Critical pairs in column chromatography

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LC-GC North America , 09/01/2003 21 9

Critical pairs in column chromatography: a primer for pharmaceutical method validation.(Advertising Supplement) Webster, Gregory K. *~|~*Basel, Christopher L. *~|~*

COPYRIGHT 2003 Advanstar Communications, Inc.

A column chromatographer's goal is to produce separation methods that provide baseline resolution of each analyte peak in a reasonably short analysis time. One way chromatographers have defined the effectiveness of their chromatographic separations is by monitoring the separation of a critical pair of components. A critical pair represents the two components of the chromatogram with the lowest calculated resolution between them. Although the concept of a critical pair of solutes in a chromatographic separation is seemingly straightforward, it is reinterpreted or used several ways. Often, analysts mistakenly note several critical pairs in their separation, when in fact only one appears. This article reviews several common applications of the critical-pair approach to column chromatography as it is applied under pharmaceutical guidelines and validation strategies. The Holy Grail for today's column chromatographers is the quest for separation methods that provide baselin!
e resolution of each analyte response from the sample matrix in a reasonably short analysis time. This process entails the development and search for efficient methods that can be applied to samples and matrices of increasing complexity. As any seasoned chromatographer knows, this quest often concludes with lower expectations than those at the beginning of the project.
Since the pioneering works of Snyder and Kirkland (1,2) and Giddings (3), chromatographers have defined the effectiveness of their chromatographic separations by the separation of a critical pair of components. A critical pair represents the two components of a chromatogram with the lowest calculated resolution between them. Accordingly, the resolution of the critical pair is called the critical resolution, and the pair is considered baseline resolved if the resolution ([R.sub.s]) between them is greater than or equal to 1.5. For simple applications, when the critical pair is separated, the remainder of the peaks in the chromatogram also will be separated. Whether this holds true under changing method conditions is specific to the robustness of the analytical method.
Although the concept of a critical pair of solutes in a chromatographic separation seemingly is straightforward, it has been our experience that the concept gets reinterpreted or used in several ways (4-9). Most separations have a single critical pair in their chromatographic profile, yet several critical pairs often are addressed in development. This error generally is the result of an analyst citing several close peaks as being critical. However, because these peak pairs behave correspondingly to changes in chromatographic conditions, the solutes with the minimum resolution are representative of the profile as a whole and should be the only assigned critical pair. Other resolution pairs in the chromarographic profile are important, but it is possible to profile a separation based on a single or the true critical pair--the solutes with the minimum resolution. Analysts often mistakenly note several critical pairs in their separation when in fact only one appears. If a sing!
le pair can represent the resolution necessary to sustain the integrity of the profile, it should be established as such.
Complex matrices can have more than one critical pair, if the solute pair with the critical resolution is nonindicative of the profile as a whole. As discussed below, this situation generally can be confirmed by robustness studies. As changes to the chromatographic conditions are applied, some solute peaks might not respond in the same manner. In this case, it is acceptable to assign additional pairs as critical. When inure than one critical pair exists, it often is indicative of complex matrices that involve analytes of varying chemistries.
In the pharmaceutical industry, the critical-pair approach takes on added accountability as methods are validated using industry guidelines (10-16). Chromatographers routinely use a method's critical pairs to conform to the policies of regulatory agencies. Critical pairs are either directly present or indirectly modeled in the resolution criteria for the analytical method and are an integral part of the establishment of system suitability. In addition, whether a method can serve as a stability-indicating method is established by the effectiveness of addressing the critical pair or pairs used with the active pharmaceutical ingredient and drug formulations.
The article reviews several common applications of the critical-pair approach to column chromatography as it is applied under pharmaceutical guidelines and validation strategies. The application of the critical-pair strategy is shown for a simple suitability standard and progresses to its application in robustness, degradation, and complex sample applications.
Critical Pairs and Pharmaceutical Method Guidelines
In its simplest case, the critical pair in a chromatographic separation is the two analytes with the minimum resolution between them. That minimum resolution is defined as the critical resolution. Obviously, a single-peak elution profile has no critical pair; for two components, analysts can find resolution only between the two responses. Interestingly, the current industry norm for single-component chromatographic investigations is to develop a method resolution standard by spiking a suitable standard into the standard matrix so that a resolution profile with a critical pair can be established and monitored. Although it is hard to say exactly how the term critical pair became part of the chromatographic vernacular, the extensive work of Snyder and Kirkland in this field certainly has been quite influential and should be credited.
Figure 1 is an example of a chromatogram produced by gradient liquid chromatography (LC). The three analytes (peaks 1-3) are fully resolved, and each of the peaks has a neighbor with a resolution greater than 1.5 (Table I). In this simple case, it is easy to discern that the critical pair for this separation comprises peaks 2 and 3. What does this designation tell us? At this point, the defining critical pair simply tells us that as long as the resolution is maintained at 2.1 between peak 2 and peak 3, the suitability of the separation for the profile is intact or as designed.
[FIGURE 1 OMITTED]
Current method validation guidelines--such as the International Conference on Harmonization of Technical Requirements for the Registration of Drugs for Human Use (ICH), the U.S. Pharmacopeia, and the European Pharmacopoeia--do not reference the term critical pair. The U.S. Food and Drug Administration (FDA) indirectly refers to this term in discussing selectivity for LC, gas chromatography, and capillary electrophoresis methods:
