Using bespoke downstream processing strategies for the purification of complex novel protein-based therapeutics

by Cintia Carreira
Associate Principal Scientist, Purification Development

Addressing the purification needs of increasingly complex novel therapeutics

Over the last several decades, the biopharmaceutical industry has established reliable platform purification processes for monoclonal antibodies (mAbs). Increasing knowledge of biology and disease pathways – combined with innovation in biomolecular design – has resulted in process development for many novel, protein-based formats that are more complex and present a new set of downstream processing challenges.

During my six years at Lonza, I have had the privilege and pleasure to apply my experience in biochemistry and chromatography techniques to the design of tailored purification solutions for:

• Bispecific antibodies
• mAb-like molecules 
• Fusion proteins
• New molecular formats (e.g non-Fc-containing molecules, enzymes, etc). 
  

The challenges for process development

One of the biggest challenges when developing purification processes for these novel molecular formats is their unique nature. This means there is often little to no information available in downstream processing literature.

In addition, for many of these more complex non-mAb molecules, no commercial affinity resins are available for use off-the-shelf. These formats can also be enriched in post transcriptional modifications (PTM), have very acidic or basic isoelectric points (pI), high levels of impurities such as host-cell proteins (HCP), and may have limited stability or be unstable under certain pH conditions.

As a result, bespoke processes must be developed. These frequently use unconventional approaches driven by data to meet the processes requirements. With rapid development to clinic there is an increasing need for faster timelines and therefore toolbox solutions are also often used during process development.

Our team at Lonza has the ability to perform high-throughput chromatography supported by appropriate high-throughput analytical methods as part of our toolboxes approaches. We focus on screening chromatography resins to ascertain product chromatography behavior and select the most promising operation conditions.

Rigorous downstream process development to achieve client goals

We start any project by discussing the molecule with the customer to understand the characteristics, including impurity profile (if available), aggregation risk and any other attributes that can influence downstream processing. Based on the information available an initial plan is proposed for development. This was the case for a recent process that we supported for a unique non-fc fusion protein. In particular, this molecule exhibited exotic characteristics like low acidic pI and unique post transcriptional modifications that were essential to maintain the potency and efficacy of the product.

Figure 1: Downstream processing strategy for new molecular formats

The Capture step underwent rigorous process development, with high-throughput screening of different anion-exchange resins allowing us to identify promising candidates. This is particularly important when an affinity resin is not an available option. Upon resin selection we performed a high-resolution gradient on ÄKTA FPLC with fractionation to generate extensive information on product quality and impurities. The gradient was then converted into an isocratic elution to facilitate operations at scale. At this point, robustness of the step was explored to improve yield and product quality, including optimizing wash conditions to remove additional HCP.

Evaluation of polishing steps started with a screening of various resin types, including hydrophobic interaction (HIC), mixed mode and ion exchange resins. Suitable high-throughput analytical methods were used to support the resin screen allowing the analysis of a high number of samples while maintaining a fast turnaround. Chromatography performance, impurity profile (e.g HCP) and product quality data were then used to construct the potential final purification process: assembling the puzzle based on intelligent process design and optimal flow.

These most promising polishing resins were further evaluated through high resolution gradients followed by conversion into isocratic elutions, promoting easier implementation and higher step reproducibility at scale. In particular, the HIC step developed showed impressive benefits for the process, evidenced a 3 LRV in HCP levels and reduction of HMWS to ≤1%. The last polishing step added additional robustness for impurity reduction (DNA, HCP and viral clearance) while improving molecule-specific product quality: the enrichment of the correct PTM characteristics using a high-resolution IEX resin with a carefully developed separation scheme.

By combining Lonza toolboxes with data driven and high-throughput approaches we enable the development of optimal, tailored purification solutions for new molecular formats within tight timelines. This process development work is always performed in close collaboration with the Manufacturing Science & Technology team so that development solutions can be easily implemented into the production plant.

Figure 2: Example of a high-resolution gradient converted into an isocratic elution

Achieving optimal downstream processing to help our customers bringing their molecules to the clinic

Lonza toolboxes and data driven approaches allow us to rapidly develop new downstream processes for our customers. Implementation of high-resolution gradients and novel design of experiments (DoE) into bespoke development processes brings enhanced optimization, robustness, and functionality to the purification process.

It is incredibly rewarding to develop new processes that can be successfully transferred into our manufacturing facilities and achieve the ultimate milestones of successful GMP batch release and IND approval. I know we are helping customers bring important new medicines to patients in need.

Need support with your own downstream processing projects?

Downstream process development can be a complex procedure involving several moving parts, especially when applied to the multitude of New Molecular Formats currently being developed throughout the industry. This can soon become quite overwhelming and costly if optimal strategies and knowledge are not applied. If you need support and guidance, don’t hesitate to reach out to a member of our team so that you may learn how we can support you in the process development lifecycle.