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GENE TRANSFER MEDICINAL PRODUCTS FOR HUMAN USE

general information

        This chapter provides information and suggests general requirements applicable to the production and control of gene therapy/transfer medicinal products for human use.  For a specific medicinal product, application of these requirements and the need for any further information shall be decided by the competent authority. It is to be noted that the information is designed to be applicable to approved products; the need for application of part or all of the texts to products used during the different phases of clinical trials is decided by the competent authority.

DEFINITION

For the purposes of this appendix, gene transfer medicinal product (GTMP) shall mean a product obtained through a set of manufacturing processes aimed at the transfer, to be performed either in vivo or ex vivo, of a prophylactic, diagnostic or therapeutic gene (i.e., a piece of nucleic acid) to human/animal cells, and its subsequent expression in vivo. The gene transfer involves an expression system known as a vector, which can be of viral as well as non-viral origin. The vector can also be included in a human or animal cell.
Recombinant vectors, such as viral vectors and plasmids Recombinant vectors are either injected directly into the patient’s body (in vivo gene transfer) or transferred into host cells before administration of these genetically modified cells to the patient (ex vivo gene transfer). Viral vectors are derived from various viruses (for example, adenoviruses, poxviruses, retroviruses, lentiviruses, adeno-associated-viruses, herpesviruses). These vectors can be replicative, non-replicative or conditionally replicative. Plasmid vectors include nucleic acids in a simple formulation (for example, naked DNA) or complexed to various molecules (synthetic vectors such as lipids or polymers). Genetic material transferred by GTMPs consists of nucleotide sequences, which may notably encode gene products, antisense transcripts, or ribozymes. Chemically synthesized oligonucleotides are not within the scope of this appendix. After transfer, the genetic material may remain either cytoplasmic or episomal, or may be integrated into the host cell genome, depending on the integrating or non-integrating status of the vector.
Genetically modified cells  Genetically modified eukaryotic or bacterial cells are modified by vectors to express a product of interest.

PRODUCTION

Substances used in production. The raw materials used during the manufacturing process, including viral seed lot and cell bank establishment, where applicable, are qualified. Unless otherwise justified, all substances used are produced within a recognized quality management system using suitable production facilities. Suitable specifications are established to control notably their identity, potency (where applicable), purity, and safety in terms of microbiological quality and bacterial endotoxin contamination. The quality of water used complies with the relevant corresponding Water for Injections and Purified Water.  Where bovine serum is used, it complies with the bovine serum. The use of antibiotics is avoided wherever possible during production.
Viral safety The requirements of “Viral Safety” apply.
Transmissible spongiform encephalopathies A risk assessment of the product with respect to transmissible spongiform encephalopathies is carried out and suitable measures are taken to “Minimize the Risk”.

RECOMBINANT VECTORS

PRODUCTION

GENERAL PROVISIONS
For viral vectors, production is based on a cell bank system and a virus seed-lot system, wherever possible.
For plasmid vectors, production is based on a bacterial cell bank system.
The production method shall have been shown to yield a vector of consistent quality. Unless otherwise justified and authorized, the vector in the final product shall have undergone no more passages or subcultures from the master seed lot than were used to prepare the vector shown in clinical trials to be satisfactory with respect to safety and efficacy.

SUBSTRATE FOR VECTOR PROPAGATION
The substrates used comply with the “Chicken Flocks Free From Specified Pathogens for the Production and Quality Control of Vaccines”, “Cell Substrates for the Production of Vaccines for Human Use”, and the section Bacterial cells used for the manufacture of plasmid vectors for human use).

CHARACTERIATION OF THE VECTOR
Historical records of vector construction are documented, including the origin of the vector and its subsequent manipulation, notably deleted or modified regions.
The vector is characterized using suitable and validated methods.
The genetic stability of the vector at or beyond the maximum passage level or the maximum number of cell doublings of the cell line used for production is assessed by suitable methods.

