Products of Recombinant DNA Technology

(Ph. Eur. monograph 0784)

Ph Eur

This monograph provides general requirements for the development and manufacture of products of recombinant DNA technology. These requirements are not necessarily comprehensive in a given case and requirements complementary or additional to those prescribed in this monograph may be imposed in an individual monograph or by the competent authority.

The monograph is not applicable to modified live organisms that are intended to be used directly in man and animals, for example as live vaccines.

Products of rDNA technology are produced by genetic modification in which DNA coding for the required product is introduced, usually by means of a plasmid or a viral vector, into a suitable micro-organism or cell line, in which that DNA is expressed and translated into protein. The desired product is then recovered by extraction and purification. The cell or micro-organism before harbouring the vector is referred to as the host cell, and the stable association of the two used in the manufacturing process is referred to as the host-vector system.

Production is based on a validated seed-lot system using a host-vector combination that has been shown to be suitable to the satisfaction of the competent authority. The seed-lot system uses a master cell bank and a working cell bank derived from the master seed lot of the host-vector combination. A detailed description of cultivation, extraction and purification steps and a definition of the production batch shall be established.

Where products of rDNA technology are manufactured using materials of human or animal origin, the requirements of chapter 5.1.7. Viral safety apply.

The determination of the suitability of the host-vector combination and the validation of the seed-lot system include the following elements.

The suitability of the host-vector system, particularly as regards microbiological purity, is demonstrated by:

Characterisation of the host cell, including source, phenotype and genotype, and of the cell-culture media;

Documentation of the strategy for the cloning of the gene and characterisation of the recombinant vector, including:

i. the origin and characterisation of the gene;

ii. nucleotide-sequence analysis of the cloned gene and the flanking control regions of the expression vector; the cloned sequences are kept to a minimum and all relevant expressed sequences are clearly identified and confirmed at the RNA level; the DNA sequence of the cloned gene is normally confirmed at the seed-lot stage, up to and beyond the normal level of population doubling for full-scale fermentation; in certain systems, for example, where multiple copies of the gene are inserted into the genome of a continuous cell line, it may be inappropriate to sequence the cloned gene at the production level; under these circumstances, Southern blot analysis of total cellular DNA or sequence analysis of the messenger RNA (mRNA) may be helpful, particular attention being paid to the characterisation of the expressed protein;

iii. the construction, genetics and structure of the complete expression vector;

Characterisation of the host-vector system, including:

i. mechanism of transfer of the vector into the host cells;

ii. copy number, physical state and stability of the vector inside the host cell;

iii. measures used to promote and control the expression.

The master cell bank is a homogeneous suspension of the original cells already transformed by the expression vector containing the desired gene, distributed in equal volumes into individual containers for storage (for example, in liquid nitrogen). In some cases it may be necessary to establish separate master cell banks for the expression vector and the host cells.

The working cell bank is a homogeneous suspension of the cell material derived from the master cell bank(s) at a finite passage level, distributed in equal volumes into individual containers for storage (for example, in liquid nitrogen).

In both cell banks, all containers are treated identically during storage and, once removed from storage, the containers are not returned to the cell stock.

The cell bank may be used for production at a finite passage level or for continuous-culture production.

Production at a finite passage level  This cultivation method is defined by a limited number of passages or population doublings which must not be exceeded during production. The maximum number of cell doublings, or passage levels, during which the manufacturing process routinely meets the criteria described below must be stated.

Continuous-culture production  By this cultivation method the number of passages or population doublings is not restricted from the beginning of production. Criteria for the harvesting as well as for the termination of production have to be defined by the manufacturer. Monitoring is necessary throughout the life of the culture; the required frequency and type of monitoring will depend on the nature of the production system and the product.

Information is required on the molecular integrity of the gene being expressed and on the phenotypic and genotypic characteristics of the host cell after long-term cultivation. The acceptance of harvests for further processing must be clearly linked to the schedule of monitoring applied and a clear definition of a 'batch' of product for further processing is required.

Validation of the cell banks includes:

i. stability by measuring viability and the retention of the vector;

ii. identity of the cells by phenotypic features;

iii. where appropriate, evidence that the cell banks are free from potentially oncogenic or infective adventitious agents (viral, bacterial, fungal or mycoplasmal); special attention has to be given to viruses that can commonly contaminate the species from which the cell line has been derived; certain cell lines contain endogenous viruses, for example, retroviruses, which may not readily be eliminated; the expression of these organisms, under a variety of conditions known to cause their induction, shall be tested for;

iv. for mammalian cells, details of the tumorigenic potential of the cell bank shall be obtained.

The origin, form, storage, use and stability at the anticipated rate of use must be documented in full for all cell banks under conditions of storage and recovery. New cell banks must be fully validated.

The capacity of each step of the extraction and purification procedure to remove and/or inactivate contaminating substances derived from the host cell or culture medium, including, in particular, virus particles, proteins, nucleic acids and excipients, must be validated.

