Editorial and Review: 28th ASMS Sanibel Conference on Mass Spectrometry-Characterization of Protein Therapeutics by Mass Spectrometry
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KeywordsProtein Therapeutics Electron Capture Dissociation Electron Transfer Dissociation Eculizumab 28th ASMS
The Thursday evening plenary lecture was given by Michael Gross from Washington University in St. Louis. Michael set the stage for the rest of the conference by discussing a total structural assessment of biotherapeutics using both low and high resolution mass spectrometry. “Native” electrospray ionization MS continues to be at the forefront of intact protein analysis and, more recently, to its application to protein aggregation. Michael discussed the use of new extended mass range (EMR) technology with Orbitrap mass spectrometry for the study of superoxide dismutase-1 (SOD1) aggregation, a process that is yet to be fully understood. Expanding on native mass spectrometry in terms of primary sequence analysis, he provided examples of how electron capture dissociation (ECD) yields fragment ions of intact antibodies with flexible regions between the β-sheet rich domains. These fragment ions correlated with high B-factors by X-ray crystallography. A Fab–antigen complex, a common focus point for the biopharmaceutical industry, was studied with this technique to provide information on regions of the antibody that become more rigid upon antigen binding.
His second focus area related to protein therapeutics that he discussed was the use of footprinting to study higher-order structure. Hydrogen–deuterium exchange (HDX) and fast photo-chemical oxidation of proteins (FPOP) are orthogonal exam-ples of protein footprinting techniques that afford information regarding higher-order structure. HDX provides structural information down to the region and, with some forms of peptide fragmentation, site-specific protein backbone conformational changes. FPOP furnishes information on site-specific changes to several side chains of amino acids. These techniques have expanded beyond that of traditional applications of epitope mapping and protein–protein interactions to include virus as-sembly and antibody neutralization. Michael discussed appli-cations of these technologies for epitope mapping of multiple monoclonal antibodies (mAbs) to a malaria vaccine target and an epidermal growth factor receptor (EGFR)–adnectin com-plex. The latter example generated residue-specific information on the protein–protein interface and agreed well with the X-ray crystal structure. In terms of the future state of MS applications to biotherapeutics, Professor Gross discussed: (1) the expansion of FPOP to other radicals, such as iodide, or carbene chemistry, (2) improved kinetics analysis by varying the hydroxyl radical scavengers used during sample preparation, (3) the use of HDX to determine the affinity of proteins to the ligands, and (4) pulsed HDX to study amyloid proteins and aggregation.
The Friday morning opening session focused on Sequence Analysis and Protein Stability. The session offered discussions about state-of-the-art protein sequence determination, characterization, and its effect on stability. Scott McLuckey (Purdue University) presented his work on gas-phase ion/ion chemistry for primary structure characterization. The core focus of Scott’s research is the use of chemical reagents that elicit charge reduction to increase peak capacity on the m/z scale. Several applications of gas-phase ion/ion chemistry included the charge reduction of intact mAbs to reduce maximum entropy (MaxEnt1) deconvolution artifacts, excipient analysis of polysorbate (Tween) using carborane acid [H(CHB11Cl11)], gold chloride cleavage of disulfide bonds, and platinum’s use as a gas-phase cleavage agent C-terminal to methionine residues. Jennifer Brodbelt (University of Texas, Austin) provided a fascinating lecture on UV photodissociation (UVPD) of mAbs. A UV laser (193 nm, ArF) is positioned to provide high energy dissociation in the HCD cell of an Orbitrap mass spectrometer. UVPD was applied to intact proteins (1 pulse around 0.8 mJ) and peptides (2–3 pulses around 2.0 mJ). Her research team observed that the fragmentation was generally charge state-independent and afforded more cleavage than collision-induced dissociation (CID), electron transfer dissociation (ETD), and higher-energy collision-activated dissociation (HCD) individually. A promising application is the primary sequence analysis of complementary determining region (CDR) peptides for antibodies, whereby endoproteinase LysC digestion generates large peptides. Additionally, mAb subunit analysis through the use of the immunoglobulin-degrading enzyme from S. pyogenes (IdeS) afforded greater sequence coverage by UVPD (50%–65% bond cleavage) versus ETD (35%–45% bond cleavage). The final speaker before the coffee break was Igor Kaltashov (University of Massachusetts, Amherst), who discussed the development of a high-accuracy temperature controlled ESI probe (±2 °C) for the use of correlating protein unfolding to common techniques such as differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The first example was the observed unfolding of cytochrome c, which was within 2 °C of the reported DSC value. Several examples of SEC-MS for aggregation separation and online ion exchange (IEX) MS provide unique insights to protein folding/unfolding and aggregation analysis. For recombinant human arylsulfatase A (rhASA), the observation of unfolded dimer was detected between two eluting profiles of a folded, native tetramer and octamer by SEC-MS. Additionally, stressed interferon beta (IFNβ) was analyzed by a MS-friendly IEX separation to conclude that deamidation was responsible for degradation.
