New USP Guidelines, Standards Address Lentivirus Characterization
A conversation with Anthony J. Blaszczyk at the United States Pharmacopeia and Life Science Connect's Jon O'Connell
The United States Pharmacopeia (USP) is nearing the end of a yearslong project to provide reference standards and best practices for characterizing lentiviral vector (LVV).
Following a similar effort for adeno-associated virus characterization, the standards organization wants to give cell and gene therapy manufacturers tools for measuring viral vectors while accounting for LVV's unique complexity and stability challenges.
Anthony Blaszczyk, a principal scientist at USP, says the guidance shouldn't surprise organizations already producing LVV at scale, though the standards and guidance may still provide new efficiencies. He sees their greatest value in helping developers with new LVV candidates quickly characterize attributes like vector copy number, capsid empty/full ratio, and process-related impurities.
USP expects to release the proposed general chapter covering best practices for lentiviral manufacturing for public review and comment in early 2027 on the USP Pharmacopeial Forum (USP-PF).
We met up with Blaszczyk at Cambridge Healthtech Institute's Bioprocessing Summit, where he explained the need for standards to support these tricky vectors and what the USP hopes to accomplish. Our interview has been edited for clarity.
Where is the gap in how we characterize LVV now, and what are you trying to solve with this new general chapter and reference standards?
Blaszczyk: Everything with LVV can be challenging. Purifying it and making it can be challenging, but there's a particular need for advanced analytics.
Analytics are challenging. There's not a lot of harmonization in the field about how we're doing them. We're trying to build a bridge for that by developing a number of tools — whether they are physical standards or documentary standards — to help bridge that challenge where people can use our tools to help ease their analytics and improve the quality of their analytical testing from accuracy and precision, all the way down to robustness.
LVVs are structurally complex and fragile. How do these reference standards and the general chapter address that nuance?
Blaszczyk: LVVs are definitely challenging. They're not very stable. We have a section in our chapter dedicated specifically to the formulation and stability of LVV. It goes through some things to consider when doing formulation, because not only do you need to consider what's going to be most advantageous for the stability of your product, but you also must make sure it's going to work well when actually administered to patients, whether in vivo or ex vivo.
You also have to monitor stability because, whether it's sitting for just a few days or weeks, and even depending on the temperature, sometimes these things can break apart. Knowing what to look for and how to assess the stability, that's something else we discussed in that chapter. Ensure that your LVV is going to be working properly whenever you need it.
USP has done similar reference work for AAV. What kind of experience can you take from that characterization process for AAVs?
Blaszczyk: Yes, we really stole the playbook from AAV here at USP. What we're really trying to do with our materials is characterize them as well as possible with as many different analytical methods as possible.
We know, for example, with AAV there are many different empty/full methods. We characterized using all of them and then put all those values on the certificate. We're doing that for LVV as well, but it's even more of a challenge for LVV because what we see is there's many different methods, not just for one, but for many different CQAs.
What you'll ultimately see in some of our reference materials is we have, for one CQA, maybe up to five or six reportable values for certain CQAs because there's that many different methods being used for it. We want to give customers and labs the data that they want. One lab might prefer method A, and that might be the piece of data they want. Well, another lab might prefer method B. So, what we're trying to do is give everyone as much information as possible and characterize this as thoroughly as possible. We feel like that's the best way to help the field.
Vector copy number is a challenging CQA to nail down. How does having a USP reference material for VCN change the accuracy of comparability?
Blaszczyk: VCN is absolutely critical, and depending on what sequence you want to target, the method might be a little bit different, but what we did is target some of the conserved regions for the PCR method. We want to give people a standard they can use to ensure their method works. We offer two different reference materials — one for qPCR, one dPCR — and we also have an application note where we go through the methods we use for both dPCR and qPCR, including the primer and probes. We want people to be able to mimic our exact conditions, hopefully get the same answers, and then ultimately incorporate that into their workflow and use that, whether it’s a control system, suitability standard, or just to ensure that their method is working for them.
The VCN reference materials are both available now, as well as for residual HEK293 and residual plasma DNA, two other process impurity reference materials. Similar to the others, they're both PCR based. The HEK293 is qPCR-based, while the residual plasma could be used for either of them via positive control for dPCR, calibrant for qPCR, or even a system suitability for both of those. And just like the VCN, since it’s another PCR based method, we have application notes where we go through the method and share primer probe sequences. We're very adamant about making sure people are able to use the same method we do, because we don't want to see differences in values because they don't understand the method. We want to give them the method and the material so they can replicate it as closely as possible.
General chapter <1267> is in for review now. What do we have to look forward to next?
Blaszczyk: The chapter covers best practices for lentiviral manufacturing all the way to quality control. It's a great overview of the field and where it is, from the LVV design, through production, CQAs, and formulation.
The expert panel has been working diligently the last two years to draft this chapter. It's in a really good spot now. I think everyone at USP is pretty optimistic that it'll go up on USP-PF in early 2027. Once it's in PF, everyone, whether you're a member of USP or not, can review the chapter. Once people go through it, we address every single comment. We encourage people to make your review and, hopefully, learn something and give us feedback.
What does the adoption cycle look like? Do you expect manufacturers to incorporate the guidelines into their existing processes, or do you expect this only for novel processes?
Blaszczyk: One thing about our general chapters greater than 1,000 like this one, they're best practices and general guidance. For manufacturers that have existing product or something further along in development, I don't think anything here is going to surprise them.
We try to target these chapters for people both new in the field as well as veterans. Someone new in the field might be a little overwhelmed, but it should give them a whole LVV playbook on how to go about manufacturing, testing, and so on. But even for people more experienced in the field, it might not necessarily be something new, but it's a good reminder, a good reference to go back and make sure they're doing everything. I don't think it'll be surprising, but I think it also will be very educational, even for veterans.
About The Expert:
Anthony Blaszczyk is in the Pipeline Development group within USP’s Global Biologics department. At USP, he works with scientific experts and stakeholders to develop new standards to support biopharmaceutical quality assessment and development. Prior to USP, Anthony worked at Catalent Cell and Gene Therapy, where he managed an analytical development team responsible for the development, qualification, and transfer of methods. He obtained his Ph.D. in biochemistry from Penn State University in 2018.