Analytical methods that separate, isolate, and specifically quantify the intended active product components determine product purity. Impurities are either product- or process-related components that can be carried through to the final product. The manufacturing and purification process should be optimized to consistently remove impurities while retaining product activity. The requirement to test for a particular impurity for product lot release will depend on the following: (1) the demonstrated capability of the manufacture and purification process to remove or inactivate the impurity through process validation, and (2) the toxicity potential associated with the impurity.
Examples of process-related impurities associated with cell and gene therapy products include residual production-medium components (e.g., FBS, antibiotics, cytokines, and Escherichia coli chromosomal DNA in a plasmid product), ancillary products used in downstream processing (e.g., nucleases such as DNase I), and residual moisture for lyophilized vector products. Impurities may be bioactive (e.g., cytokines and hormones) or immunogenic (e.g., product aggregates, degradation products, plasmid-selection markers, and nonhuman-derived proteins) or they may have other deleterious effects (e.g., they may compete with the product) if administered at a dose equal to that of the product. Product-related impurities are specific to each product type. Examples include differentiated cells in a stem-cell therapy product, nicked plasmid forms in nonviral products, and defective or immature virus particles in retroviral or adenoviral vector products. Analytical methodologies to assess purity require quantitation or physical separation of intended product from its impurities. Common sense should drive the need to quantify specific impurities. It may be possible to validate the manufacturing process to the extent that specific lot-release testing for impurities will be very limited. An emphasis may be placed on demonstrating the consistency of the product-impurity profile.
Testing for impurities is often extensive during product characterization and process validation when the consistency of the manufacturing and purification process is being demonstrated. Testing for impurities as part of lot-release testing should reflect the safety risks associated with the impurity and the ability of the process to consistently remove that impurity.
CELL THERAPY PRODUCTS
One measure of purity is the percentage of viable cells in the total cell population. Another measure is the percentage of transduced cells or the percentage of cells with a specific marker. If an entire population of cells is the therapeutic agent, methods should determine the relative amounts of each subpopulation. Limits should be defined for each cell subpopulation. These assays may be based on immunological methods utilizing flow cytometry or DNA-hybridization dot blot analysis. Additional tests for process contaminants are performed depending on the specifics of the manufacturing process. For example, a quantitative ELISA for residual serum proteins may be required. If the cells are genetically modified during manufacture, then testing for residual vector may need to be performed.
For gene-modified cell therapies, determining the purity of the cell therapy product depends on the availability of reagents and methods to distinguish therapeutic cells from the other cells present in the product. As in the case of gene therapy products, gene-modified cells can be distinguished from the unmodified cells on the basis of expression of the transgene. FACS using an antibody that detects the therapeutic protein or fluorescent probes that detect expressed RNA allow the separation and quantitation of the transgene expressing and nonexpressing cells. By adding an antibody to a cell-specific phenotypic marker and by using a double-sorting technique, FACS can be used to further identify the subpopulation of cells that are modified.
Endotoxin testing is also required. Biomaterials used with cell therapy products should also be tested for their biocompatibility.
VIRAL GENE THERAPY PRODUCTS
Product-related impurities for viral vectors include aggregates and defective and immature particles that may be produced during the manufacture or purification of the recombinant vector. Aggregates of vector may form if the product is highly concentrated, stored under certain conditions (e.g., under certain pH or temperature), or reconstituted after lyophilization. Assays to detect aggregates include particle size analysis by laser light-scattering and the use of nonreducing, nondenaturing PAGE, followed by staining of the gel or transfer and detection of viral proteins by Western blot analysis. Sedimentation rate analysis also allows separation of aggregates from monomers based on size. Optical density analyses of light-scattering are also used to assess vector aggregation.
Defective particles are viral particles that do not contain the appropriate recombinant genome, that is, they contain some other nucleic acid or contain no genome at all, or the vector has some missing, defective, or otherwise altered structural component that impairs its ability to transduce a cell. For viral vector systems that have capsomeric symmetry, which requires the appropriate nucleic acid incorporation for configuration, empty particles may be readily distinguished from those carrying genomes. For enveloped viruses, empty particles may not be as readily separated from those with encapsidated nucleic acid.
For some viral vector products, active viral particles may be separated from defective particles by using analytical HPLC. Anion-exchange resins have been used to separate active adenovirus from defective virus particles. However, this method might not be useful for an adenoviral vector purified by anion-exchange chromatography unless the resin for the assay is different from that used during manufacture. Depending on the nature of the viral vector and its nonactive or defective forms, other methods of separation, such as equilibrium centrifugation in a cesium chloride density gradient, may need to precede the quantitation of the active particle. Ideally, the method of separation will allow quantitation.
Defective particles that carry a noncell-derived oncogenic gene or other undesirable genes may pose a special concern. For example, in murine-based retroviral packaging cell lines, small viral elements called VL30 sequences can be packaged in about one third of all particles. Assays may need to be developed to quantify specific defective particles if they are known to be present in quantities sufficient to pose a safety concern.
