Technical and regulatory hurdles in delivery aspects of macromolecular drugs

journal of

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Journal of Controlled Release 41 (1996) 147-155

controlled release

Technical and regulatory hurdles in delivery aspects of macromolecular drugs Andrew J.S. Jones*, Jeffrey L. Cleland Genentech Inc., 460 Pt. San Bruno Boulevard, South San Francisco, CA 94080, USA

Received 14 August 1995; revised 9 January 1996; accepted 24 January 1996

Abstract Although this meeting attests to the importance of continuing progress in the development of novel delivery systems for macromolecular pharmaceuticals, only one such product has been approved for marketing - - DNase, which is delivered by aerosol. This situation is the result of the significant number of obstacles in the commercialization of potential delivery systems• In addition to the obvious financial commitment a company must make to their development, there are new technical challenges and novel concerns from regulatory bodies who must approve the systems. For protein macromolecular drugs (here, arbitrarily defined as -->10 000 MW) the advances in analytical methodology, stimulated by regulatory requirements concerning recombinant DNA derived products, focus attention on changes in the drug caused by the chemical components or manufacturing processes in the preparation of the new dosage forms• Thus, for a marketed product for which a new delivery system is contemplated (for example, a sustained release formulation of somatropin), extensive characterization of the 'delivered' product will be required. The extent to which preclinical or clinical work needs to be repeated will depend on the changes from the existing product caused by the delivery system. In addition, altered pharmacokinetic, pharmacodynamic or distribution profiles will require additional clinical evaluation to assess equivalence or superiority, depending on the rationale behind the new delivery mode. Somewhat paradoxically, if a new macromolecular entity must be delivered by a novel delivery system in order to be effective, it will only be subject to 'single' scrutiny, on its own merits• Emerging therapies based on DNA, such as anti-sense, 'naked' DNA, complexed DNA or viral vectors, will require comparable development and application of analytical methodologies for production and quality control as was seen during the emergence of rDNA derived protein therapies. This presentation will discuss many of these principles and illustrate their application in some examples of the development of delivery systems for rDNA derived protein pharmaceuticals. K e y w o r d s : Macromolecular pharmaceuticals; Delivery systems; Clinical evaluation

1. Introduction The success o f a research effort in a drug or drug d e l i v e r y system d e v e l o p m e n t is usually m a r k e d by a series o f e x p e r i m e n t s d e m o n s t r a t i n g ' p r o o f o f concept'. This p r o o f is required before resources are Corresponding author.

applied to the full-scale d e v e l o p m e n t for marketing. The cost to the d e v e l o p e r for such an effort ranges f r o m tens to hundreds o f millions o f dollars and there is therefore a financial (as w e l l as a technical and regulatory) hurdle to bringing the n e w product to market. The c o m p e t i t i v e marketplace and the desire to bring the therapeutic benefit to the market both result in a 'rush to m a r k e t ' approach and first

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generation products can be more quickly approved for marketing if they employ existing, proven delivery systems. For parenterally delivered macromolecules this usually means the use of injected or infused solutions of drug. The development of new delivery systems takes time and is usually considered non-essential for market entry due to the time constraints. This results in a situation in which a large proportion of the development work needs to be repeated with a new delivery system to demonstrate, for example, clinical equivalence or superiority in addition to the safety of the new system. The manufacturing plant and process need to be developed, validated and approved, and often new test methods will be required for the system. These requirements mean that the cost for development and marketing of the second generation product can approach those for a new therapeutic entity. This in turn poses the obvious dilemma to a company with limited financial resources (i.e. all companies!) of 'which investment will bring the greater return?', a dilemma in which the specifics of the case will determine the outcome. It is not unlikely, however, that the choice will be in favor of a new revenue generator (new pharmaceutical) even though the new delivery system for the existing drug may be elegant or more convenient. Unless the new system increases (or assures future) market share or increases the market size, the return on the substantial investment will never be realized. These financial considerations probably account for the fact that so few 'new' delivery systems have been approved for macromolecular drugs. In the case of companies whose business is developing delivery systems, rather than the drugs, the decision to develop the system has already been made. They will therefore need to find a suitable drug and financial support for showing the suitability of the delivery system for the drug and for the costs of clinical development thereafter. For macromolecular drugs where the investment is considered appropriate, the technical and regulatory hurdles will be the subject of the rest of the discussion. These can be divided into two major categories: (a) drugs which cannot provide their therapeutic benefit without the use of a new delivery system (e.g. DNAse, see below) and (b) those which are already approved with 'traditional' delivery systems. The issues are different for these two

