IRBM S.p.A.

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QUALITY CONTROL

Close up of adenovirus based vaccine vial being held by Quality Control specialist

QUALITY CONTROL

Advaxia meets or exceeds global quality standards in every product and process.

At Advaxia, we pride ourselves on adhering to the world’s most demanding quality standards — and excelling beyond them. 

Our dedicated quality control (QC) team relentlessly seeks out opportunities for improvement to our capabilities, equipment, staff training and associated procedures, with the aim to meet or exceed the requested pharmacopeia standards. As a result, we can boast an unparalleled 100-percent successful track record in the approval and release of the vaccines and other biotechnology pharmaceuticals we manufacture for our clients.

Exceptional quality is our baseline, and perfection is our never-ending pursuit.

Our QC team upholds highly rigorous standards in all the following areas:

ENVIRONMENTAL
MONITORING
FOR CLEAN ROOMS

Advaxia utilizes clean rooms and clean air devices (clean areas), as required by the environmental cleanliness levels specified for each operational state of a particular manufacturing process.

This minimizes the risks of products or materials being contaminated by particulates or microbes. Our production rooms are classified based on viable and non-viable particle contamination (grades A, B, C and D areas) and are constantly monitored in order to assure continuous compliance with each project’s cleanliness specifications.

We also adhere to the following environmental monitoring standards:

ANALYTICAL
SUPPORT

In addition to production support, our team is able to provide analytical support for clients, including analytical method transfer, development, qualification and validation, as well as the development of cell-based potency assays.

RAW MATERIALS
MANAGEMENT

Advaxia performs intensive QC reviews of all basic raw materials for biological manufacturing, including essential components such as cell culture media and additives, enzymes, and components of buffer solutions and chromatography resins. Our QC team examines all cell banks and viral seed stocks to be used in manufacturing, and only material that pass all the checks and satisfy all the criteria are released for the use.

IN-PROCESS CONTROL
SAMPLE ANALYSIS

We collect a wide array of in-house data throughout every manufacturing run, monitoring every step of the process, material, operational environment, and piece of equipment to ensure it continues to operate effectively and efficiently.

FINISHED PRODUCT
ANALYSIS

Our QC team conducts a battery of tests on each batch of product, ensuring that its potency, purity, and other characteristics are in full alignment with the specifications provided by the client, and dictated by the regulatory authorities.

All our QC staff are trained and equipped to perform a wide variety of analytical tests for recombinant virus products, including PCR, qPCR, SDS-PAGE, ELISA, LAL, spectrophotometry (UV/visible), electrophoresis, immuno-staining assays, restriction analysis, western blot analysis and mycoplasma testing.

Our QC laboratory also performs additional assays such as pH, appearance, osmolality, extractable volume, filter integrity, and residuals (host cell protein, host cell DNA and benzonase). We have extensive experience in all the following techniques:

We’re also experienced in helping our clients choosing test methods appropriate for each process stage and product, according to validation characteristics such as accuracy, precision, repeatability, intermediate precision, specificity, detection limit, quantitation limit, linearity, and range.

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)

IDENTITY &
INTEGRITY

We regularly perform a variety of tests to verify the genetic identity and integrity of adenovirus products. In particular, we perform tests such as specific sequence amplification by PCR, DNA sequencing, and restriction enzyme mapping to identify both the transgene of interest and the vector sequences of each product.

IDENTITY TESTING BY RESTRICTION ENZYME ANALYSIS

Advaxia uses restriction enzyme analysis to check the integrity and identity of the genomic structure of the recombinant adenovirus products.

We begin by isolating viral DNA from adenovirus products, then digest the DNA with specific endo-nuclease enzymes chosen based on the vector DNA sequence in order to have a restriction pattern with separate and distinguishable bands. We then run the sample and control digests side-by-side on agarose gel, stain them with SYBR Gold dye. Finally, we compare the resulting restriction fragment patterns of each sample to their respective control digest restriction patterns, to obtain a qualitative confirmation of each sample’s identity.

