Meg Rivers is the former senior editor for 

Meg Rivers

The purpose of stability testing programs is to ensure a drug product’s safety and efficacy remain within its specifications throughout the shelf-life. To develop suitable analytical methods to detect and quantify degradation products, drug manufacturers should conduct forced-degradation studies.

“Conduct proper analytical method development by understanding the drug substance degradation pathway,” says Shailesh Vengurlekar, SVP, quality and regulatory affairs, LGM Pharma. “Use validated stability-indicating test methods to perform testing at the scheduled time period.”

Stability programs and their practices can vary widely. These differences become more intricate when comparing stability testing of small molecules versus biologic drugs and how it impacts shelf life.

“A larger company that manufactures multiple products and batches must manage numerous stability studies with many more samples to analyze,” says Patrick Nieuwenhuizen, senior manager consultant, PharmaLex. “It can be more challenging to manage the volume of samples and testing compared to a smaller company with fewer products and fewer studies. Hence, you see larger companies are making more use of integrated software programs that can manage multiple stability studies.”

Regardless of company size, there are essential practices for stability testing that all companies should implement. From study design to testing completion, all companies should have a clearly defined process for stability, explains Anna Thabit-Jones, stability manager, PCI Pharma Services. To set up and manage a stability study, a detailed protocol is essential.

Nieuwenhuizen also notes that having a robust stability program is an essential practice for stability testing and shelf life. In addition, companies should have a scheduling system in place with adherence to testing the allocated time points within the study. There are many examples of companies receiving a regulatory citation for not having their stability program organized how it should be, Nieuwenhuizen adds.

According to Vengurlekar, all companies should follow the International Council for Harmonisation (ICH) guidelines to design a proper stability program and testing intervals.

Anis H. Khimani, PhD, senior strategy leader, pharma development, life science, PerkinElmer adds, “ICH Q1E guideline provides recommendations on how to use stability data generated in accordance with the principles detailed in the ICH guideline, Q1A(R2) 

Stability Testing of New Drug Substances and Products

, to propose a retest period or shelf life in a registration application.”

For recommendations on the setting and justification of acceptance criteria, reference ICH Q6A and Q6B, Khimani specifies, whereas ICH Q1D is for the use of full- versus reduced-design studies.

Both small and large pharma organizations should consider addressing the critical quality attributes (CQAs), continues Khimani. CQAs could have an impact on purity, potency, drug release, stability, and safety. But for both small-molecule and biologic drugs, a QA review of protocols and establishment of methods and validation should be done. Khimani also confirms that a typical study can last up to two years based on the storage conditions and expected shelf life.

“A consistent and appropriate maintenance/calibration program for the stability chambers is also essential,” Thabit-Jones adds. “If you can’t trust your chambers to be working correctly and operating at the required temperature and humidity, then you can’t trust the data generated for any products stored. Depending on the size of the company’s stability program, effective sample inventory is also key to maintaining sample integrity. Testing should be conducted as soon as possible following the removal of the samples from the stability chamber. This is particularly important at the earlier time points and for accelerated conditions, as you want to capture the product stability at that point. Further degradation can occur over time.”

Stability testing for small-molecule vs. biologic drugs

Although there are similarities in stability testing between small-molecule drugs and biologics, there are distinct differences as well.

“Issues associated with instability are potentially more significant and perhaps more likely to be observed with biologics than their small-molecule counterparts,” says Vengurlekar. “Biologics have a tendency to aggregate and are prone to degradation. Thus, the methods to accurately quantitate drug substance degradation differs between small-molecule and biologic drugs. To monitor various degradants from biologics, typically, analytical techniques such as Fourier-transform infrared, nuclear magnetic resonance, chromatography, mass spectrometry, and electrophoresis are used.”

Furthermore, stability tests are performed under controlled conditions (temperature and humidity) that match how the drug will be stored, which differ for small-molecule drugs and biologics.

“For small molecules, those conditions are typically set for 25 °C and 60% relative humidity,” says Arvind Srivastava, PhD, technical fellow, Avantor. “However, biologics may be lyophilized or stored as a liquid, and the more common condition is refrigerated at 2–8 °C. Humidity control is not generally required for biologics, but it may include additional conditions such as the orientation of the stored drug (upright or inverted). Agitation may also be applied because biologics can be sensitive to shear stress. Analytical techniques to monitor simple physical tests such as appearance, osmolality, particulates, and pH are usually the same for both small-molecule drugs and biologics. However, the technology utilized for purity and degradation products can be substantially different.”

In addition to storage conditions, CQAs for biologics must also focus on cell culture and production process parameters to ascertain the stability of the product along the entire workflow, states Khimani. Whereas, for small molecules, stability evaluation can be less complex.