categories since the former has no precedent for comparison while existing drugs (and their delivery systems) are the de facto standard against which the new system for them will be compared. This paper will discuss many of the issues from a general viewpoint while using some specific examples to illustrate how these issues may be handled.

2. Delivery systems for existing approved macromolecular drugs The issues with development of additional systems for existing, approved drugs are best appreciated by considering those faced during the development of the first generation product. Marketing approval is granted only after the product has been extensively tested for safety and efficacy in human clinical trials, the 'pivotal' trials. It is rare that the production system used to demonstrate proof of concept is the one which will support the scale of production needed for marketing after approval: with production capacity an expensive capital investment, the specific productivity during the cell culture or fermentation process is a key parameter which is continually optimized until the pivotal trials begin, and may even continue thereafter. The material which is to be marketed must, however, be made by the same process as the material used in the pivotal trials, or be shown to be equivalent if any modifications are made to the process. Such changes can be at any stage of the process and the magnitude of the changes affects the extent of work which is required to demonstrate equivalence to the satisfaction of the regulatory agencies. Of particular note here is the scale of production, since the investment in a plant capable of supporting the market usually cannot be committed until there is a reasonable expectation of approval to return the investment. The complexity of macromolecular drugs requires (and has stimulated the technological advances of) current analytical methods necessary for product characterization [1]. How these methods are integrated with biopharmaceutical development and the regulatory process has recently been reviewed [2]. For this discussion it suffices to emphasize the large and continual effort needed to show that the same

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product is being produced as the yield improvement efforts, the process scale-up and possible facilities change, all raise the possibility that some aspect of the properties of the molecule have been affected. These possibilities are frequently raised during the regulatory review dialog. Such changes could alter the toxicological, safety, pharmacokinetic or efficacy profile so that clinical or preclinical data generated on early material may not be reflective of the later material. The analytical support can guide the development to minimize these potential changes and is used to demonstrate the equivalence of materials produced throughout the history of the product's evolution. These same analytical methods are the required tools for evaluation of the behavior of the product in the arena of new delivery system development. The development of dosage forms to be used for injection or infusion (i.e. liquid or lyophilized preparations) of macromolecular drugs is in itself a complex undertaking. The large number of potentially reactive groups on a macromolecule frequently leads to the 'degradation' of the product over time, most commonly in the generation of deamidated or oxidized species [3]. In many cases these forms can be shown to be fully active while in some cases the degradation product may have altered or diminished functionality. In any case, it becomes critical to monitor the biological activity of the product to understand the nature and effect of any 'analytically' detectable changes over time, such as those observed in stability studies of formulations. However, it is also quite common that the most relevant (from a clinical efficacy viewpoint) measures of activity are the least precise. Examples of this are growth hormone potency testing (weight gain in rats) or interferons (antiviral assays, etc.). This discussion will not cover the details of the analytical methods and strategies for formulation development since these are well covered in a variety of reviews. However, the goal of the formulation developer is to minimize the changes in a product, when stored under its recommended conditions, over its shelf-life and to demonstrate that any changes which do occur do not affect the safety or efficacy of the product. One of the most commonly raised safety concerns relates to the possibility of increased immunogenicity due to aggregation of the macromolecule [3].

