Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 177

7.3 Removal of altered forms of the protein of interest from the
product stream
Modifi cation of any protein will generally alter some aspect of its physicochemical character-
istics. This facilitates removal of the modifi ed form by standard chromatographic techniques
during downstream processing. Most downstream procedures for protein-based biopharma-
ceuticals include both gel-fi ltration and ion-exchange steps (Chapter 6). Aggregated forms of
the product will be effectively removed by gel fi ltration (because they now exhibit a molecu-
lar mass greater by several orders of magnitude than the native product). This technique will
also remove extensively proteolysed forms of the product. Glycoprotein variants whose car-
bohydrate moieties have been extensively degraded will also likely be removed by gel-fi ltra-
tion (or ion-exchange) chromatography. Deamidation and oxidation will generate product vari-
ants with altered surface charge characteristics, often rendering their removal by ion exchange
relatively straightforward. Incorrect disulfi de bond formation, partial denaturation and lim-
ited proteolysis can also alter the shape and surface charge of proteins, facilitating their re-
moval from the product by ion exchange or other techniques, such as hydrophobic interaction
chromatography.
The range of chromatographic techniques now available, along with improvements in the reso-
lution achievable using such techniques, renders possible the routine production of protein biop-
harmaceuticals which are in excess of 97-99 per cent pure. This level of purity represents the
typical industry standard with regard to biopharmaceutical production.
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.
7.3.1 Productpotency
Any biopharmaceutical must obviously conform to fi nal product potency specifi cations. Such
specifi cations are usually expressed in terms of 'units of activity' per vial of product (or per thera-
Table 7.2 Methods used to characterize (protein-based) fi nished product biopharmaceuticals. An
overview of most of these methods is presented over the next several sections of this chapter
Non-denaturing gel electrophoresis
Denaturing (SDS) gel electrophoresis
Two-dimensional electrophoresis
Capillary electrophoresis
Peptide mapping
HPLC (mainly RP-HPLC)
Isoelectric focusing
Mass spectrometry
Amino acid analysis
N-terminal sequencing
Circular dichroism studies
Bioassays and immunological assays