(W1230-11-64) A Comparative Study of Agitation-Induced Aggregation of Monoclonal Antibodies via Horizontal Shaking and Orbital Shaking

Purpose: Therapeutic proteins like monoclonal antibodies (mAbs) have made substantial improvements in the treatment of diseases like cancers, rheumatoid arthritis, psoriasis, etc. Consequently, there is a continual interest in improving the stability of mAb formulations in the pharmaceutical and biotechnological industries. One major stability concern is protein aggregation, which is a common issue arising from different factors like oxidative, thermal, mechanical, and interfacial stresses. Aggregation, in turn, can result in a loss of efficacy and safety in protein products. In particular, mechanical and interfacial stresses occur at the various interfaces of a finished drug product throughout its lifecycle from the steps of manufacturing, transportation, storage, and clinical administration. For example, agitation incurred during shipping can lead to a mechanical rupture of the interfacial films of amphiphilic proteins formed via molecular adsorption to the interfaces and release aggregates into the solution (Eppler, 2010). In order to develop a high-quality drug product, the industry employs scale-down methods for the quick screening and comparison of the robustness of mAb formulations. Shaking methods are one of the widely used methods but there is an absence of a standard scale-down model guideline for shaking, and different product developers may use different parameters for shaking. The type of shaking, intensity of shaking, container size and type, and formulation characteristics influence the generation of protein aggregates, particularly subvisible particle formation (Morales, 2023). Despite this, studies examining the impact of such a host of parameters on aggregation arising via agitation through shaking are limited. This study aims to assess how variations in the experimental setup impact the outcomes of shaking stress experiments, with the primary parameter under investigation being the type of shaking. Accordingly, the present work compares the agitation-induced aggregation via horizontal shaking and orbital shaking using Cetuximab and Tocilizumab.

Methods: Initially, Cetuximab was extracted and purified from the commercialized product (Erbitux) and was buffer exchanged with phosphate-buffered saline (PBS) to prepare a stock solution of 10.0 mg/mL. Furthermore, Tocilizumab was similarly extracted and purified via the removal of polysorbate 80 from the marketed product (Actemra). This was followed by buffer exchange with PBS for the preparation of a stock solution with a concentration of 10.0 mg/mL. Both the mAb solutions were filtered and diluted to 0.5 mg/mL in PBS and 2 mL of the prepared sample was filled in the screw-top glass vials under laminar flow conditions. All the samples were subjected to a variety of accelerated stress conditions in triplicates using an orbital shaker as well as a horizontal shaker. The aggregation behavior of the mAb formulations during real-time production and post-production handling was simulated by shaking at a rate of 200 RPM at 25°C for 72 h. The contents of particles and monomers were analyzed by a variety of analytics including flow imaging microscopy, along with visual inspection to compare the effects of the two types of shaking stresses. Agitated samples were compared with the unagitated controls at different time points.

Results: At 𝑡 = 0 h, the unagitated Cetuximab samples exhibited 320 particles/mL as determined by flow imaging microscopy. After subjecting the Cetuximab samples to 72 hours of shaking stress, the average particle in the vials subjected to orbital shaking was 403 particles/mL, compared to 28,150 particles/mL in the vials subjected to horizontal shaking. This indicates that horizontal shaking induces a significantly higher formation of aggregate particles in Cetuximab compared to orbital shaking. On the other hand, the unagitated Tocilizumab samples exhibited 217 particles/ mL at 𝑡 = 0 h. Post 72 hours of shaking stress, the Tocilizumab samples showed an average of 293 particles/mL in the vials exposed to orbital shaking, whereas the vials exposed to horizontal shaking had an average of 213 particles/mL. The data demonstrates that, unlike Cetuximab, Tocilizumab is less prone to aggregate formation under horizontal shaking conditions.

Conclusion: The present study demonstrated that the choice of a laboratory-scale agitation method influences the extent of the aggregate generation, including the development of subvisible particles. In this instance, Cetuximab specifically exhibited more sensitivity to horizontal shaking, highlighting that relying solely on an orbital shaking stress setup would have overlooked these stability issues. Therefore, the comparative analysis of the two types of stresses confirms the necessity of selecting appropriate shaking stress tailored for each mAb in scale-down models employed in product development.

References: (1) Eppler A. Relevant shaking stress conditions for antibody preformulation development. European journal of pharmaceutics and biopharmaceutics. 2010.
(2) Morales AM. End-to-end approach to surfactant selection, risk mitigation, and control strategies for protein-based therapeutics. The AAPS journal. 2023.