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3. Development beyond the initial delivery system Once the first generation formulation/delivery system has been approved for marketing, it is not uncommon for different configurations to be developed in response to different needs for a new indication or dosage regimen. A new indication might, for example, require a significantly larger or smaller dose and a different vial size or content would be needed. This is usually relatively simple, given the experience developed with the original product and new methods are not usually necessary to demonstrate that the new vial configuration has an acceptable stability profile. However, the stability properties of the first generation become the standard against which the new form is compared. It might be necessary to use a different excipient, for example, if a lyophilized form is being developed to replace an original liquid formulation, or even vice versa. If the new excipient is one which has been used in parenteral formulations and is 'generally recognized as safe' (GRAS), its compatibility with the product can be demonstrated with methods available to show the stability of the product. However, if the excipient has not previously been approved in a parenteral product, the safety of the new component will need to be addressed by toxicology and possibly other studies. This explains why it is rare for a new excipient component to be considered in these efforts. One example of such a problem was encountered in the evaluation of addition of methylcellulose to create a new formulation for a peptide, relaxin. While this material is commonly used in the pharmaceutical arena, and appears to be safe and non-toxic, it was found to contain oxidizing agents which degraded the peptide [4]. New delivery systems for macromolecules are usually contemplated when there is a perceived need to improve the performance or convenience of the first generation system: for example, if a targeted delivery will improve the therapeutic ratio (of beneficial effects to undesirable side-effects) or if a sustained or controlled delivery system improves efficacy or decreases injection frequency (resulting in improved patient compliance and convenience). For targeted delivery systems there are specific concerns which must be considered during the preclinical and

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clinical evaluation. Such systems are generally desirable where efficacy cannot be obtained by systemic delivery either because of adverse reaction at sites other than the target or because insufficient drug reaches the target at maximum achievable doses. In the former case the therapeutic window will be affected by the targeting efficiency. The demonstration of reproducibility of targeting efficiency will therefore be required to provide the assurance of a reproducible safety profile.

4. Controlled release d r u g delivery - - a d e v e l o p m e n t case study

We have worked on a new delivery system for hGH and, although it has not been developed to the point where it is suitable for clinical evaluation, it serves to illustrate some of the issues discussed above. It is an obvious candidate for a new delivery system because the current treatment involves daily injections over a period of many years and the patients are children. This treatment regime was developed as an efficacious one, even though to mimic the normal physiological pulsatile delivery of the hormone effectively would have required several i.v. injections per night, clearly an impractical option [5]. A sustained release formulation could provide comparable or improved efficacy relative to daily injections [6], in addition to the more obvious improved patient compliance and comfort. Our goal was a 4-week dosage form and we selected microencapsulation using poly (lactic acid/glycolic acid) copolymer (PLGA) based on its proven record of safety in resorbable sutures and suitability demonstrated by Lupron Depot® [7], a microsphere preparation which slowly releases a peptide. No protein products have been approved using this technology and the following discussion will focus on the challenges involved in this development. The current daily dose of hGH is in the range of 0.05 mg/kg per day and a 28-day dosage form would therefore need to contain 30-50 mg of active drug. With a constraint of a maximum injection volume of approximately 1 ml and a maximum practical solids content in a suspension of microspheres of 200-300 mg/ml, this results in a target

drug loading of 15% w/w. While these initial targets appear to be reasonable, they represent a considerable series of technical challenges. The process we chose for the encapsulation was the water-in-oil-in-water double emulsion method [7]. This employed methylene chloride as the preferred organic solvent even though residual solvent in the final product is a potential concern from a safety viewpoint. A solution of hGH was emulsified in a solution of polymer in organic solvent, to produce the first emulsion (water in oil). The droplets of aqueous phase needed to be sufficiently small to remain discrete and remain independent while this emulsion was dispersed in the second emulsion step in a large volume of water. This second step generated droplets of the first emulsion which would subsequently harden into microspheres of the desired size and size distribution as the methylene chloride diffused out and the polymer hardened. Many aspects of the development of this process followed chemical and process engineering pathways to optimize equipment selection and process parameters but a key constraint through the whole project was the integrity of the protein. This was incorporated from the outset because we knew that any compromise in the quality of the protein could result in lowered efficacy or increased immunogenicity. In a water-in-oil-in-water system, the payload component (drug substance) is present in the initial aqueous phase and there is a maximum volume of this phase that a fixed volume of organic phase can contain and still produce stable microspheres. Our goal therefore became to prepare the initial aqueous phase at the highest possible concentration. Systematic evaluation of substances that stabilized an aqueous solution of hGH revealed that several sugars not only minimized the methylene chloride induced aggregation [8] but also increased the amount of hGH which could be solubilized. As a result we were able to achieve solutions of hGH at well over 300 mg/ml with the aid of sugars such as trehalose at a protein to sugar weight ratio of approximately 4:1. This allowed the production of microspheres with a nominal loading of 12% hGH (w/w) of total dried solids. The microspheres were approximately 80/~m in diameter and were dispersed in a methylcellulosepolysorbate vehicle in a suspension which was sufficiently stable to be drawn into a syringe and

