Vaccines and other pharmaceutical drugs must be held to the most stringent quality standards, and an efficient Quality Control (QC) team ensures these standards are met or exceeded. As such, Quality Control is a critical part of Advent and it is a major factor in the successful manufacturing of high-quality biotechnology products.
Our dedicated QC team fully supports our production capabilities.
Clean rooms and clean air devices (clean areas) for the manufacturing of products are qualified according to the required characteristics of the pharmaceutical environment. Each manufacturing operation requires an appropriate environmental cleanliness level in the operational state, in order to minimize the risks of particulate or microbial contamination of the product or materials being handled. Our production rooms are classified on the basis of viable and non-viable particle contamination (Grades A, B, C and grade D areas) and are routinely monitored in order to assure continued compliance with the defined limits.
The production operators are qualified for working in aseptic areas and their gowning is monitored after work, including gloves.
The high quality of the Advent GMP production environment is maintained thorough an efficient cleaning plan. The cleaning of the classified rooms is entrusted to a specialized team.
An efficient changeover procedure applied between different production campaigns and supported by a strong cleaning procedure, assures the complete removal of the previous agent.
We are always ready to implement regulatory changes that could impact our environmental monitoring procedures. Analysis of the panel of the environmental monitoring results is always performed with the aim of continuous improvement.
Trend analysis will help determine if something has changed with a process (quality, production, or service). The procedure involves collecting data from monitoring, measurement, complaint handling, or other activities, and tracking these data over time, as appropriate. Analysis of data can provide indications that controls are losing effectiveness. The information generated will be essential to achieving problem resolution or problem prevention.
Raw materials management
Basic raw materials for biological manufacturing procedures are essential components, such as cell culture media and additives, enzymes or components of buffer solutions and chromatography resins. Cell banks and virus seed stocks used for the manufacturing are defined as starting materials and released by the QC before use.
In-Process Control sample analysis
In-process sampling is valuable because collecting data throughout a manufacturing run allows us to monitor and ensure that the process is operating in the expected manner.
Finished products analysis
The compliance of each batch of product with its specifications is guaranteed by our QC.
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.
Typical validation characteristics which should be considered when qualifying a test method are: Accuracy, Precision, Repeatability, Intermediate Precision, Specificity, Detection Limit, Quantitation Limit, Linearity, and Range.
Analytical methods for customers’ virus products include PCR, qPCR, SDS-PAGE, ELISA, LAL, spectrophotometry (UV/visible), electrophoresis, immuno-staining assays, restriction analysis and western blot analysis.
Our Quality Control laboratory performs additional assays such as: pH, appearance, osmolality, extractable volume, filter integrity, residuals (host cell protein, host cell DNA and benzonase).
Our Quality Control has extensive experience in many techniques:
- Cell Based assays;
- In vitro potency assays;
- Genome quantification;
- Molecular biology methods;
- Detection and quantification of impurities such as endotoxins and host cell DNA and host cell protein.
Several assays which are run on a routine basis are detailed below.
Vector Particle Concentration
Our method for quantitation of genomes of recombinant adenovirus samples uses TaqMan quantitative PCR technology. This assay offers the advantages of exquisite sensitivity and specificity allowing quantitation of monovalent components (vector DNA) in multivalent mixtures (matrix components). The method is based on real-time quantitative polymerase chain reaction (PCR), primers are used to amplify a specific target region of the Adenovirus, and a dual-labeled fluorescent probe hybridizes within the amplicon. The probe used is labeled 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 is assigned which indicates the PCR cycle at which fluorescence crosses a designated critical level. This method is based on an absolute quantification. Genome number of the test article is then interpolated against a standard curve constructed with serial dilutions of an adeno-plasmid.
Identity and integrity
The genetic identity and integrity of the adenovirus products should be assured using tests that identify both the therapeutic sequence and the vector. Such tests might include specific sequence amplification by PCR, DNA sequencing or restriction enzyme mapping.
Identity testing by restriction enzyme analysis
Advent uses restriction enzyme analysis to check the integrity and identity of the genomic structure of the recombinant adenovirus products. Viral DNA is first isolated from adenovirus products and then restricted with opportune endo-nuclease enzymes chosen based on the DNA sequence, in order to have a restriction pattern with separate and distinguishable bands. The relative pre-adeno plasmid used to generate the virus is used as control. The sample and control digests are run side-by-side on Agarose gel and stained with SYBR Gold followed by imaging with the UV trans-illuminator. The resulting restriction fragment patterns of each sample digest are compared to their respective control digest restriction patterns for a qualitative confirmation of sample identity.
Identity analysis of Adenovirus using PCR
Polymerase Chain Reaction (PCR) technique is used to checked the recombinant Adenovirus products identity. The assay is performed 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.
Identity by sequence analysis
The identity of recombinant adenovirus products is confirmed by sequence analysis (trans-gene or full genome sequence analysis). The whole–genome sequencing of adenovirus products is performed using NGS technology. Sanger sequencing is used to confirm NGS data or trans-gene sequencing.