Andrew Hanneman, PhD, scientific advisor, mass spectrometry laboratory at Charles River, adds, “In the development of biologics, specifically, an essential practice is the use of intelligent molecular design to avoid labile amino acids/sites in or around the protein’s active binding sites when practical. It is also important to initiate forced degradation investigations to support stability programs early on, even if they are not part of the formal stability program.”

Misconceptions of stability testing: small-molecule vs. biologic drugs

One of the biggest misconceptions in stability testing—especially for late clinical or commercial products—is that the stability tests will produce a clear trend, notes Thabit-Jones, which is flat, increasing, or decreasing over time. However, some products may produce variable data points at each testing time-point and may not produce a clear trend until much later in the study.

“This makes it difficult to assess the likelihood of meeting the required shelf-life early on, and a deeper review of historical data can be required to support conclusions,” Thabit-Jones continues. “In addition, stability testing can sometimes be an ‘afterthought’ for some companies, but it is critical to collect data about product stability early on. The more data available, the easier it is to assess and investigate data that may be out of specification, out of trend, or out of expectation at later stages in the product life cycle.”

Another misconception is that stability testing is routine. While the testing techniques themselves are routine, every study is different and requires different testing, time-points, conditions, and final assessment, Thabit-Jones adds.

Although some people may perceive stability testing programs as similar for small-molecule drugs and biologics, stability programs for biologics are far more complex, confirms Srivastava.

“This is due to the complexity, degradation pathway, and mechanism of action differences between the two drug modalities,” Srivastava continues. “The degradation mechanism for small-molecule drugs is simple, typically involving hydrolysis and oxidation. Biologics, in comparison, can undergo a variety of physical and chemical degradation mechanisms, including unfolding, aggregation, fragmentation, surface absorption, particulate oxidation, deamidation, hydrolysis, and disulfide exchange. Additionally, potency can be extrapolated from the purity test for small molecules; but for biologics, a specific potency assay is mandatory in addition to a purity test.”

However, Nieuwenhuizen shares another perspective. As small molecules are better known, more stable, and less difficult to characterize than biologic drugs, it is a misconception that it would be easier to manage stability studies for small molecules, as the assays would be simpler for small-molecule drugs, Nieuwenhuizen explains. These assays for small molecules could be as complex as for biologics.

Providing an example for the complexity of assays for small molecules, Nieuwenhuizen says, “Potency assays for biologics [determine] the strength or assay in small molecules, in particular when the product is an antibody–drug conjugate (ADC), which is a combination of a [monoclonal antibody] coupled to a cytotoxic agent.”

Utilizing stability testing data to ensure shelf life

How can drug manufacturers utilize all this stability testing data to ensure shelf life, and how soon should this data be collected? According to Thabit-Jones, stability data should be collected early on. The data should ideally guide product formulation and long-term storage.

“Trending and monitoring data at each time-point is essential to get a thorough understanding of how the product behaves over time. Companies should use this data to identify clear trends and understand what is unstable about the product,” Thabit-Jones says. “This may result in adjustments to the formulation to reduce the rate of degradation or allow storage at lower temperatures. The packaging type may also need to be reviewed where significant water intake is observed over time. Profile dissolutions will help in understanding delivery of the tablet/capsules once ingested and can guide adjustments in manufacture.”

Stability data can be used to monitor unintended drift in the manufacturing process, according to Srivastava. Early detection and corrective action can lead to a drug manufacturing process that facilitates a stable shelf-life.

“The quality and stability of raw materials and excipients also play a critical role on the stability and shelf-life of drug products,” Srivastava continues. “Small molecule and biologics manufacturers can work with their raw material suppliers to access stability data to assist with designing products with a stable shelf-life.”

Srivastava adds, “For both small and large molecules, partnering early on with trusted raw materials and excipients suppliers can help ensure quality and consistency of raw materials throughout the drug manufacturing process.”


Vol. 45, No. 10
October 2021
Pages: 48, 50, 56

When referring to this article, please cite it as M. Rivers, “Determining Shelf Life: Reading the Stability Testing Data,” 

Articles

Although stability testing programs for small-molecule drugs and biologics are often perceived as similar, stability programs for biologics are far more complex.

Determining Shelf Life: Reading the Stability Testing Data

Adventitious agents—microorganisms introduced during the biomanufacturing process—present significant risks to the quality and safety of biologic drugs. Prevention, detection, and removal are key elements of risk mitigation practices.

“A source of risk for viral vaccines, biologicals, [and] cell and gene therapies are adventitious agents (AA)—microorganisms that may unintentionally be introduced into the manufacturing process,” explains Archie Lovatt, scientific operations director, SGS. “Exogenous forms of AA include bacteria, yeast, chlamydia, rickettsia, mycoplasma, prion, protozoa, and viruses. There are also endogenous forms of AA, which are process- or product-related impurities. These can include endogenous retroviruses or other viruses, replication-competent retroviruses, adenoviruses, or adeno-associated viruses, depending on the manufacturing process.”