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injected through a 23-G needle. The use of larger needles for injections for children is not readily accepted. Microspheres were prepared from low molecular weight ( 1 0 - 1 4 kDa) PLG (50/50 lactide/glycolide) and contained 12% hGH (w/w). Approximately 35% of this loading was released in the first 24 h as a 'burst', yielding a net load of approximately 8% which was released slowly over the subsequent 30 days during in vitro evaluations. While this magnitude of burst release would be undesirable in a dosage form for clinical evaluation, the net loading was considered sufficient for animal studies. One consideration for 28-day dosage forms is the integrity of the protein itself in the microspheres maintained at 37°C under physiological conditions in the injection site. We confirmed that the major degradation pathways (deamidation and oxidation, neither of which affects the biological activity of the molecule [9]) were the same for hGH inside or outside the microspheres under physiological conditions [10], although a modest increase in dimerization (higher aggregates were not seen) resulted from the high protein concentrations in the rehydrated microsphere interior. The material released throughout the in vitro release studies was biologically active in a cell based bioassay. It was considered critical for the development of this dosage form that at no time during the production or use of the microspheres should the protein quality or activity be compromised. An animal study was designed to evaluate the rate and extent of release of the hGH from the microspheres and to obtain preliminary assessment of tissue reactions to the delivery system. The animal study employed four juvenile rhesus monkeys, each of which received 200 mg of hGH containing microspheres subcutaneously at one site and a comparable amount of placebo microspheres at a separate site. Blood samples were drawn and monitored for levels of hGH, insulin-like growth factor I (I,GF-I), IGF-I binding protein 3 (IGF-I BP3) and anti-hGH antibodies, all by in-house immunoassays. At several time points the placebo microsphere injection sites were excised for histology. The histology studies showed a typical foreign body giant cell reaction as expected from previous studies of in vivo microsphere degradation [11]. Fig. 1 shows the levels of

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Fig. 1. Blood levels of hGH (- - -), IGF-I ( ) and IGF-I BP3 ( ) in Rhesus monkeys after a single dose of hGH-containing microspheres at day 0. hGH, IGF-I and IGF-I BP3 obtained during the study. One animal developed a measurable, but low, level of anti-hGH antibodies after 3 weeks, while the other three animals remained negative through the whole study, indicating that the protein had not been rendered significantly immunogenic by the production process. This observation was extremely encouraging and is likely related to the attention paid to the integrity of the protein during the development of the dosage form. It also allayed concerns about potential adjuvant effects of encapsulation of proteins: it appears that encapsulation of foreign proteins may be beneficial for vaccine development [12] but with an autologous protein an immune response is not an automatic consequence of encapsulation. While hGH and rhesus hGH are not identical in sequence, the rhesus has proven itself a useful species in which to evaluate the immunogenicity of hGH. The biological effects of the hGH microspheres are evident in the data presented in Fig. 1. The hGH levels are presented only for samples from animals which were antibody free (due to possible influence on hGH levels by antigen antibody complexes altering the pharmacokinetic profile of the drug). After the initial 'burst' phase immediately after injection, the hGH levels fall to 1 or 2 n g / m l and a delay is observed in further release of drug into