PROPAGATION AND HARVEST
All processing of the cell banks and subsequent cell cultures is done in an area where no other cells or vectors are handled at the same time. Any material of human or animal origin used in the preparation of cell suspensions and culture media is qualified. The purity of the harvest is verified by suitable tests as defined in the corresponding specific sections.

PURIFIED HARVEST
The bulk of active substance is defined as a lot of purified recombinant vectors (viral vectors, or naked or complexed plasmids).

FINAL LOT
Unless otherwise justified and authorized, formulation and distribution of the final bulk are carried out under aseptic conditions using sterile “Containers”.
The stability of the final lot is assessed using stability protocols including the duration, storage conditions, number of lots to be tested, test schedule, and assays to be performed.

ASSAYS AND TESTS
The GTMPs comply with assays and tests described in the corresponding specific sections.

GENETICALLY MODIFIED CELLS

For cells to be modified with a recombinant vector, the data related to the recombinant vector are documented above, under Recombinant vectors.

PRODUCTION

CELL SUBSTRATE
For xenogeneic cell lines, including bacterial cells, a cell bank system comprising a master cell bank and working cell banks is established.
For autologous and allogeneic cells, a cell banking system comprising a master cell bank and working cell banks is established wherever possible.

TRANSFECTION / TRANSDUCTION
Cells are transfected or transduced using a recombinant vector (plasmid or viral vector) qualified as described under Recombinant vectors; the process is validated. They are handled under aseptic conditions in an area where no other cells or vectors are handled at the same time. All reagents used during cell manipulation steps are fully qualified. Antibiotics are avoided unless otherwise justified and authorized. Transfection or transduction is carried out under aseptic conditions.

FINAL LOT
In the case of frozen storage, the viability of genetically modified cells is assessed before freezing and after thawing.
If the cells are not used within a short period, stability is determined by verifying cell viability and expression of the genetic insert.
In the case of genetically modified cells encapsulated before implantation in man, any encapsulating component used is considered as part of the final product, and is therefore quality-controlled and fully characterized (for example, physical integrity, selective permeability and sterility).

ASSAYS AND TESTS
Controls of xenogeneic, allogeneic, or autologous cells include the following:
– identity, counting, and viability of cells;
– overall integrity, functionality, copies per cell, transfer, and expression efficiency of the genetic insert;
– microbiological controls, comply with the “Sterility Test” (Appendix 10.1) or “Microbiological examination of cell-based preparations”, “Test for Bacterial Endotoxins”, (Appendix 8.5), “Mycoplasmas” (Appendix ==), adventitious virus contamination and, where applicable, replicative vector generation.
The competent authority may approve a reduced testing programme where necessary because of limited availability of cells. Where necessary because of time constraints, the product may be released for use before the completion of certain tests.

 ADENOVIRUS VECTORS FOR HUMAN USE

Category  Vectors.

Adenovirus vectors for human use are freeze-dried or liquid preparations of recombinant adenoviruses, genetically modified to transfer genetic material to human somatic cells in vivo or ex vivo.

PRODUCTION

Vector construction
There are different approaches for the design and construction of an adenovirus vector. The purpose of clinical use determines which approach is optimal. A method is chosen that minimizes the risk of generating replication-competent adenovirus vectors or that effectively eliminates helper viruses that might be used during production.

Vector production
The production method shall have been shown to yield a vector of consistent quality. Unless otherwise justified and authorized, the vector in the final product shall have undergone no more passages from the master seed lot than were used to prepare the vector shown in clinical trials to be satisfactory with respect to safety and efficacy.
The genetic and phenotypic stability of the vector at or beyond the maximum passage level used for production is assessed by suitable methods.