“Within the biopharmaceutical industry, the main sources of adventitious agents are derived from the raw materials used during production, including the cell substrates, which are the starting point for the generation of biologics products,” says Alison Armstrong, global head of field technology management, MilliporeSigma. “Potential contaminants are also originating in manufacturing processes and cleaning procedures, equipment used, general production facilities used, and from direct and/or indirect human contact (i.e., personnel-related contaminations).”

The most challenging adventitious agents are new emerging viruses and viruses that can’t be assigned to a known virus family or are a distant variant of a known virus family, explains Horst Ruppach, executive director, scientific and portfolio, at Charles River. “Viruses that are not expected as a potential contaminant are especially difficult, such as the vesivirus contamination of [Chinese hamster ovary] CHO production cell lines or the porcine circovirus contamination found in rotavirus vaccines,” he says. “The latter was detected using high-throughput sequencing (HTS), mostly by chance. All typical testing assays applied during manufacturing failed to detect this virus.”

 methods to test for adventitious agents have benefits and drawbacks.

 adventitious agent tests are broad detection systems that can detect a wide range of viral contaminants,” said Armstrong. “The 

assay is the classical method of virus detection used for many years to detect specific virus families, which allowed the detection of viruses not traditionally detected in cell culture-based systems. 

 assays use small animal models that meet regulatory requirements to provide an additional level of assurance of virus safety.”

Different guidelines for risk mitigation may require animal testing, says Pascale Beurdeley, chief scientific officer at PathoQuest. Animal testing is used for starting material evaluation (such as cell banks or virus seeds/vectors) and used only minimally for lot release. But one of the key differences between these two testing methods is timing.

“The turnaround time with animal testing is the longest: up to 42 days for the experimental phase of the 

 assays, two–six months for the complete study, as opposed to one week with molecular testing or four weeks for cell-based 

testing,” Beurdeley continues. “Specificity and sensitivity of virus detection in 

assays are usually unknown and currently, if an 

 cell-based result is positive (detection of adventitious contamination), it always requires additional investigation for virus identification using 

According to Ruppach, the breadth of detection and the sensitivity for specific viral contaminants can differ. The 

 assay typically has a higher breadth of detection and is more sensitive. A few viruses, however, are detected in the 

 assays at a higher sensitivity. For example, influenza contaminations can best be detected through inoculation in embryonated eggs.

“Different to the breadth of detection or the sensitivity, there are also some important practical aspects. The cell-based 

 assays typically perform better compared to assays using adult/suckling mice, guinea pigs, or embryonated eggs,” says Ruppach. “The administration of samples 

 is tricky and sample matrix components can lead to animal death that could be misinterpreted as a viral contamination (false positive results). Both the 

 assays require specialized laboratories but the requirements for 

 assays are usually the preferred method for detecting adventitious agents.

 methods have not proven to be effective in the detection of adventitious virus in products,” says Lorraine Borland, product manager, viral vaccine and gene therapy, Sartorius. “This may be due to lack of sensitivity of the method or indeed the reduced risk profile of products with the introduction of sterile single-use manufacturing systems, chemically defined medium, removal of animal components in the manufacturing chain, and increased process control.”

Scientific advances have opened the way to alternatives to using animals for testing. In some cases, replacing the use of animals with 

“Given the range of technologies and testing strategies available today, it seems highly unlikely there are any instances where 

 testing would be considered more appropriate,” says Beurdeley. “Animal testing in general continues to fall out of favor in lieu of more novel molecular approaches like those that utilize the polymerase chain reaction, next generation sequencing (NGS), or combinations thereof.”

 found that some viruses can be better detected with 

 cell-based assays—for example, the vesicular stomatitis virus and influenza virus. But in general, cell-based 

 assays have a higher potential to detect adventitious viruses (1).

 assay was included in the regulations for adventitious agent testing from the 1950s as a broad-spectrum detection system looking for viruses that were difficult to grow in culture and, therefore, could form contamination within a vaccine production stream without affecting the vaccine cultures themselves,” says Borland. “The hypothesis was that viruses, such as coxsackie virus, required the 

 test to be detected. For when they were isolated from clinical samples, it was seldom possible to grow them in cell culture unless a panel of cells were used. Isolation of the virus in suckling mice was, therefore, recommended. However, we can detect enteroviruses using 

 methods with a panel of susceptible cells and employing molecular methods.”

 testing methods detect live contaminating viruses and have an amplification phase of contaminants, increasing the detectability of very low levels of contamination. Each testing method also has known strengths and weaknesses.

 assays, the use of selected cell lines is permissive to a wide range of viruses using several end-point test systems (i.e., cytopathic effect, haemagglutination inhibitionor haemadsorption, or immunofluorescent assays [IFA]),” says Armstrong.

