356 APPENDIX: COMMERCIAL IMPLICATIONS CONSIDERATIONS IN CHOOSING A TARGET DISEASE FOR GENE THERAPY A variety of approaches have been utilized for the introduction of nucleic acids (principally DNA) into cells These include the use of viral and nonviral methods for gene delivery Modified retroviruses, adenovirus, adenoassociated virus (AAV), and herpes virus have been investigated for virally based delivery (see Chapter 4) Naked DNA, cationic lipids, liposomes, and cationic polypeptides are being pursued as nonviral approaches for gene therapy (see Chapters and 5) Matching a gene therapy methodology to a target disease involves a number of factors The technical issues that must be considered include determining the tissue and cell specificity needed for expression of the therapeutic gene, the number of cells that need to be targeted, and therapeutic level and duration of transgene expression The delivery vehicle identifies the tissues and cell types that the therapeutic DNA can be delivered If the choice of delivery vehicles is limited, then target diseases or genes will also be limited The delivery vehicle will dictate the number of cells targeted and the duration of expression of the transgene (therapeutic gene) Therapies that require high levels of gene expression or require targeting a large percentage of cells likely require viral delivery vectors rather than nonviral delivery vectors This is because, at present, viral vectors are more efficient at delivery The delivered gene may be integrated into the host chromosome using AAV or retrovirus vectors (see Chapter 4) These may give longer duration of expression of the transgene than would be expected with adenovirus or nonviral delivery vectors However, if the gene is to be delivered multiple times during the course of treatment, nonviral vectors may avoid the development of immune responses that can occur with viral delivery systems Regulation of the therapeutic gene is another factor to consider when choosing a target gene How gene expression is regulated may determine which and how many cells need to be targeted At present, gene expression regulated at the level of transcription is less problematic than gene expression regulated posttranscriptionally Posttranscriptional gene regulation is, in most cases, less well understood The consideration of posttranscriptional regulatory mechanisms could complicate or slow the development of gene therapy As discussed later, the levels of transcription of a gene can be manipulated by modification of the plasmid or viral vector DNA Unusual requirements for gene product processing needed for activity of the expressed gene must be considered when choosing a target disease Many genes can be expressed in cell types other than the normally expressing cell types and still be therapeutic However, other gene products require special processing in a particular cell type or in a particular organelle Thus, such genes would not be effectively expressed in other cell types Still other proteins may have cofactors (proteins) that are essential for activity and must be made in close proximity (same cell or organelle) as the cofactor Another key factor in choosing a target gene is the availability of the gene Questions that should be asked are: • • Is the gene sequenced and cloned? Is it a cDNA clone or full-length gene (containing introns)? DNA PRODUCTION AND QUALITY CONTROL • 357 Does the cloned gene also contain the native promoter and regulatory sequences at the 5¢ and possibly 3¢ ends? The commercial development process is faster when maximal information is known about a targeted gene (regulation, sequence, etc.) The overall size of the gene to be delivered is also an important consideration since many viral vectors are limited in the size of DNA that can be packaged The nonviral delivery systems are less restricted in the size of DNA that can be delivered The development of a commercial gene therapy product is also facilitated by the availability of an animal model of the genetic disease being targeted Although not all human genetic diseases currently have animal models of disease, the number of transgenic and knock-out mouse strains (see Chapter 3), as well as larger animal models, has increased exponentially in the last few years These animal models prove valuable in developing effective gene therapy treatment approaches for many single-factor genetic disorders and possibly some multifactor diseases as well As for any commercial venture, patent and licensing issues for a particular gene will necessarily be important factors in choosing a target The size of the potential patient population and the accessibility of patients for a particular product are also crucial There are numerous genes that could be targeted for gene therapy, however, many of the single-factor genetic diseases are relatively rare (see Chapter 1) Diseases currently treated with recombinant proteins (severe immune deficiency, hemophilia A and B) provide larger markets where gene therapy could have an impact As with any new therapy, gene therapy approach for a disease state would need to have advantages over treatments currently in use DNA PRODUCTION AND QUALITY CONTROL Introduction The large-scale, commercial production and quality control of DNA and viral vectors to be used in clinical research protocols is critically important Assurance of purity must be provided to investigators who purchase or contract for reagents to be used in basic or clinical