Substrate for vector propagation
The vector is propagated in continuous cell lines, comply with the “Cell Substrates for the Production of Vaccines for Human Use” (Appendix ==), based on a cell bank system. The occurrence of replication-competent adenoviruses may be significant when large regions of homology exist between the viral genome and the genome of the complementation cells. This occurrence may be minimized by minimizing the homology between both genomes. The use of cells with no sequence homology with the vector is recommended for production.
Vector seed lot
Production of the vector is based on a seed-lot system.
The strain of adenovirus used is identified by historical records that include information on its origin and its subsequent manipulation, notably deleted or modified regions. A detailed description of the genetic insert(s) and the flanking control regions is established, including the nucleotide sequence. The method by which the genetic insert is introduced into the vector is documented.
Only a seed lot that complies with the following requirements may be used for vector production.

Identification The vector is identified in the master seed lot and each working seed lot by “Immunochemical Methods” (Appendix 14.5), “Nucleic Acid Amplification Techniques” (Appendix 14.6), or restriction enzyme analysis.

Genetic and phenotypic characterization The following tests are carried out.
– The entire genome of the vector is sequenced at a passage level comparable to a production batch and the analytically determined sequence is compared to the theoretical sequence based on vector construction and available databases.
– Restriction enzyme analysis is performed on the vector DNA of the master seed lot, each working seed lot, and a production batch. The viral DNA is extracted, purified, and digested with sufficient resolution. The digested fragments are separated by gel electrophoresis or capillary electrophoresis and the observed restriction pattern is compared to the theoretical restriction pattern based on vector construction.
– A suitable number of isolated sub-clones are tested for expression of the genetic insert product(s) and biological activity at a passage level comparable to a production batch. Sub-clones giving lower levels of expression or biological activity need further characterization.

Vector concentration The titre of infectious vector or the concentration of vector particles in the master seed lot and each working seed lot are determined.

Extraneous agents The master seed lot and each working seed lot comply with the tests for extraneous agents.

Replication-competent adenoviruses  Replication-competent adenoviruses are generated by homologous recombination between the recombinant viral DNA and the adenovirus sequences integrated into the genome of the complementation cells.
Detection of replication-competent adenoviruses is performed by a suitable method approved by the competent authority. It is generally performed by an infectivity assay on sensitive detector cell lines, which are not able to complement for the genes deleted from the vector. Other indicators of viral replication may be used as appropriate.
When replication-competent adenoviruses are not supposed to be present in the test sample, considering vector construction and cell lines used, at least 2, but preferably 3 or 4 successive passages are performed on the detector cell line, where applicable. Detection of a cytopathic effect at the end of the passages reveals the presence of replication-competent adenoviruses in the preparation. Positive controls are included in each assay to monitor its sensitivity.
When replication-competent adenoviruses are expected to be present in the test sample, plaque-assays or limit dilution assays on a detector cell line may be performed.

Propagation and harvest
All processing of the cell bank and subsequent cell cultures is done in an area with a suitable containment level where no other cells or vectors are handled at the same time. Any material of human or animal origin used in the preparation of cell suspensions and culture media is qualified. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration, but it is preferable to have a substrate free from antibiotics during production. Unless otherwise justified and authorized, at no stage during production is penicillin or streptomycin used. A portion of the production cell cultures is set aside as uninfected cell cultures (control cells).
Each single harvest that complies with the following requirements may be used in the preparation of the purified harvest.

Identification The vector is identified by “Immunochemical Methods” (Appendix 14.5), “Nucleic Acid Amplification Techniques” (Appendix 14.6), or restriction enzyme analysis.

Vector concentration The titre of infectious vector and the concentration of vector particles in single harvests are determined.

Extraneous agents The single harvest complies with the tests for extraneous agents.

Control cells Control cells comply with a test for identification and a test for extraneous agents.
PURIFIED HARVEST
Several single harvests may be pooled before the purification process. The purification process is validated to demonstrate the satisfactory removal of impurities.
Purified harvests that comply with the following requirements may be used in the preparation of the final bulk.

Identification  The vector is identified by “Immunochemical Methods”  (Appendix 14.5), “Nucleic Acid Amplification Techniques”  (Appendix 14.6), or restriction enzyme analysis.

Genomic integrity  Genomic integrity of the vector is verified by suitable methods such as restriction enzyme analysis.

