Putting Shelf-Life to the Test

Stability testing of bio/pharmaceutical products evaluates how environmental factors affect an API or finished drug product and are essential to established drug handling and storage conditions, as well as shelf life.

Experts at contract analytical laboratories shared insight on testing of drug-product attributes with Pharmaceutical Technology, including testing at different stages of development, special considerations for different dosage forms, special considerations for transport tests and temperature excursions, the benefits of risk-based approaches, and new instruments and test methods.

Discussion participants are Brad Rowe, director of analytical’ and Justine Kiehl, group leader in analytical services-stability, both at Charles River Contract Manufacturing; Loretta Sukhu, staff scientist and team leader, and Rowel Tobias, principal scientist and team leader, EAG Laboratories; Amy Gladson, group leader, analytical services, Metrics Contract Services; Steven Pilewski, executive director, PPD Laboratories, GMP Lab; and Hildegard Brümmer, PhD, operational laboratory manager-SGS Life Sciences Berlin.

Product attributes for testing

PharmTech: What product attributes should be assessed in stability testing? Does the testing differ based on the stage of product development?

Sukhu and Tobias (EAG Laboratories): Stability testing provides evidence of the safety, efficacy, and quality of a pharmaceutical drug product over time. In addition to its active ingredient, several important components contribute to the overall stability of a drug product including excipients, formulation, and packaging. Assessing the right product attribute is essential and must include tests that evaluate quality, strength, potency, identity, purity/impurity, and safety.

Gladson (Metrics Contract Services): Appearance testing is the first line of defense in determining degradation. Additionally, moisture, assay, degradation/profile testing, and dissolution properties are typically assessed. Other physical properties that have been shown to change over time--and are considered key attributes of the compound--include hardness, viscosity, and pH. As the product moves through the various stages of development, more rigorous testing may be required to characterize the product (e.g., morphological changes) as well to emphasize key quality attributes, such as dissolution testing. At a minimum, a stability study will include the major testing attributes of dissolution and degradation.

Rowe and Kiehl (Charles River): Typically, you test for anything that could potentially change with age, such as the appearance, potency, related substances (impurities or degradants), dissolution, water content, and depending upon the patient population or phase of clinical stage, microbial limits. For some liquid oral products, you might also want to do additional tests, such as looking at the preservative content.

[Testing does not really differ based on the stage of development], but the level of validation and scrutiny of methods do change. The biggest changes in the product development cycle are the specification limits themselves--evolving from an open-ended ‘report result’ to specific limits that tighten as more data are available--and the level of validation of the method, starting with specificity, accuracy, and precision at early phases and adding full robustness and intermediate precision at later phases.

Pilewski (PPD): During drug-product development, the primary attributes assessed during stability studies focus on the physical and chemical limits. The testing is similar while advancing through the stages of drug development; however, the amount of stability information will differ by phase. During Phase I, stability for the drug substance and product is needed to support the shelf life of drug substance used for toxicological studies and drug product that is dosed to humans. This could be three to 12 months of stability data. During Phase II, multiple investigational formulations and product strengths may be evaluated based on the information gained during previous Phase I and Phase II studies. This could be 12-24+ months of stability data. As you advance through Phase II into Phase III, you are using the information collected during each phase to further refine and select the best formulation and packaging components to achieve your final product configuration that drives your Phase III stability protocols for commercial registration.

Brümmer (SGS): In order to undertake an efficient and meaningful study, parameters should be selected that are indicative of the product’s stability, which in most cases are unique to the molecule/product and will be different for large and small molecules. In the majority of cases, physical parameters such as viscosity, or chemical parameters such as pH, water content, product appearance, assay and degradation products, dissolution, and microbial parameters can typically change. Depending on the nature of the product or drug substance, these parameters should be fixed individually--and specifically for the formulation where appropriate--and defined in a protocol within the product’s shelf-life specifications.

During product development, it is important to know whether a new formulation is stable or not, but only limited testing is possible. It is only possible to get a first impression as to whether a formulation is stable, or whether it needs to be changed. For late-phase studies, stability has to be ascertained so that products meet shelf-life specifications and that no risks exist concerning the effectiveness of material over a period of time.

Analyses approaches

PharmTech: What instruments and expertise are required to assess each product attribute?

