Now the amount of information for the C&C section that you need to submit is dependent upon the stage of the clinical investigation, the duration of the investigation, and the dosage form. And the data we have to submit to the agency is the data to allow the reviewers to allow the agency to ensure that the product has the correct identity, quality, purity, and potency for that stage of development, and the amount of this information will vary. For example, a stability data needs to be submitted, which demonstrates that the product that you are giving to patients is within specification for the duration of the clinical trial. If you’re doing a single ascending dose study for your phase one study, then your trial time may be very short, it may be a week to get all your subjects dosed, so you would only need a very short period of time for your stability study.
However, if you’re in phase three and your study is one or two years of duration, then you need to make sure that you’ve got data demonstrating that the product will remain within specification for one to two years. So this is an example of how the amount of information, and the type of information, and the length of the study, and that data that one submits to support a clinical investigation in the duration of that investigation will be a variable. Now it’s well understood that the manufacturing process, and the product itself, will undergo optimization during the clinical phases, and thus the manufacturing process will change. And as that manufacturing process changes then additional information needs to be submitted to the agency to document those changes.
Prior to phase one the focus of the agency review is on the safety to subjects. And all data that needs to be submitted for the C&C prior to phase three needs to have a focus on safety. Any data that does not affect safety can be submitted during phase three. Now the agency does have the ability to put a clinical study on hold. What this means is that the agency will not allow the study to proceed if there is a safety concern, or if there is a lack of sufficient data for the agency to make an evaluation that the product is safe. And for a chemistry manufacturing and controls, reasons for concern may include a product that is made with unknown or impure components.
One can have a product possessing chemical structures of known or highly likely toxicity. A product can have an impurity profile, which indicates a potential health hazard, or insufficiently designed, or insufficiently defined impurity profile to assess the health hazard, or one may have a poorly characterized master or working cell bank. But what is important is that one identify, monitor, and assess signals of potential human risk in prior to the clinical study, for example, that may arise due to the manufacturing process.
Now, components are ingredients intended for use in the manufacture of a drug product, including those that may not appear in such drug product. For example, for our biotechnology products that are derived via fermentation, then ingredients that we add to enable cell growth, to enable cell reproduction, to enable the production of our protein are considered components. Even though these components may not appear in the final drug product, it is very important to know the source of these components. For example, we frequently use human serum albumin as a stabilizer in the final product of four proteins. One must trace the source of that HSA, and ensure that there are no adventitious agents, et cetera, that may affect the safety of the subjects in the clinical trial.
We also use fetal bovine serum in the fermentation of our mammalian cells, and one needs to assess the risk of BSC for this product. In the CMC section, we need to provide the agency with a general method of preparation of the product, to include the reagents, solvents, and components that we used, so that they can make a determination that these are safe. It in general is better to use compendial, versus non-compendial components. Compendial components being components that have monograph in the USP, the European Pharmacopoeia, or some sort of national formulary designation.
One must ensure that the product that one gives to subjects in a clinical trial has known chemical structures. Also, one must make sure that the structures are not likely to develop into toxicities in the subjects. That’s a lot more important with the small molecules than it is for the large.
What we need to do for the CMC section, in this regard, is we do a structural analyses looking at not only the amino acid sequence, but also at the peptides of the product, and we determine identity down to that level. If there’s a glycosylated chain, a glycosylated structure on the molecule, we need to understand the oligosaccharide pattern of that change, and the site of the glycosylation.
Physical chemical properties are also important. We measure molecular weight or molecular size through SE-HPLC, SDS-PAGE, mass spec. We look at isoform patterns to gain an understanding of what that range will be. We use electrophoretic patterns to look at identity of the pattern, to look at homogeneity and purity of the product itself, and we use various liquid chromatographic patterns, such as SE-HPLC, RP-HPLC, in order to gain an understanding of the physical chemical properties of the product.
I get asked a lot what does a typical protein characterization look like. On the previous slide, we looked at molecular characterization in general. To what level does a molecule have to be characterized? There’s a lot one can do.
This slide shows what a typical protein characterization would look like. One verifies amino acid sequence by using peptide mapping. Identity impurity can be confirmed using isoelectric focusing. Molecular weight can be determined using mass spec. SDS-PAGE is a good way to confirm the molecular mass and amino acid sequence, et cetera.
One thing I do want to mention about biologics is that the term efficacy is one that is not presented in the PHS Act, which is one of the laws for which most of the biologics in the United States are approved under. We use the term potency, and for biologic potency means a biological activity. Potency is usually determined by using a binding assay, such as an ELISA.
