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A tremendous amount of analytical testing is required to support a biopharmaceutical product from discovery, development, and clinical trials, through manufacturing and marketing. Numerous methods are used to fully characterize large molecules because of their complexity-characterizing them is significantly more difficult than it is for small molecules. Biopharmaceuticals are produced via living systems, i.e., E. coli, yeast, or mammalian cells, which require additional testing matrices.
A tremendous amount of analytical testing is required to support a biopharmaceutical product from discovery, development, and clinical trials, through manufacturing and marketing. Numerous methods are used to fully characterize large molecules because of their complexity—characterizing them is significantly more difficult than it is for small molecules. Biopharmaceuticals are produced via living systems, i.e., E. coli, yeast, or mammalian cells, which require additional testing matrices. Further, FDA requires orthogonal techniques when available to better reveal the structure and stability of a biopharmaceutical. Currently, more than half of the drug candidates in the discovery stage are biologics, including proteins, peptides, monoclonal antibodies, etc. As the overall number of these large molecules in the pipeline increases, many drug manufacturers may need to consider outsourcing portions of their analytical testing to fill voids in capacity or to meet timelines. This article describes some of the analytical methods used to support a typical biopharmaceutical product throughout its lifecycle from discovery and early development through stability and release testing. Table 1 lists some of the techniques commonly applied to biopharmaceuticals.
Biologics or biopharmaceuticals are inherently more complex than small-molecule drugs. For example, the cholesterol-lowering medicine simvastatin can be identified and fully characterized using a relatively small number of tests because of the drug's well-defined structure (C25H38O5) and associated molecular weight of 418.5722 daltons. However, biologics such as proteins can be thousands of times more massive (typical antibody molecular weight is ~150 kDa), with secondary and higher order structures. Additionally, there is the potential for molecular aggregation. The impact of the protein's structure on therapeutic efficacy and safety must be evaluated and the researcher must use various analytical tools to characterize these structures. For example, the primary structure of a protein is the specific amino acid sequence that makes up the peptide chains. This is typically determined using a technique such as high-performance liquid chromatography (HPLC).
Table 1. Common analytical techniques used for biopharmaceuticals testing
The folding of peptide chains into α-helices, β-sheets, and random coils in a protein defines its secondary structure. Fourier-transform infrared spectroscopy (FTIR) can be used to obtain secondary structure information by analyzing the amide bands. This can be used in conjunction with circular dichroism spectroscopy (CD). Therapeutic proteins and antibodies also are commonly glycosylated or PEGylated. This is the term used when proteins are modified or conjugated by attaching carbohydrates (glycosylated) or synthetic polyethylene glycol (PEG) to them. This results in different properties of the resulting product and greatly increases the challenge of fully characterizing the structure. Liquid chromatography or mass spectrometry (LC-MS) may be required to obtain a complete picture of the structure.
During the discovery phase, some unique analytical tools are used. DNA microarrays can be used to identify disease genes and show how they respond to drugs. The 3-D structure of proteins can be studied for binding sites using X-ray crystallography and NMR spectroscopy. Luminescence and fluorescence-based assays are developed to monitor specific aspects, such as binding, signaling, and other drug-receptor or enzyme interactions. Immunogenicity testing and G-protein coupled receptors (GPCR) assays may be carried out here.
Validation Through the Product Lifecycle
Based on the results of these studies, successful drug candidates may move into early development or preclinical activities, which emphasize safety and efficacy. Assays to support pharmacodynamics (PD), pharmacokinetics (PK), and toxicity with preclinical GLP animal studies are used.
In conjunction with the more expensive and lengthy in vivo animal studies, simpler in vitro test, such as cytochrome P-450 enzymes and Ames tests are increasingly being applied. P450 enzymes make up a superfamily of hemoproteins with highly diverse functions, including drug metabolism in humans; whereas, the Ames test can be used to determine mutagenic potential of drugs. The goal of these early development studies is to examine the drug in living systems before going to human clinical trials.
As the drug candidate moves later into the process development phase, assays are needed to support formulation of drug product, including ingredients and excipients. Many excipients have associated methods described in the various compendia: United States Pharmacopoeia (USP), European Pharmacopoeia EP, British Pharmacopoeia (BP), and Japanese Pharmacopoeia (JP); while other will need new methods developed and validated or supplied by the vendor.
