Potency & Purity
VECTOR PARTICLE CONCENTRATION
Advaxia uses TaqMan quantitative polymerase chain reaction (PCR) technology for quantitation of the genomes of recombinant adenovirus samples. This assay offers the advantages of exquisite sensitivity and specificity, enabling our QC specialists to quantitate monovalent components (vector DNA) in multivalent mixtures (matrix components).
The TaqMan method is based on a real-time quantitative polymerase chain reaction (PCR). We utilize primers to amplify a specific target region of the adenovirus, and a dual-labeled fluorescent probe to hybridize within the amplicon. We label the probe with a reporter dye at the 5′ end and a quencher dye at the 3′ end. Upon successful amplification of the target region, the reporter dye is spatially released from the quencher due to the 5′-3′ exonuclease activity of Taq DNA polymerase, resulting in the emission of a fluorescence signal.
Because the intensity of fluorescence is directly correlated with DNA amplification, a “cycle threshold” (CT) value can be assigned, indicating the PCR cycle at which fluorescence crosses a designated critical level, based on an absolute quantification. We then interpolate the genomes number of the test article against a standard curve constructed with serial dilutions of an adeno-plasmid, resulting in a precise quantitation of monovalent virus components.
In addition to the TaqMan technology, we have implemented a new technique for the quantitation of genomes using a Droplet Digital Polymerase Chain Reaction (ddPCR).
Droplet Digital Polymerase Chain Reaction (ddPCR) technology measures the absolute quantity of nucleic acid in the test articles with high precision and without the need of reference standard curve. The ddPCR system consists of two main instruments, Droplet Generator and Droplet reader. The method is based on the use of water-in-oil emulsion droplet system. The nucleic acid samples are partitioned into thousands of nanoliter sized droplets (20000 droplets) that contain template DNA molecules. The target and background sequences are randomly distributed into the droplets during the partitioning process.
Following the droplet generation process, samples are transferred to thermocycler and end-point PCR amplification is carried out within each droplet. Subsequently, the 96-well plate with PCR product is transferred to Droplet Reader to determine the fraction of PCR positive droplets in the original sample. The reader analyses each droplet individually using a two-color detection system.
The QuantaSoft software counts the positive and negative droplets. Positive droplets containing at least one copy of the target sequence exhibit increased fluorescence signal over the negative droplets. At the end, the fraction of positive droplets is analyzed according to Poisson distribution in order to determine the absolute concentration of the sample in the input (units: copies/uL)
We test each product for residual levels of contaminants of cellular origin, such as host cell protein (HCP) or DNA (HCDNA) from the packaging cell line, as well as raw materials that may have been used during the production process, such as benzonase.
RESIDUAL HOST CELL
BY REAL-TIME PCR
Quantification of residual DNA from the producing cell line is a crucial component of purity testing for any adenovirus vaccine.
The presence of host cell DNA in the final product is of significant concern due to the potential transfer of activated cellular and/or viral oncogenes, the production of infectious viruses from viral DNA, and aberrant gene expression by insertion of sequence into sensitive control regions of genes. International guidelines stipulate 10ng as the maximum acceptable level of DNA per dose of vector.
Advaxia uses real-time quantitative polymerase chain reaction (RT-PCR) technology, which employs TaqMan reporter-quencher dye chemistry, to quantitate the residuals of host cell DNA present in recombinant adenovirus samples. In this system, primers are used to amplify a specific target region of the E1 amplicon gene, and a dual labeled fluorescent probe hybridizes within the amplicon. This method is based on an absolute quantification: a standard curve is prepared by genomic DNA isolated from the producing cell line, and the quantity of contaminant DNA in the test articles is calculated by interpolation of their CT values against the standard curve.
The result is a residual host cell DNA quantitation, which we use to maintain each sample’s rigorous compliance with national and international purity standards.
Expression of viral vectors in the host cell line as HEK 293 cells is a widely used procedure for obtaining sufficient quantities of a desired virus. However, the processes of manufacturing and purifying these products may leave behind impurities in the form of host cell proteins (HCPs), which can result in adverse toxic or immunological reactions. Thus, it is crucial to reduce these impurities to the lowest levels.
