Addressing Bottlenecks in Monoclonal Antibody Purification

While technology has improved the complexity and quantity of therapeutic monoclonal antibodies (mAbs) that can be cultured in parallel, simultaneous purification of many different antibody constructs during the early development phase has been a limiting step. Recently published research reviews a new protocol utilizing 96-well filter plate technology as a high-throughput solution.

Over the last two decades mAbs have come to play an increasingly important role in front line therapies for a range of disorders. Seven of the top ten drugs sold in 2018 were mAbs, with combined sales in excess of $120 billion⁽¹⁾. Just 35 years after the first mAb therapeutic was approved by the FDA these biologics now account for almost one fifth of new drug approvals each year. 2021 also marked an important milestone as the FDA approved its 100^th mAb based therapeutic⁽²⁾.

Therapeutic antibody production constraints and bottlenecks

However, it’s not all plain sailing, the manufacture of mAbs is a complex multistep process with two phases. An upstream phase, that includes all of the activities required to construct a new mAb and culture the desired quantities, and a downstream phase that includes all of the processes required to clean and purify the raw mAb-containing media into a pure, therapeutic-grade solution suitable for research use.

The rapid rise of therapeutic mAbs has driven an expansion of the tools and techniques for the creation and culture of mAbs, with advances in the complexity and quantity of therapeutic mAbs that can be created in parallel. However, this increase in upstream capacity has been frustrated by its reliance on traditional methods for downstream purification.

While this problem has been addressed for the commercial-scale manufacturing of individual mAbs through the use of continuous flow purification, this type of solution is not amenable to the development stage where large numbers of different mAb variants must be produced at small to intermediate scale.  These small volumes require a different approach to purification, one that can cope with the parallel processing of many different constructs at the same time.

Addressing the bottlenecks: A high-throughput protocol for antibody purification

In a recent 2020 publication⁽³⁾ Alan Matte, a Senior Research Officer with Human Health Therapeutics in Montreal, Canada, reviewed the current strategies and processes for high throughput, parallelized and automated purification of therapeutic antibodies, providing some excellent insights into the tools and techniques that can be used to address the downstream processing bottleneck.

Early-stage development often requires the production and purification of many constructs at the small to intermediate milligram scale in order to evaluate product attributes and identify manufacturing liabilities via panels of in vitro and in vivo assays. Speed, cost, and simplicity of operation are all important factors to consider in the development of a production workflow.

The author discusses the benefits of plate-based membrane chemistries, including the AcroPrep^TM Advance 96-well filter plates with Mustang^® Q membranes. At these small to intermediate scales, it is common to produce mAbs in 96-well culture plates, enabling the parallel production of high numbers of related constructs for evaluation. Given that upstream production is already utilizing a 96-well format, there are obvious advantages to utilizing the same format for the downstream purification process. Reducing time-consuming liquid handling steps to a simple transfer of media from the 96-well culture plate to a 96-well filter plate, an activity that is easily automated on any liquid handling robot.

By using a membrane to perform the purification step, rather than bead-based resins, 96-well filter plates provide a second significant advantage in that the mass transfer is convection rather than diffusion limited, resulting in much faster binding kinetics at higher flow rates.  Plates also enable the elution of products with a much smaller volume of elution buffer, resulting in a more concentrated sample prior to desalting or other post purification treatments. 

Taken together each of these small advantages add up to create significant savings in terms of hands on and total time required for downstream processing of therapeutic antibodies. The maintenance of the same 96-well format throughout upstream and downstream processing also reduces complexity and the risk of error or contamination. The application of 96-well filter plates to the downstream processing of therapeutic antibodies provides the means of addressing the purification bottleneck, enabling a higher rate of throughput and a greater ability to exploit the advantages of upstream capacity for the generation of multiple constructs in parallel.

Learn more about the AcroPrep Advance 96-well filter plates incorporating Mustang Q ion exchange membranes.