Choosing the Right Resins for Viral Vector Affinity Chromatography

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BioPharm International, BioPharm International, January 2023, Volume 36, Issue 01

Affinity chromatography resins must perform well under mild elution conditions yet withstand robust cleaning and sanitization protocols.

Affinity chromatography is a powerful separation technique employed to capture targets from complex and challenging feed streams, which makes it ideal for use in the purification of viral vectors. Most viral-vector harvest fluids, however, contain low titers of viral vectors and comparatively high impurity levels, and that places significant demands on affinity-resin performance. The sensitivity of viral vectors and their variability with respect to size and other characteristics adds to the challenges associated with affinity chromatography process development, including selection of the right chromatography matrices (resin/monoliths/membranes/fibers) and ligands.

Sensitivity and size issues

Unlike traditional biologic drug substances, including recombinant proteins and antibodies, viral vectors can be highly sensitive to high- and low-pH levels, high- and low-salt concentrations, and high-shear environments.

“This instability is a big differentiator for viral vectors and presents some challenges when it comes to developing effective affinity ligands for use in the production of affinity-chromatography resins,” observes Ian Scanlon, a subject matter expert for cell and gene therapy at Astrea Bioseparations. More specifically, affinity ligands are required to have a good binding behavior toward the target viral vector, and at the same time, have relatively mild elution conditions to avoid a pH shift that could result in loss of infectivity of the viral particles during the process, according to Piergiuseppe Nestola, manager of process technology consultants with Sartorius.

In addition, different types of viral vectors have different sizes spanning from 20 to 200 nm and even higher for some oncolytic viral vectors, according to Nestola. “Viral particle size affects the dynamic binding capacity (DBC) of an affinity resin in an inverse relationship. Thus, for larger viral vectors, larger-column affinity-chromatography systems are needed, leading to an increase in the overall cost of the unit operation,” he says.

Some viral vectors, particularly adeno-associated viral (AAV) vectors, are generally formed as mixtures, with some containing the full genetic material and others containing none, partial or even incorrect (e.g., host-cell) DNA, according to Laurens Sierkstra, business segment leader in the BioProduction Group at Thermo Fisher Scientific.

Sanitation and cleaning challenges

The size and sensitivity of some viral vectors also creates issues for bioburden removal, particularly viral clearance, as typically 0.22 µM filtration cannot always be employed, adds Scanlon. “As a result, the importance of cleaning and sanitization of adsorbents used for affinity capture increases,” he notes. That can be problematic, as some of the proteins and peptides used as affinity ligands are not compatible with sanitization-in-place (SIP) protocols.

Limited commercial offerings

Generally, affinity chromatography for viral vectors can only be performed when commercially viable ligands are available, Nestola comments. Until recently, only ligands designed for use in affinity-chromatography resins for adeno-associated viral (AAV)-vector purification have been available. New resins for purification of lentiviral (LV) vectors are now on the market (vide infra).

One challenge to commercialization of affinity-chromatography resins relates to the size of viral vectors relative to the pore sizes of commercially available adsorbents, according to Scanlon. “Current offerings are often designed for optimal processing of antibodies—usually around 10–15nm. Many viral vectors are larger (adenovirus at ~90–100nm, gamma retrovirus and lentivirus at ~80–100nm) and thus excluded from accessing binding surfaces within pores. As a result, implementing bead-based approaches for these larger targets may yield lower capacities,” he explains.

Another difficulty is that affinity resins often must be specific for a given viral vector (LV vs. AAV vectors, for instance), and in the case of AAV, specific serotypes (AAV2, AAV8, AAV9, etc.). LV vectors are enveloped viruses, while AAV vectors are non-enveloped viruses. “In general, non-enveloped viruses are more stable and resistant than enveloped viruses, which means that from a chromatography condition point of view, elution conditions and shear stress for LV vectors are a much bigger issue than AAV,” Sierkstra notes. In addition, while empty/full capsid ratios are an issue with AAV vectors, for enveloped viruses like LV vectors, the production of a plethora of non-infective lenti resembling particles (e.g., exosomes) presents purification difficulties, she adds.

