Seamless Method Transfer from an Agilent 1260 Infinity Bio-inert LC to an Agilent 1260 Infinity II Bio-inert LC

Significance of the topic

Biopharmaceutical monoclonal antibodies can exhibit minor charge differences that influence their stability, efficacy and immunogenicity. Accurate profiling of charge variants is essential for quality control and safety assessment of both original biologics and biosimilars.

Study Objectives and Overview

This application note focuses on (1) comparing the charge variant profiles of innovator and biosimilar rituximab and (2) demonstrating seamless method transfer between two Agilent bio-inert liquid chromatography platforms: the 1260 Infinity and the 1260 Infinity II systems.

Methodology

A weak cation exchange chromatography method was optimized using dynamic gradient mixing. Phosphate buffer gradients spanning pH 6.2 to 7.2 were generated by Agilent Buffer Advisor software to find the ideal separation at pH 6.8. Key chromatographic parameters included:

• Gradient: 0–200 mM NaCl in 30 mM sodium phosphate buffer at pH 6.8
• Flow rate: 0.25 mL/min, injection volume: 2 µL
• Column temperature: 25 °C, sample temperature: 10 °C
• Detection: diode array at 280 nm

Instrumentation

Two bio-inert LC systems were employed: the Agilent 1260 Infinity and the Agilent 1260 Infinity II, each equipped with quaternary pumps, multisamplers with cooling, thermostatted column compartments, and diode array detectors. Data acquisition and control were managed by Agilent OpenLAB CDS software integrated with Buffer Advisor.

Main Results and Discussion

Dynamic pH scouting identified pH 6.8 as optimal for resolving rituximab charge variants. Both innovator and biosimilar samples yielded similar elution patterns, while the biosimilar exhibited higher intensity in certain basic variant peaks, previously assigned to C-terminal lysine forms. Method transfer between the two LC platforms produced retention time deviations below 0.5 % and demonstrated high precision with relative standard deviations below 0.15 % for retention times and below 0.7 % for peak areas.

Benefits and Practical Applications of the Method

• Enables reliable comparison between innovator and biosimilar products
• Simplifies buffer preparation through dynamic mixing, reducing manual steps
• Allows laboratories to modernize instrumentation without revalidating existing methods
• Supports routine quality control of biotherapeutics

Future Trends and Applications

Advances in automated buffer optimization, integration with multidimensional LC-MS workflows and application of machine learning for method development will further streamline charge variant analysis. Continuous manufacturing and real-time monitoring of biopharmaceutical attributes are emerging areas that can leverage these technologies.

Conclusion

The study confirms that a dynamically mixed phosphate buffer gradient at pH 6.8 effectively separates rituximab charge variants and that methods can be transferred seamlessly between Agilent bio-inert LC platforms, maintaining high precision and consistency.

References

1. ICH Topic Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.
2. Roy P.S. et al. Comparison of efficacy and safety of Rituximab (MabThera) and its biosimilar (Reditux) in diffuse large B-cell lymphoma. Indian J. Med. Paediatr Oncol. 2013;34(4):292–298.
3. Agilent 1290 Infinity with ISET User Manual, Agilent Technologies, 2015.
4. Schneider S., Strassner J. Optimizing protein separations with cation exchange chromatography using Agilent Buffer Advisor. Agilent Technologies Application Note 5991-0565EN, 2012.
5. Schneider S. 2D-LC/MS Characterization of Charge Variants Using Ion Exchange and Reversed-Phase Chromatography. Agilent Application Note 5991-6673EN, 2016.