Pilewski (PPD): There are too many required instruments to list everything that applies to the variety of different product types. Two standards for all dosage forms are high-performance liquid chromatography (HPLC)/ultra-high pressure liquid chromatography (UHPLC) with either ultraviolet (UV), fluorescent (FL), refractive index (RI), mass spectrometry (MS), charged aerosol detector (CAD), or diode array detectors (DAD), along with ICH-configured stability chambers. Other common instrumentation requirements for the various dosage forms are: dissolution apparatus, cascade impactors, microscopes, laser particle size equipment, moisture determination equipment, pH, osmolality, viscosity, X-Ray powder diffraction (XRPD), and gas chromatography

Gladson (Metrics Contract Services): Most drug substance and drug product forms require an assay, which is usually analyzed via HPLC. Knowledge of current industry standards and good manufacturing practices (GMPs)--as well as basic understanding of HPLC instrumentation and theory--is critical. Similar knowledge of the various dissolution apparatuses (typically United States Pharmacopeia [USP] Apparatus I and II) and Karl Fischer titration instrumentation used to support standard drug product forms is a must. Additionally, it’s best to know the compound’s degradation path in order to identify potential stability limiting factors.

Rowe and Kiehl (Charles River): Most analyses, namely those that assess how potent and uniform the product is, or how well it dissolves, can be tested on HPLC. For water content, you would use a Karl Fischer titrator, which is an automated electro-chemical titration device that can precisely detect water levels even at very low amounts. Dissolution tests also rely on a separate apparatus that mimics the in-vivo performance of how the product dissolves when ingested. Potency, uniformity, and water content tests are product attribute-based, while the dissolution test is a measure of product performance and one of the key tests scrutinized by the regulatory agencies.

Brümmer (SGS): There is no fixed list of equipment or prescribed lists of test that fit all products because of the number of variable attributes with individual products. However, for most products, chromatographic systems (e.g., HPLC, gas chromatography [GC]) will be used for testing the assay, with purity, dissolution, and water content usually tested by a Karl-Fischer titration system. Solutions will be analyzed, for example, by a laser diffraction technique for particle size distribution or aggregation in the case of large molecules.

A variety of different instruments need to be in place for these analyses, and also large molecules will require specific equipment depending on the attributes being assessed. These include molecular characterization, molecular size, charge, and hydrophobicity using techniques such as, gel electrophoresis, isoelectric focusing, and HPLC-MS. However, it is crucial in all cases when assessing the drug products that any instrument being used has to be qualified and meet GMP requirements.

Sukhu and Tobias (EAG Laboratories): Laboratories must be equipped with state-of-the-art instrumentation that can support a wide array of tests required for biopharmaceutical analysis. Instruments must be properly qualified for use and include pH meters, osmometer and particle counter (testing for appearance and color, visual particles, sub-visible particles, osmolality and other compendial methods); spectrophotometer (protein concentration); HPLC, electrophoresis, imaging capillary isoelectric focusing and capillary electrophoresis to confirm product identity and purity; and cell-culture capabilities with instruments to assess absorbance, fluorescence, luminescence, and electro-chemiluminescence endpoints to measure product potency. Scientific expertise is needed for the proper design, execution, and interpretation of results from the myriad tests. Analytical scientists must have solid backgrounds in protein chemistry/biochemistry, immunology/cell biology, pharmacology, and the regulatory process.

Dosage forms requirements

PharmTech: What are special considerations for different dosage forms?

Rowe and Kiehl (Charles River): The most important things to consider are the delivery mechanisms and targets for the product. For instance, a sustained release product--such as acetaminophen extended-release tablets--needs a test that can monitor dispersal of the material over a long and defined period of time to ensure it meets the exposure needs of whatever disease the product is being prescribed for. Other products labeled as delayed release may target a different absorption area such as the lower intestinal track for better performance or efficacy. Likewise, these products need to be monitored such that the release of drug is withheld for a period of time after consumption, again simulated through in-vitro experiments. For low-dose products, uniformity of potency values between individual units is typically a critical attribute to target in product development and production.

Sukhu and Tobias (EAG Laboratories): The stability of a drug product in its finished form requires a number of tests to verify its safety and efficacy. There are a number of dosage forms that require additional tests to address its specific configuration. For example, capsules require tests for dissolution rates and inspection for leaks and brittleness. A drug product in suspension form may require testing for viscosity, resuspendability, and effects of freezing.

Gladson (Metrics Contract Services): Drug-product samples in both tablet and capsule form may require special handling or may react differently based on coating and/or capsule formulation. With capsule formulations, a scientist must consider potential cross-linking of gelatin in the capsule shell, which is not typically an issue with tablet formulations. Additional considerations with respect to the storage container system and storage conditions should be made for drug-substance material and, in some cases, drug-product forms as well. Unique testing is required for liquid, gel, or foam dosage forms for attributes such as volume, pH, clarity of solution, and solution viscosity.

Brümmer (SGS): Some examples where specific tests may be necessary for certain dosage forms include, but are not limited to:

• Injectable solutions. Particle size distribution by a laser diffraction technique to detect changes in particle size, which can have an effect on the quality of a product

• Inhaler. Specific particle counting techniques such as Andersen impactor or next-generation impactor to ensure particles are not too large to be efficacious

• Emulsions. The zeta potential of a product can determine the stability of an emulsion throughout its shelf life

• Drug substances. X-ray powder diffraction (X-RPD) can show any changes or modifications that may affect the efficacy of a product

• Drug products. Specific detection systems such as an electrochemical detector can be used for assaying antibiotics to determine impurities.

