Many pharmaceutical companies make use of Contract Development and Manufacturing Organizations (CDMOs) to help them develop new products. Within the CDMO, Formulation Development and Analytical Development Scientists work together as teams to help client companies to achieve the best product designs for their APIs. Speed is often considered to be an important factor here as clients require that we get their products to the market very quickly. This is understandable as there are patients out there waiting for these therapies to come available and we should certainly minimise delays.
I believe that an important aspect of our CDMO activities is to conduct thorough data interpretations of the experimental results that we receive during our development studies. This is essential to enable us to provide explanations to our clients on the meaning of those results and therefore make recommendations on how to continue the development program. Unfortunately, sometimes the demand for speed in the delivery of results leads to a misunderstanding by scientists that rapid delivery of results is the best service that we can provide when it is the provision of knowledge on how to proceed that represents our best service.
Data Interpretation may be thought of as a relationship flow from Data through Information to Knowledge. For example, for a stability study time point on an experimental formulation, Data might be the analytical results in the form of chromatograms and tables indicating retention times and estimated quantities of related substances; Information could include the chemical structures of the various components observed and explanations for their presence; Knowledge is then the details of the best formulation and manufacturing process for the target product.
The following case study provides an example of the application of Data Interpretation to demonstrate what can be achieved. This was a real Excipient Compatibility Study case which is an example of an early-stage stability study but in order to protect client confidentiality, some details e.g. chemical structure of API and some product information have been modified.
Case Study
Study Details
The product concerned was a tablet formulation of a new chemical entity (NCE). The tablet was required to be Delayed Release so included an enteric coating. The client particularly wanted this enteric coating to be constructed using a Cellulose Acetate Phthalate/Polyvinyl Acetate Phthalate combination. This is unusual but not completely unknown composition for a tablet enteric coat, but we were not aware of reasons for this choice.
The study concerned here was an early Excipient Compatibility Study (ECS) that was designed to indicate the possibilities of interactions that may occur between API and potential excipient components. Binary mixtures of API and individual components were prepared and held at 40℃/75%RH. Samples were then pulled and analyzed at the following time points; T=0, 4, 8, 12 weeks. Samples were analyzed by reversed phase HPLC for potency assay and related substances but only the related substances data is relevant for this case study. Excipients selected for this study included many of the commonly used tablet excipients plus Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate. Interesting results were observed in the 8-week timepoint samples. Although the primary purpose of this study was to establish which excipients would be suitable for further development, we conducted some data interpretation which enabled us to provide much further advice to our client.
The Data
The API was a relatively stable molecule, and no interactions were observed with any of the excipients in this study apart from the Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate. In both cases we received similar chromatograms as shown in Figure 1.
For API mixed with both Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate 4 related substances peaks were observed to develop over time as summarized in Table 1 for API + Polyvinyl Acetate Phthalate
A number of control samples including neat API and individual neat excipients were also held at 45℃/75%RH to help us to explain the results shown above.
The related substances chromatogram for neat API is shown in Fig 2.
The chromatogram in Fig 2 shows the main API peak and then a single small peak with retention time of 12.61min.
Related substances chromatograms for control samples of Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate are shown in Figs 3 and 4 respectively.
Figs 3 and 4 show 2 very similar chromatograms. Note that since these 2 compounds are very insoluble, we see no peaks attributable to the main components, but we do see 4 related substances peaks that include an intense peak at 9.85/9.84min and 3 less intense peaks at 10.73/10.72min, 11.30/11.28min and 12.59/12.57min.
At this point we could just send all this data to our client, but this would not be a good example of client service. Surely a better approach would be to make some effort to figure out what may be happening here and send that to our client together with the data. This would at least provide our client with some information and is demonstrated in the next section.
The Information
To help follow this explanation, 4 chromatograms are shown together in Fig 5.
It should be noted that at this stage of early development, we did not have any product specification that indicated acceptable levels for related substances so the results that we received as previously summarized would not trigger any Out of Specification Investigation. However, when conducting this type of study, it is recommended to follow the advice provided in ICH Guideline Q3B(R2) that indicates how to establish specification limits for related substances in drug products. In guideline Q3B(R2), acceptable limits for related substances are based on maximum daily doses of the drug product. For the category of our client’s product, if any new impurities exceed a level of 0.2%, they would require structural identification and also qualification (toxicological safety evaluation). Higher levels for the impurity may then be accepted but they would still be considered quite undesirable. So, when we were seeing reported levels at around 2.5% and 4.5% in just 8 weeks, this was a definite concern. Even though the storage temperature was 40℃/75%RH, it could be anticipated that under ambient temperature/%RH conditions, we still would not get much of a shelf life for this product.
So, it was important to recognize this and provide our client with some recommendations.
The chromatograms shown in Fig 5 include the following features that require some explanation:
- All chromatograms show a peak that elutes at around 12.6min and the intensity of this peak is about the same in each chromatogram. This peak was also observed in blank injections and is almost certainly a system peak. The chromatographic system for this analysis includes a gradient composition of the mobile phase and this peak is a refractive index effect due to the change in mobile phase composition. So, no further explanation is required for this peak.
- There is a quite intense peak that elutes at around 9.9min and shows in chromatograms of Cellulose Acetate Phthalate + API and Polyvinyl Acetate Phthalate + API as well as chromatograms of these excipients without API
- Chromatograms of Cellulose Acetate Phthalate + API and Polyvinyl Acetate Phthalate + API show a peak at around 13.4min that does not show in chromatograms of the excipients without API
- There is also a peak that elutes at around 11.6min the shows in chromatograms of Cellulose Acetate Phthalate + API and Polyvinyl Acetate Phthalate + API
- Chromatograms of Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate show small peaks at around 10.7 and 11.3min. But might that peak at 11.3min be due to the same component that showed the 11.6min peaks in excipient + API chromatograms or is it something quite different?
