INNOVATOR AND BIOSIMILAR INFLIXIMAB: COMPARABILITY ASSESSMENT OF THE HOST CELL PROTEINS AND PROTEIN HIGHER ORDER STRUCTURE

Significance of the Topic

Comparability studies of innovator and biosimilar monoclonal antibodies (mAbs) are critical to ensure safety, efficacy and regulatory compliance. Host cell protein (HCP) impurities and subtle variations in higher order structure (HOS) can impact immunogenicity, stability and therapeutic performance. Robust analytical tools are required to assess these critical quality attributes during biosimilar development and quality control.

Objectives and Study Overview

This work compares the HCP profiles and protein tertiary structures of an innovator infliximab and its biosimilar Inflectra. The goals were to identify and quantify residual HCP species using a two-dimensional liquid chromatography with data-independent high-definition mass spectrometry (2D-LC/HDMSE) workflow, and to probe conformational similarity by hydrogen/deuterium exchange mass spectrometry (HDX MS).

Methodology

The HCP analysis employed a high-pH/low-pH reversed-phase 2D-LC method on an ACQUITY UPLC M-Class system. Peptides were separated at pH 10 in the first dimension on an XBridge C18 column, diluted online, trapped on a C18 cartridge and eluted at pH 2.4 on a CSH C18 column in ten fraction steps. A SYNAPT G2-S HDMS instrument collected alternate low- and high-energy scans with ion mobility separation. Data were processed with ProteinLynx Global Server 3.0.2.
HDX MS experiments used three innovator lots and one biosimilar lot. Samples were deuterium-labeled for defined time points, quenched, digested online on an Enzymate column, and separated at 0 °C on a BEH C18 column. Deuterium uptake was measured on a Synapt G2-S HDMS platform, with DynamX 3.0 for peptide identification and uptake calculation.

Used Instrumentation

• ACQUITY UPLC M-Class system with 2D-LC technology
• SYNAPT G2-S HDMS mass spectrometer
• HDX-2 Automation with PAL RTC robots and Chronos timing
• Enzymate online digestion column and ACQUITY UPLC BEH C18 analytical column

Main Results and Discussion

Two HCPs—epidermal growth‐factor like protein 8 and WD repeat containing protein 37—were consistently detected in both products. Quantitative comparison revealed a 2–4-fold higher total HCP content in the biosimilar. HDX MS profiles demonstrated nearly identical deuterium uptake across complementarity‐determining regions and most framework segments. A minor difference was observed in the CH2 domain of the Fc region, likely reflecting glycosylation microheterogeneity or sample handling.

Benefits and Practical Applications of the Method

• High sensitivity and resolution enable low-abundance HCP detection and precise quantification.
• Two-dimensional separation improves peptide selectivity and coverage.
• HDX MS reveals site-specific conformational changes relevant to function and stability.
• Combined workflows support regulatory comparability requirements for biosimilar approval.

Future Trends and Potential Applications

Integration of ion mobility and advanced fragmentation will further enhance impurity profiling. Emerging software with machine learning may automate data interpretation and predict immunogenic hotspots. Expanded HDX MS coverage and real-time monitoring could accelerate biosimilar screening and formulation optimization.

Conclusion

This study demonstrates that a 2D-LC/HDMSE platform and HDX MS provide complementary insights into protein purity and structure. The biosimilar exhibited slightly elevated HCP levels but overall structural comparability to the innovator infliximab, supporting its quality and functionality. These analytical strategies are well suited for biosimilar development and quality assurance.

References

No formal literature list provided in the source document.