A ddPCR primer: advantages, challenges, and key considerations when adopting Droplet Digital PCR for viral vector manufacture

Q Firstly, could you talk about the specific applications where you each employ ddPCR?

FD: For recombinant adeno-associated viral vector (AAV) the most important assay for vector characterization and batch release is the vector genome (VG) titration. Vector genome is considered the active component of the product, and the genome copy numbers are irrespectively used in preclinical and clinical studies. ddPCR, due to its high accuracy and precision, is a powerful tool for quantifying the vector genome.

LD: In addition to vector genome titration, we also use ddPCR to quantify residual host cell and plasmid DNA in our samples. These product-related impurities are a natural byproduct of our manufacturing process, because the AAV capsid can unintentionally package these non-target DNA species. We strive to minimize the presence of non-target DNA in our process, as it doesn’t provide any therapeutic benefit and can lead to an unwanted immune response in patients.

We also use ddPCR as a readout for both vector infectivity and gene expression assays to assess our product potency from an initial screening either in an in vitro or in vivo system.

SP: Following on the idea of host cell DNA, one of the key targets can be genes that we know are present in the host cell that may have oncogenic potential.

For example, one of the major cells lines we work with is the HEK293 cell line, which is transformed by adenovirus. One of the gene products, E1A, is a potential oncogene, and we want to make sure we have the lowest level possible of those DNA fragments in our product.

Looking at animal distribution studies, we can use ddPCR to measure how much genome is present in different tissues. We can even look at how much genome transcription is occurring by using some of the new reverse transcription ddPCR (RT-ddPCR) techniques.

BS: For a strictly quality control (QC) operation, which we have been involved with at this time, ddPCR applications include identity and vector copy number due to its preciseness and sensitivity, but also for safety tests. Mycoplasma is an ideal candidate due to its selectivity and precision to be able to find low levels of this. So we are also evaluating it in a safety test as well.

MW: This is in line with where Bio-Rad is trying to support the industry – vector copy numbers and vital titers being at the top of the list where most people are starting. As Bryan said, we are now offering a kit for mycoplasma detection. Some of the other areas such as residual DNA, plasmid, and potency are all developing areas that we are watching and hopefully we will be supporting in the future as well.

Q What are some of the key advantages and disadvantages of ddPCR over alternative tools for these applications?

LD: I will start with some of the advantages of ddPCR over traditional quantitative PCR (qPCR) methods.

ddPCR is an absolute quantification method. What this means is that we don’t have to rely on a standard curve to determine the concentration of our samples. It offers greater sensitivity for detection of low levels of target DNA, and it’s also highly precise and more tolerant to traditional PCR inhibitors.

In addition, we are able to obtain equal reaction efficiency when we test unpurified crude lysate samples or purified final drug products. Because of all of these qualities, we observe less assay variability run to run, between different analysts, and also over time.

The disadvantages to ddPCR are relatively minor, in my opinion. First, the method has a more limited range than qPCR, which means that we may need to perform additional dilutions to ensure we are still within the assay’s dynamic range.

In terms of assay throughput, ddPCR can handle a maximum of 96 wells at a time, versus a potential of up to a 384-well format for qPCR techniques. This translates into a slightly higher cost per sample run when compared to traditional testing with qPCR.

BS: Lauren has struck on some of the well-known advantages of ddPCR, so I can’t add much more there.

In terms of disadvantages it is a volume-based assay, so in terms of QC cost it can be a more expensive assay. You are going to have to weigh out the volume you are going to use it on pretty carefully. Certainly as this becomes more prominent in a clinical setting, and certainly as Mark can attest in the commercial setting, this works out okay in terms of the throughput. But it is something you have to consider.

We’ll get to this in a minute, but I would add the upfront investment into ddPCR. By that I mean the assay development itself. Clean, accurate, correct primer probes are key. You really have to do your upfront work to make sure you have got the right set for the assay, and that it is well controlled from that point of view.