Two perspectives from the biopharmaceutical industry were provided by Dingyi Wen from Biogen and Taylor Zhang from Genentech. Dingyi discussed proper disulfide bond determination, with a focus on alkylation and digestion below pH 7. A common approach in their lab is to compare reduced and alkylated proteolytic mapping to a non-reduced sample profile. The observation of trisulfides in mAbs has been a hot topic in the industry. This modification is thought to be mediated by cell culture and primarily occurs between two regions of a mAb: (1) light and heavy interchain disulfide connectivity, and (2) heavy–heavy interchain disulfide connectivity. The Biogen group compared mAbs containing low and high levels of trisulfides and did not observe an effect on antigen binding activity. For difficult proteins containing Cys residues near one another that form intricate disulfide pairing arrangements, an MS3 approach can discern expected and unexpected disulfide linkages. An MS/ETD/CID MS3 approach was used to discern the linkages of six Cys sites in the sequence of the protein Nogo-66. Taylor Zhang discussed the well-known and the less well-known deamidation sites in mAbs. Asparagine-glycine (NG) and asparagine-serine (NS) sequence motifs tend to be the most reactive and watched for during protein degradation studies; however, other sites may lead to a drop in antigen binding or decreases in effector function. He provided an example of an observed decrease in CDC and ADCC effector function with no observed deamidation change by LysC/trypsin proteolytic mapping. A small peptide containing VSNK was later observed through chymotryptic analysis to undergo deamidation and isoaspartate formation. It was observed that more deamidation was observed at pH 5.3 versus 7.0, which goes against common hypotheses that deamidation occurs at higher pH. The deamidation and isoaspartate formation of the VSNK sequence, which is near binding site of the FcɣIII complex, was directly correlated to decreased CDC activity. In summary, the use of mass spectrometry is indispensable for the study of primary structure and protein stability of biopharmaceuticals. New fragmentation techniques, MS-friendly mobile phases for non-reversed phase separations and sample preparation techniques all expand the utility, and information gathered on ensuring proteins are produced at high quality and high reproducibility.
The Friday afternoon discussion focused on Product/Process-related characterization approaches. Li Tao from Bristol-Myers Squibb provided insight on the molecular variants possible in biotherapeutics. He introduced the audience to biologic process development, from upstream and downstream drug substance (DS) manufacturing all the way to the clinical drug product (DP). He highlighted the thousands of variants possible and the need to understand relationships between function and the nature of variants. Forced degradation is a common approach to assess the variants formed under stressed conditions. These conditions include high temperature (30–60 °C), low (3–6) and high (8–10) pH, agitation, freeze/thaw, UV exposure, oxidation, and the presence of metals and silicon oil. The coloration of drug substances has been linked to tryptophan oxidation by UV and metal catalysis. Carlito Lebrilla (University of California–Davis) followed up with an overview of where and how glycosylation occurs in cells. He discussed how asparagine-linked (N-linked) glycans are built up, degraded, and built up again between the endoplasmic reticulum and Golgi apparatus. Chromatographic separations and the use of sequential exoglycosidases are the key to characterizing N-linked glycosylation. Ultimately, a comparison of glycopeptide analysis to that of labeled/unlabeled released glycans can show subtle differences in abundance.
