Host-cell proteins

Trace contaminants such as host cell proteins (HCPs) and DNA are deterrnined by more specialized techniques. Host cell proteins are generally deterrnined using an immunochemical assay, in which an antibody preparation, raised against a mixture of the HCPs, is used to selectively detect the total level of HCPs in the product. DNA can be deterrnined using a labeled mixture, or probe, of complimentary DNA from the host cell. 

Many Viruses Co-opt the Host Cell Protein Synthesis Machinery... 

Several key issues have to be addressed in the downstream processing of biopharmaceuticals regardless of the expression system. The removal of host cell proteins and nucleic acids, as well as other product- or process-related or adventitious contaminants, is laid down in the regulations and will not differ between the individual expression hosts. The identity, activity and stability of the end product has to be demonstrated regardless of the production system. The need for pharmaceutical quality assurance, validation of processes, analytical methods and cleaning procedures are essentially the same. 

Hoffman advises, Relying solely on a process-specific assay is ill advised and can result in failure to detect atypical process contaminants. In cases with a defined, persistent, and problematic host cell protein impurity, a down-stream process-specific assay may be justified. It is critical that the immunoassay be capable of detecting every possible host cell protein contaminant. 13... 

The Western blot method is often used in the analysis of host cell impurities. It can be used to identify a recurring impurity. O Keefe et al. used a Western blot to identify an E. coli protein impurity in the preparation of the recombinant fibroblast growth factor (aFGF).29 By using specific antisera to the E. coli host cell proteins, they were able to isolate the impurity and determine its N-terminus amino acid sequence to confirm its identity. Antibodies could be used to determine the concentration of this impurity in sample preparations. 

Hoffman, K. (2000). Strategies for host cell protein analysis. Biopharm-Appl TBio 13(5) 38 15. 

About 1.5-2 hours after infection the first viral structural proteins can be detected in cells efficiently infected by SFV (see Kaariainen and S6-derlund, 1978). By 3-4 hours the synthesis of host cell proteins is shut off. The mechanism of host protein shutoff is not yet known, but studies by van Steeg (1982) suggest that it is the viral capsid protein itself which is responsible for the selective inhibition of host protein synthesis. The capsid protein seems to reduce the activity of the initiation factors elF-4B and CAP binding protein below levels necessary for the formation of the 80 S initiation complex from host mRNA. Translation of the viral 26 S RNA is, however, unaffected. More studies are clearly needed to substantiate this interesting possibility. 

Special attention has been paid by sevo groups to cloning xylanase genes from thermophilic microorganisms as a source of thermostable xylanases. Cloning of such genes in mesophilic recepients offers a convenient way to purify xylanases simply by a heat denaturation of the more heat-labile proteins of the host (54). Applications are limited to those enzymes that possess thermostability considerably higher than the majority of the host cell proteins. 

Chakrabarty et al. discovered that the host cell protein vasodilator-stimulated phosphoprotein (VASP) concentrates at the back of motile Listeria at the bacteria-tail junction. VASP docks onto the surface of Listeria by binding to the oligoproline sequences of ActA concentrated on the rearward pole of moving bacteria. In uninfected host cells, VASP is normally found in focal con-... 

BASIC FEATURES OF THE ACTIN-BASED MOTOR COMPLEX. These experiments support the following model for Listeria actin-based motility. The Listeria surface protein ActA uses its oligoproline sequences (FEFPPPPTDE) to bind the host cell protein VASP. VASP in turn deploys its own larger set of GPPPPP sequences to bind profilin. 

FIGURE 11.5 (Panel a) Reversed phase separation of rHuBDNF feedstock showing the positions of variants and host cell proteins. (Panel b) Ion exchange separation of the same feedstock on a 50 pm cation exchange resin. Loading was 20 pg of rHuBDNF. (Reprinted with permission from Elsevier from Zhao, G.F. and Sun, Y., J. Chromatogr., 1165, 109, 2007. Copyright.)... 

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 ... 

Polishing final purification step (invariably using chromatography) to remove dose product-related impurities, residual of host cell proteins (HCPs) and endotoxins. 

Host cell proteins characterization and selective removal resulting in simpler purification process... 

One subunit (Mr 65,000) is the product of the replicase gene encoded by the viral RNA and has the active site for replication. The other three subunits are host proteins normally involved in host-cell protein synthesis the E. coli elongation factors Tu (Mt 30,000) and Ts (Mr 45,000)... 

Achneider, R. J., and T. Shenk, Impact of virus infection on host cell protein synthesis. Ann. Rev. Biochem. 56 317— 332, 1987. 

Endotoxins are found in some bacterial sources, such as E. coli. For other products they are considered a contaminant that should not be present and can be controlled by adherence to good manufacturing practices (GMPs). Nucleic acids, once considered a significant risk, are now thought of as cellular impurities, and their removal should be validated [2,3]. Proteins that pose a potential risk (e.g., immunogenicity) include host cell proteins, aberrant protein product, proteins used in cell culture, and those associated with the process (e.g., protein A affinity ligands or nucleases employed to reduce viscosity). 

The inability to pick up all host cell proteins is another problem faced in development and implementation of these assays. 

For firms that manufacture several products from one source (e.g., Chinese hamster ovary [CHO] cells), a generic approach may suffice. Regulatory authorities in some countries are accepting this approach [13]. There are also some generic kits on the market that can be used to study host cell protein clearance during development, and may be considered acceptable for licensure. 

The specificity of PCR should make validation efforts for the clearance of viruses, DNA, and host cell proteins more efficient by combining studies and increasing the speed of the assays. In the case of DNA, the assay sensitivity may enable validation to be performed at full scale and eliminate the need for more costly and less accurate small-scale spiking studies. 

Lupker, J. H. Residual host cell protein from continuous cell lines. Dev Bio Stand 93 61-64 (1998). 

Ehrenfeld, E. (1982). Poliovirus-induced inhibition of host-cell protein synthesis. Cell 28, 435-436. 

In addition to analyzing a protein for characteristics dictated by its specific nature, routine analytical assays are performed namely, amino acid analyses and amino- and carboxy-terminal sequencing. If necessary, disulfide assignments are made. A search is ordinarily conducted for oxidations and/or deamidations. In the case of recombinant proteins particular attention is paid to detecting signal sequences or proteolytically degraded species. In contrast, deletions and chemical modifications can be a consequence of proteins prepared by chemical synthesis. Lastly, tests are applied to determine if the protein is correctly folded into its native three dimensional structure. In addition, with recombinant proteins, sensitive immunological procedures are performed to ensure that host cell proteins have not been copurified with the protein of interest. 

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