Advaxia uses a quantitative ELISA system to quantify residual HCP in adenovirus products. We begin by conducting a two-site immunoenzymetric HCP assay, in which samples containing HCPs are reacted in microtiter strips coated with an affinity purified capture antibody. A HRP labeled antibody reacts simultaneously, forming a sandwich complex of solid phase antibody- HCP enzyme-labeled antibody. The substrate is then reacted. The amount of hydrolyzed substrate is directly proportional to the concentration of HCPs present, and by reading this substrate on a microtiter plate reader, we obtain a precise quantification of HCP levels in the product.
Advaxia performs a wide array of tests to detect product-related impurities, such as non-functional forms of a viral vector, or the presence of co-packaged unwanted genetic sequences in the final product. We control for empty particle counts, aggregates, and replication competent vectors, ensuring that all these impurities are contained within the product’s specifications and acceptability limits.
CONTAMINATION CONTROL BY NESTED PCR
Advaxia produces several adenovirus vectors. To prevent cross-contamination from one production run to the next, we utilize a nested PCR assay to verify that no adenovirus is present after the production environment has been cleaned. We also use this same technique to control the product for cross-contamination with preceding vectors produced in the same environment.
The nested PCR method consists of two consecutive PCRs. The first PCR is performed using an outer set of primers. Part of the first PCR amplification product is then taken into the second PCR for a consecutive amplification with an inner set of primers. The primer sets are specific for the last adenoviral vector produced. We then set up a standard curve to determine the potential detection limit of each PCR run. A positive control adenovirus vector is prepared in serial dilutions, amplified in the same reaction conditions as the test samples, and assessed along a curve composed of the adenoviral vector for which the contamination control is performed.
This enables us to verify that no cross-contamination has occurred between production runs, and that each batch is of the highest possible purity.
We perform potency assays to measure the potency of every adenovirus vector produced in our facility. This assay includes an evaluation of the efficiency of gene transfer (infectivity/transduction/delivery), as well as the level of expression of the therapeutic sequence or its direct activity.
TITER BY HEXON
Accurate measurement of adenovirus titer is critical for effective gene delivery. Advaxia uses a hexon immuno-staining assay to determine the infectious virus titer of adenovirus vectors by infectious forming unit (IFU) calculation. This cell-based assay is able to measure the biological activity and potency of the adenovirus vectors. It uses a primary antibody against hexon protein (the hexon protein is a major coat protein found in adenoviruses), which recognizes all 41 serotypes of adenoviruses by immunocytochemistry.
We begin by diluting adenovirus products, then adding the dilution to host cells previously seeded in 24-well pales and incubated at 37 °C for at least 48 hours. At the end of the incubation, cells are fixed and stained with anti-hexon antibodies. Then, labeled cells are treated with a secondary antibody conjugated with horseradish peroxidase. Detection is accomplished when the horseradish peroxidase enzyme label reacts with a specific substrate resulting in a dark brown product. The labeled, dark brown cells are then analyzed by light microscopy. The number of brown (infected) cells is counted, and the viral titer is calculated accordingly. Results are reported as infectious virus units (IFU)/mL.
In addition, we have implemented the same Infectious virus titer assay described above targeted to the transgene of interest. Fixed cells can be stained with an antibody against the transgene region to determine the infectious particles that express the transgene.
Using the results of both assays, the total infectious particles (derived from the Hexon staining assay) and the infectious particles that express the transgene (derived from the anti-transgene staining assay) are calculated and compared.
The ratio of VP to IFU in a viral product is a crucial measure of the efficiency of the encapsidation process, and the ability of the production process to yield a fully functional (infectious) virus.
We use western blot analysis to evaluate transgenic expression of recombinant adenoviruses and to determine the expression level of transgene proteins in cells infected by Adenoviruses preparations.
Protein levels are evaluated by SDS–PAGE. Cells are infected with adenovirus at the appropriate concentration, then protein extracts from infected cells are loaded on gel. After being subjected to electrophoresis, the proteins are transferred to a nitrocellulose membrane, to be probed using specific antibodies against the transgene target protein.