Targeted CQA analytical control strategy for commercial antibody products: Replacing ion-exchange chromatography methods for charge heterogeneity with multi-attribute monitoring.

Peptide mapping with mass spectrometry (MS) is an important tool for protein characterization in the biopharmaceutical industry. Historically, peptide mapping monitors post-translational modifications (PTMs) of protein products and process intermediates during development. Multi-attribute monitoring (MAM) methods have been used previously in commercial release and stability testing panels to ensure control of selected critical quality attributes (CQAs). Our goal is to use MAM methods as part of an overall analytical testing strategy specifically focused on CQAs, while removing or replacing historical separation methods that do not effectively distinguish CQAs from non-CQAs due to co-elution. For example, in this study, we developed a strategy to replace a profile-based ion-exchange chromatography (IEC) method using a MAM method in combination with traditional purity methods to ensure control of charge variant CQAs for a commercial antibody (mAb) drug product (DP). To support this change in commercial testing strategy, the charge variant CQAs were identified and characterized during development by high-resolution LC-MS and LC-MS/MS. The charge variant CQAs included PTMs, high molecular weight species, and low molecular weight species. Thus, removal of the IEC method from the DP specification was achieved using a validated LC-MS MAM method on a QDa system to directly measure the charge variant PTM CQAs in combination with size exclusion chromatography (SE-HPLC) and capillary electrophoresis (CE-SDS) to measure the non-PTM charge variant CQAs. Bridging data between the MAM, IEC, and SE-HPLC methods were included in the commercial marketing application to justify removing IEC from the DP specification. We have also used this MAM method as a test for identity to reduce the number of QC assays. This strategy has received approvals from several health authorities.

ID-MAM: A Validated Identity and Multi-Attribute Monitoring Method for Commercial Release and Stability Testing of a Bispecific Antibody.

Post-translational modifications (PTMs) that impact the safety or efficacy of protein therapeutics are critical quality attributes (CQAs) that need to be controlled to ensure product quality. Peptide mapping with online mass spectrometry (MS) is a powerful tool that has been used for many years to monitor PTM CQAs during product development. However, operating peptide mapping methods with high-resolution mass spectrometers in GMP compliant, commercial quality control (QC) labs can be difficult. Peptide mapping is also required as an identity test in several countries. To address these two different needs, we utilized high-resolution peptide mapping for comprehensive characterization during development and then developed and validated a targeted multi-attribute monitoring (MAM) method using the low-resolution Waters QDa MS system with a fully automated data processing workflow that is suitable for identity (ID) testing, sequence variant control, and CQA quantitation in commercial QC labs. The ID-MAM method was validated for the quantitation of three selected PTM CQAs (CDR isomerization, Fc Met oxidation, and CDR Met oxidation) to ensure control of the oxidation and isomerization degradation pathways of a bispecific antibody (BsAb). This ID-MAM method was successfully validated in six labs (three analytical development and three QC labs) across four countries for commercial release and stability testing of a BsAb. CQA results obtained with the ID-MAM method were similar to results obtained using high-resolution peptide mapping, and the method was robust and reproducible. To our knowledge, this ID-MAM method is the first MS-based peptide mapping method implemented in GMP compliant QC labs for commercial release and stability testing of a biotherapeutic.

High-throughput LC-MS quantitation of cell culture metabolites.

Monitoring cell culture metabolites, including media components and cellular byproducts, during bio manufacturing is critical for gaining insights into cell growth, productivity and product quality. Historically, cell culture metabolite analysis was a complicated process requiring several orthogonal methods to cover the large number of metabolites with diverse properties over wide concentration ranges. These off-line analyses are time consuming and not suitable for real time bioreactor monitoring. In this study, we present a high-throughput LC-MS method with a 17-min cycle time that is capable of simultaneously monitoring 93 cell culture metabolites, including amino acids, nucleic acids, vitamins, sugars and others. This method has high precision and accuracy and has been successfully applied to the daily profiling of bioreactors and raw material qualification. Information obtained in these studies has been used to identify limiting amino acids during production, which guided adjustments to the feed strategy that prevented the potential misincorporation of amino acids. This type of metabolite profiling can be further utilized to build predictive process models for adaptive feedback control and pave the road for continuous manufacturing and real-time release testing.

Using bispecific antibodies in forced degradation studies to analyze the structure-function relationships of symmetrically and asymmetrically modified antibodies.

Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT).

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer's disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

MS(3)-based quantitative proteomics using pulsed-Q dissociation.

RATIONALE: Isobaric tagging reagents, such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ), are high-throughput methods that allow the analysis of multiple samples simultaneously, which reduces instrument time and error. Accuracy and precision of isobaric tags are limited, however, in tandem mass spectrometry (MS/MS) acquisition due to co-isolation and co-fragmentation of neighboring peptide peaks in precursor scans. Here we present a MS(3) method using pulsed-Q dissociation (PQD) in ion trap and Orbitrap instrumentation as a means to improve ratio distortion and maintain high numbers of identified and quantified proteins. METHODS: Mouse brain protein digests were labeled with TMT-128, 129, 130, 131 reagents, mixed in the following molar ratios 1:1:2:5, respectively, and analyzed using HCD-MS(3) and PQD-MS(3) methods. The most intense fragment ion (termed as HCD-MS(3)-top ion or PQD-MS(3)-top ion) or y1 ion (i.e., lysine-TMT tag ion; termed as HCD-MS(3)-y1 or PQD-MS(3)-y1) in collision-induced dissociation (CID) MS/MS was selected for MS(3). RESULTS: Calculated protein ratios obtained in HCD-MS(3)-top ion and PQD-MS(3)-top ion, HCD-MS(3)-y1, and PQD-MS(3)-y1 are accurate and PQD-MS(3) methods resulted in higher numbers of identified and quantified peptide spectral counts and proteins. CONCLUSIONS: PQD-MS(3) methods increase the amount of MS/MS spectra collected and number of quantified proteins and are accessible to those researchers with not only an orbitrap but also an ion trap mass spectrometer.

