Improving Efficiency of rAAV Production and Purification with Chromatography

Since the discovery of the adeno-associated virus (AAV) in 1965, its use in gene therapy as a transfer vector has increased along with demand for characterization, production, and purification. Recombinant adeno-associated virus (rAAV), the smallest particulate gene delivery vector at 20 nm in diameter, is derived from a small, nonenveloped icosahedral virus with a linear single-stranded DNA genome of 4.7–6 kilobases (kb). The small size of the virion permits the packaging of only 5 kb of exogenous DNA, including the promoter. More recent work has exploited the unique ability of AAV to link together in doublets or strings to bypass this size limitation [1,2].

More than twelve naturally occurring serotypes of AAV have been characterized, each with unique surface proteins and glycosylation patterns [3,4]. Novel serotypes have also been developed. Advantages of rAAV include non-pathogenicity, low immunogenicity, and the ability to transduce specific target tissues (i.e., tissue tropism) and establish long-term gene expression. Understanding these differences in tropism is critical when choosing a specific gene delivery vector to achieve a successful therapeutic outcome [5,6]. With a better understanding of AAV structure and biology, new vectors with modifications to the genome or capsids may be designed to facilitate more specific transduction of cells for gene therapy. Also, structural differences in surface proteins and aminoglycans may affect efficient approaches for vector purification.

Production of rAAV typically follows a streamlined workflow of plasmid production, cell expansion and plasmid transfection, viral vector production, and viral recovery and purification before the final fill-finish (Figure 1). Each step in the workflow influences the quality of material produced and the efficiency of the process. Downstream purification depends on the clearance of impurities such as host cell proteins, debris, media components, exogenous DNA, and empty vector particles. Designing an efficient and effective downstream purification system involves using advantages of different chromatography types with the least amount of processing steps to minimize viral vector loss. Approaches to vector purification exploit the viral particle physical characteristics such as size, surface charge, and hydrophobicity. Viral vectors vary greatly in physical characteristics depending on the type of virus and for the same virus, can vary depending on serotype and transgene inserts. Greater purity of the final vector can reduce immunogenic responses and increase the efficiency of gene transfer. However, if purification is not ideal, rAAV may lose essential structural or genetic components [7,8].

Overview of AAV production workflow for gene therapy

Figure 1. Typical workflow for rAAV production.

One primary consideration when moving to clinical manufacturing for gene therapy is how to increase production scale. At larger scales, the purification methods need to accommodate large volumes with high efficiency and speed. Historical purification methods involved tedious and unscalable ultracentrifugation or lengthy density gradient steps, which were a bottleneck to rAAV production. Recent advances in production methods now focus on scalable chromatography [7,9]. AAV virions are relatively stable to heat, nonionic detergents, and proteolytic enzymes, making them good candidates for chromatographic purification [10]. Two standard modes of chromatography applicable to downstream purification include affinity and ion exchange. As with all chromatography, results are sensitive to buffer and mobile phase composition. The importance of precise buffer systems will be discussed in relation to these different types of chromatography.

Affinity chromatography

Affinity chromatography relies on the interaction of the external capsid structure of a viral particle with a ligand. While AAV serotypes share a common capsid morphology, the presence of specific amino acids in certain regions determines whether antibodies are able to bind to them [11]. Different types of affinity resins interact with unique capsid structures present in different serotypes, offering options for AAV vector–specific purification. Novel AAV variants that are mosaics of capsid subunits from different serotypes are being developed to help evade preexisting neutralizing antibodies generated as a result of the humoral immune response to natural infection or prior treatment with AAV-based vectors [8]. Affinity resins may be used to efficiently purify these new types of AAV.

However, during purification of both naturally occurring serotypes and in mosaic AAV, non-specific binding of impurities can occur, decreasing the efficiency of product production. Another limitation of affinity chromatography is that it cannot easily separate full vectors from empty capsids. This process requires additional purification steps that may involve ion-exchange chromatography or ultracentrifugation. Centrifugation purification has limited utility because it is difficult to scale-up and is relatively harsh on the virus capsid.

