As final products in biotechnological and pharmaceutical industry, the antibodies, protein-based therapeutics, and other recombinant proteins must have their purity and heterogeneity determined.
That's because contaminants and modified forms of the product during the production process will drastically alter the biological activity of these proteins. The contaminants and modified forms of the product should be removed to ensure the high purity and then the safety and efficiency of the protein drugs. To successfully evaluate each step of the production system, it is important to develop sensitive and accurate analytical methods for protein purity and homogeneity analysis.
Fig1. Protein Drug ImpuritiesThe impurities can be categorized into two kinds: modified forms or the product and contaminants coming from the host, the culture medium, and the separation processes such as affinity chromatography. In the cell, the adverse culture conditions, the wrong proteolytic cleaving, incorrect folding or incorrect paired disulfide bonds all can introduce impurities. Impurities can also be introduced in other steps. In purification process, the contact with specific reagents may result in conformation change and the limited proteolytic degradation of the product may lead to impurities. During the production or storage, chemical modification such as deamination of asparagine and glutamine residues and oxidation of methionine to methionine sulfoxide can also occur.
The purity and homogeneity of proteins are usually determined by SDS-PAGE, which separates proteins depending on their different sizes. Isoelectric focusing, IEF, is a more sensitive method for protein purity and homogeneity detection, based on charge differences. HPLC is a simple and fast analytical procedures for purity and homogeneity without any particular pretreatment of the samples. Different determination methods are used in different stages for different purposes.
- Protein Purity Analysis (SDS-PAGE)
- Protein Polymer Analysis (HPLC)
- Protein Fragment Analysis (HPLC)
- Protein Isoform Analysis (cIEF)
- Host Cell Proteins Analysis
- Host Cell Residual DNA Analysis
- Protein Homogeneity Analysis
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