Pilewski (PPD): In general, assay, degradation products, and appearance will be evaluated for all types of dosage forms. Critical quality attributes and methodologies for controlling these need to be identified for each product. For example, with solid oral drugs you will need to evaluate moisture, dissolution properties, color, friability, and odor. Special consideration should be given to products that utilize devices to deliver the medicine. With inhaled products: [pressurized metered-dose inhaler] (pMDI), dry-powder inhaler (DPI), nasal, and nebulizers could include testing for co-solvents, moisture, leak rate, aerodynamic particle size distribution, shot weight, microbial limits, and delivered dose, to name a few.

An additional consideration is the impact of intermediate and final packaging materials. There are increasing requirements for developing methods to understand possible extractables from primary packaging materials and the impact of these materials on leachables during drug product storage and stability. Another attribute to consider is the ability to detect and measure the amount of foreign particulate matter to assure patient safety.

Scheduling stability tests

PharmTech: When should stability tests be conducted to assess distribution and transport conditions?

Gladson (Metrics Contract Services): It’s important to have a good understanding of risks associated with transport and to determine possible areas of degradation to ensure the integrity of the product. Accelerated stability storage conditions--as well as temperature, humidity, light, and forced degradation studies--provide good information in building this preliminary data set. Simulated stability studies may be performed in which material is shipped or stored at extreme conditions. The product also may be agitated to ensure the drug product remains unaffected despite adverse shipping conditions. Chemical tests such as assay and degradation products--as well as physical tests such as appearance and tablet friability--are critical when assessing transport conditions.

Sukhu and Tobias (EAG Laboratories): Stability tests can be conducted as soon as a biopharmaceutical drug substance is available to assess sensitivity to temperature, light, pH, humidity, and oxidation during early-phase drug development. The findings of these studies are useful both when transporting the drug substance and in determining suitable tests to control transport conditions for the drug product. Stability studies are also conducted when the drug substance is formulated into a drug product with excipients and container parts to acquire adequate data to support the distribution and transport conditions during drug development.

Brümmer (SGS): The same parameters should be used as for long-term stability, because these are fixed in the product’s specification, for example, appearance, assay, purity (the most important), and more general tests such as pH, water content, and other stability indicating parameters.

Rowe and Kiehl (Charles River): Typically, these tests are conducted during Phase III trials, or during registration planning and process validation preparations, when clients are confident they have a marketable drug. If you are manufacturing for early-stage clinical trials--not mass distributing across different regions for efficacy trials or anticipation of mass-marketing--individual shipments are easier to manage, even if cold-chain shipment in insulated containers is required if there are concerns about temperature sensitivity. For commercial distribution, these transport studies are critical to understand allowable excursions and mitigate recall actions.

Pilewski (PPD): Formal transportation and distribution studies should be conducted once the final primary and secondary packaging configurations are finalized. These will drive understanding of how your product may change under the influence of time, temperature, and vibrations once it’s left your control. Generally, these are conducted during Phase III while working toward marketing authorization and commercialization.

Testing for temperature excursions

PharmTech: What tests should be conducted to assess the impact of temperature excursions?

Brümmer (SGS): On the whole, the same parameters as for transportation conditions, but it is also important to determine any visual examinations that may be appropriate, such as changes in color or clarity.

Rowe and Kiehl (Charles River): [Tests include] anything that could potentially change upon distribution, usually tests for potency and dissolution. If humidity excursions are also a threat, testing for water content and possibly microbial limits is considered.

Pilewski (PPD): This would be product specific and require an understanding of critical quality attributes impacted by the length and extent of the temperature excursion. All product types will require evaluation of assay, degradation products, and physical appearance. Solution and suspension products will need further evaluation to particle growth, mean particle size change, redispersibility, or changes to rheological properties. Inhaled-drug products will be further evaluated for leak rates, aerodynamic particle size properties, delivered dose changes, and co-solvent changes.

Sukhu and Tobias (EAG Laboratories): Accelerated degradation, storage, and stress studies should be conducted to assess the impact of temperature excursions during the transport of pharmaceutical products. In these studies, temperature ranges outside of the product-specified ranges, including freeze/thaw, must be tested by stability-indicating methods to ensure that product safety and quality are maintained during these stress conditions. At a minimum, these tests should include potency, safety, and quality attributes of the drug.

Gladson (Metrics Contract Services): As the product moves into the later phases of drug development, more thorough studies should be conducted to simulate aberrant storage situations and temperature excursions. Short-term freeze/thaw, heat/cool, and wet/dry studies usually fulfill this requirement. As with distribution and transport conditions, accelerated condition stability and forced degradation studies also can be used to set allowable temperature excursions. Similar to a standard stability study, assay, degradation, and dissolution (for drug product) should be assessed. Additionally, testing such as appearance, hardness, moisture, and viscosity may be critical at extreme storage conditions.