SP: The only thing I would add is that ddPCR tends to take a little longer to set up and perform in many cases than qPCR.

This becomes a slight disadvantage, especially in a QC environment. Manufacturing wants to have an answer for how they are doing right away, and we can’t promise to deliver that answer in the same timeframe. It changes the schedule of testing a little bit.

But I think the advantages that have been described by my colleagues here do outweigh that, overall.

FD: I am also in line with what has been said. I would add that ddPCR is a very good method with a good inter-laboratory precision. This is very important for when you perform analytical transfer to a CMO – comparing results from different labs is an important thing to help with this.

Q Mark, do the comments you’ve heard resemble what you typically hear from customers you are working with?

MW: Yes, definitely. What it really comes down to with everyone we work with is the data quality.

While it can be an additional cost to implement ddPCR over some other methods, there is the reliability of getting high-quality data and having tight coefficient of variation – especially regarding what Fabien mentioned about transferring between one lab and another. We know many of our customers now start their process in-house and then they move it out to scale up.

Having that go smoothly and not taking 6–12 months is really important. This is where a lot of companies that have really embraced ddPCR have thought about the entire cost of ownership of the assay. Not just in their group, but as they move through the entire clinical process. That calculation really brings it home regarding why this has become such a gold standard in the gene therapy realm. The accuracy of the viral titer measurement determines what you dose your patient with, so data quality is paramount in that measurement in particular.

Q Could the panel share some practical tips or practices on how to optimally set up your ddPCR?

BS: I touched on this above: the upfront work with the primary probe set. What we have found is that the work, study and data you need to put into that is pretty substantial. You have to make sure that you ultimately have the critical quality attributes of the assay which are precision, specificity, and accuracy. That takes time. Along with that what you have to really consider is bringing your QC analysts along with you in that journey. Training is very important.

Although this is a highly automated assay, the pre-work necessary in terms of clean lab bench, appropriate set up, and training of the analysts on the different aspects and critical parameters of the assay up front before you put it in the machine, are critical. We have had some painful experiences trying to work through issues that relate to the technical training of the individuals.

Now, the automation and the design of the instrumentation of the assay doesn’t require a PhD, but it certainly does require a QC analyst with a four-year degree, and with certain attributes, who of course understands the science behind these things.

Lastly, it is necessary to make sure that you really are focused on what you are going after, how you are going after it, and what you are using it for. That is applicable in terms of not only the method development and method qualification, but in the actual method that analysts use day to day.

Q Steven, does that strike a chord with you in terms of training being one of the critical aspects you think of when looking to implement this successfully?

SP: Absolutely – because of its high sensitivity, one has to be extremely careful that we are not introducing any potential cross contamination. This involves training of the analyst on how to properly segregate the activities in setting up a ddPCR run, having the right stations that segregate those activities, and maintaining the cleanliness.

Another aspect of this training is how to set up your assay in such a way that you have some control over whether or not the run is successful. When you run qPCR we have that standard curve and we can look at how the standard curve behaved, and whether it met certain parameters.

With ddPCR we are not using a standard curve, so we have other parameters of the set-up we can monitor. But having a standard we can run every time and making sure that is meeting our expectations is also very important. As we said earlier with this possible excess pipetting we have to do because we have to dilute, it is confirming that we are doing this correctly every time, and that our pipettes are accurate, because that could add an error in this type of a procedure.

FD: As we know, you have to dilute your vector a lot to be in a good range for ddPCR. Sample preparation is a key step before the ddPCR method in order to limit the sticking of AAV or DNA to plastics, which can occur during DNAse treatment or during the serial dilution steps, for example. Sample preparation is key to a reproductive and robust assay.

LD: I agree with what has been said by the previous speakers. One thing I’d like to add is that during optimization of your ddPCR assay it is also important to screen your primer-probe sets for each gene target you are going to look at.

