Atypical Asparagine Deamidation of NW Motif Significantly Attenuates the Biological Activities of an Antibody Drug Conjugate.

Asparagine deamidation is a post-translational modification (PTM) that converts asparagine residues into iso-aspartate and/or aspartate. Non-enzymatic asparagine deamidation is observed frequently during the manufacturing, processing, and/or storage of biotherapeutic proteins. Depending on the site of deamidation, this PTM can significantly impact the therapeutic's potency, stability, and/or immunogenicity. Thus, deamidation is routinely monitored as a potential critical quality attribute. The initial evaluation of an asparagine's potential to deamidate begins with identifying sequence liabilities, in which the n + 1 amino acid is of particular interest. NW is one motif that occurs frequently within the complementarity-determining region (CDR) of therapeutic antibodies, but according to the published literature, has a very low risk of deamidating. Here we report an unusual case of this NW motif readily deamidating within the CDR of an antibody drug conjugate (ADC), which greatly impacts the ADC's biological activities. Furthermore, this NW motif solely deamidates into iso-aspartate, rather than the typical mixture of iso-aspartate and aspartate. Interestingly, biological activities are more severely impacted by the conversion of asparagine into iso-aspartate via deamidation than by conversion into aspartate via mutagenesis. Here, we detail the discovery of this unusual NW deamidation occurrence, characterize its impact on biological activities, and utilize structural data and modeling to explain why conversion to iso-aspartate is favored and impacts biological activities more severely.

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.

Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody.

Site-specific cysteine engineering, along with other genetic mutations, is broadly implemented in bispecific antibodies (bsAb). Thus far, homodimer, half hole antibody, one-light chain mispaired and light chain swapped variants have been reported as chain-pairing variants for the asymmetric IgG-like bispecific antibodies. Here we report a novel mispair in which the CH3 engineered cysteine on the hole heavy chain (HC) of a knob-into-hole (KiH) bsAb is linked to the engineered cysteine in CL through a disulfide bond, forming a LHL species in a bsAb construct. Due to its impact on bioactivity, it is critical to implement an analytical strategy to monitor this CQA and mitigate risk for the future products. A set of orthogonal physicochemical assays that include hydrophobic interaction chromatography (HIC), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), reverse phase liquid chromatography ultra-performance chromatography mass spectrometry (RP-UPLC MS) and disulfide bond mapping have been utilized to monitor and characterize this chain-pairing impurity for manufacturing process control and product release. Our data shows the LHL mispair in condition medium (CM) is approximately 1.3 - 1.9%. LambdaFabSelect affinity chromatography removes two major chain-pairing variants in CM - i.e. the hole-hole homodimer and hole half-antibody, while retaining the LHL species. Process improvement in Capto Q (anion exchange) and HS50 (cation exchange) chromatography steps removes LHL to as low as 0.2% in the final product. We have demonstrated an orthogonal analytical methodology that is capable of characterizing and monitoring bsAb mispairing, suitable for use in manufacturing process control and product release, and can be potentially implemented for similar bsAb constructs with engineered disulfide bonds.

Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common CH2 cleavages in IgGs and IgG-like bispecific antibodies.

Fragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Although fragments due to cleavage in CH2 domains linked by intrachain disulfide bonds are common and can be detected by reduced reversed-phase - liquid chromatography mass spectrometry (RP-LCMS) and reduced CE-SDS methods, their separation in nonreduced CE-SDS (nrCE-SDS) has not been reported but speculated as comigrating with intact IgG. A shoulder peak in nrCE-SDS was observed in the stability samples of an IgG-like bispecific antibody and was determined to be mainly caused by fragments from clipping at the C-terminus of leucine (L)306 or L309 (EU numbering) in the CH2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the CH1 domain of the knob HC. Subunit LCMS analysis verified that the crystallizable fragment contained variants with one or multiple mass additions of ~18 Da due to clipping. Further investigation revealed that CH2 clippings at L306 and L309 were largely due to proteolytic activity, and cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied. Our study shows that CH2 domain cleavages, with complementary fragments still linked by intrachain disulfide, can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Given the high occurrence of CH2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies.

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.