Ashley Roberts

	A number of factors can influence stability, especially with highly-complex biomolecules. The increased risk of instability requires more stringent practices to maintain stability throughout the shelf life of the product. In addition to this, measures need to be taken to minimize exposure to environmental factors that can affect the integrity and efficacy of a product.

	Kerry Bradford, analytical projects manager, and Ashleigh Wake, biopharmaceutical services leader, both at Intertek; Kim Cheung, senior director of quality at Genzyme; and Niall Dinwoodie, global coordinator of analytical testing, biologics testing solutions at Charles River, spoke with 

about the challenges in maintaining a stable environment for biologics, how to determine shelf life, the effects of upstream processing techniques on the end product, and biologics versus small-molecule drugs and the importance of stability testing.
	
	

: What standard methods are used to ensure cGMP requirements during stability testing?

: The standard test methods for stability testing are, on the whole, traditional quality control techniques for biologics. They include size exclusion; ion exchange and reversed phase high-performace liquid chromatography; sodium dodecyl sulfate polyacrylamide gel electrophoresis or capillary electrophoresis; potency assays; and physicochemical measurements such as appearance, pH, and particle size. Of these, particle-size measurements are gaining increasing focus, but the first test to fail is still often appearance.
	
	

: What are some of the challenges of maintaining a stable environment throughout testing?
	
	

: The main challenge here is the need for constant monitoring. Modern stability chambers are able to maintain conditions within the International Conference on Harmonization tolerances with little input, however, excursions caused by mechanical failure need to be quickly identified in order to prevent them from exceeding 24 hours. This requires a 24/7 alarm system to monitor all chambers and a dedicated team of staff to respond to emergencies and fix any breakdowns.

: The biggest challenge is controlling the condition of shipments, from stability chambers to testing locations and storage at these locations. Tracking chain of custody for stability samples, and maintaining sufficient back-up chambers and capacity ensures stability studies are not impacted by equipment malfunction or physical capacity constraints.

: Biologics present a number of challenges in ensuring a stable environment and comparability of results across time points. The glass transition point for highly concentrated protein products can occur within the normal operating range of some freezers. While this is relevant to real storage on the market, it can lead to a sudden change that has no reflection on the period of storage. Also, for frozen storage, consistency in thawing approaches are vital for data comparison between time points.

: What are some of the advantages associated with accelerated stability tests vs. real-time stability tests?

: According to the Arrhenius equation, samples undergo the same degradation after 32 days at 25 °C as after one year at 5 °C. There are obvious cost benefits to accelerated time periods, particularly to quickly eliminate poor candidates when at the early stages of development of either a new drug substance, drug product, or package. The accelerated tests can also be used to submit early data to regulatory authorities; however, accelerated storage data must always be backed up with real-time data in the long term.

: Accelerated tests can demonstrate comparability for material associated with process changes in a shorter period of time (e.g., six months) than long-term real-time studies. The tests can assess whether a degradation profile for an attribute is expected to be linear or non-linear. When temperature excursions happen during transport, there is a specific procedure that is followed to determine whether the product can be used or whether it must be discarded. The accelerated data are leveraged within that procedure in which we’ve documented the allowable excursion temperatures and times during processing, shipping, and handling, and the data are used to respond to physicians’/patients’ questions about mishandling.

: Accelerated tests are commonly used to confirm that the test methods being applied to the product indicate stability. The methods are qualified early on in a stability program to ensure that the real-time data reflect the true stability of the product.

: What are some of the challenges or disadvantages associated with using accelerated stability tests for the determination of kinetic degradation?

: While use of accelerated stability tests in conjunction with Arrhenius calculations is a good predictor of degradation rates for straightforward reaction kinetics, it does not account for physical changes in the sample, or more complex systems such as emulsions and suspensions. For example, elevated temperatures may promote slight solubility of a suspended drug particle, leading to significant degradation that would not occur in the equivalent room-temperature sample.

: For biologics, degradation observed under accelerated conditions is not necessarily predictive of long-term conditions.