Virus quality and the comparability of preparations can also be assessed by measuring selected structural proteins with known molecular masses and known copy numbers within the virion. For this method, the virus is lysed, and the structural proteins are separated by using reverse-phase HPLC or some other high-recovery chromatographic procedure. The chromatographic separation should be validated and the identity of the selected structural proteins verified by methods such as SDS-PAGE, peptide sequencing, or mass spectroscopy. One can fingerprint the batch based on quantification of the selected structural proteins and comparison to a reference standard. When the method incorporates the use of mass spectroscopy, impurities such as structural variants can also be identified. For adenovirus preparations, some precursor and most mature virion proteins can be monitored, thus allowing monitoring of the product and of the immature virion forms.
Host cell-derived proteins may be considered impurities for some viral vector products and may be separated and quantified by PAGE or HPLC or detected by amino acid analysis, Western blot, or immunoassay-based methods. However, for enveloped viruses such as retroviruses, host cell-derived membrane proteins are an integral part of the product. In those vector systems, it may be difficult to determine the presence of contaminating exogenous host-derived proteins.
Presence of specific process-related impurities depends on the manufacture and purification process of each vector or product type. However, most products will need to be tested for residual endotoxin (see Bacterial Endotoxins Test 85
). Acceptable limits of endotoxins have been determined and can be directly applied to viral vector products.
Although genomic DNA derived from continuous cell substrates used to manufacture biological product has been considered historically as potentially tumorigenic, recent studies suggest that the risks are very low. However, every attempt should be made during process development to reduce contaminating DNA levels. The need to test for residual DNA as part of product lot release should be evaluated on a case-by-case basis and may be dependent upon the size distribution of the DNA, its association with the product or its formulation components, and the route of administration of the product. Quantitative PCR assays have been developed to analyze the amount of residual host-cell DNA by using primers designed to amplify evolutionarily conserved and abundant target sequences, such as 18S for 293 cells.
Quantitation of residual serum components such as bovine serum albumin (BSA) can be achieved by using ELISA and a BSA reference standard. Specific functional or immunological methods may need to be developed for other ancillary products including other culture media or purification process components such as cytokines or enzymes (e.g., nucleases such as DNase I or benzon nuclease).
NONVIRAL GENE THERAPY PRODUCTS
Testing is usually performed on the individual components, the plasmid DNA, the lipid or lipoplex components and (recombinant) protein components if any are present in the formulation. Plasmid DNA is characterized for a variety of impurities including residual host-cell DNA, residual RNA, and residual protein. Residual protein testing is frequently included in lot-release testing. Optical density ratios, usually the ratio of the measurement at 260 nm to that at 280 nm, are frequently used in purity specifications for plasmid DNA.
In addition, the plasmid DNA should also be characterized with regard to its form. Plasmid DNA forms include monomeric supercoiled, relaxed monomer, and linear forms. The profile of forms needs to be monitored for product consistency. Additionally, it may be possible to correlate form with in vivo transfection behavior. While monomeric supercoiled plasmid has been shown to be more efficient than relaxed monomer, linear, or multimeric forms of the plasmid in transfecting cell lines in vitro, in vitro transfection has been shown to not always predict in vivo behavior. Formulation, delivery method, and route may impact in vivo transfection. Agarose gel electrophoresis can resolve these forms of plasmid, which are then detected by UV after ethidium bromide staining. This method provides information about the relative levels of the plasmid forms, but it is not highly quantitative for the individual species. Analytical anion-exchange HPLC can be used as a quantitative assay for monomeric supercoil and percentage of other forms, including concatamers. Other sophisticated methods, such as capillary zone electrophoresis, linear-flow dichroism, and atomic-force microscopy have been proposed as replacements for agarose gel analysis. Until they are validated, these analytical methods may be more appropriate for characterization studies in support of process development and validation rather than for lot-release testing. The appropriate methods for lot release will depend on what effect these alternate plasmid forms have on the product potency. Many of these methods, such as HPLC, are also applicable to the assessment of the purity of antisense-oligonucleotide products and the determination of the level of by-products.
Tests for process-related impurities, such as cesium chloride, must also be conducted. In the case of antisense-oligonucleotide products, residual solvents must be quantified. Lipid and lipoplex formulation components must also be tested for their chemical purity. Testing for specific chemical impurities is commonly performed by using gas chromatographymass spectroscopy (GCMS), high-pressure liquid chromatography (HPLC), or thin-layer chromatography (TLC) methods.
Bacterial protein, DNA, RNA, and endotoxins are the major types of host-derived process contaminants. Standard protein assays (e.g., Lowry, Bradford, or Coomassie), PAGE followed by silver staining or Western blot analysis, or ELISA can be used to detect residual host protein in the nanogram range. Host chromosomal DNA may be detected by slot blot hybridization (detection in picogram range) or by PCR using highly conserved target sequences (e.g., 18S for Escherichia coli). However, low background may be unavoidable in PCR-based assays because the recombinant polymerases used for the amplification of target also contain residual bacterial DNA. PAGE or agarose gel electrophoresis followed by fluorescent dye staining may be used to detect residual RNA. Quantitation may not be required given the labile nature of RNA and the low-level toxicity associated with it.
Certain antibiotics, such as kanamycin, that may be used during the fermentation process must be removed during the process, and validation of the process or lot-release testing must be performed to confirm removal during the purification of the plasmid. HPLC is one method that can be used to detect low-level residual antibiotic.