Melissa Alvarez from Genentech provided a fascinating overview of sequence variant analysis (SVA). Sequence variants are unintended amino acid substitutions and considered to be product-related variants. Their platform approach is to utilize trypsin/thermolysin proteolytic digestion, reversed-phase separation, CID MS/MS, and a Mascot error tolerant search (ETS) for the discovery and relative quantitation of sequence variants. This workflow has numerous challenges that mostly relate to the amount of information collected and the time-consuming data analysis with multiple pieces of software. They validated assay performance with a positive control: mAb1 spiked into mAb2, which afforded ~20 unique peptides with varying size and elution. Some false positives associated with SVA include amino acid substitutions that match other modifications, which make interpretation difficult (i.e., carboxymethylation of Cys is isobaric to a glycine → aspartic acid amino acid substitution). The NIST mAb standard was analyzed and ~32 variants were detected at 0.1% or lower relative abundance. Finally, Justin Sperry from Pfizer provided an overview of host cell proteins (HCPs), which are considered process-related species. The detection and relative quantitation of HCPs follow a traditional proteomics workflow with optimized Orbitrap settings to expand the detection dynamic range to 5–6 orders of magnitude. The dynamic range was demonstrated through the use of a commercially available Universal Proteomics Standard containing 48 proteins. All proteins were detected down to a level of 10 ng protein per 1 mg of mAb (10 ppm). Some proteins, but not all, were detected between 1 and 10 ppm. The method was applied to process clearance studies, where one tracks the removal of HCPs through the multistep downstream process. One particular case study centered on the observed degradation of a mAb–peptide conjugate. The conjugated peptide contained a Phe–Met bond that was cleaved at low pH and various temperatures during stability studies. Upstream analysis of process samples identified the potential culprit as cathepsin D. The observation that pepstatin inhibited the cleavage led to the development of an immobilized pepstatin column to enrich for the co-purified protease for positive identification.
“Late breaking” short talks were interspersed with the primary sessions. Late breaking talks were giving by: Sunny Zhou (Northeastern University), “Meet the Challenge of Discovering Unknowns: Protein Modifications in Biotherapeutics;” Joomi Ahn (MedImmune), “Investigation of IgG4 Monoclonal Antibody Aggregation by HDX MS and Other Biophysical Tools;” Tawnya Flick (Amgen), “Applications of Mass Spectrometry in Process Development of Antibody Drug Conjugates and Novel Bioconjugates;” Richard Vachet (University of Massachusetts, Amherst), “Investigating Protein Therapeutic Structure with Covalent Labeling and Mass Spectrometry;” Jason Tang (Eli Lilly), “High Order Structure Characterization of Engineered Bispecific Antibodies by LC-MS - based Methods;” Sarah Rogstad (FDA), “The Use of Mass Spectrometry in FDA Applications for Biotherapeutics: A Retrospective Review.”
A shift from primary structure analysis to higher-order structure analysis was the focus of the Saturday morning session. Allessandra Luchini (George Mason University) discussed her desire to illuminate hot spots in protein interfaces and large interaction areas. The focus of her research utilizes surface binding molecules (organic dyes) with high affinity to various regions of a protein, even in a denatured state. The sites of dye occupancy are resistant to proteolytic cleavage when bound. A primary system of study in her laboratory was the hot topic field of immune-oncology and the use of Programmed Death-1 (PD1) and Programmed Death Ligand-1 (PD-L1) for treatment. She then discussed the use of peptide inhibitors that affect the protein interface.