research As can be seen from the recent events, poor quality control of reagents can lead to the cessation of clinical trails of gene therapy protocols (see Chapter 13) Laboratory Scale Purification As the clinical aspects of gene therapy continue to grow, one of the challenges facing industry is the large-scale purification of plasmid DNA Within the typical research laboratory, plasmids continue to be routinely obtained by the standard method of CsCl–ethidium bromide density gradient ultracentrifugation CsCl–ethidium bromide gradients are popular since large numbers of different plasmid preparations can be processed simultaneously The approach applies to both plasmid and viral DNA of varying sizes; and a single band in the density gradient contains the monomeric, supercoiled form of the DNA partially resolved from the intrinsic host cell contaminants (protein, DNA, and RNA) But there are numerous drawbacks 358 APPENDIX: COMMERCIAL IMPLICATIONS and limitations to this process For the researcher at the lab bench, it is time consuming, labor intensive, and expensive For the biotechnology company, however, this method is completely unacceptable for the production of clinical-grade materials because of its use of mutagenic reagents and its inherent inability to be a process of scale Recently, a number of companies have initiated market-adapted micropreparative methods for the production of larger quantities of plasmid DNA These modified “mini-prep” kits, make use of the alkaline lysis method for cell disruption followed by a chromatographic cartridge purification The composition of the stationary phase used in these kits varies Some kits use a silica-based stationary phase, while others are based on an agarose stationary phase In most cases, the mechanism of binding is anion exchange These kits are aimed at a particular market niche: the production of small quantities (milligram or less) of research-grade material for molecular biology applications They not meet the rigorous requirements for the development of a highly controlled drug manufacturing process and most not have a Drug Master File (DMF) Purity in these applications is usually evaluated by agarose gel electrophoresis Trace impurities such as endotoxin and host DNA are not as thoroughly investigated as needed for human clinical use The Food and Drug Administration’s (FDA’s) “Points to Consider” on plasmid DNA was drafted in October, 1996, and provides the U.S approach to regulation of plasmid preventative vaccines (see Chapter 13) The same general criteria that guide the manufacture of recombinant protein pharmaceuticals apply to the development of processes for the production of plasmid DNA for human clinical investigations The common thread linking these processes is the basis of well-documented research This basis allows for the final product to meet defined quality standards supported by validated analytical methods and controlled unit operations All components of the process must be generally recognized as safe and must meet all applicable regulatory standards It is precisely because of these reasons that plasmid DNAs used for clinical investigations are not produced using kits intended for laboratory research LARGE-SCALE PRODUCTION: AN OVERVIEW To proceed to advanced clinical trials and ultimately gain regulatory approval, the pharmaceutical development of gene therapy products need to meet the requirements for cGMP (current good manufacturing practices) production While the “c” ostensibly stands for “current,” when actually following the spirit and intent of the FDA guidelines the “c” represents control of the process and characterization of the product GMP is defined as “the part of quality assurance that medicinal products are consistently produced and controlled to the quality standards appropriate to their intended use and as required by the Marketing Authorization or product specification.” There are two main components of cGMP, comprising both production and quality controls Production control is concerned with manufacturing This includes the suitability of facility and staff for the manufacture of product, development of standard operating procedures (SOPs), and record keeping Quality control is concerned with sampling, specifications, testing, and with documentation and release procedures ensuring satisfactory quality of the final product LARGE-SCALE PRODUCTION: THE PURIFICATION PROCESS 359 For a typical production conducted under the principles of cGMP, the major points to consider in the manufacture of plasmid DNA are: • • • • • • • SOPs are in place to ensure the control and consistency of the entire production cycle starting from the initial receipt of raw materials to the final formulated drug product All raw materials used in the manufacturing process are put on a testing program based on the U.S Pharmacopeia A master cell bank (MCB) and manufacturer’s working cell bank (MWCB) has been prepared under conditions of quarantine to ensure the purity and identity of the fermentation seed pools Thorough vector characterization has been carried out, including a detailed history on the construction of the vector, complete nucleic acid sequence determination, and plasmid stability within the host strain MCBs should be shown to be free of adventitious agents Details of the fermentation process must be elucidated and consistency data generated Several commercial media have been designed for plasmid production, but a defined medium