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.