Affinity chromatography combined with ion-exchange chromatography is commonly implemented to maximize AAV purification efficiency [9,12,13].

Ion exchange chromatography

Ion exchange chromatography involves the separation of ionizable molecules based on their total charge. Ion exchange resins consist of two main types: cation exchange resins that exchange positively charged ions and anion exchange resins that exchange negatively charged ions. The charge on the molecule of interest may be manipulated by changing buffer pH or salt concentration, allowing separation of biological molecules such as proteins, peptides, and rAAV.

Surface proteins on the capsids of different AAV serotypes have charges that allow binding to ion-exchange chromatographic resins [6]. It is important to note that when working outside of their optimal pH range, some resins may rapidly lose capacity and resolution. When the pH is higher than the isoelectric point (pI) of the AAV capsid, the net charge on the protein is negative, allowing binding to an anion exchanger. Conversely, when the pH is lower than the pI, the net charge on a protein is positive, and it can bind to a cation exchanger [8,14].

Ion exchange resins work well when used with low-salt buffers. Higher salt concentrations may impede binding to the resins and cause osmotic shock, resulting in ruptured viral capsids [9]. The inclusion of buffer-exchange steps before ion-exchange chromatography may prevent the osmotic shock issue. Other methods require the use of salt gradients that are optimized to separate viruses from impurities.

The principles of ion exchange chromatography can be used in either a column or membrane format. Membranes characteristically have a binding ligand immobilized on their absorbing surface that allows protein binding. Although membranes generally have a lower binding capacity, they have a larger surface area, which allows the use of faster flow rates. In some cases, the use of membranes may result in greater productivity [15,16].


Precise buffer composition is critical for reliable and efficient rAAV purification. Due to specific differences in capsid morphology, complete buffer systems designed for each AAV serotype are of great importance. Capsid sequences of different AAV serotypes show electrostatic differences that are reflected in their pI. Optimizing the affinity of the viral particles for the resin will allow better recovery rates of purified material.

Multiple buffers are used throughout the purification process, and the composition of each will affect the final result. A buffer containing the wrong counterion can prevent binding of the protein of interest to the column resin. The charged species in buffers used for ion-exchange chromatography should generally have the same charge as the resin. For example, although phosphate buffers are commonly used for protein purification, they are not appropriate for anion exchange chromatography because the phosphate ion interacts strongly with positively charged anion exchange resins [8,14].

Ion exchange resins and membranes used in the purification process may lose binding capacity and resolution outside their optimal pH range [15]. Anionic and cationic beads and membranes must be matched with a buffer or mobile phase of appropriate pH. Depending on their environment, proteins may carry a net positive charge, a net negative charge, or no charge. In addition to monitoring pH, measuring the conductivity of solutions is another way to ensure consistency in buffer composition and performance.

Looking beyond the purification process, the effectiveness of the final rAAV product is influenced by the same buffer variables discussed earlier. Some AAV serotypes have functional proteases, enzymes that catalyze autolytic cleavage of the capsid as well as external substrates [17]. Outside of their ideal pH range, AAV proteases also show reduced function. These proteases induced significant autocleavage of capsid proteins at pH 5.5, while no autocleavage was seen at pH 7.5. Precise pH ranges have been shown to be essential for productive AAV infections [17].


Gene therapy products, as with many biologics, are quite complex and difficult to characterize. Current FDA guidance for gene therapy notes that for purity, the main concerns are impurities that can be process-related and include residual DNA, protein, or culture reagents. Consistency in the manufacturing process is critical to producing a consistent product. Having reliable sources for all production components, especially buffers, will allow greater control of the process and permit focus on the more variable aspects of rAAV production.


