Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography-tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.

Eliminating protein oxidation artifacts during High Performance Liquid Chromatography peak fractionation processes.

Therapeutic monoclonal antibodies (mAbs) are inherently heterogeneous and hence generally studied and controlled by an array of orthogonal separation methods. During drug candidate development, fractionation by HPLC is regularly employed to assist peak identification and product understanding. One overlooked challenge is the protein oxidation introduced by the fractionation process. In this study, we report the extent of fractionation-induced protein oxidation, which tends to complicate data interpretation and peak assignments. Higher-energy detectors such as fluorescence detectors and lower fraction concentration were found to exacerbate the oxidation artifacts. Other contributing factors than the detector-induced photostress were also found to contribute significantly to protein oxidation. Furthermore, our study showed that collecting fractions into a solution with oxidation scavengers, such as histidine and methionine, was effective in eliminating the oxidation artifacts introduced by detector exposure and fraction processing steps. Through an example, we demonstrate that the modified fractionation workflow improves the accuracy of peak assignments.

Characterization of N-Terminal Glutamate Cyclization in Monoclonal Antibody and Bispecific Antibody Using Charge Heterogeneity Assays and Hydrophobic Interaction Chromatography.

N-terminal glutamate (E) cyclization to form pyroglutamate (pE) generates charge heterogeneities for mAbs and proteins. Thus far, pE formation rate in lyophilized formulation as compared to in liquid formulation has not been reported. Impact of pE on antibody biological activity has only been predicted or assessed using stressed samples that may contain other confounding degradations besides pE. Additionally, application of hydrophobic interaction chromatography (HIC) to separate pE has not been reported. In our study, N-terminal E cyclization was identified as the major degradation pathway in lyophilized formulation at elevated temperature for both monoclonal antibody (mAb-A) and IgG-like bispecific antibody (bsAb-A). pE was enriched in salt-gradient ion exchange chromatography (IEC) as pre-peak and in HIC as post-peak for both mAb-A and bsAb-A. Structure-function studies with pE-enriched IEC and HIC fractions confirmed that pE did not affect binding activities for mAb-A and bsAb-A. In vitro incubation of bsAb-A in serum and PBS revealed that the serum matrix may play a role in pE conversion in human serum, in contrast to the chemical reaction mechanism reported. These techniques can help in characterization of N-terminal E-to-pE cyclization and quality attribute severity assessment during therapeutic protein product development.

Deamidation in Moxetumomab Pasudotox Leading to Conformational Change and Immunotoxin Activity Loss.

Asparagine (Asn) deamidation is a common posttranslational modification in which Asn is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kDa truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which often leads to the conclusion that it has a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide an explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest the approach that can be used for process control to ensure product quality and process consistency.

Analytical characterization of coformulated antibodies as combination therapy.

When two therapeutic agents are combined in a single formulation, i.e., coformulated, the quality and safety of the individual agents must be preserved. Here we describe an approach to evaluate the quality attributes of two individual monoclonal antibodies (mAbs), designated mAb-A and mAb-B, in coformulation. The mAbs were fractionated from heat-stressed coformulated drug product (DP) by hydrophobic interaction chromatography. Each purified mAb fraction was then compared with mAb-A and mAb-B in their individual formulations from the same drug substance sources used to make the coformulated DP lot, which was subjected to the same stress conditions. Product variants were evaluated and compared by using several analytical tests, including high-performance size exclusion chromatography (HPSEC), reducing and nonreducing gel electrophoresis, ion-exchange chromatography, capillary isoelectric focusing, and peptide mapping with mass spectrometry. Intermolecular interactions in coformulated and photostressed DPs were studied by evaluating aggregates fractionated from coformulated DP by HPSEC. Aggregate fractions of coformulated DP contained dimers, but not coaggregates, of mAb-A or mAb-B. Moreover, extensive assays for higher-order structure and biological interactions confirmed that there was no interaction between the two mAb molecules in the coformulation. These results demonstrate that the two coformulated therapeutic mAbs had the same quality attributes as the individually formulated mAb-A and mAb-B, no new quality attributes were formed, and no physicochemical, intermolecular, or biological interactions occurred between the two components. The approach described here can be used to monitor the product quality of other coformulated antibodies.

