Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody
| Autor: | Greg Kilby, Xiaoyu Chen, Matthew Aspelund, Conner Parthemore, Alan K. Hunter, Yang Jiao, Mingyan Cao, Samuel Korman |
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| Rok vydání: | 2021 |
| Předmět: |
Chromatography
Ion exchange Chemistry Hydrophilic interaction chromatography Pharmaceutical Science 02 engineering and technology Reversed-phase chromatography 021001 nanoscience & nanotechnology Immunoglobulin light chain 030226 pharmacology & pharmacy Combinatorial chemistry Mass Spectrometry 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Capillary electrophoresis Affinity chromatography Immunoglobulin G Antibodies Bispecific Cysteine Sodium dodecyl sulfate 0210 nano-technology |
| Zdroj: | Journal of pharmaceutical sciences. 110(8) |
| ISSN: | 1520-6017 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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