(M1030-01-04) Impact of Handling and Transfer on Aggregation of a Trastuzumab Biosimilar in an In-Use Study

Purpose: Biologics have attained remarkable progress in scientific research and clinical applications, revolutionizing the treatment of cancers, autoimmune conditions, and inflammatory disorders. Accordingly, there is a sustained focus within the pharmaceutical and biotechnology industries on improving the stability of monoclonal antibody (mAb) formulations. Protein drug products (DP) are greatly susceptible to physical and chemical degradation during post-production handling, leading to the generation of protein aggregates. This presents a significant stability challenge as aggregates can subsequently lead to reduced efficacy and compromised safety of biologics and biosimilars. Protein stability can be compromised by multiple handling steps including reconstitution, syringe transfer, and infusion bag preparation of protein therapeutics intended for intravenous (IV) administrations. Practices in a hospital like vigorous agitation, repeated syringe plunger movements, and improper IV bag handling introduce environmental stresses like shear forces, mechanical shocks, and exposure to interfaces exacerbating the challenges of maintaining DP stability (Nejadnik, 2018). Expelling protein therapeutics from syringes by pushing down the plunger leads to sweeping of the adsorbed protein on the syringe barrel surface resulting in greater aggregation (Yoneda, 2021). Multiple loading and unloading of the syringe and pooling of product from multiple vials is practiced in clinical trials for investigational mAbs while the impact of the procedure on the quality of product has not been reported. In this regard, this study aims to evaluate the impact of such handling and transfer processes on the aggregation of a trastuzumab biosimilar, addressing a knowledge gap for stability in clinical use.

Methods: Trastuzumab was extracted and purified by removing polysorbate from the marketed biosimilar (Herzuma) and buffer exchanged with ultrapure water to prepare a stock solution of 10 mg/mL. The mAb solution was then filtered and diluted to 1 mg/mL in 0.9% sodium chloride (NaCl) solution under laminar flow conditions. Subsequently, 2.0 mL of the prepared biosimilar solution was drawn into a 3.0 mL syringe equipped with an 18G x 1-1/2" (1.27 mm x 38 mm) hypodermic needle. Then, the plunger was pulled back and pushed to its original position to apply stress to the solution. This cycle of drawing and stressing was repeated 15 times, after which the stressed solution was collected into a screw-top glass vial for further analysis. A second type of syringe transfer and stress method involved drawing the solution into the syringe, applying stress by pulling and pushing the plunger, and then transferring the solution back into the vial. The solution was then drawn again into the syringe, and this cycle of drawing, stressing, and transferring was repeated 15 times. The final stressed solution was subsequently collected in a screw-top glass vial for analysis. The samples were analyzed using a wide variety of analytics including flow imaging microscopy (FIM) and high-pressure size exclusion chromatography (HPSEC), to evaluate the effects of the syringe stresses. Additionally, the syringe-stressed samples were compared with the unstressed samples serving as controls.

Results: As measured by FIM, the unstressed Trastuzumab samples contained 246.61 ± 106.90 particles/mL. After subjecting the samples to the first type of syringe stress with drawing and stressing, the particle count increased to 5,902.01 ± 3,426.92 particles/mL. In comparison, the second type of syringe stress, which involved a cycle of drawing, stressing, and transferring, resulted in a higher particle count of 78,295.73 ± 21,564.31 particles/mL. Besides, the HPSEC analysis indicated that the percentage of the high molecular weight species (HMWS) in the unstressed samples was 0.42 ± 0.01. The %HMWS increased to 0.53 ± 0.01 for the first type of syringe stress (drawing and stressing), whereas the %HMWS increased further to 0.74 ± 0.13 for the second type of syringe stress (drawing, stressing, and transferring).

Conclusion: The present work highlights the differences between the two types of syringe stresses, resulting in increased aggregates from the DP. Thus, this in-use study highlights the necessity of rigorous handling studies and optimized transfer protocols when pooling products from different vials to ensure the stability of protein therapeutics. These findings may offer actionable insights to improve the safety and efficacy of biologics and biosimilars during clinical use.

References: (1) Nejadnik MR. Postproduction handling and administration of protein pharmaceuticals and potential instability issues. Journal of pharmaceutical sciences. 2018.
(2) Yoneda S. Influence of protein adsorption on aggregation in prefilled syringes. Journal of Pharmaceutical Sciences. 2021.