Learn more about Teknova Resources for Gene Therapy. To consult with our rAAV specialists about your project, email us at


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Viral Transport Medium—From Formulation to US FDA Notification in Six Months

A Case Study to Highlight Capabilities to Quickly Produce Custom Products

The astonishing speed at which the SARS-CoV-2 virus spread across the globe triggered a demand for rapid development of diagnostics for mass population testing. Seeing this need, we at Teknova moved two novel formulations of viral transport medium from development to FDA notification, then to manufacturing at scale within 6 months in 2020—an achievement that is enabling greater access to COVID-19 testing worldwide. In this case study, Scientific Director Bella Neufeld, and Vice President of Engineering Jose Olague, discuss the development journey of Teknova Active Viral Transport Medium (ATM). They explain how its unique formulation addresses the challenges posed by mass testing and explore how they were able to develop and scale up production of this medium so quickly.


Early in the COVID-19 pandemic, production of the standard medium for viral sample collection—known as universal transport medium (UTM)—was disrupted, and the product became unavailable. This problem led the US Centers for Disease Control and Prevention (CDC) to release the formulation and manufacturing instructions for viral transport medium (VTM).

In April 2020, we used our experience as a custom reagent manufacturer to begin production of VTM, under the name Teknova Viral Transport Medium (Figure 1). It soon became clear, however, that there were some challenges with the CDC formulation, as Bella explains: “We got many phone calls and emails asking if the VTM had to be refrigerated. The need for the medium to be stored cold—even before vials have been inoculated—was a requirement that many healthcare providers couldn’t meet, and this affected testing capacity. Our team at Teknova had a crisis meeting, and in July 2020, decided we would develop a new formulation of VTM that could meet the challenges of widespread COVID-19 testing.”

Timeline for production of sample transport medium

Figure 1. Timeline for Development and Production of Sample Transport Medium.

Optimizing and Making the Medium

The team identified several characteristics of a sample transport medium that would improve the logistics and efficacy of testing during the pandemic. According to Bella: “We knew that the optimized medium had to be room-temperature stable, as this would eliminate the need for cold storage and transport. We also realized that there were issues with the use of fetal bovine serum (FBS)—a reagent with variable components that could include RNases, which have the potential to degrade the viral SARS-CoV-2 RNA before tests are run. This is why we chose bovine serum albumin (BSA) in place of FBS for ATM, as BSA is more chemically defined and eliminates the risk of sample degradation.”

After determining additional features for the formulation, the team started development of ATM, guided by their experience with custom reagent manufacturing. On this stage, Jose said: “One of the reasons we were able to develop this product so quickly was because we built a dedicated ATM clean room from scratch. Building to a design that was specific to ATM enabled us to streamline product development and ensured that minimal process changes were needed further down the line.”

Making ATM also required us to install new equipment and use entirely new analysis techniques—but using processes with which we had extensive experience. According to Bella: “We regularly implement new testing methods when manufacturing custom products, which definitely gave us a head start with the ATM. Our extensive QC measures are applied throughout the manufacturing process and knowing how to adapt these to new products helped us speed up development of ATM significantly.”

Rapid Scale Up of ATM to Meet Viral Testing Needs

To scale up production of ATM, we expanded our manufacturing capacity. New equipment , including automated lines for liquid handling, bottling, and capping, saved time and massively increased throughput. On this, Jose stated: “The automation has enabled a huge increase in ATM production capacity, from zero to 300,000 tubes per week at the end of 2020, and will reach a capacity of up to 1 million tubes per week by June 2021 with the addition of a new cleanroom. Automation also allows us to better control contamination risks, giving users added confidence in the sterility of the product.”

Having a robust supply chain is also critical to ensuring successful product development, especially during the COVID-19 pandemic. As Bella explains: “Because Teknova has been doing in-coming QC on raw materials for so many years, we have a wealth of data that enables us to rapidly switch vendors and purchase different materials that we know have the same level of quality. This makes us more flexible and enables us to manufacture consistently high-quality products without interruption.”

While cleanrooms, equipment, and QC have all played a role in the journey of ATM development and manufacturing at Teknova, Bella also attributes Teknova’s agile response, in large part, to the company ethos: “There is extremely strong communication between departments at every level, resulting in potential issues being dealt with very quickly. Our emphasis on GMP manufacturing and ISO 13485 processes promotes a company mindset that allows us to adapt to manufacturing different products with confidence and gives us the conviction that we can achieve anything.”

