High-Performance Biopharma Analysis - Application compendium

Significance of the Topic

Modern biopharmaceuticals such as monoclonal antibodies, antibody–drug conjugates, peptides, glycans, and oligonucleotides demand highly reliable, biocompatible liquid chromatography platforms to monitor critical quality attributes and process parameters. The Agilent InfinityLab Bio LC Solutions provide a suite of bio-inert, high-performance LC systems specifically designed to withstand corrosive buffers, high salt concentrations, and sensitive biomolecules. Optimized for use from discovery to QA/QC, these platforms ensure robust, reproducible, and high-resolution analysis across diverse biopharma workflows.

Goals and Study Overview

This compendium presents a series of application case studies demonstrating the versatility and performance of InfinityLab Bio LC instruments. Applications include: chromatographic separation of mAb charge variants, aggregates, and fragments; characterization of peptides and glycan profiles; high‐resolution ADC drug‐to‐antibody ratio (DAR) determination; dual‐mode bioprocess analytics combining nutrient and mAb titer assays; and analysis of oligonucleotide building blocks. The objective is to showcase method robustness, method transferability, and superior chromatographic performance under challenging bioanalytical conditions.

Methodology and Instrumentation

All studies employed bio-inert LC flow paths (MP35N alloy) and high-speed binary/quaternary pumps to deliver precise solvent mixing, even with corrosive salts and organic modifiers. Key systems include: • Agilent 1290 Infinity II Bio LC with High-Speed Pump for high‐end biochromatography • Agilent 1260 Infinity II Prime Bio LC with Bio Flexible Pump for quaternary mixing and at-line analytics • Agilent 1260 Infinity II Bio-Inert LC for integrated nutrient/mAb analyses using a column-selection valve Detectors ranged from UV (variable‐wavelength and fluorescence) to MS (6545XT AdvanceBio LC/Q-TOF), enabling orthogonal detection and structural confirmation.

Main Results and Discussion

• Monoclonal antibody charge variants and glycan profiles were separated with high resolution and retention‐time reproducibility (RT RSD < 0.06%) using pH and salt gradients, including sub-1% B/min shallow gradients.
• Size‐exclusion chromatography on bio-inert UHPLC instruments resolved mAb aggregates and enabled accurate molecular‐weight determination via direct calibration and GPC/SEC software.
• Peptide mapping of reduced mAbs achieved >90% sequence coverage with UV and All Ions MS/MS, showcasing <0.04% RT RSD and seamless method transfer between Bio LC and standard LC systems.
• ADC DAR analysis by hydrophobic interaction chromatography (HIC) delivered reproducible quantitation of drug‐loaded species (DAR ~3.7) in high‐salt ternary gradients containing isopropanol.
• Dual bioprocess analytics combined hydrophilic interaction LC and protein A affinity LC in sequence on a single Bio-Inert LC using an automated column valve for nutrient profiling and mAb titer determination.
• Fast and robust HILIC methods for spent‐media analysis quantified polar nutrients with <1% LODs and R2 > 0.99 in Auto Acquire mode on LC/MSD iQ.
• Oligonucleotide raw materials (phosphoramidites) were analyzed in ternary gradients with superb RT and area precision (< 0.05% RT RSD, < 0.3% area RSD) and >66% runtime reduction.
• Cross‐platform method compatibility studies confirmed retention‐time deviations < 0.2% for isocratic and fast gradients; shallow gradients sensitive to minor solvent composition changes.

Benefits and Practical Applications

InfinityLab Bio LC Solutions deliver:

• High‐confidence data under harsh biochromatography conditions (high salt, extreme pH).
• Streamlined buffer preparation and method development via Buffer Advisor and quaternary mixing.
• Direct method transfer to QC environments using UV or MS detectors.
• Flexible multi‐dimensional workflows from at-line PAT to in-depth structural characterization.
• Reduced maintenance thanks to iron-free, biocompatible flow paths.

Future Trends and Possibilities

Emerging directions include:

• Integration of multi-dimensional LC (2D/3D/4D) with high-resolution MS for comprehensive biotherapeutic characterization.
• Further miniaturization and automation for high-throughput peptide/glycan mapping.
• Advanced PAT tools combining real-time LC analysis with process control systems.
• Expanding HILIC and mixed‐mode columns for complex biomolecule and metabolite profiling.
• Cloud-based data analytics and machine learning for deeper insights into chromatographic data.

Conclusion

Agilent InfinityLab Bio LC Systems represent a new standard in biocompatible, high‐performance LC, addressing critical needs across the biopharma workflow. With unrivaled solvent‐mixing precision, bio-inert flow paths, and flexible pump architectures, these platforms enable robust, reproducible analyses from raw-material screening to advanced peptide, glycan, and protein conjugate characterization, driving faster method development and reliable QC implementation.

References

1. Hughes, R.A.; Ellington, A.D. Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology. Cold Spring Harb. Perspect. Biol. 2017, 9, a023812.
2. Walsh, G. Therapeutic Glycoprotein Development. Biotechnol. Lett. 2015, 37, 213–220.
3. Yan, J.; Jones, A. Streamlined Workflows for N-Glycan Analysis of Biotherapeutics Using AdvanceBio Gly-X. Agilent Technol. App. Note 2019.
4. Stoll, D.R.; Schoenmaker, P.J. Liquid Chromatography of Biopharmaceuticals. Anal. Chem. 2019, 91, 210–234.
5. Beaucage, S.L.; Caruthers, M.H. Deoxynucleoside Phosphoramidites—A New Class of Key Intermediates for Deoxypolynucleotide Synthesis. Tetrahedron Lett. 1981, 22, 1859–1862.