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circulation. The short-lived peak of hGH at day 1 results in responses in both markers of hGH activity (IGF-I and IGF-I BP3): they both demonstrate a short-lived peak a day or so after the hGH, consistent with the known delay in their response to hGH [13]. At approximately day 12, the hGH levels in serum begin to rise and the IGF-I and IGF-I BP3 levels also begin to rise, again followed by a 1-2 day lag. These observations suggest that levels of around 3-5 ng/ml hGH may represent a threshold above which a response is initiated. Similarly, at day 42-45, when the hGH levels start to drop below this 'threshold', the IGF-I and IGF-I BP3 levels also begin to drop from their plateau. This information is useful as a guide to future studies and provides a rational basis for targeting steady-state hGH levels in the future studies which would be needed to optimize dosing strategies as part of the further development of the product. The hGH levels remained above 5 ng/ml for approximately 30 days (day 12-42) and the IGF-I and IGF-I BP3 levels remained at their plateau levels of approximately 3 × baseline values for this period, indicating that the hGH being released during this whole period was bioactive. Both of these markers are regulated such that a 3-fold increase from baseline is the maximum elevation observable, and the fluctuations in hGH concentrations (ranging from 7 to 17 ng/ml) do not cause corresponding fluctuations in the secondary responses. The rhesus study was designed to evaluate the release characteristics of the preparation in vivo and the large dose was employed to ensure that the hGH levels were accurately measured. Further studies would be required to obtain information on the optimal steady-state level of hGH needed to cause the responses of the IGF-I and IGF-I BP3 to remain under suitable control. The study characterized the hGH release profile in vivo and emphasized the importance of choosing suitable in vitro methods for assessing release profiles. The original in vitro method of evaluating the hGH release did not predict the lag of 10-12 days and improvement of that method was aided by the animal data [14]. The level of hGH during the major release period from days 12 to 42 was approximately 1l + 5 ng/ml. The fluctuations indicate that the release rate was not absolutely constant. Modifications in the production method may be able to yield microspheres with more truly 'steady state sustained release' characteristics.

In summary, the animal study demonstrated that a sustained release preparation of a macromolecule of 22 kDa can be made in such a way as to maintain its biological activity, to maintain it in a state where it is not rendered significantly immunogenic and to provide steady levels of drug over a 30-day period. Future work would be necessary to demonstrate consistency of manufacturing, release profile and to establish the optimal sustained drug level for promoting growth. For development into a marketed product, it is anticipated that extended clinical trials, possibly 1-2 years, would be necessary to demonstrate safety and efficacy parameters equivalent to the daily injection dosage form.

5. Regulatory issues The clinical demonstration of safety and efficacy for a new pharmaceutical needed for registration, approval and marketing generally comes from 'pivotal' trials. A common requirement has been for the manufacturer to perform two randomized, doubleblind trials controlled with either a placebo group or a group treated with a pharmaceutical approved for the same indication. Such trials are expected to show a meaningful benefit to the patient with appropriate safety parameters. These issues are evaluated on a 'case-by-case' basis, with the agencies balancing the risks and benefits of the new treatment. These evaluations (and consequent requirements) are performed by the agencies in each country where the drug is to be marketed and, although 'harmonization' discussions have simplified the task of the manufacturers to meet the individual requirements of separate countries, it is not uncommon for the data from a trial in one country to be insufficient for approval in another country. 'Worldwide' approval to market a new drug therefore requires approval in each target country. In addition to the assessment of the performance of the drug in human subjects, the manufacturers must demonstrate that the manufacturing and control process is sufficient to assure a consistently safe and efficacious drug. The design, maintenance and operation of a manufacturing plant are subject of extensive guidelines, and inspections play a significant role in the enforcement of the appropriate regulations. The test methods and final product specifications (which