Looking to the Future

Teknova’s CEO, Stephen Gunstream, has big plans to expand production capacity, a vision shared by Jose: “We are really moving the company forward, introducing new technologies that not only enable us to make better products, but products that are also sensitive to contamination or changes in environment. The ability to quickly change our fill-finish manufacturing capabilities is really one of Teknova’s key strengths.”

On the direction of the company, Bella commented: “While we are sure to expand further, Stephen also understands the benefit of being a medium-sized company, and the edge that this can give in terms of flexibility—whether that be rapidly changing filling volumes and containers from milliliter to microliter level or adapting existing products to changing customer needs.”

While scale up of ATM is ongoing, the product now has FDA notification, and we are also filing a 510(k), which will extend availability of the medium beyond the COVID-19 pandemic. Products such as ATM highlight our ability to handle rapid product development and scale up for customers, bringing high-quality media and reagents to market when they are needed the most.


For more information about how we can help you with your next project, email

The Importance of Reagent Purity Grades

Purity grades are standards that indicate that a substance meets pre-determined chemical and biological purity specifications, denoting suitability for use in specific research or manufacturing applications. Regulations define the appropriate purity grades for the manufacture of food, drugs, and medicinal products. There are also separate purity grades for products intended for laboratory and analytical applications.

An understanding of different reagent purity grades is vital when designing products for diagnostic or therapeutic purposes, and using raw materials of the appropriate grade during an early research phase can eliminate lengthy re-validation at the commercialization stage.

In this article, we introduce the main reagent purity grades and highlight specialty grades offered for specific molecular biology applications. We also explore considerations for choosing purity grades, especially for projects that may progress past research toward commercial manufacturing.

Main Purity Grades and Associated Applications

A material or reagent of a specific purity grade must meet criteria set out in a monograph, which is a document released by the relevant regulatory agency containing requirements for the ingredients or preparation, handling, and specifications of the substance. The specifications define which tests should be performed, as well as the acceptance criteria that enable a substance to be labelled with a particular grade.

There are several international agencies that provide purity grade regulations for reagents used in the manufacture of food, drug, and medicinal products. In the ranking of chemical purity, the ACS (American Chemical Society) and Reagent grade chemicals are highest (Table 1, download PDF), with both having a similar purity of ≥95%. United States Pharmacopeia (USP) and National Formulary (NF) grade reagents are also suitable for manufacture of foods, drugs, and medicinal products (Table 1). Every year these organizations publish a joint compendium called USP-NF, which provides standards for drugs, dosages, chemicals, and preparations. With a few exceptions (e.g., HPLC-grade materials, which are used in analytical and preparative separations), materials used for manufacturing at Teknova are of one of these four high-purity grades.

Specialty Grades from Teknova

In addition to the general grades outlined above, we offer specialty grades for a range of reagents. Molecular biology grade reagents have equivalent purity to ultra-pure chemicals, and include reagents, enzymes, and buffers (e.g., agar, RNase A, and SSC/SSPE buffers for Southern and northern hybridization procedures) (Table 1). Our molecular biology–grade, PCR-certified water is tested for DNase, RNase, and protease activity, as well as PCR-verified for the absence of bacteria.

Our ultra-pure grade reagents include fundamental biochemistry reagents such as Tris base, sodium chloride, TEMED, and SDS. In addition, Teknova provides specialty grades as guidelines for specific applications, such as DNA sequencing grade reagents, electrophoresis grade reagents, and histology grade ethanol.

