September 15, 2008 (Vol. 28, No. 16)
David Daniels, Ph.D.
Strategies for Overcoming Critical Issues Like Aggregation and Stability
The protein therapeutics market totaled $63 billion in 2007 ($39 billion in the U.S. alone) and is expected to reach $87 billion by 2010, according to a report by Kalorama Information—“Protein Therapeutics: The Science and Business of a Growing Sector.” Although the protein therapeutics market is significantly smaller than the pharmaceutical market, the relative growth of the protein therapeutics market is substantially higher than the overall pharmaceutical market growth.
This market explosion can be traced back 20 years, according to the report. Researchers have been building a knowledge base and conducting research in proteomics and genomics while gaining a broader understanding of the molecular basis of disease.
The release of recombinant insulin in the early 1980s launched the protein therapeutics industry. Since then, the sector has undergone a rapid metamorphosis with the introduction of new therapies like interferons and interleukins in the 1990s and monoclonal antibodies in the 2000s. Such strong innovation has put the protein therapeutics sector in the driver’s seat, accelerating growth in the drug industry.
There are many challenges for scientists working in the realm of protein therapeutics, and formulation is chief among them. IBC’s upcoming “Formulation Strategies for Protein Therapeutics” will address some of the most pressing issues. This article previews several presentations, revealing strategies to handle problems dealing with aggregation, drug stability, and extractables and leachables.
Christian Rischel, Ph.D., is a senior scientist in the protein structure and biophysics group at Novo Nordisk, one of the leading producers of insulin. The company also makes several other therapeutic proteins including human growth hormone and Factor VIIa.
At the meeting, Dr. Rischel will talk about the fundamental aspects of protein aggregation, an important process to avoid when producing therapeutic proteins. The model for this work is glucagon, a peptide hormone that triggers the liver to release sugar, raising blood glucose levels following subcutaneous injection. Novo Nordisk has commercialized this therapeutic in GlucaGen® HypoKit® to treat severe hypoglycemic reactions to insulin therapy.
In a collaboration with the University of Aarhus in Denmark, Dr. Rischel and his team are studying the factors that influence the formation of glucagon fibrils. Changes in pH and salt concentration and peptide concentration can all affect the rate of fibrillation. Of these factors, the researchers have focused on the unusual influence of peptide concentration on fibrillation of glucagon.
“We recently found that fibril morphology depended on concentration,” Dr. Rischel explained. “At concentrations of less than 1 mg/mL, the fibrils took on the morphology of well-twisted ropes. Surprisingly, the process initially went slower the more peptide we added. But at concentrations above 1.5 mg/mL, the fibrils formed much faster and had the morphology of raw spaghetti. We think that at higher concentrations peptide trimers form and lock the fibril into the observed morphological shape.”
During purification and production of the therapeutic, protein fibrillation can result in loss of material due to precipitation that may clog pumps and filters. During storage, any conditions that stimulate fibrillation could result in inactivation of the protein therapeutic. Understanding these conditions is essential for commercial success.
Tim Kelly, Ph.D., vp of biopharmaceutical development at KBI Biopharma , will discuss a variety of approaches for evaluating protein drug stability during the early stages of formulation development. The goal of formulation development, at KBI and elsewhere, is to determine a final dosage form that results in a safe, efficacious product that remains stable over the course of its intended use. The stability of candidate formulations is evaluated during development via a combination of real-time and accelerated studies. Among drug companies, there are diverging opinions about the accelerated stability data deemed appropriate to support formulation development during preclinical and Phase I clinical development.
At the meeting, Dr. Kelly will present a case study that compares conservative and aggressive approaches used on different proteins to evaluate stability of candidate protein formulations. Therapeutic antibodies are the largest class of protein therapeutics today, but this group also includes enzymes, cytokines, hormones, and other proteins.
The analyses used to evaluate real-time and accelerated stability typically include purity assays such as SEC-HPLC, IEX-HPLC, RP-HPLC, SDS-PAGE, IEF, SDS-CGE, and cIEF to quantify product and process-related impurities such as deamidation, oxidation, aggregates, clipped species, and host-cell proteins. In addition, such studies often include assays to monitor the activity of the product, which may include ligand-binding ELISA methods or cell-based assays to measure proliferation, cell death, or cytokine release.
A conservative approach for evaluating formulation stability may involve placing 10 or more candidate formulations in a long-term stability study under real-time (e.g., 5°C) and accelerated (e.g., 30°C/65% relative humidity conditions. The duration for such a study may range from six months to two years, with samples from each of the candidate formulations analyzed in one- to three-month intervals over the study duration.
At the conclusion of the study, the candidate formulation that exhibits the best overall conservation of native purity and activity is chosen and utilized for manufacturing of clinical trial material. This conservative approach is most commonly utilized by large pharma companies that have the time and resources to permit this approach.
The main advantage of this approach is that the formulation decision is based on real-time stability data at the intended product storage temperature. Therefore, drug developers may have a high degree of confidence that the product will remain stable throughout the duration of its intended use in clinical studies.
