Host-cell protein impurity

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... 

A. Identification of a Host-Cell Protein Impurity in Recombinant Acidic Fibroblast Growth Factor... 

In addition to ELISA and Western blots for detecting host-cell protein impurities there is a third immunoassay now available to the bioanalyst for this purpose the immunoligand assay (ILA). Like the ELISA, it is configured as a double-antibody sandwich. The anti-host-cell protein antibodies are separately conjugated to biotin and to fluorescein. A tripartite immune complex is formed between host-cell protein impurities and these two anti-... 

For the majority of host-cell protein impurities, the most direct way of identification is through N-terminal sequence analysis. The automated... 

Protocol 14 Isolation of Host-Cell Protein Impurities by Electroblotting... 

O Keefe, D. O., and Will, M. L. (1992). Chromatographic analysis of host-cell-protein impurities in pharmaceuticals derived from recombinant DNA. ACS Symp. Ser. 512, 121-134. 

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. 

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. 

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 ideal reference preparation would contain exactly those host cell proteins, medium proteins and process raw materials (i.e., a monoclonal antibody) that are present in any particular final product lot. Further, the reference impurities would be present in the same distribution and biochemical quality as those in the final product. Without knowledge of the... 

Figure 1. Key steps in the development of protein impurity assays. The reference impurities may be obtained by a process specific purification of host cell proteins arising from a blank run or a production run. While the production run is a more accurate population of potential impurities, the product removal step involves significant technical difficulty.

Impurities Endotoxins Host cell proteins Other protein impurities DNA... 

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... 

Unlike ELISA, which can detect and quantitate host-cell proteins as a group, Western blots detect single protein impurities. Western blot analysis starts with an SDS-PAGE protocol but does not include a final colorimetric staining step. Instead after electrophoresis the proteins are electrotransferred (blotted) from the gel onto a thin membrane. Membranes made of nitrocellulose or PVDF are most often used. Once the transfer is complete, the membrane is incubated with a nonspecific protein solution to saturate and to... 

Host-cell protein ELISA have the advantage of quantitating host protein impurities. The disadvantage, however, is that the quantitation is of a group of impurities. Western blot analysis, on the other hand, provides the analyst with a relative level of an individual impurity compared to other impurities. If the level of one or more host protein impurities appears to be excessive based on the intended use of the drug product then it may be necessary to identify those impurities. This can provide assurance that the impurity is innocuous and it can also define the physicochemical properties of the impurity such that the process can be modified to reduce its presence in future production lots. The identification can also lead to the development of a quantitative assay for monitoring the individual impurity in every lot. 

Although the capture step dramatically enriches a targeted protein and removes some impurities, bulk impurities such as host cell protein, DNA, endotoxin, virus, and leaching ligand remain in the eluted pool. Additional procedures are needed to eliminate these impurities. 

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. 

The most common sources of impurities are derived from the fermentation process and would include such factors as host cell protein or DNA, or components from cell culture growth media. In addition, endogenous retroviruses from hybrido-mas in monoclonal antibody production can also be present as impurities, all of which should be removed and tested for in the final product. [1], These impurities can be present as lipopolysaccharides, oligonucleotides, and leachates from containers. The most common tests that are conducted for the reference standard and production batches are shown in Table 9.2. 

---