New Purification Techniques in Biopharmaceuticals

Biotherapeutics are produced using a multitude of technologies and vary depending on the indication. Monoclonal antibodies (mAbs) have been on the market for decades and have successfully treated cancers and autoimmune diseases. The use of viral vectors as gene therapies has recently gained attraction through new techniques, allowing the treatment of genetic disorders, infectious diseases and cancers. The use of new vaccine technology, such as messenger RNA (mRNA), has allowed the production of potent immune responses to mitigate infections, as seen with the COVID-19 pandemic.

Purification methodologies between different biotherapeutics vary drastically and, depending on the product, require a nuanced approach to enable or more efficiently allow for the purification of the product after upstream production. New technology and workflows are looking to enable the more efficient or cheaper purification of these products for downstream processing.  

Purification and processing of mAbs

The purification and downstream processing of mAbs is now relatively mature. "The platform technology is quite developed for mAb manufacturing. After production of the antibody by cell culture, the supernatant is captured by protein A chromatography and held at low pH for activation. Then follows a series of polishing steps through cation exchange or ionic exchange before the formulation of the final product," said Dr. Lukas Gerstweiler, lecturer in bioprocess engineering at the University of Adelaide, Australia. "I think the major challenges of today are to integrate the processes to work continuously as well as maybe finding ways to bring down the costs for production." 

Continuous bioprocessing poses unique challenges for antibody purification as a truly integrated line between upstream and downstream processing is still being investigated. "The past couple of years have seen the use of rapid cycling technology, which bolsters productivity," said Gerstweiler. Indeed, the use of protein A membrane adsorbers, however, has shown promise as an alternative to resin for process intensification. These absorbers eliminate the need for column packing, which lowers equipment cost, decreases fouling, pressure drops, challenging and stationary phase compression and has even been shown to decrease host cell protein contaminants.^1,2 However, it does come at the cost of increased buffer volumes. However, this technology can help move mAb processing from batch-based purification to semi-continuous purification.

Finding methodologies to decrease purification costs would also be helpful for less developed countries or companies looking to decrease the costs associated with downstream purification. One option is through precipitation in a coiled flow inverter reactor, leading to mAb capture by cation exchange multimodal chromatography that then polishes the protein in an ion exchange membrane.^3,4   

While mAb-based downstream processing has been around for decades and the purification modalities have matured, there is still a need for a better understanding of the processes to enable continuous processing and the nuances within the process to allow for better manufacturing economics.

Viral vectors for gene therapy

Downstream purification of viral vectors poses tough challenges due to technology limitations. While many different practices exist for their purification, they suffer from drawbacks such as poor scalability or inefficiencies in early purification steps, which make them cost-prohibitive due to yield losses.⁵

"The current state of the art for viral vector purification starts with density gradient centrifugation. The advantage of doing this is that not only can you purify your viral vector from everything else in your cell culture, but you can also separate empty and full capsids," said Dr. Caryn Heldt, director of the Health Research Institute, the James and Lorna Mack Chair in Continuous Processing and professor in the Department of Chemical Engineering at Michigan Technological University. "But the issue with this is that it is not feasible for commercial scale as it requires scaling out, meaning that more ultracentrifuges would be required to increase throughput. So, what can be done? Well, another approach is through chromatography, which works well on a lab scale but on the commercial scale, besides costs, it can cause issues as these chromatography columns produce large pressure drops which can affect purification."  

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However, there are new advances in purification technology that could alter the current purification paradigm. "The vast majority of the newer techniques are now in chromatography. A major focus is on new ligand development and resin design," said Heldt.

Affinity chromatography has not yet been developed for large-scale manufacturing. There currently exists only a handful of targeted affinity resins that are specific for only one type of virus serotype. To make the resins economically feasible, they must be reusable and bind to multiple viral serotypes. Some proprietary resins are currently being investigated and found to bind to multiple adeno-associated virus (AAV) serotypes.⁶ However, they still struggle with the copurification of empty capsids along with the product.

A recent approach being investigated by multiple groups is to functionalize peptides to chromatography columns to allow for universal binding of virus serotypes, namely "serotype-agnostic" resins.⁷ The production of these protein ligands is based on mimicking the anti-AAV antibody A20 by abstracting target peptide sequences that target regions of the capsid, which are highly conserved across stereotypes of various clades.⁸ These peptide-ligands have comparable binding capacity with commercial adsorbents but also reduce host cell protein contaminants and increase stability as they allow for AAV elution at physiological pHs instead of  acidic pHs.⁹ 

Where is the future of viral vector gene therapy going? "The two big things I would like to see in the future are continuous manufacturing and a better understanding of the viral vector when it is loaded with gene versus when it is not," said Heldt. Indeed, understanding the process in which these capsids bind to chromatography columns between full and empty columns would allow for more rational engineering of materials that would bias against the empty capsid products, whether this is done by engineering the resin material or the capsid itself.

Purification technology for mRNA vaccines

mRNA-based biotherapeutics are emerging as a promising technology for vaccine development. On both laboratory and manufacturing scale production, the use of chromatographic separation has been selected as a primary technique for purification based on selectivity, adaptability and scalability.¹⁰ The shift from more conventional protein-based therapeutic products towards a nucleic acid-based platform brings other challenges towards purification as the physiological properties, structure and even impurity composition vary drastically, requiring a selection of nuanced chromatographic resins. This also accounts for large inefficiencies that are present today in the downstream processing of mRNA vaccines.¹¹ Such inefficiencies are due to limitations in the stationary phases used to purify the therapeutic. Current methodologies rely on the use of proprietary polymer resin materials and the use of monolithic columns, typically based on size, charge, hydrophilicity and affinity.¹²

As mRNA is negatively charged, anion exchange chromatography is typically utilized to purify mRNA from impurities. The selectivity on the column is based on molecular weight and sequence and is prone to produce aggregates, which can be mitigated with denaturing agents or organic reagents – although this is not favorable for large-scale manufacture due to safety concerns. The approach to limit this concern is through multimodal ion exchange/hydrogen bonding chromatography. An approach by modifying a monolithic column, can enable a high yield of mRNA from in vitro transcription products, enabling purification independent of construct size or the poly-A tail of the vaccine.¹³ 

Affinity chromatography is another attractive option for purification. The use of oligo-dT affinity chromatography can capture the poly-A tail of mRNA transition through A-T pairing. This can effectively remove impurities such as the DNA template, nucleotide substrates, enzymes and buffer components. The immobilization of oligo-dT to an electrospun polymer nanofiber adsorbent has been shown to increase both the yield and allow for higher flow rates to decrease processing time.¹⁴  The largest criticism of oligo-dT-based chromatography is that it cannot distinguish single- and double-stranded RNA. Nevertheless, this technology has been used for the purification of SARS-CoV-2 mRNA.¹⁵ In reality, the processing of mRNA may require the use of both chromatography and membrane filtration. Membrane systems may be investigated further to enable continuous processing in the future.¹⁶

The processing of mRNA-based therapeutics is still inefficient, and a better understanding of the impurities and separation modalities is required for more efficient purification technology. This could be through size exclusion, ion exchange, hydrophilic interaction, affinity interactions or another modality.