Assay protein impurities

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... 

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.

The validity of any statement about the purity of a protein is directly linked to the quality of the analytical method used. The validation of immunoassay systems to detect protein impurities in rDNA pharmaceuticals must be achieved by careful production and characterization of the assay reagents. The studies presented here demonstrate that the blank run approach is reasonable for the isolation of reference materials and that high quality broad spectrum antisera can be produced to these mixtures. Significant improvements in assay sensitivity approaching the ppb level are attainable and should provide the methods to further improve product purity. 

Certain circumstances might arise where the bioanalyst wants to examine a recombinant protein preparation for low level protein impurities but a suitable antiserum is unavailable and the variability and nonspecificity of silver staining is unacceptable. An additional assay would be a Western blot analysis after derivatization of the protein mixture with Sanger s reagent... 

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-... 

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. 

Applications in Biotechnology. Biopharmaceutical products must be well characterized by means of analytical techniques. RP-HPLC with UV detection is suitable for the content assay and impurity profiling because of its high resolution. However, the bioactivity of protein-based pharmaceuticals not only relies on the primary structure but also on the higher order structure, i.e., the molecular confirmation, which is not revealed by UV absorbance. Circular dichroism and intrinsic fluorescence detection have been combined with UV detection in RP-HPLC to monitor the conformational properties of active compounds and impurities in biopharmaceutical products [57]. 

This part will mainly focus on host cell proteins (HCPs) quantification. Process-specific HCP assays are in general targeted to be in place prior to the initiation of phase III clinical trials. Immunoassays are the most specific and sensitive techniques available for detecting and quantifying protein impurities. There are two methods commonly employed to quantify protein impurities in biopharmaceuticals enzyme-linked immunosorbent assays (ELISA) and immunoligand assays (ILA). Both methods are able to detect very low ppm level of impurities. ELISA have been developed to measure host protein impurities in a number of recombinant proteins including human growth hormone (Anicetti et ah, 1986), insulin (Baker et al, 1981) and staphylokinase (Wan et ah, 2002). ILA assays have been used to detect protein impurities in recombinant bovine somatotropin (Whitmire and Eaton,... 

There are many examples of ELIS As used for detecting host cell impurities in the literature. Pauly et al.12 developed an ELISA to detect impurities in erythropoietin that had a detection limit of around 0.05 ng/ml. SDS polyacrylamide gel and Western blot analysis were used to confirm the spectrum of proteins detected and to demonstrate the specificity of the antibody preparation. Anicetti et al.14 describe an assay for the detection of E. coli proteins in recombinant DNA-derived human growth hormone. Whitmire and Eaton15 report on an immuno-ligand assay for quantitation of process-specific E. coli host cell contaminant proteins in a recombinant bovine somatotropin. 

Other common impurities, such as immunoglobulins and protein A, result from the immunoaffinity purification of recombinant proteins or MAbs.16 If affinity chromatography is used to purify an antigen, then an ELISA can be used to detect contaminating levels of MAbs leached from the column. An assay for the antibody needs to detect the antibody in the presence and absence of its specific antigen. 

CE is applied to two major categories of quality release testing identity and impurity testing. Identification assays are intended to ensure the unique identity of an analyte in the sample. This is normally achieved by comparison of a property of the sample (e.g., spectrum, chromatographic behavior, chemical reactivity, etc.) to that of a reference standard. As shown in Figure 9, CZE can be used to determine identity for monoclonal antibodies and proteins based on their unique electrophoretic profile. 

The ALIS quench method for dissociation rate measurement uses little protein and requires no biochemical assay for its implementation, yet the method readily yields quantitative values for the dissociation rates of the protein-ligand complexes. The technique can be used with pools of ligands to provide a quantitative rank ordering of the dissociation rates of all the components of the mixture. Since it is not necessary to know the exact concentrations of the ligands under study, the dissociation rate assessment can be performed using impure compounds, such as unpurified compound mixtures derived from combinatorial chemistry synthesis. The method does not require a foreknowledge of active protein concentration to measure and rank ligands based on their rates of dissociation. As such, the technique is self-contained and does not rely upon an external measure of protein activity as one of its input parameters. 

The production and isolation of the reference impurities will ultimately determine the validity of the assay. The reference material will define which impurities are detected because it is used to produce the antibodies used in for the assay. Also, because it is the standard against which the final product impurities are quantitated the distribution of proteins in the reference material should closely approximate that of final product material. 

This strategy is attractive because the impurities will reflect exactly those produced by the host cell during product expression and which copurify with the product through the process. The immunoabsorption procedures required for this approach, however, are difficult to perform and validate at the part-per-million level (i.e., ng of impurity per mg of protein product). All of the product, product fragments and product-impurity complexes must be removed without the loss of any of the impurities. Any residual product will produce antibodies during immunization and render the assay nonspecific. Conversely, the immunoabsorbtion step must not remove any of the impurities. An exact demonstration of these criteria at the ppm level is not possible with currently available non-antibody based analytical methods. 

The detection and accurate quantitation of any protein in an immunoassay requires that a condition of antibody excess exist. This is required for each protein in the reference impurity preparation. The acquisition and characterization of broad spectrum antisera against complex protein mixtures, therefore, is a fundamental goal in the development of these assay systems. 

The continuing refinement in the selection of reference materials and the production of antibodies to complex protein mixtures has resulted in immunoassay systems of remarkable sensitivity and specificity. In particular, the selection and enrichment of the antibody population by immunoaffinity purification against the reference impurities has afforded an additional level of control over the production and validation of these reagents and served to improve the assay range and sensitivity (6,17). This normalization of the antibody population to a stoichiometric relationship with the reference impurities has suggested the term Antigen Selected Immunoassay (ASIA) for these methods. 

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