Case study: Effective removal of host cell proteins in high pI recombinant protein production

The production of complex recombinant proteins presents unique challenges, particularly when dealing with proteins with high molecular weight and high isoelectric points (pI). We recently undertook a project that exemplifies these challenges. Our task was to produce a recombinant protein characterised by a high molecular weight (> 200 kDa) and a pI around 9, necessitating a robust and meticulous downstream process (DSP) development.

The challenge: High molecular weight and Host Cell Proteins (HCP)

Our primary goal was to develop a three-step purification strategy that would efficiently purify the target molecule while minimising the presence of host cell proteins (HCP). Difficulties arise during the polishing step. Unidentified HCPs were persistently co-purified with the target protein, necessitating extensive optimisation efforts.

• Optimising Washing Conditions: The first step in overcoming the HCP challenge was to optimise the washing conditions during the initial capture step. We tested eight different washing conditions, adjusting variables such as pH, buffering salts, and additives like detergents and glycerol.
• Refining Loading Conditions: Loading conditions, including the pH, salt concentration, and flow rate, often play a critical role in HCP removal. However, in this project, none of the tested loading conditions led to a significant reduction in HCP levels. This highlighted the complexity of the protein and the necessity for further refinement in other purification stages.
• Enhancing Elution Strategies: Elution strategies were another focal point of our optimisation efforts. By adjusting the elution conditions and strategically collecting fractions, we aimed to separate the target protein from the HCPs more effectively.
• Screening Alternative Polishing Media: Polishing is a critical step in ensuring the final purity of the protein product. We conducted a comprehensive screening of alternative media to identify the most effective option for HCP removal.

Achieving Purity Through Rigorous DSP Optimisation

The successful production of a complex recombinant protein with a high molecular weight and challenging pI was achieved through a meticulous and iterative process of DSP optimisation. By fine-tuning washing conditions, refining loading and elution strategies, and innovatively rethinking the polishing steps, GTP Bioways was able to deliver a product that met stringent purity requirements.

This case study offers a detailed blueprint for tackling similar challenges faced by professionals in protein purification. The key takeaway is the importance of flexibility and innovation in purification strategies to overcome the inherent complexities of high molecular weight proteins.