchain cysteinyl-linked ADCs are either not amenable to online mass spectrometry or result in the denaturing dissociation of conjugated HC and LC during chromatographic separation and subsequent mass measurement. We have avoided this outcome with our method and have successfully and routinely obtained intact mass measurement of IgG1 mAbs conjugated with maleimidocaproyl-monomethyl Auristatin F (mcMMAF) and valine-citrulline-monomethyl Auristatin E (vcMMAE) at interchain cysteine residues. Our results thus represent the first reported direct measurement of the intact mass of an ADC conjugated at interchain cysteine residues.

Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics.

Novel acetone and aldimine covalent adducts were identified on the N-termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled (13)C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified-free glycine and found to correspond to acetone linked to the N-terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from (13)C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO(2) through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers.

Resolving disulfide structural isoforms of IgG2 monoclonal antibodies by ion mobility mass spectrometry.

Recombinant monoclonal antibodies are an important class of therapeutic agents that have found widespread use for the treatment of many human diseases. Here, we have examined the utility of ion mobility mass spectrometry (IMMS) for the rapid characterization of disulfide variants in intact IgG2 monoclonal antibodies. It is shown that IMMS reveals 2 to 3 gas-phase conformer populations for IgG2s. In contrast, a single gas-phase conformer is revealed using IMMS for both an IgG1 antibody and a Cys-232 --> Ser mutant IgG2, both of which are homogeneous with respect to disulfide bonding. This provides strong evidence that the observed IgG2 gas-phase conformers are related to disulfide bond heterogeneity. Additionally, IMMS analysis of redox enriched disulfide isoforms allows assignment of the mobility peaks to established disulfide bonding patterns. These data clearly illustrate how IMMS can be used to quickly provide information on the higher order structure of antibody therapeutics.

Glutamine-linked and non-consensus asparagine-linked oligosaccharides present in human recombinant antibodies define novel protein glycosylation motifs.

We report the presence of oligosaccharide structures on a glutamine residue present in the V(L) domain sequence of a recombinant human IgG2 molecule. Residue Gln-106, present in the QGT sequence following the rule of an asparagine-linked consensus motif, was modified with biantennary fucosylated oligosaccharide structures. In addition to the glycosylated glutamine, analysis of a lectin-enriched antibody population showed that 4 asparagine residues: heavy chain Asn-162, Asn-360, and light chain Asn-164, both of which are present in the IgG1 and IgG2 constant domain sequences, and Asn-35, which was present in CDR(L)1, were also modified with oligosaccharide structures at low levels. The primary sequences around these modified residues do not adhere to the N-linked consensus sequon, NX(S/T). Modeling of these residues from known antibody crystal structures and sequence homology comparison indicates that non-consensus glycosylation occurs on Asn residues in the context of a reverse consensus motif (S/T)XN located on highly flexile turns within 3 residues of a conformational change. Taken together our results indicate that protein glycosylation is governed by more diversified requirements than previously appreciated.