Executive Summary. clinical supply services

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1 clinical supply services case study Development and NDA-level validation of quantitative polymerase chain reaction (qpcr) procedure for detection and quantification of residual E.coli genomic DNA Executive Summary A client sought Catalent s expertise to develop an improved test for quantifying residual host cell DNA (Escherichia coli; E.coli) for release of the drug product. The existing assay was not as sensitive as the regulatory agency required. Biotinylated Membrane Single-stranded DNA Urease-Conjugated Streptavidin Anti-DNA Biotinylated Single-stranded DNA Binding Protein figure 1 Representation of Threshold DNA assay Biopharmaceutical products are generally produced via cloning the therapeutic molecule into a host cell, be it bacteria, yeast or eukaryotic cells. The cells are grown in continuous or batch cultures, and the therapeutic moiety is then purified from the cell culture broth. By their very nature, biopharmaceuticals produced by recombinant means contain some level of residual host cell impurities at the time they are harvested from bioreactors. Impurities, such as residual DNA must be removed during the purification process for the production of the final drug substance and product. The level of remaining residual host cell DNA in the drug product must be assessed prior to release for clinical or commercial use. Guidance for allowable levels of residual DNA has been provided by some regulatory agencies, suggesting a target of 100 pg (FDA) or less, or 10ng (WHO and EU) or less of residual DNA per dose. The actual allowable level is case-by-case, and depends upon both the source of the residual DNA and the product s route of administration. Quantification of residual DNA in biopharmaceutical drug product has classically been done using Threshold. Threshold technology is based upon the ability of a biotinylated single-stranded-dna binding protein to bind the test (residual DNA), and then be concentrated via a biotinylated membrane (see schematic in figure 1). The client was using this assessment, which has a level of detection of approximately 3-4 pg DNA and a level of quantification 10 pg DNA, for testing residual DNA in the drug product. They were told by a regulatory agency that this level of sensitivity for their drug product was insufficient for approval of an NDA. A more sensitive assay was required. Additionally, for the client, the Threshold assay was insufficiently precise, accurate, robust and rapid. DEVELOPMENT DELIVERY SUPPLY

2 The Challenge Develop and optimize an assay for ultra-sensitive detection and quantification of residual E.coli DNA, and validate the assay for NDA-level submission. The Catalent Solution We chose to use TaqMan real time quantitative Polymerase chain reaction (qpcr) as the basis for developing an ultra-sensitive, quantitative, accurate, precise and robust assay for residual E.coli DNA. The following strategy was utilized to address the challenge. 1. Critical reagents DNA template, primer sets and probes were identified, based on most conserved, highly expressed, genetic regions of the E.coli genome. The primer sets and probes were designed and targeted to prime and amplify short sequences inside one of the 7 copies of ribosomal RNA gene (a stretch of 4,885 nucleotides) Several end-point PCR experiments were performed to address the following aspects: Usefulness of E.coli conserved region chosen as template for amplification. Therefore, the entire genomic DNA from a strain of E.coli such as strain K-12, could be used as positive control, as well as to construct standard dilution curve for qpcr analysis. Ability of primers to prime and amplify targeted sequences under chosen experimental conditions, such as amount of starting target (1 ng and 100 pg, respectively). Primer efficiency - ability of primer sets to amplify from trace amounts of E.coli genomic DNA template and establish visual limit of detection. Primer specificity ability of primers to prime and amplify targets in E.coli genome only. usefulness of e.coli conserved region chosen as template for amplification 1 ng E.coli gdna template 100 pg E.coli gdna template figure 2 Amplification efficiencies of five PCR primer sets. Starting DNA template 1ng and 100 pg E.coli gdna, respectively. Primers were capable to prime and amplify specific sequences inside the chosen target gene of the E.coli genomic DNA under specific experimental conditions 2

3 primer efficiency A B figure 3 Primer efficiencies. Purpose: 1. Assess ability of two qpcr primer pairs, 1F/1R (A) and 2F/2R (B), to prime and amplify from a serial dilution of E.coli gdna ranging from 1 ng (10-9g) in lane 4 to 1 fg (10-15g) in lane Assess limit of detection the lowest amount of starting DNA template from which the two primer sets can prime and amplify, by visual gel detection. 3. Target amplicons expected: 122bp in gel A, and 91 bp in gel B, respectively. based on these results, the visual limit of detection was determined to be 1 fg genomic dna, the equivalent of 0.2 copies of e.coli gdna. 2. Critical Reagents Qualification Once critical reagents had been identified, they were qualified for use through a series of qualitative tests including UV spectrophotometry, agarose gel electrophoresis, and PCR. 3. The Catalent qpcr Method A real-time qpcr method for determination and quantification of residual E.coli host-cell DNA was established. Absolute quantification approach was used to determine how much (e.g, ng DNA) of a target gene is present in a particular sample without reference to other samples. Absolute quantification is conceptually simple and the mathematical calculations are easy to perform. It involves comparing the CT values of test samples to those of standards of known quantity plotted on a standard curve. In absolute quantification, the quantity of the unknown sample is interpolated from a range of standards of known quantity. To construct a standard curve, a template with known concentration is required. This template is diluted to create a range of standard concentrations. The unknown test samples are amplified in parallel with the standards in the same experimental run. The standard curve constructed from the diluted standard template can then be used to determine the target quantity in the unknown sample by interpolation (similarly to the way molecular size standards are used to determine the molecular size of an unknown DNA band on an agarose gel). Usually, the quantity is normalized to a unit amount of sample such total amount of nucleic acid. 3