Now impurities can also result in safety issues that need to be monitored during clinical development. There are two types of impurities. There’s process related impurities and product related impurities. Process related impurities are impurities that are derived from the manufacturing process. They may be derived from self substrates, such as host cell proteins that the cells naturally produce, or host cell DNA that’s naturally produced by the cells. They can be derived from cell culture, such as inducers that we use to initiate a protein production, or they can be derived from downstream processing, such as leachables from the columns that we use for purification.
Product related impurities are molecular variants of a desired product. And these molecular variants do not have properties comparable to those of the desired product, with respect to activity, efficacy, and safety. So these molecular variants are truncated forms, deaminated forms, and they may be oxidized forms, and they can lead to the development of immunogenicity due to the formation of aggregates or neoepitopes.
Now impurities may be of known structure. They may be partially characterized, or they may be unidentified, but one needs to ensure that they are characterized to the extent possible. If possible, biological activity should be evaluated for the impurity, and there are many methods available to do that, such as HPLC, SDS Page, OMS Spec, immuno acids hybridization techniques, et cetera.
Now, a certificate of analysis for a drug product to be used in a clinical trial will contain usually the following tests. The certificate of analysis separates these tests into the areas of: quality, potency, purity, identity, and safety.
The typical quality tests are appearance, such as color and visual particulates and pH. For the potency assay, typically an ELISA is used. Purity may be determined using SEC, HPLC, SDS-PAGE, reverse phase HPLCs. There are many ways to determine identity such as peptide mapping and terminal sequencing, et cetera. And safety testing consists of sterility testing, testing for endotoxins, and also looking for host cell DNA.
Now, the master cell bank, and the working cell bank from which cells are taken and used in the production of biotech products can lead to a safety issue during clinical development. And in order to ensure that an IND is not put on hold, due to a CMC issues, one of the issues that the agency does look at, is a master or working cell bank to ensure that it’s been adequately characterized. That one has an understanding of the identity, purity, and suitability of the cell substrate and that that has been confirmed.
I’m giving you two definitions here, and this is all taken from ICH Q5B and Q5D. A master cell bank is an aliquot of a single pool of cells, which generally has been prepared from the selected cell clone. It’s dispensed into multiple containers and stored under defined conditions. And from this master cell bank, one derives a working cell bank. And that working cell bank is used on an every day basis.
Now, a company may or may not have the cell banks. It is completely appropriate to have solely a master cell work and not have a working cell bank. But one needs to confirm the identity, the purity, and the suitability of the cell substrate. For purity, one needs to ensure that the cell banks are free from adventitious agents and contaminates. If one has a mammalian cell bank, one is looking at bioburden, microplasma levels, and the level of viruses. For microbial cells, one does a visual examination to see if there’s any contaminating colonies. Looks for the presence of bacteriophage, gram-staining is done to ensure the identity, and to ensure also that the purity.
Identity in this sense means that the bank cell is what it is represented to be. And there’s a number of tests that one can use for that. For microbial cells, one grows the cells on selective media, ensuring that they are pure, and ensuring that they are what they’re supposed to be. And one can also do partial genotyping.
For mammalian cells, one looks for markers that may be unique to the mammalian cells when they do a DNA analysis and a morphological analysis. A genetic analysis is also done, looking to ensure that the genetic construct has been retained and that the DNA sequence of the construct is also there. So that’s the type of level that one needs to characterize the master and working cell banks, prior to entering clinical development.
Now, that’s the last slide that I have for the CMC section, and what’s required for the CMC section. Just to recap, chemistry manufacturing and controls can result in a safety concern to the agency that would result in a clinical holder that could result in a clinical hold being put on a clinical trial. So one needs to make sure that the manufacturing process is not introducing any additional safety concerns. Because when we get into clinical development, the safety of the subjects is the number one priority and the number one concern.
And these reasons for concern may include unknown or impure components. It may include a product possessing chemical structures of known or highly likely toxicity. It could include an impurity profile that may lead to a potential health hazard or it could be an impurity profile that hasn’t been sufficiently defined or sufficiently characterized. Or one could have a poorly characterized master or working cell bank. And all of these issues may result in the agency not being able to determine if the product is safe for use in clinical studies.
So now we’re going to-
The lecture today covered a lot of material. We covered preclinical from an in vitro and an in vivo perspective and we looked at setting the maximum [CMC 00:00:07] safe starting dose for our first in human study. Next week we’re going to look at [Faith One 00:00:29] studies. We’re going to look at clinical trials in general and get more into a clinical development. Please don’t hesitate to contact me either by e-mail or by phone if you have any questions. Thank you