Five of the top seven and six of the top twenty biological products in 2010 (in terms of revenue) were monoclonal antibodies (mAbs) (1). Several new mAb products and mAb biosimilars are in development, and all require extensive characterization to obtain the necessary approvals for clinical trials and to eventually be released onto the market.
The most recent regulatory document covering characterization of mAbs was published by the European Medicines Agency (EMA) in July 2009 (2). This EMA guideline entitled Development, Production, Characterization and Specifications for Monoclonal Antibodies and Related Products states that the mA should be characterized thoroughly (2). This characterization should include the determination of physicochemical and structural properties, purity, impurities and quantity of the mAb, in line with International Conference on Harmoniaation (ICH) guideline Q6B (3).
ICH Q6B provides a uniform set of internationally accepted principles for characterization of biotechnological products to support new marketing applications. The document suggests that analyses be performed to provide the following information for biological or biopharmaceutical products:Structural characterization
The techniques used to provide the analytical data required by these guidelines are discussed below.
AMINO ACID COMPOSITION AND DETERMINATION OF THE EXTINCTION COEFFICIENT
ICH Q6B states that "The overall amino acid composition is determined using various hydrolytic and analytical procedures, and compared with the amino acid composition deduced from the gene sequence for the desired product, or the natural counterpart, if considered necessary. In many cases amino acid composition analysis provides some structural information for peptides and small proteins, but such data are generally less definitive for large proteins. Quantitative amino acid analysis data can also be used to determine protein content in many cases."
Amino acid composition analysis using reverse-phase high-performance liquid chromatography (RP–HPLC) with pre-column derivatization or ion exchange chromatography with post-column derivatization are now routinely used to determine the amino acid composition of biopharmaceutical products. Provided that the molar ratios of amino acids per mole of protein and the overall molecular weight of the protein are known, the protein content can be calculated from the amount of each robust amino acid detected in a hydrolysate of the product. Also, if the optical density (at 280 nm) for the solution from which the aliquot(s) for amino acid analysis were taken is known, the Beer–Lambert law (absorbance [OD] = ε.c.l; where ε is the extinction coefficient, c is the concentration in M/L, and l is the path length in cm) allows calculation of the extinction coefficient for the product, meaning that spectrophotometry at 280 nm can be used to quantify the product in solution.