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Analytical Procedures for Viral Vectored Vaccine Quality (Draft guidelines)

Type of Posting: General Announcement

Posting Date: 29–Apr–2022

Input Deadline: 31–Jul–2022

Expert Committee: Biologics Monograph 3 – Complex Biologics & Vaccines

Proposed Title: Analytical Procedures for Viral Vectored Vaccine Quality (Draft guidelines)

Suggested audience: Suppliers and manufacturers of viral vectored vaccine drug substances and drug products, contract manufacturing organizations, drug testing organizations, regulatory agencies, and QA/QC specialists.

Background and objective: Viral vectors are an effective mechanism for delivering cargo DNA to targeted cells, and significant resources have been invested to develop and manufacture viral vectors for gene therapy and vaccine applications. In recent years, several viral vectored vaccines, including Zabdeno®, ERVEBO® and Mvabea®, have been developed for use against Ebola. Viral vectors are particularly useful for vaccines because they can induce a robust immune response to foreign or virus-infected cells without the need for adjuvants. Modified versions of adenovirus are the most commonly used viral vectors for vaccines, but modified measles and vaccinia viruses have also been used in vaccines. With the successful development of viral vectored vaccines to fight COVID-19, the global demand for these types of products is expected to grow.

Appropriate analytical techniques are critical. There are significant complex challenges in upstream and downstream processing of viral vectored vaccines. Viral vectored vaccines have two major components: the DNA transgene encoding the protein of interest responsible for eliciting an immune response, and the viral vector, which serves as a delivery vehicle to get the DNA into the cells. Each component has its own set of quality attributes and potential impurities that need to be analyzed. Furthermore, when the viral vector and DNA are combined, it is also important to assess the percentage of capsids that contain the transgene (full capsids) vs. empty capsids that lack the desired DNA cargo. Therefore, appropriate and discerning analytical methods need to be used throughout product development and manufacturing to ensure the quality and safety of the final product. Additionally, translating small-scale virus production into large-scale commercial production is a complex process, and developing a robust analytical strategy that supports the entire product development and manufacturing cycle is crucial to being able to deliver consistent, stable products to the patient.

In order to aid the global need for quality assurance of viral vectored vaccines, USP intends to develop a new general guidelines with methods to support the testing of quality attributes for viral vector-based vaccines.

Proposal and stakeholder engagement: USP and global stakeholders have identified a need for analytical procedures and best practices to support the assessment of common quality attributes of viral vectored vaccines. A standard set of analytical methods would support vaccine developers, manufacturers, regulatory agencies, and national control laboratories worldwide by providing methods to help accelerate the development and release of safe and effective vaccines and guard against substandard and falsified vaccine products.

As a first step towards developing a procedural chapter on the testing of viral vectored vaccines, USP’s BIO3 Expert Committee has developed a draft guidance containing methods to support testing of viral vector quality attributes. Methods described in the draft guidance have been adapted from publicly available sources and have not yet been verified or validated by USP.

USP and our BIO3 Expert Committee are releasing an early draft of the guidance for public comments. By pursuing the early release, USP wants to solicit feedback from stakeholders on the methods described in the referenced document and encourage the submission of any alternative methods and any additional supporting documentation, including validation documents, related to the methods presented in the draft guidance.

Preliminary outline of the proposed guidance: The following table outlines the proposed methods for assessing quality attributes for viral vectored bulk drug substances and drug products. The draft guidance includes methods to evaluate the identity, potency, quantity, purity, and safety parameters.

Quality Attribute Recommended Analytical Method
Identity DNA sequence Quantitative polymerase chain reaction (qPCR)
Next generation sequencing (NGS)
Viral Capsid Western blot
Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)
Liquid Chromatography - Mass Spectrometry (LC-MS
Potency Transgene expression ELISA
Physical viral titer Quantitative polymerase chain reaction (qPCR)
Infectivity assay (flow cytometry)
Tissue culture infection dose (TCID50) assay
Vector genome titer assay (ddPCR)
Quantity Total viral capsid particles Size-exclusion chromatography with multi-angle light scattering (SEC-MALS)
Anion-exchange High-performance Liquid Chromatography (AEX-HPLC)
Safety Replication competent virus Replication-competent adenovirus (RCA) assay
Adventitious agents In vitro adventitious virus assay
Bioburden USP <1115>
aSterility USP <71>
Endotoxin USP <85>
General tests Appearance USP <1>, <790>
pH USP <791>
aOsmolality USP <785>

aOnly tested on drug product.

For more information and to download the draft guideline please click here.

Deadline for Submitting Comments

USP welcomes your feedback and encourages you to submit your supporting data within 90 days from the post of this announcement by email as described below.

Submitting your comments

Please submit your comments and supporting documents to

USP contact information

For further information, please contact Sarita K. Acharya, Principal Scientist, USP Biologics, or


USP looks forward to continued engagement from industry, regulators, and other stakeholders.