that has been empirically developed for a specific strain plasmid is preferable This should assist in achieving a reproducible wellcontrolled process Bacterial strains should be compatible with high copy number plasmids, high biomass fermentations, and the selection system cannot be ampicillin based Purification processes must be developed to meet the challenges inherent with a high cell density fermentation process Plasmid DNA purification kits routinely fail when challenged with high cell density starting feed streams Recovery and purification must be controlled and validated Special attention must be paid to the removal of host cell proteins, DNA, and endotoxin Documented reproducible removal of key host-cell-derived impurities is essential for setting accurate limits and specifications on the bulk drug product Appropriate analytical assays must be developed for both the monitoring of the production cycle as well as for final quality control release criteria The FDA’s “Points to Consider” lists some of the tests needed to confirm purity, identity, safety, and potency of plasmid DNA A functional in vivo or in vitro bioassay that measures the biological activity of the expressed gene product, not merely its presence, should be developed Measuring the relative purity and concentration of plasmid DNA by agarose gel electrophoresis or by highpressure liquid chromatography (HPLC) is only a small part of the battery of analytical measurements necessary to confirm product quality All assays must be fully validated Ongoing stability and efficacy testing must be conducted on the product in support of the ongoing clinical trials This data is critical in eventually determining product shelf life for the approved drug LARGE-SCALE PRODUCTION: THE PURIFICATION PROCESS The basic unit operations for the manufacture of plasmid DNA are basically the same as those for the production of any recombinant biopharmaceutical (see Fig A.1) Typical process steps for the production of plasmid DNA include initial vector 360 APPENDIX: COMMERCIAL IMPLICATIONS Cell culture MCB/MWCB Fermentation Cell lysis/ clarification Cell harvest Purification/ chromatography FIGURE A.1 Shake flask Sterile filtration formulation filling Steps in a typical large-scale biotechnology process design, fermentation, cell harvesting, alkaline lysis precipitation, chromatographic purification, formulation, and filling The process cannot rely on the use of animalderived enzymes such as lysozyme, proteinase K, and RNAase Use of these reagents in any manufacturing process for a drug substance raises regulatory concerns about residuals in the final product Disregarding such purity issues would increase the difficulty in process validation and ultimately putting final regulatory approval at risk The process should also not include toxic organic extractions The various forms of plasmid DNA including supercoiled, relaxed, and concatamers should be separated The final product must be free of contaminating nucleic acids, endotoxins, and host-derived proteins Fermentation is generally considered the starting point in designing the purification process By careful selection and control of the variables associated with the fermentation process, the subsequent purification may be greatly simplified Various fermentation feed strategies (batch, fed-batch, continuous) should be explored While somewhat more difficult to optimize, as well as document, continuous fermentations may offer several advantages in terms of production cycle times Normally, fed-batch fermentations allow quicker process development times, simpler process control and sufficiently high biomass The growth stage at which the fermentation is harvested must also be tightly controlled since it will greatly impact on the final yield of purified plasmid Harvesting too early will result in suboptimal final yields Harvesting too late in the fermentation cycle will not only result in low yields but also plasmid of poor quality The optimal stage of harvest is late log phase The monitoring of fermentation process parameters including temperature, glucose addition, dissolved oxygen, and carbon dioxide evolution are critical for the development of a reproducible process By manipulation of these parameters or through the use of an inducible plasmid system, the growth characteristics of a strain can be effectively changed, resulting in an increase in the plasmid-to-biomass ratio Any increase in this ratio will aid in the design of the purification process As well, it can result in higher final yields of plasmid Chloramphenicol has been traditionally used just for this purpose The host cell and plasmid are the most important starting materials in the production fermentation The key parameters in choosing a host strain are a low endogenous endotoxin, the capability of growing to high biomass, and relevant genotypic markers These markers could be recA1, endA1, and deoR: recA1 pre- LARGE-SCALE PRODUCTION: THE PURIFICATION PROCESS 361 vents recombination and improves stability of plasmid inserts; deoR allows for the uptake of large plasmids; endA1 improves plasmid quality The plasmid should be structurally as well as segregationally stable and have a high copy number origin of replication Typically it is pUC derived Scale-up for the purification of plasmid DNA is a definite issue Chromatography is the tool that has enabled the biotechnology industry to achieve the purity levels required for today’s biotherapeutics, diagnostics, and