Fast and scalable purification of a therapeutic full‐length antibody based on process crystallization

Abstract

The potential of process crystallization for purification of a therapeutic monoclonal IgG1 antibody was studied. The purified antibody was crystallized in non-agitated micro-batch experiments for the first time. A direct crystallization from clarified CHO cell culture harvest was inhibited by high salt concentrations. The salt concentration of the harvest was reduced by a simple pretreatment step. The crystallization process from pretreated harvest was successfully transferred to stirred tanks and scaled-up from the mL-scale to the 1 L-scale for the first time. The crystallization yield after 24 h was 88-90%. A high purity of 98.5% was reached after a single recrystallization step. A 17-fold host cell protein reduction was achieved and DNA content was reduced below the detection limit. High biological activity of the therapeutic antibody was maintained during the crystallization, dissolving, and recrystallization steps. Crystallization was also performed with impure solutions from intermediate steps of a standard monoclonal antibody purification process. It was shown that process crystallization has a strong potential to replace Protein A chromatography. Fast dissolution of the crystals was possible. Furthermore, it was shown that crystallization can be used as a concentrating step and can replace several ultra-/diafiltration steps. Molecular modeling suggested that a negative electrostatic region with interspersed exposed hydrophobic residues on the Fv domain of this antibody is responsible for the high crystallization propensity. As a result, process crystallization, following the identification of highly crystallizable antibodies using molecular modeling tools, can be recognized as an efficient, scalable, fast, and inexpensive alternative to key steps of a standard purification process for therapeutic antibodies.

Current Trends and Advances in Bulk Crystallization and Freeze-Drying of Biopharmaceuticals

“…Hekmat and coworkers describe the successful transfer of vapor diffusion-based crystallization to agitated batch crystallization at milliliter scale using lysozyme as a model protein [19]. The same research group also describes a stirred-tank crystallization-based approach [53] towards successful scale-up of batch crystallization conditions using lysozyme, lipase [18], and therapeutic mAbs [17,52] as model proteins. Smejkal et al describe a maximum local energy dissipation-based scale-up approach for stirred crystallization using lysozyme and Canakinumab Fab-fragment as model proteins.…”

“…Crystallization happens to be a relatively mature industrial processing technique with several applications. Several studies describe successful purification and polishing of proteins such as aprotinin [42], mAbs [17,52], and lysozyme [19,24,25] by bulk crystallography approach. 4.…”

Technology advancements in antibody purification

“…39,40 Crystallization is a highly useful tool as it may aid concentration, purification, and stabilization of the antibody product, without the need for affinity tags or affinity tag removal. 1,41,42 Advancements in these methodologies will be discussed in greater detail in the "Non-chromatographic methods" section.…”

“…Smejkal et al 41 recently purified a therapeutic full-length mAb using a crystallization-based process. A purification yield of 88%-90% was obtained with 98% purity.…”

“…The crystallization method was also successfully scaled-up from 6 mL to 1 L and was found to be reproducible. 41 …”