Guodong Chen (Bristol Myers Squibb) provided an overview of epitope mapping and HDX. He highlighted their use for probing protein–protein interactions in discovery and the challenges associated with characterization from primary to quaternary structure, specifically regarding linear and conformation epitopes. Most antibody–antigen complexes are conformational epitopes. Epitope selection in biopharmaceuticals is critical to the proposed mechanism of action (MOA), structure/activity relationship, freedom-to-operate, and patentability. Several methodologies to characterize and understand higher-order structure include immunochemical analysis, computational analysis, X-ray crystallography, NMR, and limited proteolysis, with each having limitations and benefits. HDX-MS has good throughput and resolution and is applicable to many types of protein–protein interactions, including protein therapeutics and antigens. He presented a study of the IL-23 hetero-dimeric p19 domain with five epitope regions mapped by HDX. This study was compared with traditional alanine (Ala) mutational analysis. ETD was utilized to generate site-specific residue information—down to 3–4 residues on any individual peptide. Jeffrey Hudgens from NIST spoke of HDX harmonization efforts using the NIST mAb reference material. His team created a Fab standard for an inter-laboratory study to test reliability of the HDX measurement (pH, temperature, ionic strength, and pressure). Reproducibility over many labs (18 labs, with 10 in biopharma, and distributed over five countries) was summarized. A full experimental kit was provided for proteomics and HDX. Approximately 58,000 measurements yielded an average peptide length of 16 amino acids across all data submitted. Digestion was inconsistent across groups, with only seven identical peptides observed across all groups. The observed precision at t = 0 was 0.25 Da and at t = 14000 s was 1.0 Da.
The final two speakers in the morning focused on formulation development and comparability assessment of biopharmaceuticals. David Weis (University of Kansas) discussed the practical applications of HDX for formulation and aggregation assessment. A primary focus of his research is mAb–excipient interactions and monitoring of destabilization of the CH2 domain, the domain that typically unfolds first under thermal stress as indicated by differential scanning calorimetry. Purposeful destabilizers (arginine and thiocyanate) and stabilizers (sucrose) were utilized to monitor deuterium uptake and changes in an aggregation-prone hot-spot region of a mAb (…VFLFPPKPD….). His second topic centered on reversible self-association, typically observed as an increase in hydrodynamic diameter with increasing concentration. Direct dissolution of a lyophilized cake with D2O was utilized for high concentration HDX measurements. Both protected and deprotected residues associated with reversible self-association were observed. Finally, George Bou-Assaf from Biogen discussed the use of HDX-MS for comparability assessments in support of regulatory filings and process development. A comparability exercise is generally required of all biopharmaceuticals in development and involves the demonstration of manufacturing consistency through a comparison of batches and assessment of all deviations during the manufacturing process. Scale changes (i.e., 2000 L to 15000 L bioreactor) are a major focus of comparability and, in their assessments, no changes in HDX were observed.
The Saturday afternoon session focused on hot topics in the field, including Antibody-Drug Conjugates (ADCs) and In Vivo Quantitation. Lisa Marzilli from Pfizer provided an overview of the many types of ADCs in development, including those based on lysine chemistry, cysteine chemistry, and site-specific chemistry (mutation or enzymatic). After characterizing several lysine-conjugated ADCs, predictable sites were found to be consistently conjugated in the Fc region of the antibody. Conjugation chemistry targeted to Cys residues, after partial reduction of the antibody, are generally directed to the hinge and interchain LC/HC and site-specific at engineered Cys residues. She described a multi-faceted approach to characterization: (1) intact assessment for extent of modification, and (2) proteolytic mapping for sites of conjugation; mAb sequence liabilities (deamidation, oxidation, etc) are important to monitor. Critical to this is the use of methods that ensure minimal method-induced artifacts generated during the digestion step. She provided an example of an NY sequence motif (prone to deamidation, but not as prone as NG or NS) in the heavy chain CDR-affected target binding region. Acid labile linkers can be affected by HPLC mobile phases that lead to sample degradation prior to UV or MS analysis. Native ESI-MS assessments of intact Cys ADCs are now routine. The measured drug-to-antibody ratio (DAR) afforded from this agrees well with traditional hydrophobic interaction chromatography profiles. Tim Olah (Bristol-Myers Squibb) discussed the in vivo quantitation of ADCs, specifically focusing on the understanding of drug/target interactions. He mentioned that ADCs are complex, but the mechanism of action is too, and thus the disposition of ADCs throughout the body needs to be characterized. Bioanalytical focus areas include structure/function with appropriate ADC standards of linker-payload catabolites. He discussed some general needs for analytical development. Specifically, most center on total mAb concentration, total conjugated mAb, unconjugated payload, and conjugated payload. His group’s approach is affinity enrichment with enzymatic cleavage of protein and payload. The ability to account for payload metabolism is crucial for proper distribution analysis. Surinder Kaur from Genentech continued the discussion on the fate of ADCs and their biotransformation in vivo. She highlighted the huge expansion in the ADC field, with over 250 clinical trials and the trend toward site-specific conjugation. Specific challenges highlighted in her presentation focused on the function of in vivo catabolites, and pharmacokinetics of a mixture (mAb and payload). She provided many examples of biotransformations in vivo; linker deconjugation, or partial drug cleavage (or combination of both) complicate the mass spectra. Finally, Paula Ladwig from the Mayo Clinic discussed the clinical quantitation of eculizumab by monitoring intact light chain. Eculizumab binds complement component 5 and is one of the most expensive monoclonal antibody therapies on the market. Her studies highlighted enrichment down to 75 μg/mL with a S/N of 3 and with an IgG4 enrichment down to 2 μg/mL with S/N 4.8.