Vector concentration  The titre of infectious vector and the concentration of vector particles in purified harvests are determined.

Residual host-cell protein  The concentration of residual host-cell protein is determined by a suitable “Immunochemical Methods” (Appendix 14.5), unless the process has been validated to demonstrate suitable clearance.

Residual host-cell DNA  The content of residual host-cell DNA is determined using a suitable method, unless the process has been validated to demonstrate suitable clearance. Quantitative polymerase chain reaction (PCR) is recommended for its sensitivity and specificity, but other suitable techniques may also be used.

Residual reagents  Where reagents are used during the production process, tests for these substances are carried out on the purified harvest, unless the process has been validated to demonstrate suitable clearance.

Residual antibiotics  Where antibiotics are used during the production process, their residual concentration is determined by a microbiological assay (adapted from general method) or by other suitable methods (for example, liquid chromatography), unless the process has been validated to demonstrate suitable clearance.
FINAL BULK
Several purified harvests may be pooled during preparation of the final bulk. A stabilizer and other excipients may be added. The formulated product is filtered through a bacteria-retentive filter.
Only a final bulk that complies with the following requirement may be used in the preparation of the final lot.

Sterility It complies with the “Sterility Test” (Appendix 10.1).
FINAL LOT
Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use.
Provided that the tests for bovine serum albumin (when bovine serum is used to manufacture the vector) and replication-competent adenoviruses have been carried out with satisfactory results on the final bulk, they may be omitted on the final lot.

Identification  

The vector is identified by “Immunochemical Methods” (Appendix 14.5), “Nucleic Acid Amplification Techniques” (Appendix 14.6), or restriction enzyme analysis.

TESTS

Osmolality  Within the limits approved for the particular preparation (Appendix 4.35).

pH  Within the limits approved for the particular preparation (Appendix 4.11).

Extractable volume  It complies with the test for extractable volume.

Residual moisture  Within the limits approved for the particular freeze-dried preparation.

Bovine serum albumin  Not more than the limit approved for the particular preparation, determined by a suitable “Immunochemical Methods” (Appendix 14.5), where bovine serum has been used during production.

Replication-competent adenovirus concentration  Within the limits approved for the particular preparation.

Vector aggregates  Vector aggregates are determined by suitable methods (for example, light scattering).

Sterility  It complies with the “Sterility Test” (Appendix 10.1).

Bacterial endotoxins  Less than the limit approved for the particular preparation (Appendix 8.5).

Thermal stability  Maintain samples of the vector final lot at a temperature and for a length of time that are adapted and authorized for the particular preparation.  Determine the total infectious vector concentration after heating, as described below under Assay.  Determine in parallel the vector concentration of a non-heated sample.  The estimation of the difference between the total vector concentration without heating and after heating is within the limits approved for the particular preparation.

ASSAY

Vector particle concentration  Physical titration is performed by a suitable technique (for example, liquid chromatography, absorbance measurement or “Nucleic Acid Amplification Techniques” (Appendix 14.6), use an appropriate vector reference standard to validate each assay.
The vector particle concentration of the preparation to be examined is not less than the concentration stated on the label.

Infectious vector titre  Titrate the preparation to be examined by inoculation into cell cultures. Titrate an appropriate vector reference standard to validate each assay.
The assay is invalid if:
– the confidence interval (P = 0.95) of the logarithm of the vector concentration is greater than a value authorized by the competent authority;
– the infectious vector titre of the reference standard is outside limit values defined by a control chart.

Ratio of vector particle concentration to infectious vector titre  Within the limits approved for the particular preparation.

Expression of the genetic insert product  The expression of the genetic insert product(s) is determined wherever possible, following inoculation of cell cultures with the particular preparation at a predetermined multiplicity of infection, by suitable “Immunochemical Methods” (Appendix 14.5), or biochemical assays or by flow cytometry.

Biological activity  Unless otherwise justified and authorized, biological activity is determined by a suitable in vitro or in vivo test.