The drug delivery system also has to be developed. Because of stability issues, biopharmaceuticals are typically not formulated as tablets, capsules, or the other more common vehicles used for small, stable, active pharmaceutical ingredients. An area of increasing concern and scrutiny for FDA is the potential adulteration of drug products by extractable and leachable compounds—contamination from the container, closure system, device, etc., that comes into contact with the drug formulation.
Extractables are compounds that can be extracted from a component under exaggerated conditions such as in the presence of harsh solvents or at elevated temperatures. These compounds have the potential to contaminate the drug product. Leachables are compounds that leach into the drug product formulation from the component as a result of direct contact with the formulation under normal conditions. Leachables are typically a subset of extractables. Sources of these compounds include plastic components, elastomers, coatings, accelerants, antioxidants, inks, and vulcanizing agents. Phthalates are one specific example.
These potential carcinogens are added to plastics to make them more flexible and can be found throughout the manufacturing process, as well as in packaging materials. Other examples are nitrosamines and polynuclear hydrocarbons (PAHs) which are classes of carcinogenic compounds found in rubber. Many drug products are distributed or administered in packages made of plastic and rubber components, and therefore, phthalates, PAHs, and nitrosamines could potentially come into contact with the drug product and be passed on to the patient.
Evaluation of extractables and leachables can be an arduous task. Studies must be properly designed, and a wide array of potential contaminants must be screened for using approaches like those described in Table 2.
Table 2. Testing must be conducted to identify extractables and leachables that may contaminate the drug product.
Extractables and leachables issues should be addressed early in the process to avoid regulatory delays for the drug manufacturer. The development of unique packaging and delivery systems required for biopharmaceuticals has intensified this issue due to the growing possibilities of "foreign" materials coming into contact with drug products.
Material will initially be produced on the laboratory scale. As more material is needed for clinical trials, the manufacturing moves to pilot scale. Successful drugs will then progress to a large-scale commercial production environment, and all aspects of this environment must be validated, including water, air, and cleanliness of equipment.
GMP regulations require the testing of water for injection (WFI) and purified water (PW). An example of the battery of test performed is shown in Table 3.
Table 3. GMP regulations require testing of water for injection (WFI) for common contaminants.
During these various phases, cell line selections, cell culture processing, harvesting, and purification are all studied and optimized. GMP regulations require that these processes be validated. Therefore, assays must be developed and large amounts of data must be collected on critical parameters such as purity, contamination, degradation, etc.
Cleaning validations may also be carried out on equipment, surfaces, etc., to check for residual detergents, product, or microorganisms. In many cases, a simple, nonspecific assay such as total organic carbon (TOC) can be used. However, in other situations, specific analyses (one that can determine the concentration of a specific chemical) are performed using HPLC. Moreover, an increasing number of applications that use single-use or disposables, such as filters, tubing, and bags for biopharmaceuticals can introduce unwanted extractables into the final product. The qualification and quality control of all components coming into contact with the drug formulation has become an integral part of any FDA application process.
Process validation is a legal requirement in the industry to demonstrate through appropriate testing and documentation that the finished biopharmaceutical produced by a specified process meets all release requirements for quality. Successful process validation requires thorough process development, identification of controlled and critical parameters, and establishment of specifications throughout the process. A minimum of three consecutive lots of product meeting the established quality specifications is necessary to validate a process. Many of the tests described here are used during process validation. These validations may entail collection and testing of large volumes of samples.
Once the drug is approved there will be ongoing testing to support stability and release of the product. This typically covers critical parameters such as characterization, identity, concentration, purity, excipient testing, potency, sterility, and safety. An example of a specific testing matrix is shown in Table 4.
Table 4. Ongoing stability and release tests like these are conducted after drug approval.
In conclusion, an enormous amount of analytical testing is required to support a biopharmaceutical product throughout its lifecycle, from discovery and early development through stability and release testing. Due to their complexity, an extensive number of methods are required to fully characterize them. Many biopharmaceutical organizations may need to consider outsourcing portions of their analytical testing to remain competitive.
Jon S. Kauffman, PhD, is the director of Method Development and Validation and Biopharmaceutical Services at Lancaster Laboratories, Lancaster, PA, 717.656.2300, firstname.lastname@example.org