Since the introduction of protein therapeutics in the pharmaceutical industry, the demand for the development and release of novel biologics into the market has grown substantially. More and more new start-up, small, and large biotechnology companies are making efforts to develop medicinal products that tackle diseases and chronic conditions, in order to offer millions of people the right to enjoy a long and high-quality life. These products can vary from monoclonal antibodies and recombinant proteins, to polypeptides, conjugated proteins etc.
Drug development is a long process; it takes years of planning and demands continuous engagement from all personnel involved in order to achieve the desired outcome. During the process, drug developers may form collaborations with other companies to support the manufacturing, analytical, and clinical testing of their product. The development costs are tremendous, the timelines tight, and people’s lives are at stake. Compliance with the regulations is not an option. ‘Which regulations need to be followed?’ ’Which tests need to be undertaken and when do they need to be done?’ There are so many questions drug developers need to answer, and quite often they rely on their sub-contractors to provide the solution.
The Main Purpose of Stability Testing
As part of the drug development process, regulators require stability studies that prove the product’s continuous safety and efficacy throughout its proposed shelf-life. When your stability testing is complete, you’ll have documentation of your product specifications that will dictate storage conditions, final container / presentation, and shelf-life. Ultimately, all of these limits are meant to ensure the product’s safety.
Although stability studies are heavily regulated to ensure compliance with ICH and GMP guidelines, and require several years to complete, drug developers often begin their planning downstream in their drug development program. However, given the long timelines for the studies and the regulations surrounding them, drug developers frequently underestimate the possibility of things going wrong during stability studies, and the consequences that such events can have on delays in project completion and product launch. The diagram in Figure 1 demonstrates the various analytical steps required for a smooth outcome in the drug development process. The following sections discuss the stability of clinical product batches, not as a stand-alone part of the process, but as a directly dependant part on the pre-clinical stages and related analytical studies.
As a provider of stability testing, our laboratories encounter a variety of questions from drug developers relating to problems that have emerged during their stability studies. Therefore, the objective of this article is to provide a complete, holistic view of stability studies on biologics, with emphasis on key parts that all drug developers should consider. The end goal is that it will help drug developers save unnecessary costs and delays in their timelines, and facilitate getting safer medicines to patients.
Each protein therapeutic has its own properties and it is essential that its Critical Quality Attributes (CQAs) are understood and maintained throughout its shelf life. Example CQAs are protein structural conformation, PTM levels of glycosylation / sialylation and presence of co-factors / conjugates. Determining and maintaining CQAs can depend on a variety of factors, for example:
- presentation of the final product (final formulation, concentration / dosage, primary and secondary container)
- long-term storage conditions
- route of administration of the product
- transportation / shipment of the product
It has become apparent that some of the questions drugs developers need to answer during their development program are:
- Do we have a good understanding of our molecule? (product characterization)
- Have we determined the right environment for it? (formulation development)
- Which degrading factors is our molecule susceptible to? Can we transport the product in liquid or frozen form? (early forced degradation study)
- Are our analytical methods technically robust and fit for purpose? (Method validation)
- Is the reference standard material fully characterized and available?
- Is collaboration / sub-contracting required for stability testing? Is the contractor accredited to perform GMP testing?
- Is it required to monitor any of the CQAs/PTMs during stability studies?
If the above questions have not been fully answered prior to stability study planning, there is a high likelihood that severe problems may arise that can compromise the outcome of the studies and cause them to fail.
What to Do Before Starting Stability Studies
Understand your molecule. As simple as it may sound, drug developers often perform the minimum analyses required to construct the complete ‘picture’ of their product in order to avoid costs and decrease their timelines; however, if it is observed that a molecule ‘tends’ to aggregate, oxidize or deamidate, or is susceptible to degrading conditions, then it is advised to invest in more detailed characterization of the molecule. ‘Which site shows increased oxidation / deamidation?’ ‘Which conformational changes occur in the product when exposed to agitation?’
There is a wide variety of biophysical (eg. FT-IR, Circular Dichroism, Dynamic Light scattering, AUC), physicochemical (eg. icIEF, Differential Scanning Calorimetry, Amino Acid Analysis, N-terminal sequencing), and higher-order techniques such as mass spectrometry (MS), that can be utilized to build a composite ‘picture’ of the molecule.
Ensure the right final formulation is selected for the molecule. If the product is not in a suitable environment, it will possibly degrade quickly and fail your product specification as early as two to three months into stability (eg. protein may aggregate, precipitate and change color). It is recommended that formulation development studies are performed as soon as early development / lab-scale material is available. This will ensure that 1) the product remains stable when used in the lab, during shipments and during stability studies 2) method development and validation is accurate 3) product characterization data reflect final formulation 4) the reference standard is presented in the same formulation as the drug product and 5) no additional delays and costs are required for additional formulation development later on.
