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Characterization of bispecific antigen-binding biotherapeutic fragmentation sites using microfluidic capillary
Arnik Shah1,2, Ruhi Desai1, Weidong Cui1
1Amgen Inc, Attribute Science, 360 Binney Street, Cambridge, Massachusetts 02141, USA.
Abstract:
Fragmentation of therapeutic proteins is a potential critical quality attribute (CQA) that can occur in vivo or during manufacturing or storage due to enzymatic and non-enzymatic degradation pathways, such as hydrolysis, peroxide mediation, and acid/metal catalysis. Characterization of the fragmentation pattern of a therapeutic protein is traditionally accomplished using capillary gel electrophoresis with UV detection under both non-reducing and reducing conditions (nrCGE and rCGE). However, such methods are incompatible with direct coupling to mass spectrometry (MS) due to the use of anionic surfactants, e.g., sodium dodecyl sulfate (SDS). Here, we present a novel method to characterize size-based fragmentation variants of a new biotherapeutic kind using microfluidic ZipChip® capillary zone electrophoresis (mCZE) system interfaced with mass spectrometry (MS) to determine the molecular masses of fragments. A new modality of immuno-oncology therapy, bispecific antigen-binding biotherapeutic, was chosen to investigate its fragmentation pattern using mCZE-MS for the first time, according to our knowledge. Bispecific antigen-binding biotherapeutic samples from different stages of downstream column purification and forced degradation conditions were analyzed. The results were cross-validated with denaturing size-exclusion chromatography-mass spectrometry and conventional rSDS-CGE. In this study, we demonstrated that mCZE-MS could separate and characterize 12-40 kDa bispecific antigen-binding biotherapeutic fragments rapidly (within ≤12 minutes), with higher resolution and better sensitivity than traditional LC-MS methods.
Insights
A new microfluidic capillary zone electrophoresis-mass spectrometry (mCZE-MS) method rapidly characterizes fragmentation in bispecific antigen-binding biotherapeutics. This technique offers higher resolution and sensitivity than traditional methods for analyzing these complex protein therapeutics.
Area of Science:
- Biopharmaceutical analysis
- Protein characterization
- Analytical chemistry
Background:
- Therapeutic protein fragmentation is a critical quality attribute impacting efficacy and safety.
- Traditional methods like capillary gel electrophoresis (CGE) are incompatible with mass spectrometry (MS) due to surfactant use.
- Accurate characterization of protein fragments is essential for biotherapeutic development and quality control.
Purpose of the Study:
- To present and validate a novel microfluidic capillary zone electrophoresis-mass spectrometry (mCZE-MS) method for characterizing size-based fragmentation variants of bispecific antigen-binding biotherapeutics.
- To demonstrate the applicability of mCZE-MS for analyzing therapeutic proteins at different manufacturing stages and under forced degradation conditions.
- To establish a new analytical approach for rapid and sensitive assessment of therapeutic protein integrity.
Main Methods:
- Utilized a microfluidic ZipChip® capillary zone electrophoresis (mCZE) system interfaced with mass spectrometry (MS).
- Analyzed bispecific antigen-binding biotherapeutic samples from downstream purification and forced degradation studies.
- Cross-validated results with denaturing size-exclusion chromatography-mass spectrometry (SEC-MS) and conventional reducing sodium dodecyl sulfate-capillary gel electrophoresis (rSDS-CGE).
Main Results:
- Successfully separated and characterized bispecific antigen-binding biotherapeutic fragments ranging from 12-40 kDa within 12 minutes.
- Achieved higher resolution and sensitivity compared to traditional liquid chromatography-mass spectrometry (LC-MS) methods.
- Demonstrated the method's effectiveness for analyzing therapeutic proteins under various conditions.
Conclusions:
- mCZE-MS is a powerful, rapid, and sensitive technique for characterizing therapeutic protein fragmentation.
- This novel method overcomes limitations of traditional techniques, enabling better quality control for biotherapeutics.
- The findings support the adoption of mCZE-MS for routine analysis of complex biotherapeutics like bispecific antibodies.
