Rituximab Biosimilar Analysis Using the Agilent InfinityLab Pro iQ Plus LC/MS

Rituximab Biosimilar Analysis Using the Agilent InfinityLab Pro iQ Plus LC/MS — Expert Summary

Significance of the topic

Monoclonal antibodies (mAbs) are a dominant class of biotherapeutics with expanding clinical and commercial importance. As patents on originator antibodies expire, biosimilar development grows rapidly; regulatory approval requires rigorous analytical comparability to demonstrate no clinically meaningful differences in structure and critical quality attributes. Mass and glycosylation profiling are central to such comparability assessments. This report evaluates a compact, single‑quadrupole LC/MS platform for rapid intact- and subunit-level mass characterization of rituximab and two marketed biosimilars, demonstrating the instrument’s utility for routine QC and early development workflows.
Objectives and study overview

• Assess the capability of the Agilent InfinityLab Pro iQ Plus single‑quadrupole mass detector (m/z range up to 3,000) coupled to Agilent 1290 Infinity II Bio LC for intact and reduced subunit analysis of rituximab and two biosimilars.
• Compare observed molecular masses and glycoform distributions (G0F, G1F, G2F) between innovator and biosimilars to evaluate analytical comparability.
• Demonstrate practicality for QC-style applications where throughput, robustness, and cost-efficiency are important.

Methodology and workflow summary

Sample preparation

• Intact mAb samples: diluted from stock to 500 ng/µL in LC starting buffer; 1 µL injection.
• Reduced subunits: proteins denatured in 50 mM ammonium bicarbonate, 4 M guanidine HCl, 20 mM DTT, incubated 60 °C for 60 min, diluted to 250 ng/µL; 2 µL injection.

Chromatography and mass detection approach

• Reversed‑phase LC using PLRP‑S (2.1 × 50 mm, 5 µm) columns with formic acid in water and acetonitrile mobile phases; gradients adapted for intact and reduced/subunit separations; fast methods with short post‑run equilibration.
• Single‑quadrupole MS acquisition focused on high m/z windows to capture portions of the intact mAb charge envelope (intact: m/z 1,500–3,000; reduced: m/z 600–2,500) and collect stable averaged spectra for deconvolution.
• Data processing: Agilent OpenLab CDS v2.8 with spectral deconvolution (curve‑fit approach), comparison to theoretical average masses computed from NIST mass calculator.

Used instrumentation (key items reported in the study)

• Agilent 1290 Infinity II Bio LC (pump, multisampler, multicolumn thermostat)
• Agilent InfinityLab Pro iQ Plus mass detector (single‑quadrupole, G6170A)
• Agilent PLRP‑S analytical column (2.1 × 50 mm, 5 µm)
• Electrospray ionization with Agilent Jet Stream source; MS parameters tuned for high m/z sensitivity (capillary voltage ~4,500 V, nozzle voltage ~2,000 V, gas temperatures ~350–360 °C).
• OpenLab CDS software with built‑in deconvolution tools for averaged spectra.

