High-Resolution Capillary Zone Electrophoresis with Mass Spectrometry Peptide Mapping of Therapeutic Proteins: Peptide Recovery and Post-translational Modification Analysis in Monoclonal Antibodies and Antibody-Drug Conjugates.

Reversed phase liquid chromatography with mass spectrometry (RPLC-MS) peptide mapping is routinely used for interrogating molecular and structural attributes such as amino acid composition, sequence variants, and post-translational modifications (PTMs) in antibody-derived therapeutics. RPLC has some limitations that often impact the analysis of certain peptides including large hydrophobic peptides, hydrophilic di-/tripeptides and glycopeptides. Capillary zone electrophoresis with mass spectrometry (CZE-MS) has great potential for peptide mapping due to high efficiency and outstanding sensitivity. In this report we demonstrate the utility of CZE-MS as an orthogonal and complementary technique to RPLC-MS for peptide mapping analyses of antibody-drug conjugates (ADCs) and their parent antibodies. This work is based on high-resolution CZE-MS separation recently developed in our group, where a mixed aqueous-organic solvent system containing N,N-dimethylacetamide (DMA) or N,N-dimethylformamide (DMF) was used to improve the separation selectivity. The results described here show several advantages of CZE-MS for the analysis of small hydrophilic di-/tripeptides, large hydrophobic peptides, glycopeptides, and hydrophobic drug-linked peptides.

High-Resolution Capillary Zone Electrophoresis with Mass Spectrometry Peptide Mapping of Therapeutic Proteins: Improved Separation with Mixed Aqueous-Aprotic Dipolar Solvents (N,N-Dimethylacetamide and N,N-Dimethylformamide) as the Background Electrolyte.

Peptide mapping with mass spectrometry (MS) detection is a powerful technique routinely used for interrogating physicochemical properties of proteins. Peptide mapping benefits from an efficient front-end separation to increase selectivity and reduce complexity prior to MS detection. The most commonly used method for peptide mapping is based on reverse phase liquid chromatography with mass spectrometry. Capillary zone electrophoresis with mass spectrometry (CZE-MS) is an orthogonal technique with growing attention for peptide mapping of biotherapeutic proteins due to its high efficiency and sensitivity. However, that growth has been slow due to poorer peptide resolution and method robustness compared to RPLC. Here we present results from optimization of CZE-MS peptide mapping separation using mixed aqueous-aprotic dipolar solvent (N,N-dimethylacetamide (DMA) and N,N-dimethylformamide (DMF), as the background electrolyte (BGE) to improve the separation performance. Addition of DMA or DMF to the BGE impacts separation selectivity through differential change in pKa of the peptides. The CZE-MS peptide mapping method with the modified BGE produced significant improvement in resolution over the conventional CZE-MS methods. The method was evaluated with both sheathless and sheathflow CE-MS ion sources.

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.

Comparison of SEC and CE-SDS methods for monitoring hinge fragmentation in IgG1 monoclonal antibodies.

Fragmentation of monoclonal antibodies is a critical quality attribute routinely monitored to assess the purity and integrity of the product from development to commercialization. Cleavage in the upper hinge region of IgG1 monoclonal antibodies is a common fragmentation pattern widely studied by size exclusion chromatography (SEC). Capillary electrophoresis with sodium dodecylsulfate (CE-SDS) is a well-established technique commonly used for monitoring antibody fragments as well, but its comparability to SEC in monitoring hinge fragments has not been established until now. We report a characterization strategy that establishes the correlation between hinge region fragments analyzed by SEC and CE-SDS. Monoclonal antibodies with elevated hinge fragments were generated under low pH stress conditions and analyzed by SEC and CE-SDS. The masses of the fragments generated were determined by LC-MS. Electrophoretic migration of the hinge fragmentation products in CE-SDS were determined based on their mass values. Comparative assessment of fragments by SEC, and CE-SDS showed similar correlation with incubation time. This study demonstrates that CE-SDS can be employed as a surrogate technique to SEC for monitoring hinge region fragments. Most importantly, combination of these techniques can be used to obtain comprehensive understanding of fragment related characteristics of therapeutic protein products.

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.

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.

Robustness of iCIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study.

An international team including 12 laboratories from 11 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of imaged capillary isoelectric focusing for the charge heterogeneity analysis of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charged isoforms for a representative monoclonal antibody sample using the same capillary isoelectric focusing assay. Statistical evaluation of the data was performed to determine within and between laboratory consistencies and outlying information. The apparent pI data generated for each charged variant peak showed very good precision between laboratories with RSD values of less than 0.8%. Similarly, the RSD for the therapeutic monoclonal antibody charged variants percent peak area values are less than 11% across different laboratories using different analyst, different lots of ampholytes and multiple instruments. These results validate the appropriate use of imaged capillary isoelectric focusing in the biopharmaceutical industry in support of process development and regulatory submissions of therapeutic antibodies.

Charge heterogeneity of monoclonal antibodies by multiplexed imaged capillary isoelectric focusing immunoassay with chemiluminescence detection.

Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional CIEF technology with absorbance detection for purified mAbs and contaminated mAbs derived directly from host cellular extract. Upon method optimization, the automated CIEF immunoassay was applied to several mAbs of varying isoelectric points, demonstrating the suitability of NanoPro as a rugged high-throughput product characterization tool. Furthermore, qualification of detection sensitivity, precision, and dynamic range are reported with discussion of its advantages as an alternative approach to rapidly characterize charge variants during process development of mAbs.

Quantitative impurity analysis of monoclonal antibody size heterogeneity by CE-LIF: example of development and validation through a quality-by-design framework.

