Streamlining mAb Purification
With many biologics patents soon to expire, can multimodal chromatography meet the growing demand for efficient downstream biosimilar production?
There are many biologics patents in the US and EU that expire within the next two decades, paving the way for the development and approval of more affordable biosimilars. Consequently, there is high demand for solutions that will deliver efficient, flexible, and cost-effective biosimilar production.
Downstream processes for monoclonal antibody (mAb)-based biosimilar purification begin with a protein capture step, which typically involves a Protein A resin, followed by multiple polishing steps to remove remaining product and process-related impurities. Protein A resins are characterized by high specificity and affinity towards mAbs, resulting in highly purified end-products. However, there are limitations associated with using Protein A resins; for example, high production costs, which can be 50 percent higher than other types of chromatographic media (1). In addition, Protein A resins do not discriminate between functional and aggregated mAbs and they can leach into the purified sample during the acidic elution step.
The nature and downstream sequence of the polishing steps after protein capture depend on the individual protein and the types of impurities that need to be removed prior to product isolation, such as host cell DNA, endotoxins, viruses, and protein aggregates. The polishing steps consider the physicochemical properties of solutes, and can include anion-exchange chromatography followed by hydrophobic interaction chromatography.
Single-mode chromatographic processes are suboptimal for large-scale biosimilar manufacturing. The industry needs a faster, more cost-effective purification process that can remove a range of impurities. And so multimodal (or mixed-mode) chromatography has emerged as a highly selective yet robust method for target protein purification. This technique combines multiple interaction modes between the stationary (resin) and mobile (solute) phase in a single purification step. Specifically, mixed-mode resins use ligands capable of at least two modes of interaction, such as ion-exchange, affinity, size exclusion, and hydrophobic interactions. Combining these interactions purifies proteins more optimally than single-mode techniques – even when used sequentially.
Importantly, the use of multiple interaction modes and simplification of the purification process could bring down production costs, speed up the manufacturing process, increase yield, and increase protein activity. For example, combining the properties of hydrophobic interaction and anion-exchange media in a single hydrophobic anion-exchange resin can simultaneously purify and recover both basic and acidic mAbs in bind-elute or flow-through modes. Hydrophobic cation-exchange resins – which have a higher affinity for full-length mAbs compared to process impurities and by-products – can be used to achieve optimal purification and recovery during intermediary and final polishing steps.
Hydroxyapatite chromatography (HAC) can also help accelerate downstream process purification at a reduced cost and can be used at any stage – from initial capture to final polishing. The mixed-mode support of HAC can separate molecules based on calcium affinity and/or cation-exchange interactions. But remember that different HAC resins offer better binding capacity for different molecules (2).
In short, mixed-mode resins offer the following key benefits: i) separation of molecules that appear homogeneous, ii) selective single-step removal of impurities, iii) determination of optimal binding and elution conditions thanks to the large design space, iv) undiluted feedstock column loading at high conductivity thanks to robust salt tolerance, and v) high capacity for high-titer feed-streams.
The implementation of multimodal technologies for downstream purification during the development and manufacturing of biosimilars could accelerate the time to market and help to ensure therapeutic efficacy and patient safety, whilst using less resources and increasing cost efficiency. And when it comes to competition between equally safe and efficacious biosimilars – cost is king!
- F Fiedl et al., “Model based strategies towards protein A resin lifetime optimization and supervision,” Journal of Chromatography A, 1625, 461261 (2020). DOI: 10.1016/j.chroma.2020.461261
- A Kumar et al., “Design and Validation of Linkers for Site-Specific Preparation of Antibody-Drug Conjugates Carrying Multiple Drug Copies Per Cysteine Conjugation Site,” Int Journ Mol Sci, 21, 6882 (2020). DOI: 10.3390/ijms21186882
Sharon Bola is Global Product Manager, Process Chromatography, at Bio-Rad Laboratories, USA