Demand for parenteral-dose contract manufacturing services is on the rise, fueled in part by strong growth in the number of biologics in the approval process.1 With the increase in small emerging biotech companies and "virtual" pharmaceutical companies, lacking both laboratory and manufacturing facilities, contract pharmaceutical manufacturers must offer an increasing array of services.
Specifically, small companies may require formulation and analytical methods development to transform a promising therapeutic into a viable commercial product. Proteins, by their nature, require many methods for product development, product release testing, and stability testing. As the number of biotechnology-derived products produced by contact manufacturers increases, the level of analytical support required by clients will also increase. Contract manufacturers must plan for increased analytical resources in development and quality control to support product release of protein therapeutics. This is a case study of the analytical development of an early-phase protein pharmaceutical proposed by a virtual company client. We are including the false starts and dead ends lest anyone thinks this was easy.
THE RESULTS DEPEND ON THE METHOD
While small-molecule products typically require only one chromatographic release method to determine product purity, the specific molecular properties of protein therapeutics require multiple release methods encompassing a variety of techniques. Because proteins are produced biosynthetically in living organisms, the active pharmaceutical ingredient (API) may include molecular variants. These post-translational modifications, such as variations in glycosolation, oxidation, sulfation, phosphorylation, deamidation, and fragmentation may have no effect on product safety or efficacy, and can be considered product related substances. However, a "consistent pattern of product heterogeneity" should be demonstrated from lot to lot.2
Because manufacture and storage can produce additional modifications such as aggregation, methods to profile heterogeneous patterns are required to ensure lot-to-lot consistency of a drug's product and storage stability. The protein's inherent heterogeneity makes "the absolute purity of biotechnological products" difficult to quantify, and "the results are method dependent."2 Therefore, quality of the drug product is "usually estimated by a combination of methods."2
Typical product release methods applied to protein therapeutics include reverse-phase chromatography (RPC), ion-exchange chromatography, size-exclusion chromatography (SEC), capillary electrophoresis, peptide mapping by liquid chromatography combined with mass spectrometry (LC-MS), gel electrophoresis, immunoassays, western blot and isoelectric focusing (IEF). Our company has almost all of these in its bio-analytical toolbox.
The Raw API
The API was an approximately 80 kDa protein produced by a bulk-drug contract manufacturer using Escherichia coli fermentation followed by several purification steps. The protein was not glycosolated and contained no disulfide bonds.