Defining Your Product Profile and Maintaining Control Over It | A Look Back with Emily Shacter

Emily Shacter, PhD, Consultant, ThinkFDA LLC

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This is a transcript from a Q&A interview with Emily Shacter, PhD, Consultant, ThinkFDA LLC (former FDA Scientist and Regulator).

We will be talking today about the CMC Forum that was published back in 2005. We are revisiting it in the magazine to specifically update our understanding of how to maintain process control; understanding your process.

In general, how do you feel the discussions in the four-part paper from 2005 has held up after 10 years?

Emily: I think they have held up well. We spent many years developing principles through which to regulate protein products so what we discussed back in 2005 was all very well considered. Companies have also spent decades figuring out how to manufacture and control protein products. By the time we got to the 2005 forum where we talked about control of process- and product-related impurities, the field had already come a long way. There are certain details that have changed over the past decade – and we can talk about those – but the principles for how we evaluate the importance and control of process- and product-related impurities have stayed pretty much the same. So, I think it was a job well done 10 years ago.

What major aspects of process and product related impurity concerns have changed the most over that period of time?

Emily: I’m not sure that I could really pinpoint a major change, but how we understand and control impurities has evolved.

For example, we have seen an evolution and a lot more experience with using the principles of Quality-by-Design and developing a design space in which a process can operate and still result in the production of the desired product. That has certainly come a long way in the past 10 years.

In addition, concerns come up now and then about various specific impurities that we need to assess and these concerns tend to come in waves.

For example, over the past 10 years, we realized that tungsten in pre-filled syringes could lead to oxidation and aggregation of proteins. In some cases, serious adverse events could result. So, there was a wave of concern about tungsten. At around the same time, we also experienced a wave of concern over glass lamellae coming from container closure systems. More recently, we moved into a wave of focus on low endotoxin recovery which undermines the validity of endotoxin testing results; certainly an important impurity.

So, overall, I think there has been an evolution in what we know and how we address certain impurities in terms of how to measure and control them.

As more manufacturers switch to single-use systems or flexible systems, has there been a change in terms of product-related impurity concerns, like where the concerns are as it changes from raw materials to process equipment?

Emily: That’s actually a good question and a good example. I think the concerns there tend to relate more to how to validate a process when you have a single-use system and how to address leachables and extractables. For this, you move away from cleaning validation and you move towards how to ensure that each of the units of production equipment remain constant and how they are controlled.

So, yes, that shows how a change manufacturing impacts how we think about controlling impurities, mostly from concerns around process validation and the evaluation of leachables and extractables.

Generally, what has gone unchanged, surprisingly or not? It sounds like you already answered that, but is there anything that you’d like to add?

Emily: I think the principles have remained unchanged but if you look at any element of biotech manufacture, you can identify aspects that have changed and evolved. You gave a good example of a significant change. But, I think a lot has stayed the same and we’ve just improved, enhanced, and better defined what we do and how we do it. For example, we have a whole lot more experience with platform technology for monoclonal antibodies. We also have additional knowledge about critical structure function relationships in proteins and how impurities might impact clinical activity.

One other thing that has changed is that we have vastly more protein products that have been licensed or that are under development than we had 10 years ago. In the days when I was in CBER there was a more manageable number of biotech products to regulate and we could focus on each one in a more deliberative way. But now there are so many protein products, it’s increasingly hard for the FDA reviewers to keep up with everything. So, the FDA itself needs better systems for cross-pollinating what all of the different reviewers learn about the different products, for example by setting up databases for impurities, protein interactions, clinical sequelae, and how to control them. The number of products has changed a lot.

That makes sense. It’s all about data these days, anyway.

Emily: Mmmm hmmm. (Yes). And how do you keep up with those data?

So, along with Kathleen Champion, Helena Madden and John Dougherty, you were an author for Part Two that focused on host cell proteins. Immunoassays – ELISAs in particular – have been the most common approach to detecting host cell proteins. Is that still the case or are there any new methods that have been applied over the years?

Emily: That’s a good question. I think the answer is that immunoassays still remain the work horses of host cell protein analysis, and ELISAs are still the method that is used for release or in-process testing.