Labelling  The label on the container or the label on the package states at least (1) the content of active substance; (2) the recommended human dose, expressed in vector particle concentration; (3) for freeze-dried preparations, states the name or composition and the volume of the reconstituting liquid to be added and the time within which the product is to be used after reconstitution.

  

BACTERIAL CELLS USED FOR THE MANUFACTURE OF PLASMID VECTORS FOR HUMAN USE

Category  Cells used for the manufacture of plasmid vectors.

Production of plasmid vectors for human use is based on the use of a bacterial cell-bank system with generation and characterization of a master cell bank (MCB), working cell banks (WCBs) and end-of-production cells (EOPCs). A bacterial cell bank for the manufacture of plasmid vectors is a collection of vials containing bacterial cells stored under defined conditions, with uniform composition, and obtained from pooled cells derived from a single clone of a transformed host strain. The MCB has a known, documented history; it is preferably derived from a qualified repository source. The WCB is produced by expanding one or more vials of the MCB. Methods and reagents used to produce the bank and storage conditions are documented.
MCBs and WCBs are qualified by testing an aliquot of the banked material or testing a subculture of the cell bank.
The following table indicates the tests required at each stage of production.

IDENTITY AND PURITY TESTING

Viability  The number of viable cells is determined by plating a diluted aliquot of bacterial cells on an appropriate medium and counting individual colonies.

Biochemical and physiological bacterial strain characterization  Depending on the bacterial strain used for production, relevant biochemical and physiological characterization is performed to confirm cell dentity at the species level.

Genotyping/phenotyping  The genotype of bacterial cells is verified by determination of the suitable specific phenotypic markers or by appropriate genetic analysis.
Presence of the plasmid
Sequencing  The whole nucleotide sequence of the plasmid is verified.
Copy number  The plasmid DNA is isolated and purified from a known number of bacteria and the copy number determined by a suitable method such as “Nucleic Acid Amplification Techniques” (Appendix 14.6).
Restriction map  Restriction endonuclease digestion is performed with sufficient resolution to verify that the structure of the plasmid is unaltered in bacterial cells.
Percentage of cells retaining the plasmid  Bacterial elements present in the plasmid, such as selectable genetic markers, are used to define the percentage of bacteria retaining the plasmid.

 

Assay	Host strain	MCB	WCB	EOPCsa
Identity and purity
Viability	+	+	+	+
Bacterial strain characterization	+	+	–	+
Genotyping / phenotyping	+	+	–	+
Presence of the plasmid
–SequenceoftheDNAplasmid	–	+	–	+
–Copynumber	–	+	+	+
–Restrictionmap	–	+	+	+
– Percentage of cells retaining the plasmid	–	+	+	+
Adventitious agents
Purity by plating	+	+	+	+
Presence of bacteriophage	+	+	–	+

^aEOPCs are cells with a passage number at least equivalent to that used for production. The analysis has to be done once to validate each new WCB, except for purity, which has to be tested for each fermentation.

ADVENTITIOUS AGENTS AND ENDOGENOUS VIRUSES
Purity by plating  Bacterial cells are streaked out onto suitable media and incubated in the required conditions in order to detect potential bacterial contaminants.  In order to test for inhibition of the growth of contaminating organisms, additional tests in the presence of a definite amount of relevant positive control bacteria are carried out.  A suitable number of colonies is examined; no contamination is detected.

Presence of bacteriophage  Bacterial cells are plated and incubated in a medium allowing proliferation of bacteriophages, to test for bacteriophage presence.  The test is validated by the use of a reference bacteriophage strain and permissive cells as positive controls.  A suitable number of colonies are examined; no contamination is detected.

 PLASMID VECTORS FOR HUMAN USE

Category Vectors.

