(427a) A Precipitation-Based Purification Process of an Aflibercept Biosimilar

Neovascular age-related macular degeneration (wet AMD) can cause severe vision loss. A crucial role in wet AMD is played by the vascular endothelial growth factor (VEGF). Aflibercept is a drug based on VEGF inhibition, which has been utilized effectively for the treatment of wet AMD.¹ The anti-VEGF capability of aflibercept also makes it potentially an effective therapy during the metastatic state of colorectal cancer, which is among the most prevalent cancer types globally.² The Aflibercept market was estimated to be over USD 8,300 million in 2022 and is expected to rise to around USD 14,000 million by 2030.³ Aflibercept is a monoclonal antibody (mAb)-like recombinant fusion protein with the Fc region of human IgG1.⁴ In a typical mAb or Fc-fusion protein manufacturing process, affinity chromatography is a commonly employed technique for the initial capture of the protein from the upstream cell culture harvest. The widely used Protein-A columns typically result in over 90% purity in a single step due to high selectivity towards the Fc region.⁵ However, despite this advantage, affinity chromatography is typically associated with a high cost, posing a bottleneck in scaled-up manufacturing.⁶ Consequently, the development of alternative purification technologies is needed. Precipitation is a simple, low-cost, and scalable alternative that has been successfully demonstrated for the purification of mAbs in several studies, where the key aspects of mAb precipitation, such as mechanistic understanding,⁷ precipitant optimization,⁸ process development,⁹ and scale-up¹⁰ are addressed. Developing a precipitation-based purification process for a given mAb is still a challenging task, as precipitation conditions are highly specific to the molecule of interest and general guidelines are lacking. These precipitation conditions are typically identified from micro-scale, high-throughput screening experiments, from which crucial process-related information such as the final mAb recovery or the induction time cannot be extracted. Process throughput is adversely affected by a lengthy induction time, which can be particularly significant when a precipitation process is operated under relatively low precipitant concentrations to avoid likely impurity precipitation under higher concentrations. Case studies on the early process development when translating high-throughput screening results into precipitation behavior under process-like conditions of commercially relevant therapeutic proteins like aflibercept are limited, which complicates their industrial adoption. Furthermore, despite its significance, induction time has never been systematically studied for mAb precipitation to the best of our knowledge. The objective of this work is to develop and optimize a precipitation-based purification process principle of an aflibercept biosimilar under process-like conditions in terms of recovery, nucleation kinetics, and selectivity. Precipitation experiments are conducted across multiple scales ranging from several microliters to several hundred microliters to translate high throughput screening results to more process-like, stirred platforms. The impact of precipitant concentration and stirring rate on precipitation induction time is studied using in-line turbidity measurements.

First, 98 conditions differing in the type of precipitants, salts, and pH levels, which commonly have led to the crystallization of other bio-macromolecules, were investigated in hanging drop vapor diffusion experiments at room temperature with a stock solution of the aflibercept biosimilar (~8 mg/mL) for their precipitation propensity. About half of the conditions from the initial screening showed some degree of precipitate formation. The particles were too small to confirm crystal formation microscopically. Polyethylene glycol 4000 (PEG4000) and ammonium sulfate were considered the most promising types of precipitants, given that they yielded clearly distinguishable precipitate particles when observed under the microscope, regardless of the pH or the other salts in the precipitant solution. Second, those conditions that yielded larger precipitates were further optimized by varying the aflibercept and precipitant (PEG4000 and ammonium sulfate) concentrations and pH. For all these conditions, precipitate particles with a similar shape and sizes compared to the initial screen were obtained. Although solid-state form could not be determined, the particles appear amorphous-like, indicating aflibercept has a low tendency to form large crystals under the tested conditions. Third, precipitation experiments were carried out in closed systems at two larger scales (50 µL and 600 µL) at room temperature within a range of precipitant concentrations (6.25-15.0 % (w/v) and 2.0-4.0 M for PEG 4000 and ammonium sulfate, respectively). The 50 µL experiments were conducted under stagnant conditions, while the 600 µL experiments were conducted with stirring (magnetic bar, 150-500 RPM) and in-line turbidity measurements to quantify inductions (Crystal16, Technobis Crystallization Systems). The precipitate was separated with centrifugation and redissolved in a fresh buffer to assess the recovery. The aflibercept concentrations in the supernatant and in the redissolved buffer solution were determined spectrometrically. The supernatant aflibercept concentrations after 24 hours in both 50 µL and 600 µL experiments showed a gradual decrease with the precipitant concentration, as expected. The supernatant concentrations continued to drop for several days in 50 µL (unstirred) experiments, indicating that 24 hours was insufficient to reach equilibrium in the unstirred systems due to slow kinetics, similar to observations made for precipitation of mAbs by others.¹¹ A near-complete precipitation of aflibercept could be achieved when the final PEG4000 and ammonium sulfate concentrations are around 15.0 % (w/v) and 2.0 M, respectively. However, only 41% of aflibercept precipitated with PEG4000 could be recovered, possibly due to the formation of small, liquid-like droplets or colloidal particles that were difficult to separate centrifugally. On the other hand, aflibercept precipitated with ammonium sulfate could be recovered up to 89%, indicating that ammonium sulfate produces an easily separable protein-rich precipitate and, therefore, is a promising precipitant for the purification of the aflibercept. In 600 µL (stirred) experiments, indiction times were substantially long under low ammonium sulfate concentrations, while increasing the precipitant concentration from 1.40 to 1.45 M caused the induction time to drop drastically from several hours to several minutes. Precipitation became instantaneous when the ammonium sulfate concentration was higher than 1.50 M. Final supernatant aflibercept concentrations suggested that the introduction of stirring increased precipitation rates substantially compared to unstirred conditions due to increased nucleation and growth rates. At a 1.7 M ammonium sulfate concentration and 500 RPM, 85 % of the initial aflibercept amount was recovered within 3 hours. Finally, the selectivity of the chosen precipitation conditions was investigated by mixing precipitate solutions with a cell lysis solution, which could mimic typical impurities in a cell culture harvest. The cell lysis solution did not show precipitation within the ammonium sulfate concentration range investigated for aflibercept precipitation (2.0-4.0 M), indicating a good potential high selectivity. Overall, the process conditions identified in this work for the precipitation of an aflibercept biosimilar show good promise for an intensified purification process with high yield and purity. The future work will focus on the selective precipitation of aflibercept from a mixture of impurities representing an actual cell culture harvest under process-like conditions and assessing the final product purity.

Acknowledgment

This research is funded by the Innovation and Technology Commission of the Hong Kong Special Administrative Region, People’s Republic of China, under the Innovation and Technology Support Programme (Project No. ITS/009/21).

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