4 The Catalent qpcr method is based on a proprietary primer set/taqman probe combination. The specific probe was designed to anneal to a target sequence located between two PCR primers. These primers were designed to prime and amplify a short region of one of the seven Escherichia coli (E.coli) 16s ribosomal RNA genes. The probe was labeled with the fluorescent reporter dye FAM at the 5 end and with the quencher TAMRA at the 3 end. In its intact, un-cleaved state, the fluorescence of the reporter dye is quenched by the proximity of the quencher. During the extension phase of each PCR cycle the Taq DNA polymerase cleaves the annealed probe, releasing the reporter dye from the probe, thus resulting in an increase in fluorescence. This increase in fluorescence is directly proportional to the amount of amplified target DNA present in the reaction and is continually monitored, in real-time, throughout the PCR reaction. Within the linear region of amplification, the quantity of E.coli product sequence is proportional to the starting quantity of DNA. A standard curve of known quantities of E.coli genomic DNA is used to correlate the level of standard curve fluorescence to concentrations of contaminating genomic DNA in the original test sample. figure 4 Absolute quantification using a standard curve to assess the amount of Unknown Sample (spike). Primers were specific to a 122 bp amplicon inside the 16S rrna gene sequence, and a TaqMan probe was designed to anneal to a target sequence located between the two PCR primers, as the indicator chemistry. All standard dilutions and unknown sample (the spike) were assayed in triplicate on Applied Biosystems 7900HT Fast Real Time PCR System. Equation for the linear regression line: [y = mx + b, or C T = m(log quantity) + b] R 2 : ; Slope: ; Y-Intercept: : Efficiency: From the equation for the linear regression, the following equation to determine the quantity (Qty) of an unknown sample can be derived: C T b ( m ) Quantity DNA (pg) = 10 Where C T is the threshold cycle for the well and m and b are the slope and y-intercept of the standard curve, respectively. By entering the C T values of the individual replicate assays for samples A and B into this equation, the copy number for each assay can be determined. The results of these calculations are shown in table 1. 4

5 sample replicate c t quantity (pg e.coli dna) Spike (10 pg E.coli gdna) Spike (10 pg E.coli gdna) Spike (10 pg E.coli gdna) Quantity Mean (pg E.coli gdna) 9.61 ± 0.41 table 1 Absolute quantification of an Unknown sample (10 pg E.coli gdna spike). The equation of the linear regression line shown in Figure 3 was used to calculate the copy number of the unknown samples. Additionally, the Sequence Detection System (SDS) software controlling the Applied Biosystems 7900HT Fast Real Time PCR System performs regression analysis using the data from the reference standards and calculates the quantity of DNA for the Unknown samples. 4. In the second part of the strategy, the qpcr assay was optimized for sample digestion/dna extraction/reconstitution procedure, primer set/taqman probe combination, PCR cycling conditions, standard curve preparation (7-point standard curve vs 9-point standard curve), and spike amount (10 pg DNA vs 100 pg DNA). Ultimately, the Catalent qpcr method was validated per client requirements. Validation strategy included the following components: Specificity; Linearity; Accuracy; Repeatability; Precision; Detection and Quantification Limits. The validation exercise passed all acceptance criteria set for each validation component per validation protocol, in addition to assay acceptance criteria established per method. The method proved to be very specific and accurate. The final limit of detection (LOD) was 0.05 pg gdna, and the limit of quantification was (LOQ) was 0.5 pg gdna. The final assay range was 500 pg gdna 0.5 pg gdna. Conclusion In this case study we described the development, optimization and validation of a qpcr method for detection and quantification of residual E.coli genomic DNA contamination of therapeutic moieties and active pharmacological products. The method proved to be accurate, precise, robust and reproducible, with low failure rate. It can be used in early phase/late phase/registration lot/commercial release and stability, and has the potential of assaying up to 19 unspiked unknown test samples, or up to 9 unspiked/spiked test sample combinations. Discover more solutions with Catalent. Call: SOLUTION ( ) solutions@catalent.com Visit: more products. better treatments. reliably supplied. Catalent Pharma Solutions 14 Schoolhouse Road Somerset, NJ USA F Copyright 2012 Catalent Pharma Solutions, Inc. All rights reserved. dcs/css/cs6 (03/12)