other biologicals These include enzymes and plasma products Chromatographic purification of DNA presents a novel set of problems These are based on the physical characteristics of the biomolecule as well as the intrinsic impurities derived from the host cell of choice, Escherichia coli The chief culprits that hinder the purification of plasmid DNA are the large amounts of polymers of similar structure (chromosomal DNA and RNA) and high levels of endotoxin Plasmid DNA is a highly anionic polymer that is sensitive to shear and to degradation by nucleases Plasmids are as large or larger than the pores of almost all chromatographic resins Several chromatographic procedures for the purification of biologically active plasmid DNA (without the use of CsCl–ethidium bromide ultracentrifugation) have been developed, at least at laboratory scale They include gel filtration chromatography, hydroxyapaptite chromatography, acridine yellow affinity chromatography, anion exchange chromatography, reversed phase chromatography, silica membrane binding, and binding to glass powder Unfortunately, many of these methods are not well suited to the purification of large quantities of DNA In choosing the method of purification for large-scale production of plasmid DNA, there is a most important physical characteristic of the biomolecule to consider It is that DNA is a highly anionic polymer that is sensitive to shear and to degradation by nucleases Any large-scale manufacturing process must address all of these characteristics Currently, the most successful methods of extraction and purification involve large-scale alkaline lysis in sodium deodecyl-sulfate (SDS) This step efficiently removes chromosomal DNA, nuclease enzymes, and other contaminants Therefore, cell lysis conditions must be carefully optimized Low shear mixing must also be used during this step Large-scale tangential flow systems, which are routinely used for the processing of recombinant proteins, can easily nick the supercoiled form of the plasmid Cross flow rates, pump design, as well as the mixer’s impeller design must all be carefully scrutinized Plasmid extracts are primarily contaminated with low-molecular-weight cell components, process chemicals, and RNA These contaminants and trace host protein contamination may be removed by a combination of selective precipitation, anion exchange chromatography, and a final polishing step A major drawback of using anion exchange chromatography as the sole highresolution purification step in the purification of plasmid DNA is that a portion of endotoxin and pyrogen contaminants will co-purify with the plasmid Given the limitations of currently available commercial matrices and the similar structure and charge profile of biomolecule species passing over the column, anion exchange chromatography is best used as a primary capture and initial purification step A second polishing step, which is orthogonal to the principles of anion exchange, is prudent and ensures rigorous process control Historically, gel filtration has been used in the biotechnology industry as a polishing step Plasmid DNA, host cell DNA, and endotoxin resolve using gel filtration 362 APPENDIX: COMMERCIAL IMPLICATIONS chromatography This is a simple and reproducible method that also offers the advantage of simultaneously incorporating a buffer exchange step within the chromatographic process Contaminating salts and/or residual metals can thus be removed allowing for the careful control of the counter ion in the final drug product However, the main drawback in using gel filtration is that it is a very slow and volume-dependent method It is not a high throughput method and often becomes the bottleneck within a given process Reversed phase chromatography (RPC), on the other hand, can also offer excellent separation and resolution of trace contaminants as well as the removal of endotoxin It is commonly the method of choice for the purification of small pharmaceutical compounds When purifying biologically active molecules, care must be taken so that biological activity is retained Through its use of volatile solvents, RPC can also serve the function of a buffer exchange step But it is precisely this point that contributes to reversed phase’s own set of unique problems The use of combustible organic solvents (acetonitrile or ethanol) requires explosion-proof facilities This safety factor can dramatically increase the cost of waste management With the heightened awareness of environmental issues in today’s industrial nations, the cost and feasibility of waste disposal are major considerations when designing or deciding on a purification process Ion-pair RPC, while again providing excellent separation, resolution, and endotoxin removal, introduces ion-pair reagents that must be assayed for in the final product Their removal must be assured by validated methods The final crucial aspect in deciding on a chromatographic support is the necessity of cleaning in place and sanitization by cycles of caustic washing The ability to withstand repeated cycles of regeneration, sterilization, and sanitization with 0.5 N NaOH while maintaining run-to-run reproducibility of the column profile is an important consideration in manufacturing pharmaceutical-grade plasmid DNA in accordance with cGMP manufacturing guidelines LARGE-SCALE PRODUCTION: QUALITY CONTROL Recombinant proteins and plasmid DNA are both derived from E.