5 Different Types of Chromatography Resins, How to Choose?

Chromatography technology plays a critical role in the biopharmaceutical sector, by offering an efficient approach to achieve the separation of complex components. Chromatography resin consists of ligands and a base matrix, which serves as a critical factor in the downstream processing by determining the productivity and quality of pharmaceutical ingredients purification. 

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Chromatography resins can be categorized into affinity resin, ion exchange (IEX) chromatography resin, hydrophobic interaction chromatography (HIC) resin, Size exclusion chromatography (SEC) resin, and Mix-mode resin in terms of separation mode.

From this article you will learn:

1. Overview of chromatography resins
2. Introduction to 5 common types of chromatography resins
3. How do you choose between different types of chromatography resins?
4. Outsource your resin needs to Bestchrom

Overview of chromatography resins

Chromatography resins are a class of key materials used in chromatography processes and are widely used in the pharmaceutical, biotechnology and chemical fields. These resins are often highly selective and can effectively separate compounds in mixtures.

The choice of chromatography resin depends on the desired separation properties, including molecular size, charge, hydrophilicity and hydrophobicity, etc. Common chromatography resin types include ion exchange resins, hydrophilic and hydrophobic chromatography resins, and metal chelate chromatography resins.

These resins play a key role in the preparation and purification of biomolecules, drugs and compounds, improving the efficiency and effectiveness of chromatography processes in laboratories and industry.

Introduction to 5 common types of chromatography resins

There are different types of chromatography resins, and each resin has its own advantages and functions according to user requirements.

Affinity chromatography

Affinity chromatography is a chromatography method separating biomolecules based on the specific interaction among biomolecules. Thanks to its high selectivity, affinity resin is able to capture protein from complexes at a purity higher than 90% via one-step purification. 

Affinity resin is widely applicable in the efficient purification of antibodies, tag proteins, and other bio-molecules with specific adsorption.

Advantages: High selectivity, excellent purity outcomes, and suitability for purifying specific target molecules.

Ion exchange (IEX) chromatography

Ion exchange (IEX) chromatography is a separation method based on the different mass and quantity of electric charges on biomolecules. Enjoying advantages including high selectivity, high binding capacity, high yield, and convenient operation, the versatile chromatography method can be used in the initial capture step as well as intermediate purification and polishing. 

Therefore, it is widely applicable in the purification of electric-charged bio-molecules including amino acids, peptides, antibodies, proteins, saccharides, viruses, and nucleotides.

Advantages: High specificity for charged molecules, effective separation of biomolecules, and applicability in both analytical and preparative chromatography.

Hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is a widely used method in the separation of macro-biomolecules based on the hydrophobicity difference on the molecule surface.  The biggest merit lies in its mild interaction with proteins, which enables the maintenance of natural structure and bio-activity in macro-biomolecules. 

HIC adopts an adsorption mode in terms of high salinity sample loading and low salinity elution, which makes it an ideal purification option after eluting with high salinity. HIC provides an efficient method for the separation of macro-molecules including antibodies, recombinant proteins, vaccines, peptides, and nucleic acid.

Advantages: Gentle separation conditions, effective purification of proteins, and compatibility with proteins that denature in other chromatographic methods.

Size exclusion chromatography (SEC)

Size exclusion chromatography (SEC), also known as gel filtration, is a non-adsorption chromatography method. It achieves chromatography separation based on the size and shape of biomolecules, which means the key to SEC resin selection lies in the right fractionation range. 

SEC enjoys the advantage of easy operation, which enables the completion of chromatography separation via a single buffer. Therefore, it is an ideal option in the desalting, buffer exchange, and polishing step of chromatography.

Advantages: Non-destructive separation method, compatibility with a wide range of biomolecules, and suitability for determining molecular weight.

Mixed-mode chromatography

Mixed-mode chromatography is an innovative chromatography method that can simultaneously provide different interactions for the binding between ligands and macro-biomolecules. Dominant interactions in this category include electric charge interaction and hydrophobic interaction. 

Mixed-mode chromatography enjoys wider binding condition and a bigger operation room, which simplifies process step and boost productivity. It is widely applicable in the purification of viruses, antibodies, peptides, recombinant proteins, and nucleic acids, especially in providing effective solutions to challenges faced in the purification process.

Advantages: Versatility in separating complex samples, increased selectivity, and the ability to handle challenging separation tasks.

How do you choose between different types of chromatography resins?

1. Affinity Chromatography:

Use When: High specificity is required for the separation of target molecules based on specific binding interactions.

Ideal for: Purification of proteins, antibodies, and enzymes where a strong affinity between the target and ligand is present.

2. Ion Exchange (IEX) Chromatography:

Use When: Separation based on charge differences is needed, making it suitable for molecules with varying ionization states.

Ideal for: Purification of proteins, peptides, and nucleic acids by exploiting differences in charge.

3. Hydrophobic Interaction Chromatography:

Use When: Separation is desired based on hydrophobicity, offering a milder condition compared to other hydrophobic methods.

Ideal for: Purifying proteins that are sensitive to high salt concentrations or denaturation.

4. Size Exclusion Chromatography (SEC):

Use When: Separation based on molecular size is crucial, allowing for the analysis of size distributions and purification of biomolecules.

Ideal for: Determining molecular weights and separating biomolecules based on their size without denaturation.

5. Mixed-Mode Chromatography:

Use When: Enhanced selectivity is needed by combining different separation mechanisms like ion exchange, hydrophobic interaction, and affinity.

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Ideal for: Complex samples requiring versatile approaches, providing increased selectivity in bioseparation processes.

Chromatography resin plays a critical role in chromatography purification. Different combinations of functional groups and base matrices provide various functions to resins, which enables the efficient purification of complex components via a selection of the right resin. 

When choosing suitable resins, the following factors should be taken into account:  matrix property (polymer-based matrix provides a high flow rate due to its good mechanical property; Agarose-based resin enjoys better non-specific adsorption due to its excellent hydrophilicity), bead size(finer beads provide high selectivity while big beads can endure high flow rate), resin binding capacity (which depends on ligand types and ligand concentration) as well as scalability. 

In addition, evaluation results in terms of sample yield and quality should also be considered when making the resin purchase decision.

Outsource your resin needs to Bestchrom

Bestchrom Biosciences focuses on the process development and product R&D required in bio-pharmaceutical downstream processing. We take our core strength in product quality and production capacity, which enable us to continuously provide chromatography resins with more variety, better performance, and cost-effectiveness. 

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References

Ion exchange resin

Affinity chromatography

Mixed-mode Resins

Size exclusion resins

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 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.

References

1. Rieser, R.; Koch, J.; Faccioli, G.;et al., Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors, Pharmaceutics, , 13 (5), 748.
2. Repligen. Repligen Launches AAV Affinity Resins for Gene Therapy Purification. Press Release, Feb. 15, .
3. Thermo Fisher Scientific. Cell and Gene Therapy Purification Solutions. Resource page [accessed Nov. 21, ].
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., , 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, .

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
Pages: 19-21

Citation

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

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