Table 1. Common Reagent Purity Grades. (download PDF)

Grade Description Application
ACS Meets or exceeds the purity standards set by the American Chemical Society (ACS). Considered the highest purity chemical grade (≥95% purity). Can be used for food, drug, and medical uses and general procedures that require strict quality specifications.
Reagent Describes high purity chemicals for which no established specifications exist. Usually considered equivalent to ACS grade. Can be used for food, drug, and medical uses and general procedures that require strict quality specifications. Very often these are solutions or dilutions of ACS grade materials.
USP Meets or exceeds requirements set by the United States Pharmacopeia (USP). Can be used for food, drug, and medical purposes, and also, for most laboratory purposes.
NF Meets or exceeds requirements of the National Formulary (NF). The USP-NF publishes a book with the standards for chemicals.
BP Meets or exceeds requirements set by the British Pharmacopeia (BP).
JP Meets or exceeds requirements set by the Japanese Pharmacopeia (JP).
PhEur Meets or exceeds requirements set by the European Pharmacopeia (PhEur or EP).
Multi-compendial Meets or exceeds requirements set by more than one pharmacopeia.
Specialty Grades
Molecular biology Tested for DNase, RNase, and protease activity. Can be used in molecular biology research applications.
HPLC High purity chemicals for use in analytical and preparative separations that are tested at one or more UV wavelengths. Used in high-performance liquid chromatography, gel-permeation chromatography, and UV-spectrophotometric analysis.

* ACS, Reagent, USP, NF grades are often interchangeable but applicable regulatory requirements should be reviewed to confirm that appropriate standards are met for your application.

In contrast, the following three grades are not suitable for food, drug, or medicinal use: laboratory, purified, and technical grade. Substances of these grades are mainly used for industrial or educational purposes where high purity is not required.

How to Choose Reagent Grades

Ultimately, your application and end goal (i.e., research vs. therapeutic or diagnostic) determine what reagent grade you need. You can reference some of our specialty grade designations to help guide product selection. In addition, note that we only use high-purity raw materials that allow us to make consistently high-quality research use only (RUO) products. We manufacture all products using ISO 13485:2016 certified processes, which provides an advantage if you are pursuing clinical application of your products.

Good Manufacturing Practices for Clinical Products

RUO reagents are often used during the early stages of diagnostic or therapeutic product development, due to their widespread availability and low cost. These reagents can be manufactured in compliance with either ISO 13485—the standard for medical devices—or the more general ISO 9001. However, before diagnostic or therapeutic products can be commercialized, FDA and EMA regulations require manufacturers to switch from using RUO reagents to those produced according to good manufacturing practices (GMP). GMP reagent production is subject to more stringent requirements for material traceability, equipment maintenance, environmental control, and staff training, which ultimately ensure the safety of the end user.

As mentioned above, all Teknova manufacturing facilities are ISO 13485:2016 certified and integrate extensive raw material analysis, bioburden testing, and quality control processes into production of reagents, enabling seamless transition from RUO to GMP manufacturing. The high standards imposed by ISO 13485 align well with the requirements of GMP, making transitioning these reagents to GMP faster and more flexible than moving from ISO 9001. Using ISO 13485 certified processes from the outset also enables protocol modifications that mitigate costs, such as replacing enzymes with less expensive alternatives.

High-Quality Water for Use in Research and Pharmaceutical Development

Another important consideration for obtaining high-quality end products is to use high purity, sterile water to manufacture reagents intended for research and clinical applications. Standards for the purity and sterility of water are defined by different agencies, including the US Pharmacopeia (USP) and the American Society for Testing and Materials (ASTM).

USP standards for “purified water” and “water for injection” (WFI) are the most relevant for use in laboratories and manufacturing. Of the two, WFI is of higher purity and the standard for pharmaceutical and diagnostic development. ASTM purity standards are comprised of four categories, with I being the highest purity and IV the least. Categories I to III include standards for sterility, whereas type IV does not. Learn more about USP and ASTM water in this overview of water standards.

All Teknova water meets USP WFI and ASTM Type I standards. We also offer four specialty grades of water that have additional testing, depending on use—three for specific applications, such as cell and tissue culture, RNA work, and PCR applications, and one general reagent grade for various buffer and media applications. In addition, water products can be manufactured under GMP, eliminating the need for transitioning reagents upon product commercialization. You can learn more about Teknova’s rigorous two-stage manufacturing and quality control procedures for water.