Back in 2005, we talked about using 2D gels and Western blotting to verify that the primary antibody is suitable for its intended purpose; i.e., that it can detect a wide spectrum of host cell proteins in a sample. I think we have evolved in that regard. The FDA has become more clear and precise about what it wants sponsors to do to verify that the host cell protein assay is doing what it is supposed to do, which is to detect (at least) those proteins that induce an immune response in an animal so that you can make an antibody and then use it detect those host cell proteins in your product. The method is still blind to those host cell proteins that are not immunogenic, but sponsors are using other means to try to boost up the number of antibodies that they generate – improving the polyclonality – so that they can see more of the host cell proteins. In addition, the FDA has become more precise and more prescriptive about what it expects to see.

For example, asking sponsors to run 2D gels and silver stain (or use another stain of high sensitivity) on one gel while doing an immunoblot on a duplicate gel. This allows you to look at the spectrum of proteins detectible in the silver stained gel and then determine which of those proteins is also seen by the immunoassay. If the antibody only detects 10 or 20% of the proteins that you can see in a silver stained gel, it’s not suitable. But, if it can detect 70-80% of the proteins that are present, well then that’s pretty good. That’s probably acceptable.

So, I’d say that the approach and regulatory requirements for using an ELISA for quantification of HCPs has become better defined over the last 10 years, and the FDA communicates these expectations routinely to sponsors.

The other approach that has come up more – and we started talking about it back in 2004/2005 – is the use of 2D LC-MS (2-Dimensional Liquid Chromatography-Mass Spectrometry) to identify the host cell proteins that are present in the product. In this case, you need to have the proteomic tools and the mass spectrometer sensitivity to be able to identify the different proteins that are there. You are not relying on the immunogenicity of the host cell proteins. So, that’s a very complementary method to enable you to evaluate and be very specific about what host cell proteins are present. Instead of measuring a combination of proteins in a spot on an ELISA plate, you are actually measuring the exact protein impurities that you are identifying.

This approach has advanced quite a bit over the past decade, and sometimes the FDA is even asking sponsors to use this approach in order to have a better understanding of what the host cell proteins are that are in a protein product.

Over the years we’ve seen some discussion about the value of commercially available generic host cell protein assays. What’s your take on that?

Emily: We certainly see them used quite a bit. In all cases, sponsors need to verify that the kit is suitable for detecting host cell proteins in a product derived from a host cell that was not the cell against which the antibodies were generated to form the primary antibodies of the kit. So, in other words, a company uses, let’s say, E. coli or a CHO cell to express their protein product. To detect the residual host cell protein impurities using an immunoassay, ideally they would immunize animals to extracts from those same cells (usually minus the protein-specific expression system) and generate a primary antibody that they can then use to detects the proteins that are in their host cell. But typically the host cell used to generate the primary antibody in a commercial kit does not use the same host cell that a protein product manufacturer uses to make their protein product. So, how do you know if that commercial antibody is going to detect the host cell proteins in your product?

You have to verify that the commercial kit is suitable for its intended purpose and this will typically involve what we mentioned before, which is to use 2D gels and immunoblots to see the spectrum of proteins that are detected by the primary antibody in that kit.

More often than not, I would say that the kit misses detecting a lot of the proteins in a protein product, and for understandable reasons. The same cell line was not used and the cells weren’t grown in the same way. Some of the proteins – especially many of the housekeeping proteins – will be the same, but there will be many other proteins that are different due to differences in the strain and in the culture conditions. Sponsors like to use the kits, and they are suitable for early development when you don’t even know if you’re going to have a commercial product in the end, which is dependent upon the success of the clinical trials. But I think that, over time, it is wise for sponsors to develop an in-house immunoassay to be able to detect the host cell proteins in their protein products. They know a lot about their process and they know that when the process changes, the host cell protein profile might change. You have a whole lot better control over the detection system and can make sure that you get an antibody that is suitable for its intended purpose when you make the antibody in-house.