-coli-based expression systems This results in a fair degree of similarity in their contaminant profiles The FDA has presented a general list of contaminants that should be quantified in all biopharmaceutical products They include pyrogen, nucleic acid, antigen, and microbial and residual contamination Most assays that have been developed for the quality control of recombinant protein drug substances need only slight adaptation for the quality control of plasmid DNA production The most challenging assays in terms of unique or specific analytical tests for plasmid DNA bulk products are the measurements of protein (antigen), nonplasmid DNA, and RNA trace contaminants Documentation and validation of all assays must adhere to cGMP guidelines Fermentation cultures need to be routinely monitored for microbial contamination Sterility checks should be performed on inoculation flasks, the fermentor, and the fermentation media The presence of contaminating organisms will alter the production levels of plasmid produced and thereby invalidate data on the levels of contaminating impurities within the final DNA product LARGE-SCALE PRODUCTION: QUALITY CONTROL 363 The most common and routine analysis of plasmid DNA is through the use of ethidium-bromide-stained agarose gels In research settings, this assay is usually used as a standalone technique for determining RNA contamination, residual genomic DNA, as well as quantifying the relative amounts of supercoiled plasmid in relation to the relaxed or nicked form It is well known, though, that ethidium differentially stains linear, nicked, and supercoiled plasmid DNA as well as host cell RNA Thus, care must be used when using this assay as the sole tool for judging relative amounts of DNA or in determining residual RNA levels To accurately characterize the purified product (and monitor in-process samples) an array of electrophoretic, chromatographic, and spectrophotometric assays should be employed In particular, the use of analytical high-resolution HPLC can avoid the detection and quantitation problems associated with ethidium bromide staining of plasmid DNA since detection is based on ultraviolet absorption Another common quality control test for plasmid DNA used in most research laboratories is the A260/A280 absorbance ratio assay It highlights the discrepancies between true cGMP production and laboratory-scale purification The test was originally designed to measure enzyme concentrations in the presence of low levels of nucleic acid contamination The original usage has been corrupted, however, and now it is routinely used in molecular biology laboratories to assess DNA purity An A260/A280 absorbance ratio of 1.8 to 2.0 is generally considered “pure.” In fact, when one does the actual calculation using the true extinction coefficients of nucleic acids and proteins (nucleic acids have extinction coefficients on the order of 50 times higher than proteins), it becomes obvious that an A260/A280 = 1.8 can contain as much as 60% protein contamination Therefore, this method can only be used as a functional test and cannot in itself be used to determine DNA purity Equally critical for achieving pharmaceutical-grade plasmid DNA is the monitoring of any chemical reagents introduced into the manufacturing process If alcohol is used in a precipitation step in the process, an assay must be included to determine the residual trace levels of alcohol that remain in the final product If antifoam (a common fermentation additive) has been used, an analytical assay must be in place for its determination as well as a final release specification for its concentration Choosing the appropriate analyses in this area requires careful control and sourcing of all raw materials One of the hallmarks of a fully FDA-compliant production process is the use of well-characterized reference standards These are necessary for the completion of analytical assay assessment and for use in ongoing validation studies The most critical reference standard is the plasmid DNA Ideally, the plasmid should be fully characterized and be derived from a manufacturing batch that has been clinically evaluated Having a well-characterized reference standard greatly aids in the successful evaluation of product stability testing With the proper appropriate supporting data, background information and supplementary studies, the development of a minimal panel of characterizing assays can be put in place These would provide the necessary level of confidence to reliably determine identity, purity, potency, and stability of the manufactured plasmid DNA (Table A.1) The foundation that makes this possible is rooted in the compliance to cGMP throughout the entire plasmid production cycle While there may be differences in the specific physiochemical assays for the determination of identity and purity, pharmaceutical-grade plasmid DNA and recombinant-protein-based therapeutics share a similar quality control characterization strategy 364 APPENDIX: COMMERCIAL IMPLICATIONS TABLE A.1 Sample Plasmid Specifications and Test Methods Assay Method Specification >95% supercoiled plasmid Potency 1% agarose gel electrophoresis Anion exchange HPLC Slot blot hybridization Slot blot hybridization LAL kinetic Restriction digest followed by 1% agarose gel electrophoresis USP membrane A260/A280 A260/A230 Optical density scan BCA microtiter Transfection assay Residual ethanol Gas chromatography DNA homogeneity E coli chromosomal DNA RNA Endotoxin Identity Sterility Purity Protein contamination