What we want to see is good separation between the positive and negative droplets. Some practical tips we like to employ during our screening process are to look at temperature gradients and investigate increasing the annealing time to further optimize our ddPCR reactions.

Q Mark, listening to the panel, are these pain points or challenges you are looking to address, or are you working with clients to support them on these?

MW: Yes, these are all the transition things to think about. It is interesting that as we support customers doing the transition, often they have to unlearn some things that they know that are fundamental to qPCR. It is a very different way of measuring.

We are measuring individual molecules in a droplet, so some of the controls that are important are different, as was mentioned by the panel. For example, the source of the primer probe sets – you can have contaminating templates in primer probe sets, so looking at where they are made and doing some quality control on the way in is actually much more important for ddPCR. Bio-Rad has spent a lot of time and energy on making sure that what we offer for ddPCR is clean of any contaminating DNA. Our team has experience with all of that, and we’ve got multiple white papers, as well as experts on staff who can help with that transition.

It is definitely a short learning curve, and again circling back to what Steven said earlier, the benefits outweigh the drawbacks once you get across that start up and assay development phase and start running. The benefits justify that transition.

Q Next, let’s do a deeper dive into the important considerations when transitioning into ddPCR from a different platform. What additional insights can you share?

SP: As has been mentioned, determining the optimal primer probe set is paramount for getting a good ddPCR, and looking at the best separation between positive and negative controls. I have had cases where we have had a qPCR reaction and we tried to use the same primer probe set, and we didn’t have a good optimization.

Additionally, have a good reference material that you have properly titered. When you run it in each ddPCR assay, by achieving the same result every time you are running it, you can be confident the assay was performed correctly.

Another thing we found a problem with at one time, and I think you’ve got to consider, is duplexing. An advantage of these methods is that you can do more than one type of reaction at the same time. By duplexing you could be looking at two different targets in your genome, and trying to get an idea of what partials you might have in your material. When we set up one of our duplex reactions we found that we were having a bit of a problem with it, and had to go to a slightly different buffer than normal in order to get that to work properly. These are some of the considerations you have to consider every time you are going to be transferring to this kind of method.

BS: When I think about this question I also think of the analyst element to it. You really have to really hone in on some of the essential and principle GMP compliance aspects of this as you move this through, hopefully into the success of your product and filing.

It is precision; it is accuracy, it is control of reagents – critical reagent control. There is, and should be, an expectation of the stability of the primer probe set, and stability of some of these critical solutions. It is not up to the manufacturer of the primer-probe set, or Bio-Rad, to demonstrate this. When the FDA comes to your shop, it is up to you to have the data for your product.

It is absolutely essential to be thinking not only of the analytical benefits of the assay but also to consider the GMP compliance aspects and make sure you are prepared to put these things in place. The benefits outweigh the effort here, especially as you get into more volume throughput through your laboratory. But it is a key aspect to remember.

MW: Thinking about the compliance, we spent a lot of effort on the Bio-Rad side building in parts to the software to enable our customers to be compliant. Then, like Brian said, it is really up to our customers to validate everything and have that package.

What we are trying to do on the Bio-Rad side is make sure that it is as easy and seamless as possible for our customers, and make sure that we have really high-quality products going in. For some of our newer assays that we have on the market, we are also developing data packages that can help guide our customers on how to put that package together themselves. This means they have a starting point and some material to reference; essentially a step-by-step. They can see the data that Bio-Rad put together and understand what they need to replicate in their lab.

This is another key area, and why droplet digital from Bio-Rad has moved quickly into this space. We are keenly aware of those challenges you all are going through, and trying to build up our support structure to enable that.

BS: I would add that some things don’t change; some things are foundational. In terms of assay qualification and assay state of control, the EMA and FDA have plenty of information on their websites on potential pitfalls, or they do surveys of 43s or observations. There is plenty of information out there from those leading agencies, and others as well, on the expectation of assay control.