Although both assay types detect infectious viruses, neither are usually specific, says Ruppach. Ruppach adds that a viral contaminant isn’t automatically identified in a positive result. The exception is when applying specific read-out methods like ELISA or polymerase chain reaction (PCR), which are designed for specific viruses.

 approaches except if a specific contaminant is assumed and known to be best detectable through animals,” says Ruppach.

Current adventitious agent tests can detect a broad range of viruses at a low limit of detection of about 1 TCID

, Armstrong explains. Because these test methods have a limit for the sensitivity they can detect, any contaminants below that level are challenging to detect. Armstrong further adds that known limitations for cell-culture-based systems include the fact that not all virus agents grow well in cell-culture-based systems. Therefore, according to Armstrong, there is a need for alternative methods in adventitious agent testing—for example, molecular-based technologies including degenerate PCR methods, advanced molecular technologies such as NGS/HTS or IFA systems, which allow detection of a wider range of known and emerging adventitious agents.

“Latent or silent virus infections can be also difficult to detect and require special testing regimes” says Armstrong. “Cell culture systems are particularly susceptible to matrix-related toxicity, as well as to viral infection-related cytopathic effects.”

Knowing the benefits and shortcomings of adventitious agent testing, what is next for the industry? NGS or HTS technologies are considered suitable supplements or even replacements of 

 assays, says Ruppach. In a given sample, these technologies detect nucleic acids—including virus-specific nucleic acids—and analyze the nucleotide sequence. Regulators have recognized the impact of state-of-the-art technologies and have provided guidance on the use of such technologies (WHO TR978, recent EP chapters 2.6.16, 5.2.3, and 5.2.14).

“The major benefit is that these methods can detect and identify all viruses (even unknown viruses) at a sensitivity compared to PCR or even beyond. In fact, there are ongoing discussions that NGS/HTS may ultimately replace the 

 methods for both 3Rs considerations, and the limitations outlined above,” Ruppach says. “The major difference between NGS/HTS to the 

 methods is that NGS/HTS methods detect both infectious and non-infectious viruses. Non-infectious viruses or related nucleic acids could be a left over from a treatment, and—while not typically a risk—they can lead to a ‘false’ positive result with an NGS/HTS approach. However, while NGS/HTS methods have been developed to differentiate infectious from non-infectious viruses, which could make these technologies universal for viral contamination detection moving forward, there are still misconceptions about their effectiveness [according to the literature (2)].”

 cell-based assays] were initially assessed as broad range virus detection assays. This approach has been challenged with the advent of truly broad molecular test methods, such as NGS, which does not require prior knowledge or suspicion of the adventitious agent. Moreover, our alternative NGS approaches can distinguish replicative—thus infectious—from non-replicative viruses. As such, NGS can detect previously undetected or undetectable replicative virus contaminants [citing an article in the 

Only time will tell what technologies will emerge most effective for detecting adventitious agents and other contaminants.

A. Brussel, “The Emergence of Next Gen Sequencing for Analyzing Biologics,”

Alternatives to animal testing and in-vivo assays are occurring as a result of recent scientific advances.

In Vivo Falls Behind In Vitro for Adventitious Agent Testing

As the COVID-19 pandemic continues, one pressing concern for the industry is ensuring the supply of legitimate COVID-19 vaccines. Now more than ever, the supply chain needs to be secure to ensure the population can have access to legitimate vaccines. But substandard and falsified COVID-19 vaccines—commonly referred to as counterfeit vaccines—continue to be a threat globally.

Pharmaceutical Technology’s September 2021 Regulatory Sourcebook eBook

The Real Danger of Substandard and Counterfeit COVID-19 Vaccines

Pharmaceutical Technology’s Regulatory Sourcebook eBook.

, September 2021
September 2021
Pages: 10-14

When referring to this article, please cite it as M. Rivers, “The Real Danger of Substandard and Counterfeit COVID-19 Vaccines," 

Pharmaceutical Technology Regulatory Sourcebook 

Low- and middle-income countries are at the greatest risk of receiving substandard and falsified COVID-19 vaccines.

Drug shortages were a major concern before the COVID-19 pandemic, which only served to further add pressure to an imperfect supply chain. The COVID-19 pandemic placed added pressure on bio/pharma’s imperfect supply chain, contributing to ongoing drug shortages and creating some new challenges. What has the industry learned from the pandemic and will practices and supply-related decisions change going forward?

Pharmaceutical Technology's September 2021 Regulatory Sourcebook

Pharmaceutical Technology's Regulatory Sourcebook eBook

, September 2021
September 2021
Pages: 16-22

When referring to this article, please cite it as M. Rivers, “Changing the Course of Drug Shortages," 

The COVID-19 pandemic has further impacted ongoing drug shortage issues; essential action is needed to avoid repeating previous mistakes.