March 15, 2005 (Vol. 25, No. 6)

Forced Degradation Studies Predict Effects on Bioproducts in Drug Development and Manufacture

Forced degradation studies involve exposing a drug substance to harsher conditions than the product would be expected to experience and determining at what point and how it degrades.

Identifying strategies to minimize the effects of variable temperature, humidity, and lighting conditions on the structural integrity and biological activity of bioproducts during drug development, manufacturing, storage, and administration is another important aspect of this field of study.

Stability testing of biological drugs should be more comprehensive than that of small molecule compounds, states Duu-Gong Wu, Ph.D., senior director, PharmaNet Consulting at PharmaNet (Washington, D.C.).

“It should cover many more aspects of drug development and manufacturing,” including holding time, shipping, freeze/thaw cycles, temperature, exposure to oxygen and light, physical stress, and excipient and container compatibility.

“Some characterization work should be done early in the development of a drug to identify the degradation pathways so that potential problems associated with stability, storage conditions, and formulation design can be avoided during clinical trials,” he says.

Dr. Wu also makes the point that a good understanding of product stability early on can help companies make assessments and comparisons on product comparability following manufacturing changes.

The scientific principles of biostability testing, as stipulated in the International Conference on Harmonization (ICH) guidelines Q5C, Q6B, and Q1AR, have not changed substantially in recent years, according to Dr. Wu. However, the FDA is focusing more keenly on certain aspects of product stability “due to recent incidents with some products.”

One area of interest he identifies is the development of more complex protein products, such as monoclonal antibodies, “which have many more potential sites for degradation due to oxidation, deamidation, etc., and this has the potential to affect product quality. How to identify, characterize, and control each of these presents a real challenge,” says Dr. Wu.

Modified products such as PEGylated compounds also present challenges for stability testing. New product formulations, including different dosage forms such as liquid formulations and extended release drugs, as well as novel delivery systems such as needleless injection systems, pre-filled syringes, and other combination devices, introduce additional uncertainty and extra work when assessing product stability.


F”Aggregation has recently attracted a lot of attention from the FDA due to a number of incidents in which stability problems were caused by container leachables/ extractables or changes in excipients, as suggested in the case of Eprex (epoetin alfa, Johnson & Johnson; New Brunswick, NJ),” comments Dr. Wu.

Since aggregation can induce immunogenic reactions with potentially severe consequences, the FDA is requiring manufacturers to pay more attention.

Bioproducts that are administered in high doses, require high concentration formulations, and are stored in vials, such as monoclonal antibodies or enzyme replacement therapies, are more prone to aggregation. Bioproducts are, in general, more complex and require specialized analytical techniques.

Stability Issues for Mabs

There are nearly 200 antibodies (constituting 20% of all protein therapeutics) in clinical trials today. Amgen (Thousand Oaks, CA) has several new therapeutic antibodies in the pipeline to treat conditions such as osteoporosis, cancer, inflammation, and autoimmune diseases.

Aggregation is typically the primary degradation pathway for antibodies, which are often formulated at high concentrations as high dosage volumes. Sometimes four orders of magnitude higher than conventional protein therapeutics are required to achieve the stochiometry necessary for them to be effective.

“Understanding the protein degradation mechanisms of different classes of antibodies is critical for developing stable, potent drug products,” says Sampath Krishnan, Ph.D., a research scientist in the department of pharmaceutics at Amgen.

Antibody aggregation relates directly to the shelf stability and activity of these biomolecules. Particle formation due to aggregation is a major issue, notes Dr. Krishnan, as it can cause increased immunogenicity, clogging of intravenous lines and filters, and altered serum half-life. It can also have detrimental effects on protein activity, particularly if aggregation involves regions of the protein necessary for substrate binding interactions.

“The mechanisms of aggregation of proteins in general are not very well understood,” Dr. Krishnan says. Identifying the specific mechanism of antibody aggregation is essential in order to be able to change the formulation in a way that minimizes aggregation.

Antibodies are useful therapeutic molecules in part because they have naturally evolved to associate with a variety of targets with high affinity. They can also, however, self-associate and aggregate through these same domain interactions.

“Antibodies can undergo covalent or noncovalent association that is highly dependent on the solution conditions, including pH, ionic strength, and excipients,” explains Dr. Krishnan.

Antibodies also have multiple intra-domain and inter-domain linkages, and these linkages can undergo disulfide shuffling during processing, “leading to product heterogeneity and aggregation. Antibodies are also susceptible to photo oxidation that can lead to aggregation.”