New technologies advance stability studies

PharmTech: What new technologies or methods have advanced drug stability studies?

Gladson (Metrics Contract Services): Several advances in technology have had a significant impact on drug stability studies by improving the turnaround time and the quality of the data generated. UHPLC, charged aerosol detector (CAD), improvements in LC-MS and gas chromatography (GC)-MS capabilities and improvements in dissolution sampling all have helped to streamline the stability testing process, deliver results more quickly, and provide accurate testing of compounds that previously were considered a challenge. Additionally, improvements in data collection software and laboratory information management systems (LIMS) have created the ability for increasing stability storage and organization gains to understand, track, trend, and predict data generated.

Sukhu and Tobias (EAG Laboratories): [Technologies include] MS (i.e., multiple-reaction monitored [MRM]-MS assay), electrochemiluminescence-based immunoassays, and technologies to improve sensitivity in current methods.

Pilewski (PPD): The introduction of UHPLC technology has advanced stability studies by helping improve chromatography sensitivity, speed, and resolution. The higher separation efficiencies achieved by UHPLC have allowed chromatographers to greatly reduce runtimes for complex related substance assays from 90 min to 15 min in some cases. The growing interest with combination products containing two or more active ingredients has, and will continue to benefit, from UHPLC technology.

Rowe and Kiehl (Charles River): UHPLC is a next-generation version of HPLC. It’s a similar base technology; however, superior equipment design allows for higher pressure load on the system, which in turn allows for selection of finer particle size in separation columns and superior resolution. This offers greater specificity of active ingredients and related compounds detection in your products. Water activity monitoring is another emerging tool that is being used specifically to assess whether the environment in the product is able to support microbial growth. If proven that the water level is insufficient for microbial growth, such as bacteria or fungi, this eliminates the need for microbial limits testing. This method is much faster than microbial limits testing, which requires defined incubation times up to or exceeding one week. Microbial limits testing also requires working with live organisms as standard controls, which sometimes don’t always grow properly. You would still be expected to prove the equivalency of water activity as a supplement to microbial limits testing before defending the data to regulatory authorities.

Brümmer (SGS): These changes mainly affect large molecules, and biophysical analysis has become increasingly important for monitoring biopharmaceutical drug stability. Changes in the higher-order structure of the protein can have a critical impact on potency and activity and, together with degradation and aggregation, should be assessed. Multiple orthogonal analytical techniques are now being used more routinely. Methods include circular dichroism (CD) spectroscopy and Fourier transform infrared spectroscopy (FTIR), thermodynamic-differential scanning calorimetry (DSC), and a variety of approaches to assess aggregation such as size-exclusion chromatography with multi-angle light scattering (SEC-MALS), dynamic light scattering (DLS), field flow fractionation, and sedimentation velocity analytical ultracentrifugation (SV-AUC). More recently, techniques have been emerging from research environments such as hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and 2D protein nuclear magnetic resonance (NMR).

Adopting a risk-based approach to stability testing

PharmTech: What are the benefits of a risk-based approach to stability testing?

Brümmer (SGS): This approach saves money and time by reducing the number of batches and tests, as well as reducing the number of test intervals.

Pilewski (PPD): Using a risk-based approach allows prioritization and focus of the aspects of the stability program that are likely to have the largest impact. For example, performing risk assessment on analytical methods helps to identify risk (e.g., robustness affecting precision) that can be further studied in a systematic fashion.

Gladson (Metrics Contract Services): A risk-based approach to stability testing can save both money and time, if staged correctly. A robust understanding of the interactive and degradation properties of the compound’s active pharmaceutical ingredient can reveal the stability properties. This would allow for a reduction in the number of time points needed or the use of matrix designs, because the point of critical degradation already would be known. Also, the storage conditions can be optimized to focus on expected conditions for which the product would be distributed and consumed, such as climatic zones. Overall, the process would be more streamlined and the data generated would be fine-tuned to give a good overall picture of the drug profile.

Sukhu and Tobias (EAG Laboratories): Stability is a central issue in the pharmaceutical industry that can have a significant impact on the safety and efficacy of drug products. Stability includes characteristics of the manufacturing process, drug substance and excipient, type of formulation, and container closure system. Appropriate testing must be conducted to ensure the pharmaceutical products’ stability. A risk-based approach offers the capability to conduct effective and efficient testing without compromising the drug products’ safety and quality. A knowledge base, previous work, and familiarity of methodologies allows the manufacturer to conduct a rational approach to testing that can save time and be cost effective without forfeiting the drug products’ safety and quality.