Mostafa Zarei - headshot 


by Mostafa Zarei

Senior Principal Scientist, Protein Mass Spectrometry, Drug Product Services

For those who work in gene therapy, it’s no secret why adenoviruses (AdVs) and adeno-associated viruses (AAVs) have become widely used vectors:

  • They have a substantial track record of safety and efficiency due to their low toxicity, availability of viral serotypes, and stable gene expression.
  • Both AdV and AAV vectors are able to infect a wide range of host cells, including dividing and non-dividing cells, without integrating with the host genome.
  • AAVs trigger very low levels of immune response (unlike their AdV counterparts).

Mreover, recent advances in gene transfer technology have enabled the development of various AdV-based vaccines against HIV, influenza, Ebola virus, and SARS-CoV-2, as well as AdV- and AAV-derived DNA therapeutics, further enhancing the clinical value of these useful viruses.

The Need for Robust, Reliable Analytical Assays

As with any bioengineered product, quality control is essential to the safe and effective use of AdV and AAV vectors. Given that the viral proteins of AdV and AAV capsids are the main components and key players in the initial stages of infection by the virus particles, their heterogeneity and content must be evaluated to ensure product and process consistency.

The U.S. Food and Drug Administration (FDA) recommends the identification of gene therapy products (GTPs), such as AAV serotypes, from other products in the same facility “to assure product safety, identity, quality, purity, and strength (including potency) of the investigational product.”[1] The Agency’s recommendations include providing information on empty to full capsid ratio, double- or single-stranded DNA, proteins encapsulated within a viral capsid, and protein level. Hence there is a need for robust, user-friendly analytical methods to determine the heterogeneity and composition of the capsid viral proteins (VPs).

The Power of Peptide Mapping

As gene therapy developers strive to optimize their products, peptide mapping has emerged as an alternative approach to identifying and characterizing AdV VPs and AAV serotypes. Among its advantages, peptide mapping:

  • enables clear distinction of capsid protein amino acid sequences, facilitating confirmation of capsid identity with high confidence
  • can provide detailed information on amino acid sequences of VPs used in AdV capsids, as well as on site-specific post-translational modifications (PTMs).

Notably, differences in production cell systems, storage conditions, or production scale can lead to differences in the PTMs of VPs that affect AAV infectivity and vector potency. Comprehensive characterization of AdV and AAV proteins, including their amino acid sequences and PTMs, is therefore crucial to ensuring the quality and consistency of GTPs.

An Unexpected Inspiration Leads to Collaboration

As is often the case in drug development and manufacturing, inspiration can come from unexpected sources. My moment of inspiration came in May 2020 when I had an insightful conversation with my daughter about how my expertise could be applied to the COVID-19 pandemic. That conversation sparked my outreach to several Lonza colleagues to explore various methods of characterizing AdV and AAV capsid proteins. Through our cross-divisional collaboration, we led the development of a fast and efficient peptide mapping protocol[2] that helps to control the protein composition within viral capsids used in vaccines and GTPs, ensuring quality control of the final product.

Refining the Process

Upon receiving the first AAV samples, we discovered that AAVs are totally different from the monoclonal antibody-based biologics and biopharmaceuticals we were accustomed to working with. For example, AAVs require the use of a detergent (e.g. poloxamer) to enable downstream processing. Without the poloxamer, sample recovery can decrease by more than 80%.

We found the poloxamer contained in our samples interfered with the liquid chromatography/mass spectrometry (LC-MS) signal, creating a lot of background noise. We applied protein precipitation, which allowed us to fully remove the sample matrix, thus resolving the noise issue. In standard workflow systems, sample preparation starts by adding an acid to break the viral capsid and release the proteins. We applied a different approach by using organic solvents, which can break the capsid without the need for an acid, eliminating the need to neutralize the acid in the next step.

As a result, we compared two processes:

  • Single-phase precipitation: directly adding acetone (an organic solvent)
  • Two-phase precipitation: adding chloroform water and methanol, allowing the precipitated proteins to remain between the organic and aqueous phases

Adding Simple, Effective Workflows to Lonza’s Toolbox

Our collaborative approach resulted in a novel AdV and AAV VP analysis workflow.[3] One of its main benefits is a substantial reduction in analysis times compared to traditional peptide mapping methods, which require the use of several proteases and long sample processing for sufficient in-depth characterization.

In addition to enhancing analytical performance, Lonza’s method minimizes sample preparation steps. This benefit reduces the risk of protein or peptide losses and enables high-throughput analysis when numerous samples must be rapidly characterized to support process development, stability testing, and characterization of GTPs.

Lonza’s workflow yields comprehensive information that can provide valuable mechanistic insights into a viral infection that are difficult to obtain via other approaches. Our method is highly reproducible, robust, and suitable for the proteomic study of AdV VPs and AAV serotypes. Moreover, it is not labor-intensive and can easily be adapted for both high and low quantities of starting materials.

Last but not least, Lonza’s peptide mapping capability exemplifies the benefits of cross-divisional collaboration facilitated through state-of-the-art instrumentation, software, and expertise. Those attributes will serve our clients and us well as we strive to optimize the development and manufacturing of innovative gene therapies that advance human health.


[1] Food and Drug Administration. Guidance for industry: Chemistry, Manufacturing, and Control (CMC) information for human gene therapy Investigational New Drug applications (INDs). Published January 2020. Accessed September 20, 2021.

[2] Zarei, M, Wang, P, Jonveaux, J, et al. A novel protocol for in-depth analysis of recombinant adeno-associated virus capsid proteins using UHPLC–MS/MS. Rapid Commun Mass Spectrom. 2022; 36( 6):e9247. doi:10.1002/rcm.9247

[3] Mostafa Zarei, Jérôme Jonveaux, Peng Wang, Friedrich M. Haller, Bingnan Gu, Atanas V. Koulov, and Michael Jahn, ACS Omega 2022 7 (41), 36825-36835. doi:10.1021/acsomega.2c05325

Additional Information and Disclaimer

Lonza Group Ltd has its headquarters in Basel, Switzerland, and is listed on the SIX Swiss Exchange. It has a secondary listing on the Singapore Exchange Securities Trading Limited (“SGX-ST”). Lonza Group Ltd is not subject to the SGX-ST’s continuing listing requirements but remains subject to Rules 217 and 751 of the SGX-ST Listing Manual.

Certain matters discussed in these articles may constitute forward-looking statements. These statements are based on current expectations and estimates of Lonza Group Ltd, although Lonza Group Ltd can give no assurance that these expectations and estimates will be achieved. Investors are cautioned that all forward-looking statements involve risks and uncertainty and are qualified in their entirety. The actual results may differ materially in the future from the forward-looking statements included in these articles due to various factors. Furthermore, except as otherwise required by law, Lonza Group Ltd disclaims any intention or obligation to update the statements contained in these articles.

Latest content
Latest briefing from the Knowledge Center