Unless process validation data has been provided to demonstrate that such residues are consistently reduced to acceptable levels. Tests should be developed and relevant (upper) limits set to monitor the residual levels of contaminants of cellular origin, e.g. host cell protein or DNA from the packaging cell line, as well as raw materials that may have been used during the production process such as benzonase or resins. Other process-related impurities may include impurities such as residual animal serum proteins (e.g. BSA) used in production.
Residual Host cell DNA Quantitation by real time PCR
Recombinant adenovirus vectors are routinely prepared on cultures of immortalized cells. Quantification of residual DNA from the producing cell line is a part of the purity tests of the adenovirus vaccine. Stringent guidelines stipulate 10ng as maximum acceptable level of DNA per dose of vector. 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.
To quantify residual Host Cell DNA in Adenovirus products; Advent uses real-time quantitative polymerase chain reaction (RT-PCR) which employs TaqMan reporter-quencher dye chemistry. In this system, primers are used to amplify a specific target region of E1 Amplicon gene and a dual labelled 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 from the standard curve.
Residual Host cell protein (HCP)
Expression of viral vectors in host cell line as HEK 293 cells is a widely used procedure for obtaining sufficient quantities of a desired virus. The manufacturing and purification process of these products leaves the potential for impurities by host cell proteins (HCPs). HCP impurities can result in adverse toxic or immunological reactions and thus it is desirable to reduce these impurities to the lowest levels practical. To quantify residual HCP in adenovirus products, Advent uses quantitative ELISA system. The Host Cell Protein assay is a two-site immunoenzymetric assay, samples containing HCPs are reacted in microtiter strips coated with an affinity purified capture antibody. A HRP labeled anti-host cell 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 read on a microtiter plate reader and is directly proportional to the concentration of HCPs present.
Determination of Endotoxin Level in Adenovirus preparations
Endotoxin released from gram-negative bacteria is the main reason for contamination of medical products, Therefore, an endotoxin test is normally performed on the sterile product, medical device or pharmaceutical product which is to be injected or implanted into the body.. This test is used to confirm that there has been no endotoxin carry-over through the manufacturing process from any starting materials. To quantify the endotoxin level in adenovirus preparations, Advent uses the quantitative endpoint LAL Chromogenic test. Samples are mixed with the LAL reagent and chromogenic substrate reagent over a short incubation period, and read 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.
The presence of product-related impurities such as non-functional forms of the vector, or the presence of co-packaged unwanted genetic sequences should be included in the specification and acceptance limits set to exclude or limit these impurities as appropriate and justified. For viral vectors, empty particle number, aggregates and replication competent vectors should be controlled.
Contamination control by Nested PCR
Advent is producing several different Adenovirus vectors. To avoid cross contamination from one production to the next a Nested PCR is performed to verify that no adenovirus is present after facility cleaning. The same technique is used to control the product for cross contamination with preceding production vectors. This method consists in two consecutive PCRs. A 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. To determine the potential detection limit of each PCR a standard curve is set up. A positive control Adenovirus vector is prepared in serial dilutions and amplified in the same reaction conditions as the test samples. This curve should be composed of the adenoviral vector for which the contamination control is performed.
A suitable measure of the potency of the Adenovirus products should be established. The potency assay should normally encompass an evaluation of the efficiency of gene transfer (infectivity/transduction/delivery) and the level of expression of the therapeutic sequence or its direct activity.
Infectious Virus Titer by Hexon Immunostaining:
Accurate measurement of adenovirus titer is critical for gene delivery. The hexon immuno-staining assay is used to determine the infectious virus titer of the adenovirus vector by IFU (Infectious forming unit) calculation. This cell-based assay is able to measure the biological activity and potency of the adenovirus vectors. This test 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. In particular, Adenovirus products are diluted and added 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 by using anti-hexon antibody. 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.
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. The ratio of Vp to IFU is thus a measure of the efficiency of the encapsidation process and the ability to produce fully functional (infectious) virus. Based on the experience of the manufacturer, the acceptance criteria has been set at ≤300 Vp/IFU to account for different vectors which may show different encapsidation/infection efficiencies.
Western Blot Analysis for evaluation of Trans-genes expression of recombinant Adenoviruses:
Western Blot Analysis is performed to evaluate the expression level of trans-gene proteins in cells infected by Adenoviruses preparations. Protein levels are evaluated by SDS–PAGE. Cells are infected with Adenovirus at the appropriate concentration and then protein extracts from infected cells are loaded on gel. After the electrophoresis, the proteins are transferred to a nitrocellulose membrane to be probed using specific antibodies against the trans-gene target protein.
All viral vector products manufactured at Advent undergo rigorous stability testing, and are assessed according to ICH guidelines.
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 light, and to establish a re-test period for the product or a shelf life and recommended storage conditions.
In addition to the standard stability studies, viruses can also be tested at Advent under stressed conditions and by running in-use stability testing.
Pre-formulation studies can further be set up in order to identify the best matrix.