“That means the application of such resins is less flexible, and it is difficult for viral vector manufacturers to build platform-purification processes for a range of vectors using a single affinity-chromatography resin,” Nestola says.

In addition, while both AAV serotype-specific and non-specific affinity-chromatography products are available on the market, not all serotypes bind well to affinity ligands. “For each serotype, therefore, optimization and screening should be performed to find the best affinity ligand/resin for the specific serotype,” Nestola notes.

Complicating the picture is the constant evolution of the viral-vector field, according to Nestola. For instance, he points to the ongoing development of new AAV serotypes, or chimera serotypes, that do not bind to existing off-the-shelf affinity ligands. For this reason, Scanlon observes that manufacturers are moving away from the current affinity approaches and returning to a platform charge-based approach (as outlined in reference 1). “Although more polish steps may be required to reduce process-related impurities, this platform approach has the advantages of compatibility with SIP and clean-in-place (CIP) processes, flexibility across serotype variants, and reduced cost,” he says.


Several important affinity resin attributes

As with any other affinity-chromatography resin, those for use with viral vectors should allow for clearance of impurities with high flow rates for reduced purification times to reduce costs and, in this case, also avoid loss of infectivity. These goals must be achieved, reiterates Nestola, under mild elution conditions. Affinity resins for viral vectors must also be readily scalable and have the correct build-in attributes for the viral vector of choice, according to Sierkstra.

Major impurities that must be removed include host-cell DNA and other nucleic acids, according to Scanlon. Adventitious and process-related viruses must also be cleared, which requires that helper viruses (such as adenoviruses in AAV processes) not be bound by the affinity ligand employed.

Robust separation of the target virus from impurities at high supernatant loading volumes is also essential given the often quite low titers of viral vector products, Scanlon stresses. He adds that affinity resins must also be designed to withstand strong SIP and CIP processes without compromising their performance because of the crucial role they play in bioburden removal.

Screening is fundamental

High-throughput screening is the best approach to both the development of novel ligands for affinity chromatography of viral vectors and for development of optimal affinity-chromatography purification processes for a given vector product.

“Phage-display- and library-based approaches with high-throughput process-development tools such as liquid handling and plate-based screens allow for the screening of large numbers of constructs and conditions to develop novel ligands,” Scanlon states. Thermo Fisher Scientific uses the specific attributes of a viral vector as the starting point for high-throughput development employing a quality-by-design approach. “In the initial screening phase, this technology ensures that the ligand being developed has the ability to elute at the conditions and with the specificity needed,” says Sierkstra.

Meanwhile, optimization of affinity chromatography process conditions should start with small-scale screening, normally using 96-well plates, according to Nestola, to examine different ligands/matrices and buffer systems. “Developing good buffer strategies that allow good binding of the viral vector product to the affinity resin with minimal virus aggregation is critical,” he observes. Screening is then typically validated on small-scale devices (1–5 mL) to confirm the screening results.

Further advances needed to address scalability

The need for vector—and serotype-specific affinity resins—creates an opportunity for chromatography-technology suppliers to offer a greater variety of ligand/matrix solutions. “Having a selection of off-the-shelf ligands for several viral vectors including but not limited to AAV would provide good benefit to the industry,” Nestola
contends. “Such matrices could be based on various types of ligands from antibodies to antibody fragments to synthetic peptides and thus provide options for achieving enhanced purity and selectivity for any given viral vector,” he adds.

In particular, Nestola notes that the need for caustic-stable ligands will only rise as more gene and gene-modified-cell therapies that leverage viral vectors reach commercialization, and as a result, the number of batches produced increases exponentially. He also observes that the use of different formats (e.g., membrane, monolith) for immobilization of affinity ligands other than beads/resins should be explored to achieve more rapid purification of labile viral-vector products.

Scanlon agrees that current processing times when using affinity resins for large viral-vector purification are long due to low volumetric flow rates, which directly impacts cost of goods. “Any reduction in processing time results in lowering this cost and increasing the speed of delivery to market of new therapies,” he states.

