We interviewed Educo Life Sciences trainer Nadine Ritter to discuss the most challenging analytical assays for biological products. In the interview Nadine discusses the issues scientists face with Host Cell Protein assays. She also discusses the challenges of potency assays and how early decisions can significantly impact development stages later on.

Watch the full interview below:

What is the most challenging analytical assays in biotech?

I think that people who are experts in the technologies would say theirs but looking over the pallet of tools that we have for biological products there’s two of them that cut across every biological product. One of these that is significantly analytically challenging are residual host cell proteins.

Every biological product that is coming from an expression system (which is kind of the definition of biological product), your purification team is trying to enrich downstream the molecule that you want. They are trying to remove the molecules that you don’t want that the cell needed to make the thing that you do want, and inevitably you cannot get rid of every single piece of residual host cell protein or host cell nucleic acids. The DNA assay has got some features to it that make it a bit more amenable to being more platform like because it’s a genome, but the host cell protein assay is the cells proteome and that’s a function of cell conditions, of cell stock, of a lot of things, of age of the cells, of apoptosis level of the cells in the culture.

As a result, there is a lot of variables that lead to a possible thousand or more analytes which could come downstream. We have seen already in case studies that slight changes in the upstream or downstream conditions can lead to a shifted population that appears in the drug substance. The consistency of the proteomic mixture of proteins is the challenge for analytical assays and its and even further challenge because they may be below the limit of detection of our methods. Most importantly they are not always below the limits of detections of our body. Our body is exquisitely sensitive to foreign proteins as we have evolved that way on purpose.

We have got something which could be problematic to the patient. We have seen publications where some host cell proteins are problematic to the product. There are things which can exacerbate degradation pathways that if they are residual in the product, they don’t necessarily cause biological problems in people, but they can certainly change your product quality attributes overtime.

With the assessment of host cell proteins, we have had some amazing advances in the last decade or more including tools like 2D-Dige, mass spectrometry. These are in addition to the classic tools we’ve had, such as immunoassays. Each tool has its own strengths and weaknesses, it has its own limitation, and my concern is that in the field there is a lot of misinformation. We’ve had lots of wonderful advances like commercially available HCP allies which filled a tremendous unmet need that we had in the industry. Before you got to your process specific HCPSA you’re blind, that filled a hole. But we have gotten so far so fast and there’s so much misinformation about quantitative techniques, and what you see, and don’t see, that’s the most challenging. So, I really encourage people to pay attention to their HCP characterisation and control strategy because the tools are very powerful when they are used correctly

What is the second most challenging assay in biotech?

I would think that the second most challenging one would be functional assays, potency assays, particularly potency assays. We have a statutory requirement that biological products must demonstrate a certain amount of activity, potency, for their quality attributes. It is the difference between an egg that can become a chicken and egg that is fried, so I could have the same egg but it’s not going to do it is supposed to do if I fry it. We need to have functional assessment of our biological products and many of them, not all of them, are based on a relative potency assay which requires an established calibrated stable batch, or batches, of the product itself, as a reference for potency assay.

It is extremely challenging to develop and establish a reference standard when it is the first one ever. That literally becomes a chicken and egg scenario. There are better and worse strategies at the beginning. The one major thing that people fail to recognise when they set up their CMC programme, is that that the decisions they make and the data they generate on their very first potency reference standard will live with them all the way through development. From the beginning of their clinical experience all the way through to commercialisation. And how do you go from one reference to another? That’s challenging for potency, for physical assays that’s not hard to do but for potency assays it is very challenging.

“The one major thing that people fail to recognise when they set up their CMC programme, is that that the decisions they make and the data they generate on their very first potency reference standard will live with them all the way through development”

The assays themselves can be challenging, although we have made some huge strides in the last decade or more with better control of critical reagents even to some cases ready to use cells. The assays themselves are not as challenging as they used to be, so long as we operate them in a state of control. The biggest challenge is the control and characterisation and stability of the relative potency reference standard for the product.

Where can people find information to learn about the regulations and guidance?

Unfortunately, in biologics we’ve had guidance documents that say that thou shalt have host cell protein assay to monitor postal proteins and thou shalt have reference standards that you can measure relative potency with. But none of them go into much granularity about actually how to do it. There have been some industry meetings, it’s probably obvious I’ve been associated with one of the organisations CASS for very long time. We have had some CMC forums on HCPs and on potency assays and on reference standards for potency assays. Those are case studies and they’re very interesting and important and they provide some best practises, but there’s not a lot in official guidance. This also contributes to the misunderstanding or the poor decisions that are made in CMC strategy.

Of course, since I’m talking to you I could say come to a class and I will share everything I know about these with you, but it is an accumulation of various bits and pieces and we don’t really have a very clear, consistent, comprehensive set of regulatory guidance documents drilling down into either of those subjects.

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