Key results and discussion

• Intact mAb analysis
  • Partial capture of the intact charge envelope (m/z 2,000–3,000) still provided sufficient information to deconvolute intact masses and identify major glycoforms. Deconvolution revealed five dominant glycoforms for the innovator, four for biosimilar 1, and greater heterogeneity for biosimilar 2.
  • Most intact glycoform masses agreed with theoretical average masses within approximately –17.5 to +22.9 Da, corresponding to mass errors typically within ±100 ppm; only three glycoforms exceeded ±100 ppm, largely linked to complex lysine variants in biosimilar 2.
• Reduced subunit analysis
  • Reduction simplified spectra: light chains (~23 kDa) produced single, well‑defined peaks; heavy chains (~50 kDa) resolved the main glycoforms (G0F, G1F, G2F) and, in biosimilar 2, lysine‑variant species (HC + Lys).
  • Mass accuracy for subunits was improved relative to intact analysis, with observed errors generally between –62 and +28 ppm, demonstrating reliable assignment of LC and HC glycoforms.
• Comparability conclusions
  • Biosimilar 1 showed close alignment with the innovator across major intact and subunit glycoforms, supporting high structural similarity.
  • Biosimilar 2 displayed increased heterogeneity driven primarily by variable C‑terminal lysine truncation on heavy chains; each lysine difference produced ~128.2 Da shifts, producing multiple overlapping peaks that complicate deconvolution at unit‑mass resolution.
• Practical performance
  • The single‑quadrupole detector produced reproducible results with minimal sample prep and rapid run times, making it suitable for routine screening and QC tasks where high‑resolution MS is not mandatory.
  • Limitations include partial capture of the intact envelope (due to m/z upper limit) and lower resolving power versus high‑resolution instruments, which can complicate interpretation for heavily heterogeneous species.

Benefits and practical applications

• Fast, cost‑effective molecular mass verification for intact mAbs and reduced subunits, enabling routine comparability checks during biosimilar development and manufacture.
• Appropriate for lot release screening, clone selection, and early development QC where identification of major glycoforms and gross heterogeneity (e.g., lysine variants) is required.
• Minimal method transfer effort from higher‑resolution workflows; robust operation and straightforward data processing via OpenLab CDS facilitate adoption in regulated labs.

Future trends and potential applications

• Integration of single‑quadrupole LC/MS into hybrid workflows where it provides routine screening while high‑resolution MS is reserved for detailed characterization of ambiguous or complex samples.
• Improved deconvolution algorithms and expanded instrument m/z ranges could increase intact‑mass coverage and accuracy, reducing the need for subunit digestion in many cases.
• Automation of sample prep and standardized data processing pipelines to further increase throughput and regulatory readiness for biosimilar comparability programs.
• Application expansion to process‑related impurity screening and simplified glycoform monitoring in production environments.

Conclusions

The Agilent InfinityLab Pro iQ Plus single‑quadrupole LC/MS, coupled with the 1290 Infinity II Bio LC and OpenLab CDS, delivers a rapid and practical approach for intact and subunit mass analysis of rituximab and biosimilars. The platform reliably detected major glycoforms (G0F, G1F, G2F) and provided mass accuracy adequate for routine comparability tasks. Biosimilar 1 demonstrated close structural parity with the innovator, while biosimilar 2 exhibited additional heterogeneity attributable to variable C‑terminal lysine processing. The system’s compact footprint, cost profile, and simplicity make it well suited for QC, lot release, clone screening, and early biosimilar development, with the caveat that heavily heterogeneous species may still require high‑resolution MS for definitive characterization.
References

1. Dϋbel S., Reichert J. M. Handbook of Therapeutic Antibodies, 2nd edition.
2. U.S. Public Health Service Act, Section 351(k).
3. Rituximab information booklet. Versus Arthritis, 2022.
4. Kilpatrick E. L., et al. Expression and Characterization of 15N‑Labeled Human C‑Reactive Protein in Escherichia coli and Pichia pastoris for use in Isotope‑Dilution Mass Spectrometry. Protein Expression and Purification, 2012, 85, 94–99.
5. Agilent Technologies. Monitoring Antibody Glycosylation at Intact and Subunit Levels Using a Single Quadrupole LC/MS, Application Note 5994‑8345EN, 2025.
6. Agilent Technologies. A Comparative Study of the Intact Mass, Subunit Mass, and Released Glycans of Two Rituximab Biosimilars Using High‑Resolution LC/MS, Application Note 5994‑1653EN, 2024.
7. Hu Z., et al. Carboxypeptidase D is the Only Enzyme Responsible for Antibody C‑Terminal Lysine Cleavage in Chinese Hamster Ovary (CHO) cells. Biotechnology and Bioengineering, 2016, 113, 2100–2106.