Based on the specific needs of each viral vector, Sierkstra believes that improvements in these products will require continued R&D investment regarding all aspects of their manufacturing processes. As an example, she points to the pretreatment steps often needed for AAV vectors prior to the affinity capture step. “Thermo Fisher Scientific has developed a purification solution leveraging magnetic beads that for small-scale processes offers a simpler approach,” she comments.

A few new solutions have reached the market

Two companies have recently introduced new affinity resins to the market—one set for AAV vectors and another solution focused on LV vectors. Avitide, a Repligen company, has developed three ligands specific to the major AAV gene therapy vectors used today (AAV2, AAV8, and AAV9). The AVIPure affinity chromatography resins have increased caustic stability without sacrificing high dynamic binding capacity, according to the company (2).

The latter comes from Thermo Fisher Scientific. CaptureSelect Lenti VSVG Affinity Matrix has been designed specifically for the purification of VSV-G pseudotyped lentivirus particles under mild elution conditions (3).The company also offers Poros CaptureSelect AAVX, a resin for all serotypes of AAV. “These products were developed to address the specific need for platform purification processes and the rapid growing clinical pipeline. Previous technologies and solutions lacked specificity and resulted often in purification processes that involved multiple, sometimes complex, steps, increasing the clean-room and manufacturing time as well affording low yields,” Sierkstra comments.

Heparin affinity chromatography has also been proposed by bluebird bio and collaborators as an effective method for the purification of retroviral vectors used for gene-therapy applications (4).

Astrea Bioseparations, meanwhile, has targeted its efforts at the development of a novel bioseparation material for viral-vector affinity chromatography. AstreAdept is an electrospun composite nanofiber chromatography membrane with a large open structure that provides virtually instantaneous binding of large targets, including viral vectors, to significantly larger binding surface areas, according to Scanlon. “Importantly,” he emphasizes, “this increased capacity is not compromised by the high flow rates enabled by the nanofiber’s open structure and operates without the pressure drops observed with other convective chromatography media.”

Nereus LentiHERO, the first commercial product to incorporate the AstreAdept technology, was launched in September 2022 and is optimized for primary capture of LV particles at lab scale (5). “This new non-pseudotype-specific affinity chromatography solution specifically addresses the issues of capacity and process times seen with legacy bio separation tools. Operating at physiological pH and salt concentration, it preserves the integrity and quality of the LV particles and delivers a high binding capacity and high recovery for higher yields from feedstock volumes,” Scanlon says. Scalable solutions to bring this technology to process scale are under development.


  1. Rieser, R.; Koch, J.; Faccioli, G.;et al., Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors, Pharmaceutics, 2021, 13 (5), 748.
  2. Repligen. Repligen Launches AAV Affinity Resins for Gene Therapy Purification. Press Release, Feb. 15, 2022.
  3. Thermo Fisher Scientific. Cell and Gene Therapy Purification Solutions. Resource page [accessed Nov. 21, 2022].
  4. Mercedes Segura, M.; Kamen, A.; Trudel, P.; Garnier, A., A Novel Purification Strategy for Retrovirus Gene Therapy Vectors Using Heparin Affinity Chromatography, Biotechnol. Bioeng., 2005, 90 (4) 391–404.
  5. Astrea Bioseparations. Astrea Bioseparations Introduces Nereus LentiHERO, A Fit-for-Purpose Solution for Lentiviral Vector Purification Nereus LentiHERO. Press Release, Sept. 29, 2022.

About the author

Cynthia A. Challener, PhD has been a freelance technical writer for over 20 years, leveraging her education from Stanford University (BS) and University of Chicago (PhD) and 10+ years of industry experience. She currently focuses on pharma/biopharma topics, writing technical articles, white papers, blogs, and other content for a variety of clients in addition to contributing regularly to BioPharm International and Pharmaceutical Technology.

Article details

BioPharm International
Volume 36, Number 1
January 2023
Pages: 19-21


When referring to this article, please cite it as Challener, C.A.. Choosing the Right Resins for Viral Vector Affinity Chromatography. BioPharm International 2023 36 (1).