Successful Strategies to Measure ResidualHost Cell ProteinsResidual Host Cell Protein (HCP) assays are an essential requirement for thecharacterisation of biologics and vaccine products and frequently appear in the releasespecifications of products. Regulators have specified that the amount of HCP be controlledand measured from the very earliest stage of clinical development due to the potentialfor these contaminants to affect the potency of drugs and cause side effects. HCP, as thename suggests, originate from the cells used in the manufacture of product and can beconsidered as an impurity. These proteins can be very complex in nature and will vary intheir constituency as product moves from the very crude bulk harvest material to the finaldrug bulk and product. HCP assays are performed on all bacterial, yeast and eukaryotic cells.Basic ELISA Technologies These include :- Human A-549 cells, Baby Hamster Kidney cells, Chinese Hamster Ovary Cells, Humanwww.cygnustechnologies.com Embryonic Kidney 293 cells, Human Embryonic LungDuring very early stages of product development it MRC-5 cells, Murine NS0 cells, the primate cellshas been the custom to use “off the shelf” solution line Vero, insect cell line SF-9, the yeasts Pichia andfor the measurement of HCP. The company Cygnus Saccharomyces and the bacteria PseudomonasTechnologies, based in USA, manufactures ELISA kits and E.coli.for all of the most common HCP used in the production These kits are widely used and provide sufficient data toof vaccines and biologics. be acceptable during early phase of drug development. The generic ELISA kits are widely popular because they are readily available, easy to use and highly sensitive. The kits are intended to be used for the semi-quantification of Host Cell Proteins and bioprocess contaminants. The kits are complete with all of the reagents necessary to perform the assays on a micro- titer strip well format.
Sample Qualification and samples be diluted during processing, the diluted sample will yield results which are directly proportionalAssay Validation to the extent of dilution. This assessment involvesBioOutsource has already acquired data on selected diluting the reference standard protein which is suppliedCygnus kits to demonstrate the utility of these assays by the kit or will reference provided by the sponsorin our hands; however it is essential to monitor these through a number of serial dilutions in the samplekits for their suitability with individual client samples. matrix and buffer. These dilutions are then assayed andBioOutsource also has the ability to offer these assays the HCP concentration determined. The linearity canfor the cGMP batch release of product in Europe be established by regression analysis to determine theand USA. derived equation for the dilution compared with the determined concentration. The closer this relationshipBioOutsource has created a generic study protocol is to linear (r2 = 1) the more confidence there is thatwhich will provide a comprehensive qualification of a the assay is linear. Typically in BioOutsource we wouldsponsor specific sample type in the assay of choice. expect to see an r2 of greater than 0.90 over theOur study protocol is based on the current ICH expected range of the assay.guidelines for the validation of analytical methods. Thefirst experiments in the protocol will be to qualify the The last part of qualification of these assays is tosample type for use with the Cygnus kit by spiking determine the precision of the assay, depending onknown amounts of reference standard HCP into the how the spiking and dilutional linearity experimentssponsors sample under test and assess the results have been performed there may be sufficient data toof the sample both with and without the spike. This establish the confidence in precision with the assay.spiking experiment can be performed over the expected Precision experiments are performed by the sameconcentration range of HCP in the sample, if known, technician with the same sample on different days. Thisand can be performed with 4-6 concentrations of establishes the variability of the assay using differentspike, typically performed in triplicate. The manufacturer pieces of equipment or different batches of reagent.suggests that a recovery rate of 80-120% of the spike BioOutsource would normally expect the variability ofamount can be expected, however in our experience the assay to be less than 20%. Any higher than thisthis is very dependent on sample type and preliminary and an assessment of the assay performance would beexperiments are recommended to determine the under question.suitability of sample type.The HCP assays already have a Limit Of Quantitation Advanced HCP Assays(LOQ) and Limit of Detection (LOD) when the Following early stage clinical trials, the regulatoryreference standards are tested in buffer and this requirements for HCP assays become more stringentis specified by the manufacturer. These are LOD = and there is the requirement to develop product400pg/mL to 1.1ng/mL for the CHO HCP assay and specific HCP assays rather than relying on the genericLOQ = 450pg/mL to 1.4ng/mL for CHO HCP assay. It assays. The development of a product specific assaywould be expected that the experiments would include requires an understanding of the potential proteinssamples spiked with concentrations near the LOD which could be present and contaminating in the finaland LOQ to determine the recovery expected at these product and to identify immunological reagents whichconcentrations in the sample matrix. are capable of detecting these proteins. In Europe theA second important consideration to these assays is regulation produced by the EMEA entitled ICH Topicdilutional linearity. This assessment ensures that, should Q 6 B Specifications: Test Procedures and Acceptance