Sample multiplexing with cysteine-selective approaches: cysDML and cPILOT.

Cysteine-selective proteomics approaches simplify complex protein mixtures and improve the chance of detecting low abundant proteins. It is possible that cysteinyl-peptide/protein enrichment methods could be coupled to isotopic labeling and isobaric tagging methods for quantitative proteomics analyses in as few as two or up to 10 samples, respectively. Here we present two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT). CysDML is a duplex precursor quantification technique that couples cysteinyl-peptide enrichment with on-resin stable-isotope dimethyl labeling. Cysteine-selective cPILOT is a novel 12-plex workflow based on cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues. To demonstrate the broad applicability of the approaches, we applied cysDML and cPILOT methods to liver tissues from an Alzheimer's disease (AD) mouse model and wild-type (WT) controls. From the cysDML experiments, an average of 850 proteins were identified and 594 were quantified, whereas from the cPILOT experiment, 330 and 151 proteins were identified and quantified, respectively. Overall, 2259 unique total proteins were detected from both cysDML and cPILOT experiments. There is tremendous overlap in the proteins identified and quantified between both experiments, and many proteins have AD/WT fold-change values that are within ~20% error. A total of 65 statistically significant proteins are differentially expressed in the liver proteome of AD mice relative to WT. The performance of cysDML and cPILOT are demonstrated and advantages and limitations of using multiple duplex experiments versus a single 12-plex experiment are highlighted.

Global cPILOT analysis of the APP/PS-1 mouse liver proteome.

PURPOSE: A quantitative proteomics strategy called combined precursor isotopic labeling and isobaric tagging (cPILOT) was designed to discover alterations in the amyloid precursor protein/presenilin-1 (APP/PS-1) mouse liver proteome. The multiplexing strategy allows simultaneous quantitation of 12 samples in a single experiment. EXPERIMENTAL DESIGN: For cPILOT samples, six APP/PS-1 and six heterozygous mouse livers were modified using precursor dimethylation (pH 2.5) followed by isobaric tagging (pH 8.0). Samples were pooled, fractioned with strong cation exchange, and analyzed using RPLC-MS(3) for protein identification and relative quantitation. In order to increase proteome coverage, a two-tiered data collection strategy was employed. Six duplex precursor dimethylation experiments were also performed to verify cPILOT protein quantitation. RESULTS: The combination of cPILOT with precursor dimethylation data resulted in 2437 total liver proteins identified and 77 differentially expressed proteins in APP/PS-1 liver. Differentially expressed proteins are involved in metabolic processes such as B-oxidation, pyruvate metabolism, and glucose regulation. CONCLUSIONS AND CLINICAL RELEVANCE: cPILOT expands protein quantitation using isobaric tags and can be applied to any clinical laboratory interested in enhanced multiplexing strategies. Differentially expressed proteins in APP/PS-1 mouse liver suggest the potential use of ketone bodies to alleviate metabolic dysregulation in Alzheimer's disease brain. Our work also suggests alterations in the alanine cycle potentially leading to hyperammonia production, may contribute to Alzheimer's disease pathogenesis.

Global combined precursor isotopic labeling and isobaric tagging (cPILOT) approach with selective MS(3) acquisition.

Recently, we reported a novel proteomics quantitation scheme termed "combined precursor isotopic labeling and isobaric tagging (cPILOT)" that allows for the identification and quantitation of nitrated peptides in as many as 12-16 samples in a single experiment. cPILOT offers enhanced multiplexing and posttranslational modification specificity, however excludes global quantitation for all peptides present in a mixture and underestimates reporter ion ratios similar to other isobaric tagging methods due to precursor co-isolation. Here, we present a novel chemical workflow for cPILOT that can be used for global tagging of all peptides in a mixture. Specifically, through low pH precursor dimethylation of tryptic or LysC peptides followed by high pH tandem mass tags, the same reporter ion can be used twice in a single experiment. Also, to improve triple-stage mass spectrometry (MS(3) ) data acquisition, a selective MS(3) method that focuses on product selection of the y1 fragment of lysine-terminated peptides is incorporated into the workflow. This novel cPILOT workflow has potential for global peptide quantitation that could lead to enhanced sample multiplexing and increase the number of quantifiable spectra obtained from MS(3) acquisition methods.

Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and isobaric tagging.

Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs "light" and "heavy" labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT(0), TMT(6), and iTRAQ(8) reagents and discuss limitations of the strategy.

Global effects of adriamycin treatment on mouse splenic protein levels.

Adriamycin (ADR) is a potent anticancer drug used to treat a variety of cancers. Patients treated with ADR have experienced side effects such as heart failure, cardiomyopathy, and "chemobrain", which have been correlated to changes in protein expression in the heart and brain. In order to better understand cellular responses that are disrupted following ADR treatment in immune tissues, this work focuses on spleen. Significantly reduced spleen sizes were found in ADR-treated mice. Global isotopic labeling of tryptic peptides and nanoflow reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to determine differences in the relative abundances of proteins from ADR-treated mice relative to controls. Fifty-nine proteins of the 388 unique proteins identified showed statistically significant differences in expression levels following acute ADR treatment. Differentially expressed proteins are involved in processes such as cytoskeletal structural integrity, cellular signaling and transport, transcription and translation, immune response, and Ca(2+) binding. These are the first studies to provide insight to the downstream effects of ADR treatment in a peripheral immune organ such as spleen using proteomics.