I think that the kits are really helpful for early product development. It takes a lot of time to develop an in-house HCP method; a lot of time and a lot of resources. For early development – when you don’t even know if your novel product is going to have clinical success – it may make sense to use a commercial HCP kit. But I think that by the time you start moving through Phase II and it’s looking like you are going to go to Phase III, sponsors should really start developing an in-house method, especially if they are going to be in the game for a long time and not just be a one-product manufacturer.

What is currently the most talked about type of process-related impurity and have there been any high-profile incidents that have highlighted certain issues?

Emily: I think over the past decade, we have had a lot of talk of sub-visible particles in a smaller range than we used to look due to compendial requirements (i.e., greater than 10 and greater than 25 micrometers). Because of the potential similarity between smaller sub-visible particles and microbial/viral particles – and the ability of the immune system to see them – particles in the 0.1 to 10 micrometer range – looking at those specifically has taken a lot of prominence over the past number of years.

With subvisible particles – very small subvisible particles – you need to determine whether they are process- or product-related impurities. There are methods available now to help you determine if you are looking, for example, at silicone oil droplets or dust particles or whether you are looking at protein particles. These have become an impurity that needs to be evaluated and controlled.

Also over the years, metal leachates from pre-filled syringes have become a point of focus, like tungsten because of its ability to cause protein aggregation, which was the topic of Part 4 of the Bioprocess Series. That is stil a very important and prominent issue in biotech manufacture and control.

Those come to mind. What’s most talked about right now beyond those? Oddly enough, I think that for process-related impurities, host cell proteins are probably still the most talked about. They are always there and they will always need to be controlled. So, they are still much discussed.

What about product-related impurities?

Emily: We’ve had a lot of improvement in our ability to analyze protein structure, amino acid modifications, and glycoforms through advances in analytical technologies; for example, we are now much better at analyzing and quantifying glycoforms, higher order structure, and post-translational modifications. Consequently we need to keep up with what we are able to know about the structure and function of our products, including what is a critical quality attribute and why. So, we use these advanced analytical techniques more and more. Sometimes the information that we gain is more than we can actually understand and know how to apply. Like, I may be able to detect these different glycoforms, but what does it mean for the clinical profile of my product? Because some products have so many glycoforms, it would really be hard to tease out how they combine to impact clinical performance. But if different glycoforms or protein variants are there, you need at least to be able to detect and control them, unless they are known not to have clinical impact. The advent of improvements in analytical technologies requires us to keep up with the capability of the analytics and then control what we see – even if we don’t always understand how what we see impacts clinical safety and efficacy – we need at least to have a consistent product profile.

Also we’ve seen a waning in the use of SDS polyacrylamide gel electrophoresis – or SDS PAGE – and much increased use of capillary electrophoresis (CE) with SDS in its place. So, we are seeing a phasing-out of a technique that used to be a work horse for looking at product-related impurities – SDS PAGE – and now the main technique is CE-SDS or some variation thereof. Hence, we are seeing some change in the techniques that are used routinely now to analyze impurities in our products.

Is there anything else that you would like to add about the series and changes?

Emily: I suppose the other big thing that has happened in the past 10 years is that we’ve gotten legislation and guidance for the development of biosimilars in the US, and this has required an even deeper evaluation of all product characteristics including product-related variants (substances), which have biological activity, and product-related impurities which have less or no activity. This is critical for ensuring that there will be no significant differences in the clinical performance of a biosimilar after it is licensed for commercial use based upon a relatively limited set of comparative clinical studies. As a result, we are seeing a a deeper use of analytics for the development of biosimilars, and this may be spilling into the expectations for comparability assessments as well. That’s another thing that has changed from the regulatory and scientific perspective over the past decade. The FDA has had occasion to ask biosimilars sponsors to compare the HCPs in their product to the HCPs in the reference product, and I think this is probably unrealistic and unnecessary if the levels in the biosimilar are very low. Nonetheless, it is certainly important to establish that there is no impurity in the biosimilar that might impact the safety and immunogenicity of the product.

Beyond what I have already said, I don’t think I have anything else of particular interest to mention at the moment. If I think of anything else, I’ll let you know.

Thank you so much, Emily.

Emily: Thanks for the discussion.

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