The final session of the conference was on Sunday morning and focused on Biosimilars and New Directions. Michael Boyne (BioTech Logic) kicked off the discussion on the use of MS in a regulatory setting. Multiple strategies exist, including ID/characterization, comparability/similarity, counterfitting, process monitoring, epitope/higher-order structure, PK/biotransformation, and a trend for validated MS assays in QC. A particular example of protamine sulfate and the use of ECD/ETD for sequence assignment were discussed, highlighting that no monographs to date in the US Pharmacopeia (USP) were on this topic. Alain Beck (Centre d’Immunologie Pierre Fabre) provided comprehensive MS characterization approaches for mAbs, biosimilars, mAb mixtures, and ADCs. He provided evidence of comparability using ion mobility for biosimilar candidates, capillary electrophoresis (CE)-ESI MS for peptide mapping (particularly for separation of deamidated peptides), and ion mobility for the dynamics of IgG4 arm exchange. For middle down fragmentation, his laboratory utilized a Sap9 protease for digestion at pH 5.5 for 1 h to generate 3–7 kDa fragments of an antibody. Albert Heck (Utrecht University) emphasized the importance of minimizing sample preparation prior to MS analysis. Much of his work centers on new instrument modifications for high mass complex analysis. His group members and collaborators mutated specific residues, in particular Y407, on an antibody and monitored its effect on glycan distributions by MS. Joseph Loo (University of California–Los Angeles) discussed the combination of native MS and top-down MS with high resolution Fourier-transform ion cyclotron resonance MS and ECD fragmentation. Several protein–ligand studies by ECD have demonstrated its utility for locating ligand binding sites. Differences in top-down MS sequence coverage between native and denatured proteins conditions are common. Finally, Brandon Ruotolo (University of Michigan) discussed the development and application of collision induced unfolding of monoclonal antibodies as a tool for biosimilarity assessments and for comparability exercises.
“A Retrospective Evaluation of the Use of Mass Spectrometry in FDA Biologics License Applications” by Sarah Rogstad, Anneliese Faustino, Ashley Ruth, David Keire, Michael Boyne, and Jun Park
“Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry” by Richard Y.-C. Huang, Roxana E. Iacob, Stanley R. Krystek, Mi Jin, Hui Wei, Li Tao, Tapan K. Das, Adrienne A. Tymiak, John R. Engen, and Guodong Chen
“Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery” by K. Ilker Sen, Wilfred H. Tang, Shruti Nayak, Yong J. Kil, Marshall Bern, Berk Ozoglu, Beatrix Ueberheide, Darryl Davis, and Christopher Becker
“Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry” by Paula M. Ladwig, David R. Barnidge, and Maria A.V. Willrich
“The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS)” by Majors J. Badgett, Barry Boyes, and Ron Orlando
“Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS” by Lidong He, Lissa C. Anderson, David R. Barnidge, David L. Murray, Christopher L. Hendrickson, and Alan G. Marshall
“Deciphering the Biophysical Effects of Oxidizing Sulfur-Containing Amino Acids in Interferon-beta-1a using MS and HDX-MS” by Damian J. Houde, George M. Bou-Assaf, and Steven A. Berkowitz
“Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry” by Ying Zhang, Aaron Wecksler, Patricia Molina, Galahad Deperalta, and Michael L. Gross