: Complex molecules such as biologics rarely, if ever, obey the Arrhenius equation. There are different pathways for degradation to occur, both chemically and structurally, so no single degradation rate can be determined. The accelerated conditions will also influence the degradation observed and may lead to steps being missed or inappropriate molecules being targeted as the indication of degradation.

: What factors are involved in determining shelf life?

: Understanding the potential degradation pathways of any biologic is key to gaining a true shelf life. Achieving this understanding is definitely complicated by the structural diversity of biologics. Regardless, if we are working on an antibody, peptide, or oligonucleotide, our general approach is the same, and in many ways, weighted before any sample is placed on stability. During early stage development, the material will be stressed under extreme conditions of pH, temperature (60 ºC is high), light, and oxidative conditions, as well as physical forces such as prolonged agitation, orientation of storage, and freeze-thaw. The potential pathways of degradation are then evaluated using various analytical techniques such as circular dichroism (for higher order), Fourier-transform infrared, nuclear magnetic resonance, mass spectrometry, chromatography, and electrophoresis.

	The use of orthogonal approaches is, where possible, always applied in response to the complexity of analytics. The final stability program is then designed based on the results observed and the potential degradation pathways elucidated. Stability storage is then initiated accordingly, remembering that chemical/structural integrity is not enough. All biologic evaluations of potency upon storage should also be documented. A simple ‘box ticking’/checklist approach does not work for biologics, as what works for one will not necessarily work for another.

: Do upstream processing techniques have an effect on the stability of end products?

: The short answer is yes. However, this is very dependent on the drug and the downstream process. A lot of this is covered with assessment of the drug product as well as substance.

: For biologics, yes. For example, hold times of upstream materials can impact critical quality attributes and ultimately affect the stability of drug substances and drug products. Therefore, stability studies are executed on all intermediate production materials held longer than 24 hours.

 How does stability testing of biologics and small-molecule drugs compare?

 Issues associated with instability are potentially more significant and perhaps more likely to be observed with biologics than their small-molecule counterparts. Biologics are inherently unstable, and this can manifest in many ways, but what differentiates them the most is their tendency toward aggregation, a phenomenon which is both frequently observed and yet difficult to control. Aggregation can present considerable detrimental effects to the safety as well as efficacy of a molecule. Formulations are optimized to reduce or at least slow down the process of aggregation, but often, aggregation is the predominant change to the molecule observed on storage.

Both stability testing for large-molecule and small-molecule drugs are important to ensure that a product maintains specifications throughout its shelf-life. For small-molecules, critical quality attributes and degradation products may be better understood for each product and shelf-life specifications for the degradation products can be set based on toxicology studies. In my experience, there is more lot-to-lot consistency for stability testing of small-molecules, so interpretation of results is straight forward. Also, in my experience with biologics, stability testing encompasses many more product attributes and the degradation profiles may be inconsistent from product-to-product and/or lot-to-lot, making understanding identified trends and interpreting results more difficult. Test methods may be more variable, again making it difficult to interpret results.

 There is less predictability for biologics. Similar formulations of different proteins can behave in a different manner and have quite different shelf-lives, and the concentration often has a greater effect than it does for small-molecule drugs. The industry has seen many examples where process changes have led to changes in the stability of products that could not have been predicted. The use of committed stability tests for biologics is vital.

 When referring to this article, please cite it as A. Roberts, “Maintaining the Stability of Biologics,” 

Articles

Experts discuss crucial aspects of measuring the stability of biologics, which exhibit greater variability in stability testing than do small-molecule drugs.

Best Practices for Stability Testing of Biologics

A number of factors can influence stability, especially with highly-complex biomolecules. The increased risk of instability requires more stringent practices to maintain stability throughout the shelf life of the product. In addition to this, measures need to be taken to minimize exposure to environmental factors that can affect the integrity and efficacy of a product.

Biologics exhibit greater variability in stability testing than do small-molecule drugs, and maintaining a stable test environment is crucial.

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