Preparative crystallization of a single chain antibody using an aqueous two-phase system

Area of Science:

• Biotechnology
• Chemical Engineering
• Protein Crystallization

Background:

• Process integration is crucial for efficient biopharmaceutical manufacturing.
• Simultaneous crystallization and separation can streamline downstream processing.

Purpose of the Study:

• To develop and demonstrate a simultaneous crystallization and aqueous two-phase extraction process for single-chain antibodies.
• To investigate the influence of polyethylene glycol (PEG) concentration on crystallization yield.

Main Methods:

• Utilized an aqueous two-phase system with sodium sulfate and PEG-2000 to induce simultaneous crystallization and extraction.
• Established a phase diagram to delineate zones for undersaturation, crystallization, and amorphous precipitation.
• Varied PEG concentration to assess its impact on crystallization yield.

Main Results:

• Achieved simultaneous crystallization and extraction of single-chain antibodies in a PEG-rich phase.
• Crystal nucleation occurred in the sodium sulfate-rich phase and at the phase boundary, with growth in the PEG-rich phase.
• Increased PEG concentration from 2% to 4% (w/v) boosted crystallization yield from approximately 63% to 87%.

Conclusions:

• Aqueous two-phase systems offer a viable strategy for integrated protein crystallization and extraction.
• PEG acts as an anti-solvent, significantly influencing crystallization yield.
• This integrated approach presents an opportunity to enhance bioprocess efficiency.