Physicochemical Assessment Techniques in Biopharmaceutical Development: Biosimilars

by Malvern Panalytical, Tuesday, 17th August 2021

We present the items to be evaluated at each stage of biopharmaceutical development, along with the measurement technologies of Malvern Panalytical that enable these evaluations. This page provides details on “Biosimilars”.

A biosimilar must demonstrate equivalence and sameness with the innovator. Malvern Panalytical’s measurement technologies support equivalence and sameness evaluation across multiple parameters.

Equivalence Assessment Using SEC-LS (SEC-LS)

Comparative Case Study of Particle Size and Aggregates with Bevacizumab

Bevacizumab (Bevacizumab) is a monoclonal antibody against vascular endothelial growth factor, used as an anticancer agent. We conducted measurements under conditions where size exclusion chromatography (SEC) was connected to a light scattering detector (LS) for Bevacizumab (the innovator) and its biosimilar, comparing each signal. The RI signals revealed slight differences, but similar peaks were observed. It was confirmed that the light scattering signals (RALS) were able to detect the monomer and dimer similarly. However, the biosimilar showed a peak, indicating an aggregate not present in the innovator, suggesting distinct light scattering characteristics. These were details that could not be discerned with the RI signal alone, and by connecting multiple detectors to the SEC, a more detailed comparison between the innovator and biosimilar was possible.

As demonstrated, the use of SEC-LS allows for the evaluation of even subtle aggregate presence, undetectable by UV or RI, permitting discussions on the inclusion of biosimilars.

→Detecting differences in aggregate mixing between innovator and biosimilar using SEC-LS

Equivalence Assessment Using DSC (DSC)

Comparison of Higher Order Structure Stability with Remicade

The WHO presents guidelines for the quality and safety of biopharmaceuticals derived from recombinant proteins. A need to confirm higher order structures (HOS) is indicated, with differential scanning calorimetry (DSC) being one of the methods proposed.

The figure below compares whether Renflexis (left) and Inflectra (right), biosimilars of the anti-human TNF-alpha monoclonal antibody Remicade, possess equivalent higher order structure stability as the innovator. The measurement uses the same buffer under identical formulation conditions. Both datasets show comparable profiles to the innovator Remicade (solid line), but Renflexis deviates in profile around 68–75°C (highlighted region), indicating Inflectra’s higher equivalence.

This evaluation of biosimilars using DSC allows for equivalence assessments with the innovator by using the profile as a fingerprint for comparison.

→DSC detects fingerprint pattern differences between two biosimilars and the innovator

SVP Evaluation Using NTA (NTA)

SVP Comparison Between Innovator and Biosimilar

For biosimilars, it is preferable if the innovator’s aggregates (SVP) are more reduced.

The figure below shows the SVP analysis results for two types of antibody drugs, innovator and biosimilar, using nanoparticle tracking analysis (NTA). SVP content is typically analyzed using SEC, but its upper limit is about 100 nm. NTA characteristically covers submicron SVP. The detected concentration of SVP is higher for the innovator, suggesting differences in immunogenicity risk with the biosimilar.

Thus, NTA enables quantitative evaluation of SVP, which conventional methods like SEC could not achieve.

→Quantitative evaluation of SVP content differences between innovator and biosimilar using NTA

Comprehensive Stability Evaluation (DLS・SLS・ELS・DSC・NTA)

Comprehensive Comparison of Therapeutic Antibody Innovator and Biosimilar

Using Malvern Panalytical’s technologies, we provide an overview of evaluating the stability of antibody innovators and biosimilars through various methods. The concentration dependence of the diffusion coefficient (kD) in DLS results showed that the innovator’s diffusion coefficient decreases with increasing concentration, while the biosimilar’s increases. The DLS measurement data below indicate that the biosimilar differs in shape concentration-dependently compared to the innovator, with the particle size appearing smaller. This is likely influenced by the apparent diffusion coefficient increase due to intermolecular repulsion. These suggest the dispersion stability of the biosimilar is superior. Additionally, a correlation was observed between DLS T_agg results and DSC’s T_m results, indicating higher thermal stability in biosimilars as well. The differences observed in NTA’s C_total and SVP suggest multimer formation in solution. Therefore, compared to biosimilars, innovators likely form more complex multimers, according to multiple measurement results.

Thus, Malvern Panalytical’s technologies provide a comprehensive evaluation of biosimilar significance.