Select the right primary container and container closure for your product. Are they of pharmaceutical grade? Ensure that possible extractables & leachables (E&Ls) are determined as early as possible from the final container and process contact materials (eg. tubing used in the manufacturing process). It is possible that any extractables or leachables have a toxic effect on the drug recipient? Therefore, it is important to determine how those can affect your product and the patient’s safety. Regulatory bodies have started requesting E&L data to be present when a new product BLA is submitted. It is not uncommon for drug developers to experience unexpected problems from leachables derived from the product container, even after market release. Consequently, the manufacturer needs to perform E&L studies in retrospect to satisfy the regulatory bodies. Example leachables are antioxidants and additives present in the final container. Generally, recommended vials are pharmaceutical grade USP Type I glass vials with bromo/chloro-butyl rubber stoppers.
Have the appropriate analytical methods in place. Are they fit for purpose? Are they product-specific? Are they technically sound and stability-indicating (if appropriate)? Stability studies follow ICH Q1A and Q5C guidelines as part of which, drug developers are required to demonstrate the physical characteristics, identity, purity, potency and safety of their product have remained stable over time. When developing a chromatographic product-specific analytical method to determine identity and purity for example, ensure that as part of method development, interference from the formulation buffer has been evaluated and whether the method is capable of detecting product changes has been evaluated. We advise to include forced degraded material as part of method development; if, for example, the method should be able to detect high molecular weight species, it may be worth forcing the material to aggregate using heat treatment; the forced degradation condition implemented depends on what the method should be able to detect (eg. aggregates, oxidized / deamidated species etc.)
What to Do When Things Go Wrong
Stability testing can take anywhere from one to five years to complete. Within this timeframe, the probability of things that can go wrong is very high due to the reasons previously mentioned, and tackling stability study-related problems requires a rapid and regulatory-compliant response, in order to avoid compromising the study. In such instances, drug developers often rely on the quality system and procedures of the laboratory performing the stability study (if not performed in-house) to provide the solution. Clear and frequent communication between all relevant parties is necessary to enable the problem to be solved adequately and quickly to avoid unnecessary delays in the stability study.
It is vital that the stability protocol (and technical agreements between the drug development and the stability service provider if applicable) clarify responsibilities and plans of actions when deviating from the agreed procedures and plans, or in the event of an out-of-specification result, the most common stability-related problems.
A typical example of a stability study deviation is late testing of a stability timepoint condition. Quite often, this is due to an analytical instrument issue or an error during sample shipment to another party for testing. Acknowledging, documenting, and (if necessary) communicating, the issue as soon as it’s discovered is necessary to assess its impact to the study. If and when the issue has been resolved, and the timepoint condition has been tested, it is important to assess the result and report it as reflective of the date it was tested, and not the timepoint it’s supposed to represent.
Although unexpected problems may arise in stability studies, it is quite often possible to predict some of them and prepare in advance. What is important in this case is trending of the stability data and the pass:fail ratio of the analytical methods used. Trending of stability data allows drug developers to predict when a long-term storage condition may fail the specification for the product. Likewise, trending of the performance of analytical method, or an analytical instrument, may predict possible instrument or assay failures, and if these are solved quickly, then delays in analytical testing will be avoided. In addition, trending of the acceptance criteria of a stability analytical method provides real-time robustness data and confidence on that method.
Hot Topics in Stability Testing
As both drug developers and regulatory bodies become more knowledgeable about protein therapeutics, the more concerned they become for the safety of the products. For example, regulatory bodies often require extensive data on particles that can be present in liquid formulations (eg. visible, sub-visible, aggregate particles) and their safety implications on the patients. Compendia methods for visible and sub-visible particle assessment are available in US and EU pharmacopoeias, and are recommended for use in stability studies despite requiring a large volume of material available (USP <788> and Ph.Eur. 2.9.20). Furthermore, identifying a new species in the charge isoform profile of the protein therapeutic that can have an impact on the efficacy of the product may require more in depth analysis to fully understand its properties and impact on patient safety.
Quite frequently, biotechnology companies outsource large parts of their drug development program to other parties, and maintaining control of the different stages is essential to ensure that patients receive an effective and safe therapeutic sooner rather than later. Outsourcing stability studies, or even performing them in-house, involves GMP accreditation and approval from regulatory bodies for the testing laboratory. It is advised that drug developers start planning the stability requirements of their product as soon as early development material has become available, and understanding of what can possibly go wrong will help prepare in advance and avoid unexpected costs and delays that no patient should wait for.