Recombinant proteins, analytical characterization

The MS techniques described previously for characterization of the final recombinant protein product can be applied at all stages during process development. MS might be used upstream to define clone selection, processing format, and purification steps, and downstream to characterize the final product, ascertain lotto-lot reproducibility, determine stability, and define the formulation of biopharmaceutical molecules. Presented here are some examples found either in the literature or from our own experience in which MS has been found to be a useful or necessary tool. Potential limitations of MS methods are discussed, and when appropriate, other analytical methods are mentioned that can be alternatives to MS and are also efficient tools for biopharmaceutical development. 

The number of residues identified using this method is sequence dependent. Typically 1 to 2 nmol of purified protein will allow sequencing of 3-5 (and sometimes more) cycles fi om the C-terminus. Sequencing of electroblotted samples, where less than 1 nmol of protein is present, has also been successful for 3 or more cycles. Although a few amino acids remain difficult to detect or sequence, our chemistry is especially useful for the verification of the expected C-terminus of recombinant proteins, and has provided information on possible modifications. Here we show specific examples, where the application of this chemistry can be used as an additional analytical tool for protein characterization. 

The scope of this article is to summarize the analytical methods used to characterize the purified recombinant protein in the different stages of drug development, and to summarize bioanalytical methods with focus on their validation and standardization as well as in the determination of immuno-genicity of the therapeutic protein. 

Since the number of recombinant protein drugs will substantially increase over the next decade, analytical as well as bioan-alytical methods for the characterization of such macromolecules will definitively gain in importance. Especially the vaKdation of bioanalytical methods is of key importance to allow an accurate description of protein drugs. This requires harmonized guidelines, which are not yet existing but seem to be on the way, to accelerate drug development. 

A wide range of analytical techniques is used in the characterization of recombinant proteins, including proteomics tools. By definition, proteomics is the simultaneous analysis of complex protein mixtures like tissue extracts, cell lysates, subcellular Iractions or biological fluids at a given time and under precisely defined conditions. Therefore, proteomics tools can also be applied to quantify and identify complex mixtures of proteins in a purified biopharmaceutical. Purify analysis occurs at three stages of the production process during the production (in-process controls), at the end of the purification process on the bulk material, and after the final formulation on the finished product. 

---