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must be met for each batch for sale) are also subject to review by the regulatory agencies, again with individual countries differing in their requirements. The agencies are responsible to the public they serve but they must maintain a balance: they must assure that medicines will be safe for their intended use while at the same time they do not wish to slow the approval process for a new drug with significant benefit to the patients. They are therefore generally conservative in their review of an application and attempt to minimize any risk associated with the product. The complexity of macromolecular drugs and the analytical methods needed to test and control them, together with the multiplicity of agencies which must review and approve them leads to extensive scientific as well as regulatory discussion. This nearly always results in improved understanding, on both sides, of the requirements for approval. There are occasionally, however, additional hurdles due to differences of opinion between the scientists within one agency or between different agencies concerning individual issues. Frequently, these issues can only be resolved by additional experimentation or clinical trials which are seen as essential by some but unnecessary by the majority of others. The recent development of recombinant human DNAse (deoxyribonuclease or dornase alpha) provided some examples of these issues. The drug is a glycoprotein, produced in mammalian cell culture, which is administered to degrade the DNA found in mucous secretions of cystic fibrosis patients, resulting in reduced viscosity of the material obstructing the airways [15]. It is delivered by aerosol and improves lung function, presumably by making it easier for the patients to clear their lungs of the secretions. Treatment resulted in reduced hospitalization and lowered the requirements for antibiotic treatment, due to the increased efficacy of the antibiotics in the control of chronic pulmonary infections common in these patients [16]. The glycoprotein nature of the molecule required tests for sialic acid content, phosphate content (from mannose-phosphate) and a labile deamidation site required a test for deamidation. Despite the application of these individual tests (which measure the post translational modifications which would affect the isoelectric point of the molecule), some regulatory agencies insisted on the development of a 'semi-

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quantitative' isoelectric focusing test in addition to the more informative tests. Biological pharmaceuticals were traditionally defined by the process that was used to produce them, because of the inability of the older analytical methods to characterize them adequately. Thus, materials prepared from a different manufacturing process (i.e. other than the one described in the registration document) were considered different drugs and some aspects of safety and efficacy needed to be re-evaluated before the new process could be approved. This approach and the complexity of macromolecules, such as DNAse, can result in a very conservative approach to biotechnology products. Despite the dramatic technological improvements, the perception of the utility of modem analytical methods can vary widely between different agencies. There is also ongoing discussion between manufacturers and regulatory authorities concerning changes to a manufacturing process and possible changes to the detailed properties of the product, and whether such changes could be detected. For rhDNAse, this was seen in the discussions concerning the equivalence of the material produced at different manufacturing scales. It is commonly the case for products produced by complex biotechnology methods, that the manufacturer cannot invest in capital equipment to produce sufficient quantities of product to supply market needs until there is reason to believe the product will be approved. The material used for pivotal trials will therefore be produced at a smaller scale than that ultimately required for the market, with subsequent confirmation that the material produced at both scales is equivalent. Biochemical and biological comparisons will generally be sufficient although, for parenteral drugs, a confirmatory pharmacokinetics equivalence study (either preclinical or clinical or both) may be necessary, since the factors controlling clearance and half-life are often not well understood. In the case of rhDNAse, a change in production scale was required to meet projected market needs and extensive equivalence testing using 'state-of-the-art' analytical technology demonstrated that the materials had the same properties. Even though this is a drug which is applied topically (i.e to the site of action directly in the lungs) and pharmacokinetics in circulation is irrelevant, one European country's regulatory agency required a repetition of a pivotal trial with the material from the

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scaled-up process before it would accept the equivalence of the new material. The agencies of all other countries, however, were satisfied with the biochemical and preclinical data. As noted above, the delivery system employed for a drug is considered an integral part of the drug in terms of the regulatory process. The delivery of rhDNAse is by aerosol, setting a precedent for this type of protein delivery. The consistency of the clinical benefit will be related to the ability of the nebulizer device to generate a reproducible aerosol with the correct size distribution for delivery and deposition of the drug to the appropriate parts of the airways. Extensive methods development and device evaluation showed that the product was not adversely affected by the aerosolization process and that several carefully selected devices gave comparable and consistent droplet size distribution profiles [17]. Slight differences in nebulization efficiency were detected (the three separate devices yielded values of 22 _+ 3%, 23 _+ 2% and 27 _+ 4%) and therefore a clinical evaluation of their comparability was required. The study was well designed and suitably powered to demonstrate that the groups of patients using the three devices showed statistically indistinguishable responses to treatment [18]. However, given the wide range in individual responses of the patients, this experiment might be considered analogous to assessing the equivalence of, for example, three different insulin syringes in a diabetes clinical trial setting using efficacy as the measure of equivalence. While this experiment was not onerous on the developer of the drug and patients received real benefit from the study, it represents a hurdle that was a good scientific experiment but may not have been necessary (as the authors state "In theory, different nebulizers having similar in vitro delivery characteristics should yield similar degrees of clinical improvement in vivo" [18]).