Dr. Krishnan identifies biocomparability in terms of product stability as one of the main challenges facing manufacturers of biologicals today. “In most cases, any alterations in the process used to manufacture the antibody molecules can result in wide differences in the structural and functional properties of the molecules.”

“Formulation screening must be initiated early in development, even before knowing the commercial drug dose and before the commercial process is set. Challenges include the purity of excipients used in the formulation of the antibody molecules, as this can jeopardize the consistency of the drug product, as well as the need to optimize formulation screens due to the limited protein available for early formulation studies.”

From a technology perspective, Dr. Krishnan has seen increased use of mass spectrometry coupled to chromatography to track chemical and physical degradation, and light scattering techniques to monitor the irreversible disruption of the equilibrium between monomer and soluble aggregates.

“This disruption of equilibrium precedes the formation of large insoluble aggregates and precipitates. Calorimetry and spectroscopic techniques are used to get high precision stability and structural information, respectively, on the protein.

“In addition, analytical ultracentrifugation (AUC) is increasingly being used to understand the nature of the protein interactions in aggregates and the size distributions of such aggregates.”

Freeze/Thaw Issues

At “Early Development of Biotherapeutics,”an upcoming IBC conference to be held April 2527 in Reston, VA, James Colandene, Ph.D., section head, fill-finish process development, pharmaceutics sciences at Human Genome Sciences (Rockville, MD), will speak on “Scaling Up Freezing for Protein Drug Products.”

Various factors come into play when scaling up freezing for long-term storage of bulk drug substance, and unfortunately, small-scale freeze-thaw studies may not be predictive of potential problems. Factors such as the size and composition of the storage container and a product’s compatibility with a particular freezing method may all impact a drug’s stability over time.

Bulk drug substance is typically stored frozen. Ideally, it would be maintained at -80C to stay below thermal transitions such as eutectic melting and glass transitions, but walk-in freezers capable of accommodating bulk storage containers, needed for volumes associated with Phase III clinical studies and commercial production, usually reach temperatures no lower than -20C to -40C.

The intent of freezing is to prevent product degradation, and formulation groups typically conduct studies early in development to assess the effects of freezing and freeze-thaw cycling on a product. “If they can’t get a product in a formulation that can withstand freeze-thaw, it will likely jeopardize the project,” says Dr. Colandene.

Proteins may denature during the freezing process, for example, and once unfolded may not revert properly to their native state. Unfolding of proteins also increased the chances that aggregation will occur.

A variety of cryoprotectants are available to protect drug substances from the effects of freezing: surfactants may help protect against surface adsorption at the ice-solution interface, while other cryoprotectants may help keep proteins in a more tightly bound state through preferential exclusion. Similarly, lyoprotectants such as disaccharides help protect drug substance that undergoes freeze-drying.

During preformulation studies, scientists are typically working with small amounts of product and may not take into consideration issues related to scale-up of a freeze-thaw process. In fact, it is not really feasible to model the large-scale environment when working with these small quantities.


One potential problem is bulk-scale cryoconcentration, which is affected by the time it takes for an entire system to freeze. These concentration effects can occur at small scale, too, but they may be magnified by bulk freezing. When a protein mixture is dissolved in water, the water molecules will first begin to form ice crystals, increasing the protein concentration in the remaining solution.

The effects of cryoconcentration are protein specific and may not affect the integrity of some protein solutions, but may have negative consequences for others. HPLC studies can be used to assess the extent of aggregation. A possible solution to this problem would be the use of specialized freezing methods or devices that rapidly freeze large quantities of solution.

A storage temperature of -80C helps minimize product stability problems by keeping a product below the eutectic melting temperature and below the glass transition temperature. Consider a salt solution of NaCl and water.

As the temperature drops, the water component begins to freeze first and the NaCl becomes more concentrated until it reaches a critical concentration and the salt begins to crystallize together with the remaining water molecules, a phenomenon called eutectic crystallization.

As this mixture then warms, it reaches the eutectic melting point, at which the salt goes back into solution. For a protein solution, this process can affect the structural integrity of the proteins as the ionic strength of the solution increases. Even though many protein solutions may cross the eutectic melting point as storage conditions shift from -80C to -20C, no obvious melting may be evident.

The glass transition temperature relates to the viscosity of a solution. At -20C, some solutions will be above the glass transition temperature, and their molecules will be relatively mobile, despite the solution being “frozen,” which can increase the chances for stability problems.

Dr. Colandene points to the characteristics of the storage container as another important factor. The material should be compatible with the solution it will house and should be suitable for freezing.

The size and shape of the container will affect the freezing time and, therefore, the risk of cryoconcentration. New disposable bag freezing systems offer convenient alternatives to traditional containers. Thawing can present similar problems as freezing, and the advantages of rapid-thaw strategies should be considered.

