Biopharmaceutical proteins analysis

Combe C, Tredree RL, Schellekens H Biosimilar epoetins an analysis based on recently implemented European medicines evaluation agency guidelines on comparability of biopharmaceutical proteins. Pharmacotherapy (2005) 25(7) 954—962. 

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. 

Biopharmaceuticals CE Analysis Table 2 Common protein modification and degradation pathways. 249... 

Covers characterization and analysis of biopharmaceutical proteins, as these molecules are gaining significance. 

The sulfhydryl group of the Cys residue can form a covalent disulfide bond with another sulfhydryl. The other sulfhydryl may be from another Cys residue in the same protein, or from a small molecule such as glutathione or free cysteine (glutathionylation or cysteinylation). The formation of disulfide bonds in specific configurations is often important to the structure, function, or stability of the biopharmaceutical protein. Therefore, the confirmation of the disulfide connectivity is necessary. The analysis of disulfide bond structure is discussed in more detail in Section II.A.2. 

This chapter aims to overview the manufacturing process of therapeutic proteins. It concerns itself with two major themes (1) sources of biopharmaceuticals and (2) upstream processing. The additional elements of biopharmaceutical manufacturing, i.e. downstream processing and product analysis, are discussed in Chapters 6 and 7 respectively. 

A number of different techniques may be used to characterize protein-based biopharmaceutical products, and to detect any protein-based impurities that may be present in that product (Table 7.2). Analysis for non-protein-based contaminant is described in subsequent sections. 

Isoelectric focusing also finds application in analysing the stability of biopharmaceuticals over the course of their shelf life. Repeat analysis of samples over time will detect deamidation or other degradative processes that alter protein charge characteristics. 

The speed, sensitivity, high degree of automation and ability to quantitate protein bands directly render this system ideal for biopharmaceutical analysis. 

The primary analytical applications of RPLC in the development of biopharmaceuticals are the determination of protein purity and protein identity. Purity is established by analysis of the intact protein, and RPLC is useful in detecting the presence of protein variants, degradation products, and contaminants. Protein identity is most often established by cleavage of the protein with a site-specific protease followed by resolution of the cleavage products by RPLC. This technique, termed peptide mapping, should yield a unique pattern of product peptides for a protein that is homogeneous with respect to primary sequence. 

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