Knowledge of different reagent grades and their applications is vital when conducting research and considering commercialization of a biotechnology product. As well as supplying a wide range of pre-qualified GMP reagents that meet the purity grades outlined above, we also provide services for GMP transition, which include capabilities in manufacturing, logistics and warehousing, analytical and QC procedures, and secure document control services.

Contact us to find out how we can support your projects with our custom RUO and GMP products and services.

Types of Microbial Culture Media

As a researcher working with microorganisms, you likely have varying needs for microbial cultivation. When you grow microbes under controlled circumstances in the lab, you must provide a source of nutrients for their survival. These nutrients are contained in culture media. Many types of media are available, and which one you use depends on your intended purpose (Table 1). The kinds and amounts of nutrients can vary, and other ingredients can be added to promote selective growth of desired microorganisms.

Broth vs. Agar

Liquid (broth) and solid (agar) forms of culture media provide different growth environments for microbes, where the cells are either suspended in the broth or form colonies on the agar. Broth media are used for the planktonic (free-floating) phase of microbial growth, and agar media are used for the isolated colony or large biofilm phase of microbial growth. However, biofilm studies can be conducted in broth as well, by allowing the biofilms to grow on the surface. You will likely use both broth and agar for different phases of an experiment or research application. Typically, organisms are initially grown in broth to reach a particular growth phase, at which point the cells can be plated on agar for further studies or long-term storage. (Learn more about the history of agar as a microbiology tool.)

Rich vs. Minimal Media

In many cases, you can select a rich medium that supplies a diverse range of nutrients to the microbe. However, in some situations, such as for studying metabolism, you may need to control the nutrients available to the microbe, so you would use a minimal medium, which contains only the metabolites required for microbial growth (e.g., water, carbon source, and various salts that contain essential elements required for protein and nucleic acid synthesis).

Chemically Defined (Synthetic) vs. Complex Media

Culture media can be chemically defined (synthetic), with a known composition and quantity of nutrients; or complex, with an undefined assortment and quantity of nutrients. Chemically defined media are required for metabolic studies, where you may need to include or exclude certain metabolites. They are also useful for organisms that require specific nutrients for growth. Complex media, on the other hand, provide a variety of nutrients in unspecific quantities because they are composed of extracts and enzymatic digests of living organisms (e.g., peptones from animal, plant, or yeast). Therefore, they are suitable for most everyday needs for easy-to-grow microbes except for metabolic or physiological studies due to the undefined levels of the components.

Chemically defined media can be either rich or minimal, while complex media are usually rich. However, if you formulate your own media, you can add complex nutrients (e.g., yeast extract or tryptone) at minimal levels, but determining those levels would require growth studies.

Selective, Enriched, and Differential Media

By using the appropriate media, you can inhibit the growth of undesired microbes, promote the growth of microbes of interest, or distinguish colonies of different microbes. Selective Media allow the growth of a specified microbe because they contain ingredients that inhibit the growth of undesired microbes. Difficult-to-grow, or fastidious, organisms require even more nutrients than are provided in rich media. By using enriched media you provide the extra nutrients to help these organisms grow under laboratory conditions. Differential media contain ingredients that distinguish between different microbes and can indicate the presence of a certain microbe.

Table 1 summarizes the types of culture media described here. Microbe suppliers will provide growth media recommendations for their specific organisms, and additional information about growth media is available from ATTC.

Table 1. Glossary and examples of culture media.

Type Description Example
Broth Liquid form LB broth
Agar Solid form LB agar
Rich Numerous nutrients Tryptic soy broth
Minimal Only nutrients essential for growth M9 minimal medium
Chemically defined Known assortment and quantity of nutrients MOPS EZ Rich Defined Medium
Complex Undefined assortment and quantity of nutrients Brain heart infusion (BHI) broth
Differential Ingredients that enable distinction between different microbes MacConkey agar (MCK)
Selective Ingredients that allow the growth of only a specific microbe Mannitol salt agar (MSA)
Enriched Extra nutrients to promote cultivation of difficult-to-grow (fastidious) organisms, such as Haemophilus influenzae and Neisseria spp Blood agar