(157a) Advancing Biopharmaceutical Manufacturing: Continuous Crystallization of Therapeutic Full-Length Monoclonal Antibodies

Monoclonal antibodies (mAbs) are dominating the biopharmaceutical market as a top-selling class of biotherapeutics. This is mostly due to their specific action and reduced immunogenicity [1]. The growing demand for mAbs is contradicted by the high manufacturing cost, which requires exploring more cost-effective and sustainable processes [2]. The downstream processing of mAbs traditionally relies on protein A chromatography followed by ion exchange or hydrophobic interaction chromatography for purification. Despite the high purification efficiency of protein A chromatography, additional steps are needed to meet biopharmaceutical standards, such as stability and bioactivity. Therefore, crystallization emerges as a cost-efficient purification strategy by reducing operational costs and facility footprint.

Crystallization processes for biomacromolecules are much less mature compared to small molecules due to their size, complexity, and dynamic behavior, which constitutes a challenge for the biopharmaceutical industry. For this reason, the first processes being employed were batch processes. Nowadays, these processes are relatively well-established and still the predominant technology. Batch-to-continuous transition of biopharmaceutical production has been continuously advancing and applied during the last decade to improve product quality attributes at a low manufacturing cost [3]. However, only a few studies have reported experimental methodologies to continuously crystallize biomolecules (i.e., model proteins: lysozyme, insulin).

This study highlights, to the best of our knowledge, the first integrated crystallization-based platform to crystallize full-length mAbs. The experimental apparatus comprises a series of three mixed suspension mixed product removal crystallizers (MSMPRCs) with a pressure-driven slurry transfer [4]. In addition, the MSMPRCs are equipped with process analytical technology (PAT) probes for real-time monitoring of different mAb crystallization processes, including in-line crystal size distribution (CSD), microscopy, and Raman spectroscopy. Complementary, off-line sampling consists of e.g., microscopy imaging and mAb concentration measurement (HPLC) to assess the quality and efficacy of the continuous crystallization process. Ultimately, needle-shaped crystals are produced and characterized using SAXS and Cryo-EM to ensure they are mAb crystals. Lastly, AI-based image analysis is implemented to obtain CSD over time, and mAb crystallization kinetics are quantified by Population Balance Modeling and considering a steady-state MSMPR model.

References

[1] M. Hong et al., Computers & Chemical Engineering, 2018, 110, 106–114.

[2] A. Shukla et al., Journal of Chromatography B, 2007, 848, 28–39.

[3] M. Sun Hong et al., American Pharmaceutical Review, 2020, 1–4.

[4] Y. Cui et al., Organic Process Research & Development, 2016, 20, 1276–1282.

Acknowledgements

This work was funded by FDA under the contract number 75F40121C00111.