Essentials for designing and conducting stability studies for custom GMP reagents

Stability Testing of Custom GMP Reagents to Determine Shelf Life

How to design and conduct stability studies to support your custom biomanufacturing

The demand for custom GMP reagents used to develop and manufacture novel biopharmaceuticals is increasing rapidly alongside advancements in next-generation therapies and vaccines. Not surprisingly, high-quality intermediate reagents are essential for creating safe and effective consumer products. Inadequate raw materials and reagents could chemically change, becoming a source of unsafe impurities or leading to inefficient and inconsistent manufacturing. These issues may cause expensive delays to troubleshoot and address problems that potentially could be avoided by careful selection and testing of reagents.

Why is stability testing done?

An important way to monitor the quality of customized reagents used in manufacturing processes is through stability studies, which are designed to assess whether and how the chemical breakdown of components occurs. This chemical degradation could lower concentrations of active ingredients or accumulate as toxins, including leachables. Stability studies are typically custom designed using a product's composition and its intended use as guides. The results are fundamental to defining degradation pathways, determining retest dates to confirm that a product is still suitable for use, and establishing shelf lives of products.

In this article, we focus on studies used to inform the shelf life of new, custom GMP reagents. These stability tests provide supporting evidence that intermediate GMP products used in manufacturing meet internal and external requirements and contribute to the production of safe, effective consumer products.

What is stability testing?

Stability tests measure selected critical product parameters, such as performance, conductivity, and pH, over defined intervals that can vary from a month to many years. Study samples are stored under predetermined environmental conditions that often include controlled exposure to temperature, light, and humidity.

The two main types of stability studies are real-time and accelerated testing (Figure 1). Real-time stability testing is performed under the recommended storage conditions of the final product to evaluate changes over time. In a typical study, testing time points occur at 3, 6, 9, and 12 months during the first year, 18 and 24 months during the second year, and once per year thereafter for a predetermined time. All storage conditions and time points are based on the composition of the product and are defined before the stability study begins.

Accelerated testing is conducted over a shorter period of time than real-time testing and in more extreme environments than the established storage conditions. Frequently, elevated temperature, increased light exposure, or changes in relative humidity are used to speed up the aging process and simulate long-term storage. Accelerated studies always run concurrently with real-time studies because extrapolating accelerated-study results to those using recommended storage conditions can be misleading and should be interpreted cautiously. However, the data from accelerated studies provide an early view of what might be expected from real-time stability studies, thereby reducing risks and allowing research and development to proceed before the real-time testing is completed.

Comparison of accelerated and real-time stability testing

Figure 1. A comparison of accelerated and real-time stability studies. Product samples are stored under different conditions and tested at different time points. In the accelerated study, the storage temperature is higher than recommended, and tests are performed over 6 months. In the real-time study, product samples are stored under the recommended conditions and tested for 3 years.

Additional considerations for stability study design

Types of tests

Stability studies can be used to monitor physical, chemical, biological, and microbiological attributes. The product type, constituents, and intended use determine which tests are vital to establishing stability (e.g., concentration, identity, integrity, potency, and purity). Key specifications from the certificate of analysis and QC release criteria can also influence test selection.

Frequently chosen metrics include appearance, conductivity, density, osmolality, and pH. The types of analyses used to monitor other common metrics, such as concentration, aggregation, performance, and activity, are product specific. In addition, United States Pharmacopeia (USP) chapters and monographs can be followed for additional standardization of methods used for many types of QC and stability tests, such as pH, conductivity, and sterility.

Samples

The number of product samples required for stability testing must be accounted for in the overall manufacturing plan. For accurate simulation of storage conditions, stability tests are usually conducted using products in the final packaging format, including sample volume and container type.

Facilities, equipment, and trained personnel

In addition to samples, other resources required to support stability testing can become consequential. The duration of the stability studies necessitates long-term storage facilities, such as incubators, cold rooms, and freezers, to hold samples under controlled environmental conditions. Laboratories with instruments for testing must be established and maintained. Also, staff must be trained to follow standardized procedures when running stability tests.

Case studies to illustrate stability study design

We have created two case studies that highlight the features of comprehensive stability studies.

Example 1: Custom buffer that improves yields in gene therapy manufacturing

A gene therapy manufacturing company requested a custom buffer that contained a critical component that maximizes yields of their viral production. It was important that the critical component, magnesium chloride (MgCl2), remained at a specific concentration in the buffer. Experiments were performed to confirm the concentration of MgCl2 by both analytical titration and atomic absorption spectroscopy. The first 3 lots of buffer included additional samples for both accelerated and real-time stability testing.