6. Delivery of nucleic acid based pharmaceuticals This symposium has covered many aspects of the scientific issues for therapeutics using nucleic acid based molecules in a variety of forms and the current areas of progress. While many of these possibilities and developments are very new, some gene therapy

trials have been performed in man, in particular in patients for whom there is little therapeutic alternative. Thus, while the risks associated with such therapy are little understood, the potential benefit is large and initial trials have been approved. However, it is anticipated that as products are developed and move into extensive human trials, there will be an increased focus of the regulatory agencies on analytical aspects of the candidate molecules. This was the case for protein molecules resulting from genetic engineering and indeed it stimulated great progress in the development and application of protein characterization methods for use in final product testing. The concepts of purity, potency, identity and quality as applied to proteins will probably be applied equally to gene therapy products. For example, it is quite likely that limits will be discussed/imposed in terms of allowable proportions of altered nucleic acid sequences present in a preparation of a gene therapy product of a desired sequence. Methods will need to be developed which can accurately assess such variant molecules. The routine assessment of biological activity of a particular construct or molecule will require the development of 'bioassays' for use in product control and stability testing. Freedom from impurities such as endotoxin or host-cell proteins (from the cell used to synthesize the molecule) will need to be shown to be adequate for the intended dosage. In addition to the extension of concepts already in place for product testing, the nucleic acid based therapeutics may also require new chemical entities for targeting or delivery. Thus DNA/lipid complexes may represent an attractive system for delivery of genetic information and the lipids or lipid like molecules will need to be shown to be safe and methods will be needed for purity analysis. It has also become apparent that the physical form of such complexes can play a major role in the effectiveness and biophysical methods will likely be required to show consistency and stability of such a class of therapeutics.

7. Conclusions This paper has covered many aspects of the development of new delivery systems for pharmaceutically active macromolecules after they have

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m a d e t h e t r a n s i t i o n f r o m ' r e s e a r c h ' to d e v e l o p m e n t for m a r k e t i n g . T h e d e l i v e r y s y s t e m s s h o u l d b e c o n s i d e r e d as i n t e g r a l to the d r u g i t s e l f a n d t h e c o n s t r a i n t s o r specific r e q u i r e m e n t s for the d e l i v e r y s y s t e m s h o u l d b e i n c o r p o r a t e d as e a r l y as p o s s i b l e into the d e v e l o p m e n t plan. P a r a m o u n t a m o n g t h e s e c o n s t r a i n t s is the i n t e g r i t y o f the d r u g d u r i n g its i n c o r p o r a t i o n i n t o t h e n e w s y s t e m , to the p o i n t w h e r e the s y s t e m m a y n e e d to b e d e s i g n e d a r o u n d the p r o d u c t . T h e s y s t e m c a n n o t b e d e v e l o p e d in i s o l a t i o n f r o m the drug. N e w c h e m i c a l c o m p o n e n t s o f a d e l i v e r y s y s t e m will n e e d to b e t e s t e d in t h e i r o w n r i g h t for safety as w e l l as for t h e i r p o t e n t i a l effects o n the drug. It is c o s t e f f i c i e n t to p e r f o r m the p i v o t a l trials u s i n g t h e n e w s y s t e m i f p o s s i b l e , s i n c e m a n y c o s t s will b e i n c u r r e d t w i c e i f the n e w s y s t e m is to b e i m p l e m e n t e d a f t e r m a r k e t i n g a p p r o v a l for the first generation delivery system. Emphasis on analytical c h a r a c t e r i z a t i o n m e t h o d s f r o m the i n c e p t i o n o f the p r o j e c t will p a y d i v i d e n d s d u r i n g the d e v e l o p m e n t o f subsequent delivery systems and ensure that the drug is m a i n t a i n e d in a s u i t a b l e state t h r o u g h o u t . T h e s e m e t h o d s will u s u a l l y b e sufficient to assess the p r o p e r t i e s o f t h e p r o d u c t to t h e s a t i s f a c t i o n o f the r e g u l a t o r y a g e n c i e s , e s p e c i a l l y i f d i a l o g w i t h the a g e n c i e s is f r e q u e n t a n d c o l l a b o r a t i v e .

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