Validation studies are carried out to demonstrate that the production process routinely meets the following criteria:

• — exclusion of extraneous agents from the product; studies including, for example, viruses with relevant physico-chemical features are undertaken, and a reduction capacity for such contaminants at each relevant stage of purification is established;

• — adequate removal of vector, host-cell, culture medium and reagent-derived contaminants from the product; the reduction capacity for DNA is established by spiking; the reduction of proteins of animal origin can be determined by immunochemical methods;

• — maintenance within stated limits of the yield of product from the culture;

• — adequate stability of any intermediate of production and/or manufacturing when it is intended to use intermediate storage during the process.

The identity, purity, potency and stability of the final bulk product are established initially by carrying out a wide range of chemical, physical, immunochemical and biological tests. Prior to release, each batch of the product is tested by the manufacturer for identity and purity and an appropriate assay is carried out.

Suitable tests for demonstrating the consistency of the production and purification are performed. In particular, the tests include characterisation tests, in-process controls and final-product tests as exemplified below.

Partial amino-acid sequence analysis  The sequence data permit confirmation of the correct N-terminal processing and detection of loss of the C-terminal amino acids.

Peptide mapping  Peptide mapping using chemical and/or enzymatic cleavage of the protein product and analysis by a suitable method such as two-dimensional gel electrophoresis, capillary electrophoresis or liquid chromatography must show no significant difference between the test protein and the reference preparation. Peptide mapping can also be used to demonstrate correct disulfide bonding.

Cloned-gene retention  The minimum percentage of cells containing the vector or the cloned gene after cultivation is approved by the relevant authority.

Total protein  The yield of protein is determined.

Chemical purity  The purity of the protein product is analysed in comparison with a reference preparation by a suitable method such as liquid chromatography, capillary electrophoresis or sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Host-cell-derived proteins  Host-cell-derived proteins are detected by immunochemical methods, using, for example, polyclonal antisera raised against protein components of the host-vector system used to manufacture the product, unless otherwise prescribed. The following types of procedure may be used: liquid-phase displacement assays (for example, radio-immunoassay), liquid-phase direct-binding assays and direct-binding assays using antigens immobilised on nitrocellulose (or similar) membranes (for example, dot-immunoblot assays, Western blots). General requirements for the validation of immunoassay procedures are given under 2.7.1. Immunochemical Methods. In addition, immunoassay methods for host-cell contaminants meet the following criteria.

• — Antigen preparations Antisera are raised against a preparation of antigens derived from the host organism, into which has been inserted the vector used in the manufacturing process that lacks the specific gene coding for the product. This host cell is cultured, and proteins are extracted, using conditions identical to those used for culture and extraction in the manufacturing process. Partly purified preparations of antigens, using some of the purification steps in the manufacturing process, may also be used for the preparation of antisera.

• — Calibration and standardisation Quantitative data are obtained by comparison with dose-response curves obtained using standard preparations of host-derived protein antigens. Since these preparations are mixtures of poorly defined proteins, a standard preparation is prepared and calibrated by a suitable protein determination method. This preparation is stored in a stable state suitable for use over an extended period of time.

• — Antisera Antisera contain high-avidity antibodies recognising as many different proteins in the antigen mixture as possible, and do not cross-react with the product.

Host-cell- and vector-derived DNA  Residual DNA is detected by hybridisation analysis, using suitably sensitive, sequence-independent analytical techniques or other suitably sensitive analytical techniques.

DNA in the test sample is denatured to give single-stranded DNA, immobilised on a nitrocellulose or other suitable filter and hybridised with labelled DNA prepared from the host-vector manufacturing system (DNA probes). Although a wide variety of experimental approaches is available, hybridisation methods for measurement of host-vector DNA meet the following criteria.

• — DNA probes Purified DNA is obtained from the host-vector system grown under the same conditions as those used in the manufacturing process. Host chromosomal DNA and vector DNA may be separately prepared and used as probes.

• — Calibration and standardisation Quantitative data are obtained by comparison with responses obtained using standard preparations. Chromosomal DNA probes and vector DNA probes are used with chromosomal DNA and vector DNA standards, respectively. Standard preparations are calibrated by spectroscopic measurements and stored in a state suitable for use over an extended period of time.

• — Hybridisation conditions The stringency of hybridisation conditions is such as to ensure specific hybridisation between probes and standard DNA preparations and the drug substances must not interfere with hybridisation at the concentrations used.

Suitable procedures include: detection of sulfonated cytosine residues in single-stranded DNA (where DNA is immobilised on a filter and cytosines are derivatised in situ, before detection and quantitation using an antibody directed against the sulfonated group); detection of single-stranded DNA using a fragment of single-stranded DNA bound to a protein and an antibody of this protein. Neither procedure requires the use of specific host or vector DNA as an assay standard. However, the method used must be validated to ensure parallelism with the DNA standard used, linearity of response and non-interference of either the drug substance or excipients of the formulation at the dilutions used in the assay.

The requirements with which the final product (bulk material or dose form) must comply throughout its period of validity, as well as specific test methods, are stated in the individual monograph.

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