Once developed, the chromatographic procedure can be used both to analyse the optical purity of the product and to isolate the enantiomers. In the drug development process, speed to market is vital and rapidly reaching a go / no- go decision point for the development is critical in resource allocation. Thus, as the new drug moves through the early stages of development and increasing amounts of material are needed, the initial chromatographic method can grow in scale with the needs of the project, often reaching the isolation ( under cGMP) of kilogram quantities for Phase 1 clinical trials. At this point the immediate pressure of development eases, allowing a more leisurely investigation of the possible processes to make the desired enantiomer. The focus at this point is to find the most economical procedure for the production of the material in time for Phase III, where the manufacturing process is typically locked in and all alternative processes from crystallization through asymmetric synthesis are investigated. In some cases, chromatography remains the option of choice while in others the alternative procedures are chosen. While the aim is generally to use the process that results in the lowest cost per kg of the final product, the choice may also be influenced by capital expenditure requirements or by concerns about the scalability of the process. While the latter concerns should by now be alleviated by the success of the current production scale chromatographic enantiomer separations, the capital expense of installing a large scale chromatographic system as opposed to utilization of existing tankage ( for a crystallization, for example) could result in a decision to use a more expensive but less capital intensive process. Considerations of scale. In the progression from the small scale chromatographic purification to production scale operations there are many changes made in both the chromatographic methodology and its philosophy. At the smallest scale, cost is not important and the need is to find an adequate separation method in the shortest possible time which can produce the few tens to hundreds of mg. At this scale the separation time for the isolation is short; there is little purpose in spending several days to develop an optimised separation. As the scale increases, there is increasing emphasis on the economics of the separation. Despite the high overall costs of bringing a new pharmaceutical product to market, the costs of individual steps remain under strict scrutiny and the chromatographic method frequently is optimized and in some cases may be redeveloped in order to meet the cost requirements. Much more care is taken to find a high selectivity and to optimize the separation when the scale increases to the few hundred grams needed for toxicology or the kg quantities for Phase 1 trials. The transition to large scale processing beyond Phase 1 is usually accompanied by a transition from conventional batch chromatographic separation techniques to the production- scale oriented simulated moving bed technology. This continuous chromatographic process is generally more cost effective than conventional single column chromatography, combining use of significantly less solvent and stationary phase with higher productivity, but it requires more optimization and development time than the simpler batch process. Early Stages. Separation method development time has to be short in the early stages of the development of the new product to meet the stringent time constraints. Methods are typically developed by screening a small set of enantioselective columns with the aim of finding a baseline separation quickly. Increasingly ( in the USA at least) this is done using supercritical fluid chromatography; replacement of organic solvents with a mobile phase predominantly consisting of supercritical carbon dioxide results in approximately a fourfold reduction in solvent viscosity. This allows the columns to be operated at four times the flow velocity used in corresponding HPLC methods, dramatically reducing the screening and separation time. SFC methods also result in the use of smaller volumes of organic solvent during the separation process. While efficient solvent recycling procedures minimize the environmental impact of this reduction in solvent use relative to HPLC, the products are isolated in smaller volumes ( often 5 to 10 times less) than in HPLC. This reduces the evaporation time and results in a little less energy use in the process ( though it should be noted that operation in SFC involves several phase transitions which consume more energy than simply pumping solvent as in an HPLC system). Although SFC is widely used at this stage of development, this does not mean that HPLC processing should be avoided or Preparative Chiral Separations - from Laboratory Scale to Production Geoffrey B Cox, Chiral Technologies, Inc., 800 N Five Points Rd., West Chester, PA 19380 USA . gcox@ chiraltech. com Over the past few years, preparative chromatographic separation of racemic mixtures into their individual enantiomers has become an integral part of the development process for new drug entities. This is because the number of chiral drug candidates has been increasing, a not surprising development, given the asymmetric nature of the drug receptor sites. At an early stage in development it is essential to know the differences in activity and toxicity between the two enantiomers in order to maximize the effectiveness of the product while minimizing the possible negative side effects of the new drug. At this stage of the process there is practically nothing known about the chemistry and physical properties of the molecule and the fastest and most convenient way to the pure enantiomers is usually chromatographic purification. In contrast to other possible procedures, only a few mg of product and a few hours are needed to develop a chromatographic separation method - important for these new candidates where there may be only a few hundred mg of the product in the world. 4November/ December 2009