This paper describes the framework of quality by design applied to the development, optimization and validation of a sensitive capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) assay for monitoring impurities that potentially impact drug efficacy or patient safety produced in the manufacture of therapeutic MAb products. Drug substance or drug product samples are derivatized with fluorogenic 3-(2-furoyl)quinoline-2-carboxaldehyde and nucleophilic cyanide before separation by CE-SDS coupled to LIF detection. Three design-of-experiments enabled critical labeling parameters to meet method requirements for detecting minor impurities while building precision and robustness into the assay during development. The screening design predicted optimal conditions to control labeling artifacts while two full factorial designs demonstrated method robustness through control of temperature and cyanide parameters within the normal operating range. Subsequent validation according to the guidelines of the International Committee of Harmonization showed the CE-SDS/LIF assay was specific, accurate, and precise (RSD ≤ 0.8%) for relative peak distribution and linear (R > 0.997) between the range of 0.5-1.5 mg/mL with LOD and LOQ of 10 ng/mL and 35 ng/mL, respectively. Validation confirmed the system suitability criteria used as a level of control to ensure reliable method performance.

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.

Characterization of deamidated peptide variants by micro-preparative capillary electrophoresis and mass spectrometry.

Capillary electrophoresis (CE) was compared with reversed-phase liquid chromatography for its ability to separate native and deamidated peptides. CE is shown to provide superior resolution of these peptides due to its charge-based separation mechanism. Fraction collection performed using a standard CE instrument equipped with a 96-well plate permits subsequent characterization by nanospray mass spectrometric (MS) analysis. Additional in-depth analysis by MS/MS is able to provide the location of the deamidation site based on y-ion mass shifts of 1Da.

Investigation of sample preparation artifacts formed during the enzymatic release of N-linked glycans prior to analysis by capillary electrophoresis.

In the biotechnology industry, highly sensitive and accurate methods are required for monitoring glycosylation of therapeutic recombinant monoclonal antibodies (rMAbs) due to possible effects on bioactivity. At Genentech, a method employing PNGase F digestion, fluorescent labeling of released glycans, and analysis by capillary electrophoresis (CE) is used for routine monitoring of N-linked glycosylation during process development and quality control of therapeutic glycoproteins. In our laboratory, capillary electrophoresis-mass spectrometry (CE-MS) technology was developed to identify minor glycan species in assay and it revealed several unidentified isomeric species. Additional studies indicate that these species (1-10% total glycans) are sample preparation artifacts caused by base-catalyzed epimerization of N-acetylglucosamine (GlcNAc) at the reducing terminus by following the use of commercially available PNGase F and the supplied incubation buffer (pH 7.5). As these isomeric species directly impact the accuracy of the reported results, an optimized PNGase F release step is presented which minimizes and/or eliminates the formation of these artifacts. We have found that PNGase F incubation at pH 5.5 for IgG(1) rMAbs shows no significant decrease in enzyme activity while minimizing GlcNAc epimerization. Implementation of this change has resulted in a more accurate and robust CE-laser-induced fluorescence (LIF) assay and is generally applicable to any analysis requiring PNGase F digestion of rMAbs.

On-line CE-LIF-MS technology for the direct characterization of N-linked glycans from therapeutic antibodies.

Glycan characterization of therapeutic proteins is of utmost importance due to the role of carbohydrates in protein stability, half-life, efficacy and mechanism of action. The primary assay for characterization and lot release of N-linked glycans on glycoprotein products at Genentech, Inc., is a capillary electrophoresis (CE) based assay, wherein PNGase F-released, APTS-labeled glycans are separated by CE with laser induced fluorescence (LIF) detection. With the growing number of new molecular entities in the pipeline, a fast and direct characterization approach is of increasing importance. This paper describes the development of CE-MS technology with on-line LIF detection that allows identification of major and minor glycan species (1-5% of total glycans) by providing accurate mass information. Data is presented for therapeutic rMAbs which presented previously unidentified, minor peaks during routine CE-LIF analysis. CE-LIF-MS was then used to provide accurate mass on these species, identifying CE peaks corresponding to sialylated (G1 + NANA, G2 + NANA), afucosylated (G0-GlcNAc-fucose) and low-level isomers of major APTS-labeled glycans G0, G1, G1' and G2.

Optimization and validation of a quantitative capillary electrophoresis sodium dodecyl sulfate method for quality control and stability monitoring of monoclonal antibodies.

In previous work, a capillary electrophoresis sodium dodecyl sulfate (CE-SDS) method using precolumn labeling and laser-induced fluorescence (LIF) detection was developed at Genentech Inc. as part of the control system for the quality control release of a recombinant monoclonal antibody (rMAb) (Hunt, G.; Nashabeh, W. Anal. Chem. 1999, 71, 2390-2397.). In the current work, a generic and quantitative CE-SDS assay with LIF detection of rMAbs with improved accuracy and precision is described. The implementation of an alkylating step with iodoacetamide and optimization of the incubation temperature and time, in the presence of SDS, greatly decrease any thermally induced fragmentation of nonreduced labeled rMAb samples. In addition, a quantitative study of the effects of sample buffer pH on rMAb fragmentation is also presented. Furthermore, the performance of alternative CE-SDS polymer solutions and instrumentation for quantitative analysis of rMAbs is shown in this article. The validation of this method, under the guidelines of the International Committee on Harmonization (ICH), demonstrates that the assay quantitatively determines the consistency of rMAb manufacture as it relates to size heterogeneity and product purity.

Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins.

With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE-mass spectrometry (CE-MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE-MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE-laser-induced fluorescence (CE-LIF). For each study, CE-MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE-MS for profiling biomolecules.