To explain all this, we need to refer to some chemical structures. It is vital that analytical scientists have ready access to the chemical structures of all APIs and formulation components that they are working on to enable any data interpretation activities to be conducted.
In this case, the chemical structures of relevance are of API, Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate and are shown in Fig 6. The API structure shown below has been modified to protect client confidentiality but does contain all features of relevance to this case study.
Note that although the 2 excipients, Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate are structurally very different, what they do have in common is that both polymers are very highly hydroxylated and that many of these hydroxyl groups are esterified with either Phthalic Acid or Acetic Acid.
The 8 week chromatograms of both Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate without API showed very similar chromatograms with the most intense peal eluting at 9.9min. The most likely explanation for this is hydrolysis of the phthalate ester to generate Phthalic Acid as shown for Polyvinyl Acetate Phthalate in Fig 7.
This was easily confirmed by demonstrating similar retention time and diode array detector acquired UV spectrum with an authentic sample of Phthalic Acid.
You might also expect to see a peak due to Acetic Acid. This is not the case as the UV absorption spectrum of Acetic Acid is extremely weak at the HPLC monitoring wavelength but see later for more on this.
The 8 week chromatograms for Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate without API also showed weaker peaks at around 10.7 and 11.3min that need to be explained. The Phthalic Acid used to manufacture Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate excipients is the 1,2-dicarboxylic acid isomer but this will nearly always contain traces of the 1,3- and 1,4- isomers. It was confirmed that these peaks did indeed correspond to these 2 isomeric impurities by comparison with retention times and diode array detector acquired UV spectra of authentic samples of those components.
So now turning to the 8 week chromatograms of Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate + API, the main impurity peak eluting at around 9.9min can also be assigned to Phthalic Acid resulting from hydrolysis of the 2 excipients. But now we have an additional peak showing at around 13.4min. This certainly is a result of an interaction between API and free Phthalic Acid leading to formation of a Phthalic Acid Amide impurity as shown in Fig 8.
This now just leaves the impurity peak that eluted at 11.6min observed in samples of Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate + API. The question was raised whether this might be the same component that was observed in the excipient samples without API and that eluted at 11.3min. There was also a question concerning the formation of Acetic Acid. In the excipient samples without API, it was concluded that the 11.3min impurity was one of the Phthalic Acid isomers. However, the UV spectrum of the excipient + API 11.6min peak was quite different. We concluded that the component responsible for this peak resulted from an interaction between API and Acetic Acid. So as shown in Fig 9, in similar fashion to Phthalic Acid, the API can react with Acetic Acid to generate an acetamido derivative.
This provides a reasonable explanation for the 8 week Excipient Compatibility Study chromatograms observed for samples of API + Cellulose Acetate Phthalate and Polyvinyl Acetate Phthalate so we can now move to the final stages of our data interpretation.
The Knowledge
Now that we have an explanation for those 8 week Excipient Compatibility Study results, we can create some recommendations for our client. Previously in this article, I indicated that it was a client preference to use Cellulose Acetate Phthalate/Polyvinyl Acetate Phthalate combination as the enteric coating material for this product, but they did not share the reasons, so we did not know how strongly they wanted to go in this direction. On this basis we provided the following possibilities for consideration:
- Assuming that the use of the Cellulose Acetate Phthalate/Polyvinyl Acetate Phthalate combination as the enteric coating is essential for the business success of this project, we offered some options.
- It may be that there really is no problem here. For the Excipient Compatibility Study, API and excipients were prepared as powder mixes so there was maximum contact between the components. In a tablet product, the 2 excipients will only be located in a coating layer so there will only be contact with API located at the surface of the tablet core. On this basis we would expect to see much reduced interaction.
- In spite of the situation presented in 1. above, it would be better to eliminate the opportunities for interactions. Since it was now clear that interactions were initiated by hydrolytic reactions, the use of moisture resistant barrier coating(s) could be applied in the tablet formulation as indicated in Fig 10.
As shown in Fig 10, this could be a single coat that could be applied either to the core prior to the enteric coat to prevent interactions due to moisture contained in the core; or applied after the enteric coat to prevent interactions with atmospheric moisture; or applied both before and after the enteric coat. This would certainly be a very effective way to prevent interactions but would also significantly impact manufacturing costs during development and also for eventual commercial batches.
- If the client is able to continue with this project using an alternative enteric coating material, we recommended that this should be the best route to proceed.
It turned out that the client did particularly want to proceed with the Cellulose Acetate Phthalate/Polyvinyl Acetate Phthalate combination for the product enteric coating to help them achieve a business objective, but required some intellectual property (IP) to support this plan. The use of the Cellulose Acetate Phthalate/Polyvinyl Acetate Phthalate combination coating together with the barrier layers made this possible. So, the product development proceeded with both the inner and outer barrier coats as per Fig 10. Even though this was the most expensive option it led to a successful conclusion
Conclusions
It is very important for a CDMO to provide their clients with quality data interpretation whenever they provide study results in support of any pharmaceutical product projects. A good model to follow for data interpretation incorporates a sequence of Data through Information leading to Knowledge. This process will require some additions to timelines which may not be too popular in an environment where Companies are very sensitive to applications of efficiencies wherever possible. However, for the case study described a data interpretation was conducted on results from an Excipient Compatibility Study and this led to establishment of a route forwards just based on those results. Had we not conducted this data interpretation, it is likely that a number of additional experimental batches plus some sort of stability study would have been required to get to the same stage and this would have added significant costs as well as many additional months to the timeline.
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