All of those principles apply to ddPCR. If someone is interested in making this transition – and they should be, as this is where it’s going – then there is plenty of information out there to look at in order to make sure you build in not only the scientific efforts but also the compliance efforts.

MW: To build on that, one of the things we observe on the Bio-Rad side is that as people are migrating away from the standard curve, it takes a lot of time and energy to maintain an accurate standard curve. When you have to replace it, it is a lot of work to make sure it is the same or recharacterize it. And so, there is a big benefit to not having to do that as much.

But like Steve said, you have to have a standard material that you can use to kind of bridge between lots, and all those types of things. But one of the things qPCR users find out when they move to droplet digital in this area is that if your standard curve is off, every other measurement is going to be off.

Often we have people measure their standard curve with droplet digital and find that the absolute count is quite different to what they think it is. There are lots of different reasons for that, but that’s an interesting and really detailed nuance to this whole quality control aspect that is eye-opening for people as they start using digital. I don’t know if anyone else has had that experience, but I’d be curious to hear from anybody else on that.

Q We have touched on the need for standardization. It would be great to talk about some of the key issues and requirements related to standardization that are relevant to the various specific applications of ddPCR.

BS: Standardization of an assay in general, in a quality control environment, is something that is critical to a high-throughput clinical, commercial, QC operation.

ddPCR lends itself to that because the methodology for prep and the process is pretty standard. This holds true across different genomes, different pieces of gene you are looking for, whatever your target is – whether it be copy number, identity, or whatever it may be. That is pretty straightforward. It goes again to what Steven pointed out: appropriate laboratory logistics. It’s in a clean place. It’s well organized. Some of us in the industry do a lot of 5S-ing to make sure everything is organized for this standard flow.

Obviously, with that standardization comes increased assurance of accuracy. At the end of the day that is what we are looking for: a valid, reportable result.

LD: When I think about standardization, one of the major issues we face is how unique each gene therapy product is both in terms of the capsid as well as the package transgene.

Some organizations such as the USP and NIST are working together to develop various AAV reference materials that could assist in the development of robust analytical methods, including ddPCR. Certainly, the implementation of standardized reference materials could be very helpful in initial ddPCR assay development to help ensure that our core method is accurate and robust.

However, due to the differences between each gene therapy vector, applying this to both vector genome titration assays and potency assays which are designed to be product-specific could make implementing universal AAV reference materials challenging.

SP: Reference materials would be very helpful, because especially if you are setting up training for a new group to test your material, you would be able to establish that assay is working similarly.

Another thing I have observed is just technique and pipetting. I had an example of working on a project where we were doing ddPCR to support a client, and we weren’t able to get the same values with the materials that they sent us. They sent somebody out to us to show us how to do it, so to speak, and it turns out it was a difference in pipetting technique. They were doing some things that we didn’t think were proper in a QC environment, and yet that’s what led them to get their value consistently.

We tried it their way and it worked the way they said, but not with what we thought was a better technique. So again, that consistency and training is very important. Making sure that everybody is trained on not just how to organize themselves, but on how to perform very simple tasks like proper pipetting.

MW: There is definitely a lot of talk in the field about a standard material that everyone can reference, as a way to test their process.

If you are implementing droplet digital, you can get a material from a NISS-like organization to verify that what you are doing is good. Then when you move to your custom assay, at least you know the basics of droplet digital are working in your lab. So if something isn’t working about this new assay, you start focusing on the primer probes, source material, or any inhibitors that may be unique to that material.

That is something that Bio-Rad is supporting and bringing up. What we are seeing on our side from our ITR-2 assay targeting the ITR-2 sequence, is that that was the best sequence that we could find that cuts across as many of the different products that are out there as possible. Everyone getting it from Bio-Rad is at least one step towards everybody running a similar assay as a set point or a start point.

I think it is especially important for the new labs that are moving from qPCR to digital to have one known assay that Bio-Rad knows very well, and the field knows very well. This has been valuable for everyone, so we will continue to make those.