Smaller pharma and biotech companies, especially those that do not yet have revenue streams from commercial products, often can’t afford to devote the time and resources required to execute a 1–2 year real-time stability study during formulation development. Such companies may take a more aggressive approach, with a greater emphasis on accelerated stability over a shorter duration.
KBI Biopharma frequently employs an approach where a large number (30–40) of candidate formulations are evaluated via statistical design of experiments including short term stability under real-time and accelerated conditions for 1–2 months.
The accelerated stability conditions used in such studies may range from 40–55°C to increase the rates of degradation and enhance the likelihood of observing significant differences among the candidate formulations over the short duration of the study. A long-term stability study is then performed on the actual clinical trial material prepared in the final selected formulation, in accordance with the ICH guidelines.
The main advantage of this approach over the more conservative approach is the acceleration of development timelines. The quantity of API available for such studies during pre-clinical development also impacts the duration of formulation stability studies.
A potential disadvantage of this approach is the challenge of assigning predictive value to accelerated stability data for protein therapeutics. The translation of accelerated stability data into a real-time storage shelf life is problematic as protein degradation processes are quite complex and often do not follow Arrhenius kinetics.
“We have used both approaches successfully to develop stable formulations for different therapeutic proteins” says Dr. Kelly. “I would love the opportunity to evaluate both approaches in parallel on the same protein to see if the more aggressive approach consistently and accurately predicts formulation shelf life. I expect our client partners will continue to request each approach in order to best meet their Phase I clinical trial timelines.”
West established West Analytical Services in 1999 to provide analytical testing for glass, plastic, and elastomer packaging components to help customers meet container, closure, and packaging regulatory guidelines.
Jennifer Riter, director of analytical and technical services, will talk about the challenges of pharmaceutical product development related to selecting and testing an appropriate packaging system or delivery device. The extractables and leachables testing case study that Riter is presenting at the conference is at the heart of this testing.
Extractables are chemical species that can migrate or be extracted from containers, closure systems, and other packaging components under appropriate exaggerated solvent, temperature, and time conditions, and have the potential to contaminate the drug product. Leachables are chemical species that actually migrate from the containers, closure systems, and other packaging components under normal conditions or during stability studies.
“We highly recommend that our customers determine the compatibility between their drug product and their chosen packaging and delivery device early in the drug development process,” says Riter. “Sources of extractable and leachables include glass, plastic, and elastomer-containing closure and delivery systems.”
Extractables from these materials include coatings, accelerants, antioxidants, and vulcanizing agents. Leachables may render the drug product inactive or impact the drug from a toxicity perspective. At the IBC meeting, Riter will present an approach for testing several prefilled syringe systems.
Screening studies can be completed early in development by putting the drug product/placebo and components under stress conditions to determine if there may be any gross incompatibilities between the drug and package.
Extraction studies and the identification of extractables employ the use of several analytical techniques including GC/MS, LC/MS/MS, IC, and ICP/MS. At issue is the potential for drug inactivation, protein aggregation, and toxicity via introduction of a leachable from the packaging materials upon injection of the patient.
The test for leachables is focused on drug stability over the shelf life of the product. Samples of the drug product that have been in contact with components of the packaging or delivery device are taken and analyzed for leachables using several analytical techniques.
It should be noted that leachables may be the same compounds identified during extractable studies or may be different based on reaction products or interaction with the drug product. All major components that don’t have origins in the drug product are identified.
Prolonged Therapeutic Benefit
Michiel Lodder, Ph.D., director of business development for OctoPlus, will discuss OctoPlus’ formulation approach that allows for long-acting, controlled-release versions of known protein therapeutics.
Dr. Lodder will share the success story of the development of Locteron®, the company’s controlled-release alpha interferon (IFN-a) product. This new drug formulation has been shown to significantly improve the tolerability of the treatment of chronic hepatitis C infection, with similar or even improved efficacy of the therapy for patients with this liver disease, he reports.
Locteron combines PolyActive® microspheres with BLX-883, a recombinant IFN-a produced by OctoPlus’ co-development partner Biolex Therapeutics. PolyActive biodegradable microspheres exhibit a hydrogel character, providing a natural environment for proteins. These microsphers also preserve the activity and stability of the embedded compound until release, Dr. Lodder says.
In his presentation, Dr. Lodder will elaborate on the formulation, manufacturing, and clinical development of Locteron.
“We have been able to demonstrate with Locteron that IFN-a plasma drug concentrations remain constant and within the therapeutic range during the time between injections,” he says. “This approach enables reducing the frequency of injection from once a week to once every two weeks because the drug stays in circulation longer. Phase IIa studies in Europe have been successfully completed. We will begin a Phase IIb study at the end of 2008 or early next year.”
In addition to reducing the frequency of injections, the patient also benefits from an improved side effect profile because the delayed and constant release prevents the high concentration of bolus in the blood resulting from traditional IFN-a treatment, which is often associated with severe side effects.