Plasmid vectors for human use are double-stranded circular forms of bacterial DNA that carry a gene of interest or a nucleotide sequence encoding antisense sequences or ribozymes and its expression cassette; they are amplified in bacteria extrachromosomally. They are used to transfer genetic material into human somatic cells in vivo or to genetically modify autologous, allogeneic, xenogeneic, or bacterial cells before administration to humans. Plasmid vectors may be presented as naked DNA or may be formulated with synthetic delivery systems such as lipids (lipoplexes), polymers (polyplexes), and/or peptide ligands that facilitate transfer across the cell membrane and delivery to the cell, or that target delivery via specific receptors.
Plasmids formulated with synthetic delivery systems are not within the scope of this section.

PRODUCTION
Plasmid construction
A typical plasmid vector is composed of:
– the plasmid vector backbone that contains multiple restriction endonuclease recognition sites for insertion of the genetic insert and the bacterial elements necessary for plasmid production, such as selectable genetic markers for the identification of cells that carry the recombinant vector;
– the required regulatory genetic elements to facilitate expression of the genetic insert;
– the genetic insert;
– a polyadenylation signal.
A complete description of the plasmid DNA, including its nucleotide sequence, is established with the identification, source, means of isolation and nucleotide sequence of the genetic insert. The source and function of component parts of the plasmid, such as the origin of replication, viral and eukaryotic promoters, and genes encoding selection markers, are documented.

General Provisions

Cell banks  Production of plasmid vectors is based on a bacterial cell-bank system with generation and characterization of a master cell bank (MCB), working cell banks (WCBs), and end-of-production cells (EOPCs), which comply with the section Bacterial cells used for the manufacture of plasmid vectors for human use. The raw materials used during the manufacturing process, including cell bank establishment, are qualified.
Selection techniques  Unless otherwise justified and authorized, antibiotic-resistance genes used as selectable genetic markers, particularly for clinically useful antibiotics, are not included in the vector construct. Other selection techniques for the recombinant plasmid are preferred.
Reference substances  A suitable batch of the formulated plasmid, preferably one that has been clinically evaluated, is fully characterized and retained for use as a reference standard as necessary in routine control tests.

Propagation and Harvest

Plasmid DNA is transferred to host strain bacterial cells and a single clone of transformed bacteria is expanded to create the MCB. The WCB is then derived from the MCB. The EOPCs are obtained from the WCB by fermentation in production conditions.
Plasmid DNA is isolated from harvested cells using an extraction step and is purified to obtain the bulk product.
Unless otherwise justified and authorized, caesium chloride-ethidium bromide density gradients are not used for production.

