Cell Culture Optimization Using an Agilent Bio-Monolith Protein A Column and LC/MS

Importance of Topic

Monoclonal antibodies represent a leading class of biotherapeutics, and their glycosylation profiles critically influence efficacy, stability, and immunogenicity. Matching biosimilar glycoforms to originator products is essential for regulatory approval and clinical performance.

Study Objectives and Overview

This work aimed to optimize Chinese hamster ovary (CHO) cell culture conditions for a trastuzumab biosimilar, focusing on achieving an originator-equivalent glycosylation pattern. The study varied concentrations of galactose, uridine, and manganese chloride and employed an Agilent Bio-Monolith Protein A column with LC/MS analysis to assess antibody titer and structural attributes.

Instrumentation

Bio-Monolith Protein A Analysis

• Agilent 1100 Series Quaternary Pump, Autosampler, Diode Array Detector
• Agilent 1200 Infinity Series Analytical-scale Fraction Collector

LC/MS Analysis

• Agilent 1290 Infinity Binary LC (Binary Pump, Autosampler, Thermostat)
• Agilent 6540 UHD Accurate-Mass Q-TOF with Jet Stream

Software

• OpenLAB CDS ChemStation
• MassHunter for instrument control and data analysis
• BioConfirm for glycoform deconvolution

Main Results and Discussion

Protein A chromatograms showed distinct antibody peaks under varying feed conditions. Calibration with Herceptin demonstrated excellent linearity (0.02–2 mg/mL, R²=0.9999). High-resolution MS of reduced heavy chains identified six principal glycoforms (Man5, G0, G0F, G1F, G2F, G0F-GlcNAc). Originator batches exhibited lot-to-lot average G0F and G1F contents of 44.7±6.7% and 38.6±4.8%, respectively. Biosimilar cultures with increased galactose, uridine, and manganese showed elevated galactosylation (G1F:G0F ratio) up to 13.3% G2F at the highest feed. Antibody titers decreased from 0.47 mg/mL at 4× feed to 0.14 mg/mL at 24×.Optimal feed levels (4×–8×) balanced glycan profile alignment with acceptable yields.

Method Benefits and Practical Applications

This workflow integrates rapid Protein A capture with LC/MS for simultaneous quantification and glycoform profiling, streamlining clone selection and medium optimization. It ensures biosimilar quality attributes align with regulatory requirements and supports high-throughput bioprocess development.

Future Trends and Potential Applications

Future developments may include multi-attribute LC/MS assays combining intact mass, peptide mapping, and glycopeptide analysis for deeper characterization. Real-time online MS monitoring of glycosylation and automated feed strategies could further accelerate process development for novel antibodies and biosimilars.

Conclusion

The Agilent Bio-Monolith Protein A column coupled to LC/MS effectively guided CHO culture optimization, achieving trastuzumab glycosylation within originator specifications while maintaining viable antibody titers under intermediate feed conditions.

References

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