(T1430-03-16) Temperature Variations and Impurities Preclude a Reliable Measurement of the Diffusion Interaction Parameter for Monoclonal Antibodies

Purpose: Biologics have made tremendous scientific and clinical advancements in the treatment areas like cancers, autoimmune diseases, and other immune-related disorders. As a result, there is a rising interest in improving methods involved in the developability as well as quality assessment of therapeutic proteins, such as monoclonal antibodies. In this regard, studying the dynamics and nature of protein-protein interactions (PPI) is critical to understanding the association of proteins in a solution. A variety of methods exist for the analysis of PPI with the light scattering techniques being the most extensively utilized approaches. Dynamic light scattering (DLS) is a valuable tool for high-throughput light scattering employed in the pharmaceutical industry enabling scientists to investigate protein behavior (Houen, 2022). The diffusion interaction parameter measured by DLS (kD-DLS) is derived by evaluating the translational diffusion coefficient (DC) as a function of protein concentration. The kD-DLS serves to provide significant information on the attractive or repulsive nature of intermolecular interactions in a solution. Ideally, the kD-DLS values should quantitively capture the extent and strength of PPI in a reproducible fashion (Kingsbury, 2020). However, DLS experiments and the robustness of kD-DLS analysis (stability and reliability) depend on sample-related parameters (including purity, concentration, pH, buffer composition, etc.) and experimental parameters (temperature, preparation, and handling techniques, instrument quality, etc.). Despite this, studies examining the impact of such parameters on the validity and accuracy of the kD-DLS measurements through validation studies and control experiments are limited. The present work investigates the influence of temperature variations and the presence of impurities/contamination on the robustness of kD-DLS measurements using Tocilizumab and Trastuzumab respectively.

Methods: To examine the effect of temperature variations on kD-DLS, Tocilizumab was extracted and purified via the removal of polysorbate from the commercialized product (Actemra) and buffer exchanged with 10 mM, histidine buffer (pH 6.0) to prepare a stock solution of 10 mg/mL. This stock solution was diluted to nine final concentrations ranging from 1.0 to 5.0 mg/mL. Subsequently, the DLS experiments were performed by analyzing each individual sample using a DLS plate reader at seven different temperatures ranging from 20.0 to 26.0°C. The kD-DLS value was then derived by fitting a linear regression model to the DC values as a function of the Tocilizumab concentration. Furthermore, Trastuzumab was similarly extracted and purified via the removal of polysorbate from the marketed biosimilar (Herzuma). This was followed by buffer exchange with 10 mM, histidine buffer (pH 6.0) for the preparation of a stock solution with a concentration of 10.0 mg/mL. The stock solution was further diluted to nine final concentrations ranging from 1.0 to 5.0 mg/mL, which served as the control samples for the study investigating the impact of impurities on kD-DLS. To mimic particulate impurities such as aggregates and external impurities like dust, 1 µm polystyrene microspheres were spiked into the protein dilutions, resulting in a final concentration of 2.0 million particles/mL. Subsequently, the DLS experimentation was conducted using the DLS plate reader with the analysis performed at a temperature of 20°C. Finally, the kD-DLS value was derived by fitting a linear regression model to the DC, considering the Trastuzumab concentration as the independent variable.

Results: The average DC (n=3) increased with an increase in the measurement temperature (Figure 1) as well as an increase in the Tocilizumab concentration (Figure 2). A temperature variation of a mere 1°C led to an average DC difference of 1.40E-08 per 1°C as compared to an average DC difference of 5.76E-09 per 0.5 mg/mL of Tocilizumab concentration (across all measurements). Furthermore, the study examining the effect of impurities on kD-DLS revealed that a reliable fitting of DC values for kD-DLS evaluation was not possible in the presence of impurities (see the linear fit and R-squared values in Figure 3).

Conclusion: The present study confirmed that the temperature variations and the impurities/contaminants affect the reproducibility and consistency of kD-DLS analysis. Therefore, kD-DLS assessments can only be relied on if 1) proper quality control measures and rigorous protocols are in place to monitor and ensure temperature among other parameters and 2) the sample is free of aggregates and interfering impurities. Such controls are needed to enhance confidence in the findings related to the evaluation of monoclonal antibody candidates in developability and in formulation studies, and the overall quality of biologic development.

References: (1) Houen G, editor. Therapeutic antibodies: methods and protocols. Humana Press; 2022.
(2) Kingsbury JS. A single molecular descriptor to predict solution behavior of therapeutic antibodies. Science Advances. 2020.

Acknowledgements:Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R21AI178218. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Figure 1. Average Diffusion Coefficient vs. Temperature for 5 mg/mL Tocilizumab.

Figure 2. Average Diffusion Coefficient vs. Concentration at 26°C.

Figure 3. Comparison of Average Diffusion Coefficient vs. Concentration for (A) Herzuma (control) and (B) Herzuma spiked with polystyrene beads at 20°C.