Part of PCR equipment used for adenovirus identity analysis and verification

ADENOVIRUS IDENTITY
ANALYSIS USING PCR

We use industry-standard polymerase chain reaction (PCR) techniques to verify the identity of recombinant adenovirus products.

We perform this assay using two sets of primers. The first set of primers anneals in the hexon portion of the adenovirus DNA (the PCR fragment products differ in size from vector to vector.) The second set of primers anneals in the transgene region as well as immediately out of the transgene region, determining the presence of a specific transgene in the adenovirus vector.

ADENOVIRUS IDENTITY
VERIFICATION BY
SEQUENCE ANALYSIS

We confirm the identity of recombinant adenovirus products by trans-gene and/or full genome sequence analysis.

This whole–genome sequencing of adenovirus products is performed using NGS technology.

PROCESS-RELATED
IMPURITY ANALYSIS

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
DNA QUANTITATION
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.

RESIDUAL HOST
CELL PROTEIN
(HCP) ANALYSIS

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.

DETERMINATION OF
ENDOTOXIN LEVELS
IN ADENOVIRUS
PREPARATIONS

Endotoxin released from gram-negative bacteria is a widespread cause of contamination of medical products. Therefore, it’s crucial to perform an endotoxin test on the sterile product, medical device or pharmaceutical product which is to be injected or implanted into the patient’s body, to confirm that no endotoxin has been carried over through the manufacturing process from any starting materials.

Advaxia uses a quantitative endpoint LAL chromogenic test to quantify the endotoxin level in adenovirus preparations. We begin by mixing samples with the LAL reagent and chromogenic substrate reagent over a short incubation period, then read the resulting sample on a spectrophotometer or plate reader which is capable of measuring 405-410 nm. A standard curve is created using the E. coli endotoxin standard included with each kit to calculate endotoxin levels as low as 0.1 EU/mL, where one endotoxin unit/mL (EU/mL) equals approximately 0.1ng endotoxin/mL of solution.

As a result, we arrive at a precise quantitative reading of the product’s endotoxin level, and its purity from contamination, prior to its release.

In addition, we have implemented a rapid quantitative bacterial endotoxin test assay.

The Endosafe® nexgen-PTS™ is a rapid point of use test system which provides quantitative chromogenic test results in less than 15 minutes. The technology utilizes licensed LAL reagents in a disposable cartridge using the nexgen-PTS™ for a completely contained, real-time endotoxin assay. This enables us to test a quantity of samples in a very fast timeframe and with high sensitivity, quantitative results.

PRODUCT-RELATED
IMPURITY ANALYSIS

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.

POTENCY ASSAYS

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.

Close-up of tubing connected to bioreactor equipment used for vaccine production.

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.

INFECTIOUS VIRUS
TITER BY HEXON
IMMUNOSTAINING

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.

WP/IFU RATIO
ANALYSIS

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.

The Infectious virus titer ratio (Vp/IFU) is calculated by dividing the vector particle concentration (VP/mL) result by the infectious virus titer (IFU/mL) result. The ideal result for this measure is 1 VP/IFU, meaning that every particle is essentially a full particle able to infect target cells. Based on Advaxia’s manufacturing experience, we have set the acceptance criteria at ≤300 VP/IFU, to account for the encapsidation/infection efficiencies of different vectors.

TRANSGENIC
EXPRESSION
ANALYSIS

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.

STABILITY TESTING​

The purpose of stability testing is to provide evidence on how the quality of product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and containers, and to establish a re-test period for the product or a shelf life and recommended storage conditions.​

Advaxia performs rigorous stability testing on all viral vector products we manufacture and assesses each product according to ICH guidelines. 

In addition to the standard stability studies, we also test viruses under stressed conditions, and by running in-use stability testing. We also offer pre-formulation studies, which can help further identify the ideal matrix for a virus product.