To touch on what Steven was talking about with duplexing, we built a design engine that allows people to take one of these off-the-shelf ITR-2 assays, or a few others that we’ve developed, and pair it with a completely custom assay. The design engine makes sure those will two work together, so you won’t experience what Steven experienced where when you pair them together all of a sudden things go haywire. That’s another thing we are doing to try and help advance that aspect of multiplexing in titer measurement.

Q It would be great to explore some of the key additional areas in which ddPCR is beginning to be applied, and the impact so far.

FD: ddPCR’s main advantage is to isolate vector particles and enable you to check for genome integrity, and using 2D ddPCR or duplex ddPCR for deeper characterization of the vector genome.

This could also give you key information in the early stages of your project, for example when designing your vector genome, and in the upstream process for comparison of production systems and transfection reagents, in order to improve the quality of your final product.

LD: We can also use ddPCR to measure the potency of our gene therapy products, as I alluded to earlier. Drug candidates can first be screened either in vitro, in an animal model, or in vivo using cultured cells. Then, we can use ddPCR as a readout to assess various properties of our product potency, such as vector infectivity and integration, as well as target gene expression.

For example, gene therapy products can either knock down existing bad genes, or replace missing good genes. We can assess this by coupling reverse transcription of our mRNA to DNA, and then subsequent ddPCR quantification to measure the expression of our particular target gene. This powerful technique is used in preclinical drug screening, as well as throughout the entire drug development process.

It is particularly critical for the characterization and release of our clinical material to demonstrate both our product strength as well as the batch-to-batch consistency of manufactured drug products. In the last couple of years a lot of focus has been placed on gene therapy potency assays, and I anticipate this trend will continue in the years to come.

BS: Aside from those mentioned so far, one of the interesting areas is coming out of our translational sciences function, due to the unique ability of ddPCR to measure, and its accuracy, precision and specificity in complex backgrounds.

Our translational scientists are considering and evaluating it from a clinical patient point of view. Being able to use it in pharmacokinetic studies, and asking how our therapy is working in patients. How is the CAR T expanding, how are we detecting it?

I find it fascinating that we have now moved from final product or end-process product or drug discovery, and are actually moving to the bedside with patients. I am looking forward to seeing what the future holds there.

SP: My colleagues have defined a lot of exciting things we are working on with ddPCR. Because of the specificity that we can drive it with we can go into patients or animals and look at biodistribution, and be able to distinguish between regions that don’t receive a therapy product. Our primers are specific enough to identify endogenous genes that might be present there versus those areas that are expressing and have received our gene product.

This is a very powerful tool for being able to optimize our vectors in vivo, which is clearly where we want them to be most potent. And potency is key. We are always looking at how we determine the infectivity of the vector in as accurate a way as possible. Because although the genome titer is what we dose by, we want to know that the ratio of active molecules to just genomes is going to be consistent from lot-to-lot. These are some of the indications that we are continuing to develop and expand this technology for.

MW: I think we are seeing similar things, and it is the obvious next step as people find the value in titer to ask where else we can apply this. I will highlight two areas I have seen that are exciting and up and coming.

One is incumbent plasmid equality. We do a lot of studies at Bio-Rad, and we had plasmids coming in where there was a whole duplication of the entire gene of interest, back-to-back. We could count the molecules of promoter, gene of interest, and poly A, and we saw a doubling of the gene of interest. And it had been concatenated.

When you do a restriction digest it all looks the same. With droplet digital you are counting molecules, so we could tell that this vendor had sent us a concatenated gene of interest. This is one area where multiplexing and counting molecules helps with quality.

