Process-related impurities proteins

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

Bulk and Intermediate Purification primarily for removal of process-related impurities, e.g. reagents, host cell proteins, DNA, endotoxins some product-related impurities common methods ... 

In the early days of biotechnology product development, the focus was on quality issues [4] or process-related impurities.The concerns at that time were for carryover of other cellular proteins and DNA and for contamination with endotoxins, chemicals, and viruses. Of course, these concerns still exist, but methods for purification and assays for evaluation of clearance have alleviated the need for the safety assessment scientist to focus on contaminants instead they are now asked to focus on the pharmacological activity of the molecules. An ICH guidance (Q6B Specifications Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) addresses the specific issues related to the manufacturing process [6], Other product-related issues such as impurities do need to be considered by the safety assessment scientist, for... 

Protein impurities are either process- or product-related impurities. Process-related impurities include proteins added to the culture medium, proteins used during purification, such as nucleases and chromatography ligands, and proteins from the host organism. Product-related impurities include degra-dates, aggregates, and conformational isomers of the recombinant drug product. 

Immunoassays are the most specific and sensitive techniques available for detecting protein impurities. There are two classes of protein impurities that are most often analyzed with these techniques host-cell proteins and protein additives, both of which are process-related impurities. Although protein additives are known entities and therefore amenable to other quantitative... 

Process-related impurities encompass those that are derived from the manufacturing process, that is, cell substrates (e.g., host cell proteins, host cell DNA), cell culture (e.g., inducers, antibiotics, or media components), or downstream processing. Product-related impurities (e.g., precursors and certain degradation products) are molecular variants arising during manufacture and/or storage that do not have properties comparable with those of the desired product with respect to activity, efficacy, and safety. 

Process-related impurities encompass aU possible material that is used during manufacture and which might still be present in the final product These include cell components (e.g., host cell protein, DNA and RNA) and components of the cell culture medium (e.g., antibiotics, inducers, media). In addition, possible downstream-derived components (e.g., enzymes, (bio)-chemical reagents, inorganic salts and solvents) must be considered. Any adventitiously introduced material which is not part of the manufacturing process of either DS or DP is considered as a contaminant. For viral products and processes, special attention is paid to endogenous or adventitious viruses, which should ideally not be present or at least be removed/inac-tivated by the manufacturing process. 

Leached ligands from chromatographic column (i.e., protein A) Other process-related impurities... 

A biosimilar is dehned as any therapeutic protein by its own manufacturing process that may influence the characteristics of the product itself but also introduce specihc process-related impurities. The producers of biosimilars therefore need to demonstrate consistency and robustness of their production process. They need to do formulation studies showing stability and compatibility, even if the formulation is identical to the reference product. 

The separation tasks for ion-exchange chromatography differ mainly by the type of expression system used. The most abundant systems are mammalian cells, microbial cells (yeast), and the bacterial organism E. coli. All expression systems have a common feature that they not only generate the target protein but also process related impurities, such as host cell proteins, DNA, viruses, endotoxins. In addition the downstream process has to isolate the active form of the target molecule from all its derivatives, for example, oxidated, deamidated, acetylated forms, dimers, aggregates, and unfolded proteins. 

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