Detailed Analytical Characterization of a Bispecific IgG1 CrossMab Antibody of the Knob-into-Hole Format Applying Various Stress Conditions Revealed Pronounced Stability

Abstract

In recent years, a variety of new antibody formats have been developed. One of these formats allows the binding of one type of antibody to two different epitopes. This can for example be achieved by introduction of the “knob-into-hole” format and a combined CrossMab approach. Due to their complexity, these bispecific antibodies are expected to result in an enhanced variety of different degradation products. Reports on the stability of these molecules are still largely lacking. To address this, a panel of stress conditions, including elevated temperature, pH, oxidizing agents, and forced glycation via glucose incubation, to identify and functionally evaluate critical quality attributes in the complementary-determining and conserved regions of a bispecific antibody was applied in this study. The exertion of various stress conditions combined with an assessment by size exclusion chromatography, ion exchange chromatography, LC–MS/MS peptide mapping, and functional evaluation by cell-based assays was adequate to identify chemical modification sites and assess the stability and integrity, as well as the functionality of a bispecific antibody. Stress conditions induced size variants and post-translational modifications, such as isomerization, deamidation, and oxidation, albeit to a modest extent. Of note, all the observed stress conditions largely maintained functionality. In summary, this study revealed the pronounced stability of IgG1 “knob-into-hole” bispecific CrossMab antibodies compared to already marketed antibody products.

Comprehensive Multidimensional Liquid Chromatography–Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody

“…However, as already observed for the offline peptide mapping experiments no specific peak assignment for HC2-D106-iso was feasible, since similar levels for all acidic cuts were detected. As described previously, this degradation site was identified as CQA and hence led us to a further mD-LC-MS investigation of real-time stability material which we will describe in more detail later. Likewise, the bsAb1 basic variants were verified successfully by the automated online 4D-LC-MS setup: BP1 comprises around 50% glutamine (Cut B1), whereas BP2 contains C-terminal proline amidation (∼20%, Cut B2), BP3 shows LC2-D50-iso and the respective succinimide intermediate (Cut B3, ∼7% for both sites), whereas BP4 contains the C-terminal lysine (∼3%, Cut B4).…”

“…Cuts A1–A5 are from the acidic region, cuts B1–B4 cover the basic region, and MP-A1, MP-A2, and MP-B correspond to the main peak region. Further optimization of peptide separation was required to enable assessment of the aspartate isomerization sites (LC2-D50-iso and HC2-D106-iso) that have been previously identified as CQAs while ensuring a low back pressure for the trypsin cartridge. A previous study applying thermal and physiological stress conditions showed an increase of HC2-D106-iso for the acidic region peaks (predominantly AP1 and AP2), whereas LC2-D50-iso was found to be increased in BP3 .…”

“…Further optimization of peptide separation was required to enable assessment of the aspartate isomerization sites (LC2-D50-iso and HC2-D106-iso) that have been previously identified as CQAs while ensuring a low back pressure for the trypsin cartridge. A previous study applying thermal and physiological stress conditions showed an increase of HC2-D106-iso for the acidic region peaks (predominantly AP1 and AP2), whereas LC2-D50-iso was found to be increased in BP3 . Hence, acidic peaks MP-A1 and MP-A2 were analyzed on an RPLC Poroshell 120 Bonus-RP separating HC2-D106-iso, whereas an additional 4D-LC-MS run for the basic cuts and MP-B was conducted implementing a RPLC Poroshell 120 SB-C18 column within 4 D to separate the modified peptide LC2-D50-iso from its wild type.…”

“…In order to investigate method-induced artifacts in comparison to the offline approach, an asparagine spot known to be prone to deamidation during the procedure of peptide mapping (HC1’HC2-N321’N331-deam) and two susceptible methionine oxidation spots (HC2-M124-ox, HC1’HC2-M434’M444-ox) were assessed additionally (Figure B: Additional Variants). Particularly for the most susceptible oxidation site located in CDR (HC2-M124-ox) the online approach showed quantities around 1–4%, whereas the offline approach exhibited values roughly twice as high (3.4–7.8%, Supporting Information Table S2).…”

“…A previous study applying thermal and physiological stress conditions showed an increase of HC2-D106-iso for the acidic region peaks (predominantly AP1 and AP2), whereas LC2-D50-iso was found to be increased in BP3. 24 Hence, acidic peaks MP-A1 and MP-A2 were analyzed on an RPLC Poroshell 120 Bonus-RP separating HC2-D106-iso, whereas an additional 4D-LC-MS run for the basic cuts and MP-B was conducted implementing a RPLC Poroshell 120 SB-C18 column within 4 D to separate the modified peptide LC2-D50-iso from its wild type. Be aware that some further PTMs do not separate or elute early (Figure 4B) on one of the RP columns, but the combination of the two chosen columns enables the quantification of all important bsAb1 PTMs depending on their occurrence within either the acidic or basic region.…”