Photostability Guideline

In 1997, the FDA adopted a new ICH guideline aimed at promoting international standards for regulatory requirements governing pharmaceuticals for human use. The new guideline describes the basic protocol for photostability testing of new drug substances and products (Federal Register 1997;62(95):27116).

In introducing this guideline the FDA states, “Light testing should be an integral part of stress testing,” and that “the intrinsic photostability characteristics of new drug substances and products should be evaluated to demonstrate that light exposure does not result in unacceptable change.”

Prior to the late 1990s, the FDA’s emphasis in stability testing focused on evaluating the effects of temperature and humidity on product integrity and activity, according to Bob Dotterer, applications engineer at Caron Products (Marietta, OH).

The recent focus on light stability has had a significant impact on the selection of packaging and storage containers, in Dotterer’s view. In the past, the extent of photostability testing was left up to individual companies, and the testing methods and criteria used varied greatly.

Unified standards among regulatory agencies in the U.S., Europe, and Japan give researchers a better opportunity to test the ingredients of a drug during the design phase and be able to evaluate various formulation strategies.

With regard to photostability testing, the issues and methods are basically the same regardless of whether the drug substance to be assessed is a biological or a small molecule. Because light has different kinetics than temperature, for example, photostability testing on a small scale has the advantage of being very predictive of what will occur with bulk storage.

Photostability is based on quantum yields and will vary with the spectrum of light used, the intensity of the light source, and the wavelengths absorbed by a particular molecule. In most cases, a three-day experiment using various light sources can predict what would be expected if a drug were exposed to light eight hours a day for a year.

The exception, which occurs infrequently, according to Allen Templeton, Ph.D., a research fellow at Merck & Co. (Whitehouse Station, NJ), is when dark reactions take place and exposure to light triggers a chain of events that continues to affect the drug substance even after the light source is turned off.

Photostability testing should take into account all phases of drug development, contends Templeton, from drug synthesis to manufacture, packaging, and use of the drug by the physician or patient. It is also important to focus on potential photo-degradation of excipients as this can greatly affect the physical nature of the overall product.

The FDA’s photostability guideline includes a requirement to expose a product to both visual and near-UV light and allow for one of two scenarios: use of either a single light source that emits a combination of visible and ultraviolet outputs; or use of two different lighting sources, one being a cool white fluorescent lamp and one a near-UV fluorescent lamp.

“We have seen a big trend toward two light sources (option 2),” says Dotterer. Using two separate lamps allows researchers to evaluate these variables independently and to distinguish between degradation caused by different wavelengths of light. It also helps eliminate effects due to over-exposure.

Caron Products designs and manufactures test chambers that simulate various types of environmental conditions for use in forced degradation studies. The company offers standardized and customized test chambers capable of exposing a product to temperatures ranging from -20C to +80C, relative humidity of 2% to 98%, freeze-thaw cycling, freezer storage, and various light conditions.

In February, Caron introduced a new photostability chamber targeting the European pharmaceutical industry. The Model 6540 is a bench-top instrument designed to perform near-UV and visual light testing using fluorescent lamps that emit high intensity light with a uniform distribution. The instrument offers programmable light and temperature protocols and has an optional controlled humidity system.

Forced Degredation Study Design

In a presentation entitled “Using Forced Photostability Degradation Study Data to Drive Decision-Making during Pharmaceutical Development,” given at a recent Institute for International Research conference in Washington, D.C., Dr. Templeton emphasized the importance of the thought process behind the design and implementation of forced degradation studies needed to yield high quality, relevant stability data that can drive decision-making.

The ICH guideline on photostability “provided some much needed guidance,” Dr. Templeton says, as there had been a lot of diversity in the industry in the types of light sources used and the quality of the data generated.

However, the guideline is just that: a recommendation that still leaves a lot of leeway for judgment and interpretation in terms of study design, the mechanics of photostability testing, and the light sources used, which has led to some controversy and confusion.

A central point of controversy surrounding the photostability guideline is whether data derived using option I versus option II are equivalent and comparable. Dr. Templeton explains that option I is more representative of the sun, whereas option II decouples the effects of outdoor and indoor light.

If the product is not likely to be exposed to daylight during manufacture, packaging, or regular use, how relevant are photostability data derived using an option I source?

Another issue is the lack of standardization of these light sources, as all fluorescent lamps do not emit the same light spectra. Also controversial is the definition of “acceptable change,” as used in the guideline. What represents an acceptable change and should that be based on product safety, activity of the drug substance, or compliance with ICH thresholds?

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