5 ignored. There may be advantages in an HPLC process - better selectivity or solubility, for example - which allow faster purification despite the lower flow rates typically used. Sometimes separations can be achieved using one of the techniques and not the other; thus it is worth screening both, especially where the scale of separation may be increased at some time in the near future and the most effective separation will be required. Method Development. Whether one is developing an HPLC or an SFC separation, the procedure is very similar. As there is currently no way to predict which column - mobile phase combination will give a separation of the desired product ( and it is probable that such a prediction will continue to elude scientists in this field for some years to come!) the method development process generally involves screening a number of chiral stationary phases and potential mobile phases in a systematic scheme. This is aided by statistical information which tells us that for past separations there are sets of chiral phases which will give at least an 85 to 95% chance that such a set will provide conditions suitable for the preparative separation. This is not, of course, a guarantee, especially when new molecular structures are in development. Typical sets of columns and mobile phases for primary HPLC screening are shown in Table 1. If this initial screening is not successful, typically one moves to a secondary screen, where the lesser used columns and solvents are employed, again in a similar process. Usually the column sets are mounted on switching valves in the chromatograph and the whole is operated automatically, allowing much of the screening process to be run overnight in an unattended fashion. A typical screening result is shown in Figure 1. For larger scale separations it is often most convenient to run a full screen of all available columns and mobile phases for the separation since at this point the best rather than a merely adequate separation is often required. Screening in this case can be an involved process. At Chiral Technologies, for example, a full screen involves more than 100 solvent - column combinations while a screen for an industrial process in which at least 70 to 80 additional chiral phases are investigated involves even more. Such a full screen can take a long time to complete and ways to reduce this are continuously researched. Besides the use of SFC, which as noted above reduces the analysis time by a factor of around 4 from HPLC, screening can be accelerated by use of smaller particle size columns. A column 5 cm in length packed with 3 micron particles will have higher efficiency than the 15 cm column packed with the 20 micron CSP often used for larger scale separations and can give selectivity and retention data in an order of magnitude less time. It is essential, of course, that the small particles have chromatographic properties identical with the larger particles that will be used for the separation project. Parallel chromatography systems have been developed as another approach to rapid screening. These typically use 8 channels with either conventional columns ( Sepiatech, both HPLC and SFC) or microflow columns of 0.3 mm id ( Eksigent). Such parallel systems allow a screen of 8 columns in the same time as conventionally used in screening just one. Coupled with solvent switching to allow fully automated screening gives these systems an 8- fold time advantage over the conventional single channel units. Optimisation. Once screening is complete, the separation is generally optimized to maximize the selectivity and to bring retention times into an acceptable window. This process can be more time consuming than the screening, especially as this step relies on the expertise of the chromatographer to develop the most effective procedure. For HPLC processes, it has been calculated that the optimum retention factor for the first peak in the chromatogram should have a value around 11. For SMB processes ( see below) this value should be reduced for maximum production rate 2. Optimisation also may include investigation of the sample solubility; if a solubility of only a few g/ l is attained, the preparative method will always be slow and expensive. In this respect, the use of a combination of immobilized chiral phases and mid- polarity range solvents such as dichloromethane, ethyl acetate and THF ( see Table 1) have been found to be extremely useful; many drug candidates are not especially soluble in the more conventional hexane - alcohol mobile phases employed in chiral chromatography . The method development process is completed by a loading study in which increasing quantities of the racemic compound are injected to the point where the two enantiomer peaks overlap. For small scale separations this process is stopped at the point at which the two chromatographic bands just touch. As the scale increases it may be better to sacrifice some recovery in favour of increasing the production rate of the separation by increasing load further, allowing the bands to overlap and taking the appropriate fractions which give the desired combination of purity and product yield. Particle size and column technology. At this point it is also necessary to make decisions on the particle size of the media that will be employed in the larger scale separations. Small particles, while they give high separation efficiency and allow difficult separations, produce high operating pressures. This is not an issue in small scale operations ( up to ~ 5 cm id columns) for many ( a) Immobilised polysaccharide- based phases Columns+: 1. CHIRALPAK ® IATM ( immobilized amylose tris( 3,5- dimethylphenylcarbamate)) 2. CHIRALPAK IBTM ( immobilized cellulose tris( 3,5- dimethylphenylcarbamate)) 3. CHIRALPAK ICTM ( immobilized cellulose tris( 3,5- dichlorophenylcarbamate)) + Other solvent- stable chiral columns such as Whelk- O 1 ( etc) may be included in the set. Mobile phases: 1. Hexane - 2- Propanol( 80: 20) 2 Hexane - Ethanol ( 80: 20) 3. Methyl tert- Butyl Ether - Methanol ( 98: 2) 4. Hexane - Dichloromethane - Methanol* ( 49: 49: 2) * Alternatively Hexane - THF - methanol may be used in place of the chlorinated solvent. ( b) Coated Polysaccharide- based Phases. Columns: 1. CHIRALPAK AD ® ( amylose tris( 3,5- dimethylphenylcarbamate)) 2. CHIRALCEL ® OD ® ( cellulose tris( 3,5- dimethylphenylcarbamate)) 3. CHIRALPAK AS ® ( amylose tris( S- a- methylbenzylcarbamate)) 4. CHIRALCEL OJ ® ( cellulose tris( 4- methylbenzoate)) Mobile Phases1. Hexane - 2- Propanol ( 85: 15) 2. Hexane - Ethanol ( 80: 20) 3. Methanol ( 100%) 4. Acetonitrile( 100%) The solvent strength of the mobile phases used in screening should be adjusted to obtain reasonable elution times by changing the proportion of the polar ( alcohol) modifier. ( CHIRALPAK, CHIRALCEL, AD, OD, OJ and AS are registered trademarks of Daicel Chemical Industries, Ltd.) Table 1. Screening conditions for HPLC Method Development Figure 1. Screening results for benzoin ethyl ether. Columns 250 x 4.6 mm. Mobile phase hexane : 2- propanol ( 85: 15), flow rate 1 ml/ min. Columns: 1: CHIRALCEL OD; 2: CHIRALPAK AD; 3: CHIRALPAK AS; 4: CHIRALCEL OF; 5: CHIRALCEL OB; 6: CHIRALCEL OG; 7: CHIRALCEL OJ.