Characterization of IgG1 Fc Deamidation at Asparagine 325 and Its Impact on Antibody-dependent Cell-mediated Cytotoxicity and FcγRIIIa Binding.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action for many therapeutic antibodies. A therapeutic immunoglobulin (Ig) G1 monoclonal antibody lost more than half of its ADCC activity after heat stress at 40 °C for 4 months. Size-exclusion and ion-exchange chromatography were used to fractionate various size and charge variants from the stressed IgG1. Physicochemical characterization of these fractions revealed that a rarely seen crystallizable fragment (Fc) modification, N325 deamidation, exhibited a positive correlation with the loss of ADCC activity. A further surface plasmon resonance study showed that this modification disrupted the binding between the IgG1 Fc and Fcγ receptor IIIa, resulting in decreased ADCC activity of the IgG1 antibody. Mutants of N325/D and N325/Q were made to confirm the effect of N325 deamidation on ADCC. We hypothesize that N325 deamidation altered the local three-dimensional structure, which might interfere with the binding and interaction with the effector cell. Because of its impact on biological activity, N325 deamidation is a critical quality attribute for products whose mechanism of action includes ADCC. A thorough understanding of the criticality of N325 deamidation and appropriate monitoring can help ensure the safety and efficacy of IgG1 or Fc-fusion products.

Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis.

Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ADCs yields some process-related species heterogeneity, including stereoisomers, unconjugated antibody, underconjugated species, and overconjugated species. An elevated level of size variants, such as heavy chain-light chain species (half ADC), heavy chain-heavy chain-light chain species, and light chain species, is also observed with the final site-specific ADC product. To understand the root cause of heterogeneity generated during the ADC conjugation process, we designed time-course studies for each conjugation step, including reduction, oxidation, conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample analysis. On the basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidation as a result of alternative disulfide bond arrangements. During oxidation, two hinge cysteines formed an intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in the unconjugated antibody. Time-course data also showed that the elevated level of size variants, especially heavy chain-heavy chain-light chain species and light chain species, resulted from the quenching step, where the quenching reagent engaged in a disulfide bond exchange reaction with the ADC and broke the disulfide bonds connecting the heavy chain and light chain. Underconjugated and overconjugated species arose from the equilibrium established during the conjugation reaction.

A method to directly analyze free-drug-related species in antibody-drug conjugates without sample preparation.

Residual free-drug-related species that are present in antibody-drug conjugates (ADC) are a potential safety risk to patients and are therefore categorized as a critical quality attribute that must be strictly monitored and controlled. Among the many analytical methods developed for free-drug analysis, reversed-phase liquid chromatography (RP-LC) is the most common approach. Conventional RP-LC methods for free-drug analysis, however, involve labor-intensive sample preparation. Here we present a new RP-LC method to directly analyze free-drug-related species in an ADC sample without the need for sample preparation. In our work, free-drug-related species were very well separated from ADC peaks in the chromatography gradient. Typical performance issues observed in conventional RP-LC, such as column fouling, detection interference, and carryover, were not observed or were negligible with this new method. Three options were evaluated for free-drug quantitation: Strohl (2017) [1] use of an external free drug calibration curve for determination of absolute concentration; Perez et al. (2014) [2] calculation of relative percentage based on peak area ratio between free drug and ADC at a characteristic wavelength unique for drug payload; and (Beck et al., 2017) [3] calculation of relative percentage based on peak area ratio between free drug and corrected ADC peak area (at any wavelength). The method with calibration curve provides the highest sensitivity, the best accuracy and precision for determination of free drug present in the ADC. However, the second and third options were simpler because they eliminated the need for an external calibration curve, making them worth exploring. Due to its simplicity and compatibility with mass spectrometry, the new method is also a good choice for direct analysis of stability samples.

Charge variants characterization and release assay development for co-formulated antibodies as a combination therapy.

Combination therapy is a fast-growing strategy to maximize therapeutic benefits to patients. Co-formulation of two or more therapeutic proteins has advantages over the administration of multiple medications, including reduced medication errors and convenience for patients. Characterization of co-formulated biologics can be challenging due to the high degree of similarity in the physicochemical properties of co-formulated proteins, especially at different concentrations of individual components. We present the results of a deamidation study of one monoclonal antibody component (mAb-B) in co-formulated combination antibodies (referred to as COMBO) that contain various ratios of mAb-A and mAb-B. A single deamidation site in the complementarity-determining region of mAb-B was identified as a critical quality attribute (CQA) due to its impact on biological activity. A conventional charge-based method of monitoring mAb-B deamidation presented specificity and robustness challenges, especially when mAb-B was a minor component in the COMBO, making it unsuitable for lot release and stability testing. We developed and qualified a new, quality-control-friendly, single quadrupole Dalton mass detector (QDa)-based method to monitor site-specific deamidation. Our approach can be also used as a multi-attribute method for monitoring other quality attributes in COMBO. This analytical paradigm is applicable to the identification of CQAs in combination therapeutic molecules, and to the subsequent development of a highly specific, highly sensitive, and sufficiently robust method for routine monitoring CQAs for lot release test and during stability studies.