Precipitation of non-therapeutic monoclonal antibodies with polyethylene glycol and zinc chloride: challenges and remarks from precipitation to resolubilization step

Abstract

Monoclonal antibodies (mAbs) have become fundamental across various sectors, with growing demands in both therapeutic and non-therapeutic applications. For the antibodies capture step, precipitation stands out as an alternative or precursor method to chromatographic techniques. However, research on unconventional precipitants, such as polyethylene glycol (PEG) and ZnCl₂, for diluted non-therapeutic mAbs is still scarce. This study evaluates the potential and challenges of combining PEG 6000 kDA and ZnCl₂ as precipitants to capture anti-human IgG₁ mAbs from highly diluted cell culture supernatants. We established mAbs solubility curves, identified optimal conditions for batch precipitation, fine-tuned the resolubilization step and conducted a viscosity analysis for the precipitates. We achieved 100% yield and 59% purity for the redissolution step, after using a 12% PEG 6000/3 mmol L⁻¹ ZnCl₂ precipitation condition with a 1:1 dilution ratio at pH 6.0 for resolubilization. Nevertheless, results also indicate that the resolubilization step potentially leads to mAb fragmentation and that protein aggregates might impact mAb viscosity and flow behavior. In conclusion, the combination of these agents is promising for mAbs capture, however, pre-concentration techniques are advised for severely diluted systems and caution toward ZnCl₂ is recommended, especially concerning the pH of the system and ZnCl₂ concentration.

Keywords:

• mAbs
• PEG
• precipitation
• purification
• rheology
• zinc

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the São Paulo Research Foundation (FAPESP, Brazil) [grant numbers 2019/08975-7, 2020/06538-6, 2020/12552-1, 2022/02807-8, 2022/03417-9, 2022/07322-2, and 2022/15751-0] and Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) [Finance code 001].