Another customer of ours did a very elegant study. He took a six well format vector copy number assay and reduced it to a 96-well format with no DNA isolation. They used cell lysate directly, and counted a reference gene copy number to establish the number of cells in the reaction. They also multiplexed an assay for their target vector, and with the reference assay, were able to very accurately count the number of molecules of their target compared to the number of genomes. They were able to scale up dramatically from six-well plates to 96-well plates, and the coefficient of variation came down threefold with the new assay format. Because of ddPCR’s tolerance to inhibitors, the 96-well assay format was enabled. I think there will be more creative ways to apply droplet digital going forward now that many people have it and it is becoming more standard.

Q Finally, given the regulators current priorities and likely future evolution in this regard, what is your expectation for the future application of ddPCR and other such tools? How or where will the technology need to adapt and keep improving?

MW: The one I see is the evolution of the standards of the regulatory bodies. Gene therapies are becoming much more popular, and I think the FDA has been a great partner in helping these get to the market. It is very new and there is a lot of innovation happening, and they are being quite flexible as this is going along.

There is going to be a renewed focus on what the therapeutic piece of DNA is. It’s not just the vector backbone; it’s the gene of interest itself. And it is making sure it’s not just the gene of interest, but the promoter, plus the gene, plus the poly A. That whole cassette is required to deliver the therapeutic molecule, so I could see multiplexing becoming very important. Not just a single number titer, but all the molecules making up that titer, and making sure all of the therapeutic parts are getting into your virus and into your patient.

This goes back to what Steven was talking about. If you just look at one part of your gene of interest, what happens if it is truncated, what happens if the whole thing doesn’t get in? As multiplexing is enabled there might be a higher level of scrutiny on those parts of the genome that are getting in.

Looking at identity and integrity of your plasmids, of your vectors after they are packaged, is going to be an interesting area of focus in the next 5 years.

FD: I would add that we need a method for process analytical control. By improving the sensitivity of ddPCR and the detection of the target sequence, for example with low cycle, ddPCR may be a good tool for the future of in-process technology.

SP: If we take a step back and ask what some of the other issues that we deal with in the gene therapy realm are, one is certainly confirming that the materials we want to take into patients are free of any other adventitious agents. That has been a concern for a lot of people for a long time. Now, there are some newer technologies other than the in vitro assays and animal assays, and we do things like next-generation sequencing. I would not be surprised if ddPCR can play a role in that in the future.

Its extreme sensitivity could allow you to identify potential viral genomes that might be present in material at levels where we have problems with some of these other assays. And although DNA itself doesn’t mean that you’ve got a problem, it is something the regulators are always interested in, and how we make sure things are as safe as possible. I think that’s an area of future interest.

LD: In the future, we could envision a standardized ddPCR method for titering AAV gene therapy products. This would ensure that companies are dosing their respective drug products at similar levels. So a patient dose of 1E+13 vg/kg would be the same for gene therapy product A as for gene therapy product B. What this would allow us to do is correlate dose and safety data along with other critical quality attributes across the board, from one gene therapy drug product to another. We could then apply these learnings to enhance both the safety and efficacy of future gene therapy products.

BS: I’m going to go back to the promise of it at the bedside, in terms of the clinical patient samples, and what will be utilized in clinical development, clinical studies, and biometrics. How can it advance the knowledge of the performance of the gene or CAR T-cell therapy in the patient itself?

I think in the industry we have become very savvy at characterizing manufacturing processes, certainly in the last few decades. What we haven’t become savvy at, and what we’re still exploring, is characterizing the effect of these very complex therapeutic molecules, genes, CAR Ts, in the body. I really look forward to seeing how that is further explored.

Authorship & Conflict of Interest

Contributions: All named authors take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.

Acknowledgements: None.

Disclosure and potential conflicts of interest: The authors declare that they have no conflicts of interest.

Funding declaration: The authors received no financial support for the research, authorship and/or publication of this article.

Article & copyright information

Copyright: Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.

Attribution: Copyright © 2021 Bio-Rad Laboratories. Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.

Article source: This article is based on a recorded roundtable, which can be found here.

Roundtable recorded: Nov 10 2021; Publication date: Dec 13 2021.