The accelerated study incorporated time points at 1, 3, and 6 months with samples stored at 25°C. The real-time stability study was run simultaneously to verify the accelerated stability study data and establish the shelf life. The testing time points occurred at 3, 6, 9, 12, 18, 24, and 36 months with samples stored at 4°C, which is the recommended storage temperature.

At each time point, 3 technical replicates were taken from each sample and were tested for sterility, pH, conductivity, and magnesium chloride content:

  • Sterility testing, following USP<71> guidelines, is typically included for reassurance since sterility should not change in an unopened product. Note that some studies only test sterility once per year instead of at every time point because this test primarily monitors the quality of the container closure system.
  • pH (USP <791>) and conductivity (USP <645>) are standard measures of stability in biopharma. Degradation of components could change the pH and conductivity of the solution.
  • Concentration is monitored for a critical component that is required for high performance. Here, the concentration of magnesium chloride was monitored by following the testing methodology specified by the USP monograph for magnesium chloride (Figure 2).

In this example, the magnesium chloride concentration in the buffer was stable during the accelerated study, and these findings were also observed in the data from the three-year real-time study. Therefore, data from both stability studies supported product use during development and scale-up, and data from the real-time stability study helped establish the shelf life of this unique custom buffer.

Example data, critical component concentration

Figure 2. Representative magnesium chloride concentration data. The gray area indicated the acceptable concentration range defined in the study plan. In this example, the buffer performed as expected during both accelerated stability testing (actual duration shown) and the three-year real-time stability testing.

Example 2: Custom broth for growing bacteria

Product development scientists at a microbiome company requested a custom broth to grow a specific strain of bacteria. A stock culture of the bacteria was purchased, and information about the growth characteristics of the strain was obtained. In addition, the growth range of the bacteria of interest and a control strain was determined using the requested formulation of broth and OD550 measurements. The first 3 lots of broth included additional samples for both accelerated and real-time stability testing.

The accelerated study contained time points at 3 and 6 months with samples stored at 25°C protected from light. The real-time study was done to verify the data provided by the accelerated stability study and to determine the shelf life of the new broth. Testing time points occurred at 3, 6, 9, 12, 18, 24, and 36 months with samples stored at 4°C protected from light, which are the recommended storage conditions for the broth.

At each time point, samples were tested for performance to monitor bacterial growth, as well as sterility, pH, and conductivity. Performance testing was conducted by recording OD550 measurements after an established incubation period of the specific strain of bacteria or the control bacteria in the custom broth (Figure 3).

In this example, the broth performed as expected during accelerated testing, and these findings were also observed in the data from the three-year real-time testing. Therefore, data from both stability studies supported product use during early product development stages, and data from the real-time stability study helped establish the shelf life of this unique custom broth.

Example performance data, microbial growth

Figure 3. Representative growth data. The gray area indicated the predetermined acceptable range for OD550 measurements. In this example, the broth performed as expected during both accelerated stability testing (actual duration shown) and the three-year real-time stability testing.

Understanding the scope of stability testing—and why some choose to outsource this work

The number of individual tests in a stability study can increase quickly when including the number of lots, controls, technical replicates, and tests run at each time point. Similarly, the resources needed for conducting stability studies also can grow rapidly, depending on the sample sizes, number and type of tests, and the duration of the study. For example, 30 samples and over 350 tests (not including controls) are needed to complete both accelerated and real-time studies in our two case studies (Table 1).

Navigating when and how to perform stability studies on custom reagents can take considerable planning and expertise. Like outsourcing custom GMP reagent manufacturing, outsourcing stability testing can free up time and resources to focus your expertise on product development and manufacturing processes, as well as decrease investment in capital expenditures specific for stability testing facilities and equipment.

Table 1. Number of tests in a representative stability study.

Total number
(not including controls)
Accelerated
(3 lots, 3 technical replicates, 4 tests, 3 time points)
Real-time
(3 lots, 3 technical replicates, 4 tests, 7 time points)
Samples
(lots x time points)
9 21
Tests run at each time point
(lots x replicates x tests)
36 36
Tests run during the stability study
(lots x replicates x tests x time points)
108 252

As part of our comprehensive custom GMP reagent manufacturing offering, we have established a stability studies program to help determine the shelf lives of novel reagents. When stability studies are needed, we include the volumes required for sampling in our manufacturing plans to ensure there is sufficient material for the duration of the study. In addition, when we perform the stability study, you get a true, time-zero data point by using the QC release data that can be generated with the same standardized protocols and the same instruments as your stability study. With dedicated facilities, equipment, and trained personnel, we are well positioned to efficiently support the evolving demands for custom reagent manufacturing along your development and commercialization pipeline.

Let’s talk about the manufacturing plans for your custom GMP reagents today.