Purified Plasmid

The production process is optimized to remove impurities consistently while retaining product activity. The requirement to test for a particular impurity depends on the following:
– the demonstrated capability of the manufacture and purification processes to remove or inactivate the impurity through process validation, using specific quantification methods;
– the potential toxicity associated with the impurity;
– the potential decrease of the efficacy of the genetic insert product associated with the impurity.
If selective resistance to specific antibiotics has been used for selection, data from validation studies of purification procedures are required to demonstrate the clearance capability for residual antibiotics.
Relevant in-process controls are performed to ensure that the process is continuously under control, for example, amount and form of plasmid after the extraction steps and amount of endotoxins after the extraction steps. Only a batch of purified plasmid that complies with the following requirements may be used.
Identity and integrity of the purified plasmid  Identity and integrity of the purified plasmid are established by suitable methods such as sequencing or “Nucleic Acid Amplification Techniques”  (Appendix 14.6); restriction enzyme analysis may be used where it is sufficient to detect potential critical modifications in the plasmid and confirm the plasmid identity.
Plasmid DNA The following indications are given as examples.
DNA concentrations greater than 500 ng per mL may be determined using absorbance measurement at 260 nm. A 50 µg per mL double-stranded DNA solution has an absorbance of 1 (specific absorbance 200).
DNA concentrations less than 500 ng per mL are determined following incubation with fluorescent dyes that bind specifically to double-stranded DNA, using a reference standard of DNA to establish a calibration curve.
Liquid chromatography may also be used to determine the concentration of plasmid DNA using a reference standard.  In some cases, capillary electrophoresis is also acceptable.
DNA forms  Plasmid DNA is characterized in terms of the proportions of supercoiled, multimeric, relaxed monomer and linear forms, using suitable analytical methods, examples of which are given below.  For quantification of supercoiled forms, anion-exchange “High-Performance Liquid Chromatography” (Appendix 3.5) or “Capillary Electrophoresis” (Appendix 3.8) may be used.  Capillary electrophoresis is also suitable for the quantification of other forms.
Residual host-cell DNA  The content of residual host-cell DNA is determined using a suitable method, unless the process has been validated to demonstrate suitable clearance. Quantitative PCR is recommended for its sensitivity and specificity, but other suitable techniques may also be used.
Residual RNA  The content of residual RNA is determined, unless the process has been validated to demonstrate suitable clearance.  Reversed-phase HPLC (RP-HPLC) may be used, or quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), comply with the “Nucleic Acid Amplification Techniques” (Appendix 14.6) when a lower limit of detection is required.
Residual host-cell protein The concentration of residual host-cell protein is determined using standard protein assays, comply with the “Total Protein” (Appendix ==), SDS-PAGE followed by silver staining, or specific immuno-assays such as western blot or ELISA, unless the process has been validated to demonstrate suitable clearance.
Microbiological control Depending on the preparation concerned, it complies with the “Sterility Test” (Appendix 10.1) or the bioburden is determined (2.6.12).
Bacterial endotoxins  Less than the limit approved for the particular preparation, it complies with the “Test for Bacterial Endotoxins” (Appendix 8.5).

Final Bulk
Several purified harvests may be pooled during preparation of the final bulk. A stabilizer and other excipients may be added. The formulated product is filtered through a bacteria-retentive filter.
Only a final bulk that complies with the following requirement may be used in the preparation of the final lot.
Sterility  It complies with the “Sterility Test” (Appendix 10.1).

Final Lot
Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use.

Identification  The plasmid vector is identified by restriction enzyme analysis or by sequencing. The test for biological activity also serves to identify the product.

TESTS

Appearance  According to the manufacturer.

pH  Within the limits approved for the particular preparation (Appendix 4.11).

Extractable volume  It complies with the test for extractable volume.

Residual moisture  Within the limits approved for the particular freeze-dried preparation.

DNA forms  The percentage of the specific monomeric supercoiled form is determined as described for the purified plasmid.

Sterility  It complies with the “Sterility Test” (Appendix 10.1).

Bacterial endotoxins  Less than the limit approved for the particular preparation, it complies with the “Test for Bacterial endotoxins” (Appendix 8.5).

Assay

Plasmid DNA  Not less than the quantity stated on the label, determined, for example, by one of the following methods.
DNA concentrations greater than 500 ng/mL may be determined using absorbance measurement at 260 nm. A 50 µg/mL double-stranded DNA solution has an absorbance of 1 (specific absorbance 200).
DNA concentrations less than 500 ng/mL are determined following incubation with fluorescent dyes that bind specifically to double-stranded DNA, using a reference standard of DNA to establish a calibration curve.
Liquid chromatography may also be used to determine the concentration of plasmid DNA using a reference standard. In some cases, capillary electrophoresis is also acceptable.

Biological activity  Wherever possible, biological activity is assessed through in vitro or in vivo bioassays. A well-defined, representative reference standard is required as a positive control for the assay.  Bioassays employed to assay plasmid vectors generally involve transfection of a relevant cell line in vitro, followed by some functional measure of the expressed genetic insert. Such functional assays provide information about the activity of the product encoded by the genetic insert instead of the expression level of the genetic insert itself.
It may be necessary to supplement the bioassay with western-blot and ELISA assays to assess the integrity and quantity of the expressed product.

Labelling  The label on the container or the label on the package states at least (1) the plasmid DNA concentration; (2) the recommended human dose; (3) for freeze-dried preparations; states the name and volume of the liquid to be added; the time within which the product is to be used after reconstitution.