Criteria for Biotechnological/Biological Products. Note blotting the extent of the binding of the detectionFor Guidance On Specifications: Test Procedures And antibody to the proteins detected can be established.Acceptance Criteria For Biotechnological/Biological If the stained protein profile mirrors the western blotProducts (CPMP/ICH/365/96) finalised in September profile then this should provide sufficient data to ensure1999 stated that all proteins present will be detected. If there are proteins absent from the blot then consideration should“Process related impurities, i.e., cell substrates (e.g., be made to create a new antiserum for detection ofhost cell proteins, host cell DNA), cell culture or proteins. It will be the case that some proteins will bedownstream processing. Product-related impurities (e.g., too small to elicit an immune response and thereforeprecursors, certain degradation products) are molecular may not be detected in these assays.variants arising during manufacture and/or storage,which do not have properties comparable to those of Should the development of a product specific HCPthe desired product with respect to activity, efficacy, and be required, there are typically two reagents within asafety. Further, the acceptance criteria for impurities sandwich ELISA format. The first is the binding antibodyshould be based on data obtained from lots used and the second is the detection antibody to which ain preclinical and clinical studies and manufacturing marker molecule is conjugated. HCP detection reliesconsistency lots. Individual and/or collective acceptance on the binding antibody being attached to a micro-titrecriteria for impurities (product-related and process- plate, upon addition of the sample the binding antibodyrelated) should be set, as appropriate.” will bind and hold any HCP in the sample. Following washing procedures any unbound proteins are removedIt is possible that the commercially available and the detection antibody added. The detectionimmunological reagents would be suitable to detect antibody will bind to the HCP held by the bindingthe host cell contaminants from cells such as CHO antibody and washing and addition of an appropriateor Murine NS0, however previous experience has substrate for the marker molecule then takes place.shown that the modification of cell lines to produce a Conversion of the substrate indicates the presence ofproduct does affect the profile of proteins expressed HCP. Reference standard material can be used to gaugeby the cell and this requires specific reagents to be the level of converted substrate in comparison to thecreated. Furthermore, the specific host-cell protein amount of HCP present.profile is likely to be highly dependent on the design ofthe manufacturing process. It is frequently necessarytherefore to evaluate the proteins which are present in Alternative Techniques toa production process and to determine if the reagent ELISA Technologyavailable will detect these in their entirety. In mostcases, manufacturers find it necessary to develop a Threshold Systemprocess and product-specific HCP assay for use during www.moleculardevices.combatch-release testing for Phase III clinical trial material There are alternatives to the standard ELISA technology.and licensed product. One method which has been used for a number ofThe first step in HCP evaluation is normally to years is the Threshold method, manufactured byacquire a protein profile of the contaminants. This Molecular Devices. This technology utilises the samecan be relatively easily accomplished using standard antibody detection methods as the ELISA techniquepolyacrylamide gel techniques with appropriate but has the advantage that the reaction takes place invisualisation methodologies such as silver staining. the liquid phase rather than bound to the micro-titreA western blot is performed to measure the extent plate which may affect the stochiochemistry of thethat the immunological detection reagents will pick up reaction. The binding of HCP is identified by a ureasethe different HCP seen in the gels. Following western conjugated to the detection antibody which converts
the substrate urea to ammonia and carbon dioxide. Thisis then detected by a pH change. The sensitivity of thismethod is reported to be at least 4ng of HCP per mgof product.Meso-scale Discovery Technologywww.meso-scale.comA second alternative to the ELISA technology is theMeso-scale discovery technology which is basedon the replacement of the marker on the detectionantibody with a Ruthenium ion which allows the electro-chemiluminescence to detect the presence of HCP. Thismethod is reported to achieve a sensitivity of at leastone log and possibly two logs of increased sensitivityover ELISA assays.Summary BioOutsource Service OfferingHCP determination is always critical during the BioOutsource offers a complete service for thedevelopment of a new drug. In drug substance it should detection of Residual Host Cell Proteins. Wherebe below detectable levels using a highly sensitive appropriate, we can offer the detection of HCP using allanalytical method, usually less than 100ppm. However of the available Cygnus Technologies kits. These testsno exact limit for HCP can be established because of are performed to cGMP standard and are suitable forthe differences in production processes regimes and testing early batches of clinical trials material.clinical dosing regimes. The specificity and sensitivity of For more advanced projects, BioOutsource offers theany antibody-based assay used for detection of HCP is complete development of product and process-specificdirectly related to the quality of the antibodies used to HCP assays from the immunisation of the animals withdetect the proteins themselves. The goal of the assay is the target proteins to the qualification and validation ofto detect the variety of different proteins that represent the assays to ICH standards.the HCP spectrum of the process and the product.Any immunoassay used to measure HCP should be Please contact us at firstname.lastname@example.org forevaluated and proven capable of reporting the true more information.extent of HCP contamination. Validation of suchassays will follow the ICH guidelines already discussed.Developing such an assay can require a considerableamount of time, principally due to the length of timetaken to acquire the antiserum. However it shouldalso be noted that, should there be any change in theproduction process, then the HCP assay would requirere-evaluation and possibly further development. BioOutsource Ltd • Units 3/4 Technology Terrace • Todd Campus • West of Scotland Science Park • Glasgow • G20 0XA tel: +44 (0)141 946 4222 • email: email@example.com • web: www.biooutsource.com