"If the analytical procedure is used to control their level of impurities, the minimum resolution between the active and the closest eluting impurity, or the two peaks eluting closest to each other, should be given" (17).
The referenced guidance also uses the term critical impurity, which is an impurity greater than the identification or qualification threshold. This impurity should not be confused as a component of the chromatographic critical pair, but it might be. Although the term critical pair is not directly referenced in the official guidance, critical pairs are used routinely in pharmaceutical laboratories to demonstrate that the intended separation method meets the criteria established in ICH and compendial guidances. In addition, the use of representative critical pairs in the resolution standard often overcomes the necessity to inventory impurity and degradant standards on a case-by-case basis. The concept of a representative pair is discussed in more detail below.
Routine Case--Critical Pair in a Standard or Sample
For chromatographic separations under pharmaceutical industry guidelines, separation scientists must establish the robustness of their analytical procedures. Typically, they establish robustness by two components: robustness against the variability of the column packing materials and robustness against the variability of method conditions. Variability in the column packing material can be evaluated using the resolution standard and samples representative of each stress condition in the accelerated degradation study in which degradation was observed. The column packing material is acceptable if the specificity and the resolution criterion are maintained. Variability of method conditions can be evaluated by altering the method conditions, one condition at a time, as listed below:
* [+ or -] 2% relative change in the volume of the lesser component (organic or aqueous) of the mobile phase (the larger component volume remains unchanged)
* [+ or -] 2[degrees]C change in column temperature
* [+ or -] 5% relative change in the mobile phase flow rate
* [+ or -] 0.1 pH units in mobile-phase pH
Suppose the separation in Figure 2 is a profile for a method being validated as a pharmaceutical stability-indicating method. To establish robustness under ICH guidelines, a separation scientist would inject the solution used to create Figure 2 under each of the above conditions and examine the change, if any, in the chromatographic result (Table II). The analyst would inspect the data to verify whether peaks 3 and 4 are still resolved and remain the critical pair. Assuming the critical-pair peaks (peaks 3 and 4) remain the critical pair throughout the robustness study, the minimum resolution between these critical-pair peaks from this study becomes the foundation for the resolution specification for suitability of the method. The analytical method then would specify that as long as the resolution of this pair is maintained at or above this critical value, the method is suitable for use and the integrity of the chromatographic separation is maintained. This information is !
valuable to separation scientists because it is used for establishing system suitability and as the reference for establishing that integrity of the system is maintained throughout individual runs. In addition, it can be used as a diagnostic tool for the analytical column. Typically, resolution deteriorates as a column ages, so knowing the critical pair provides reference point for establishing that the chromatographic integrity is unaffected.
[FIGURE 2 OMITTED]
Rather than injecting a sample from each stress condition in the accelerated degradation study, this experiment can be shortened greatly after establishing the critical pair in the profile of each of these stress condition and then performing the robustness stud with the solutions that represent these critical pairs. If a single critical pair is established for all the degradation conditions, the robustness study can be executed solely wit a single solution that contains this critic pair in the matrix profile. It is common for a degradation product to be in the critical pair and also to be produced under several stress conditions. Thus, it is not necessary t repeat the analysis of this degradant simply because another degradation pathway produced it.
Typically, the resolution and tailing facto criteria established for the critical pair must be met for each robustness investigation condition. In doing so, separation scientist establish a great deal of control for the analytical method. In all cases in which critical resolution values are calculated, analyst should give appropriate consideration to repeating the study and reporting a confidence interval near the critical resolution.
Critical-Pair Modeling
In the previous example, separation scientists traced the effect of the robustness conditions upon the critical pair. How the critical pair responds to each stress also should be compared with the other responses in the profile. Why? Typically, the chromatographic critical pair is established with a degradant or process impurity, often at a time before a reference standard for the compound has been produced. Thus, system suitability commonly is established using retained samples from the validation study. After the degradant or impurity standard is produced, the method suitability is established by spiking this component into the method standard matrix. Not only is this process cumbersome to the routine assay, it often is unnecessary. If the critical pair is responding in a representative manner to other peaks in the profile during the robustness studies, these other peaks (the representative pair) can be used to model the actual critical-pair components. Thus the actual c!
ritical-pair components need not be present in the method's suitability or resolution solution. By maintaining the representative pair at greater than an established minimum criterion, analysts can assure that the method will maintain the resolution of the critical pair, if it had been present. For example, a minimum criterion for resolution might be set at 10, which seems absurdly higher than 1.5. What the method author likely was conveying is that if the resolution of X and Y is greater than 10, the chromatographic profile is suitable to ensure the resolution of the components produced under stress and validation conditions, including the true critical pair.
Suppose in Figure 2 that peak 3 is a degradant produced from stressing the product sample, peaks 1 and 4 are preservatives, and peak 2 is the active pharmaceutical ingredient. The result is illustrated in Figure 3a (Table III), which leaves the remaining peaks as impurities. During the robustness investigations, the critical pair between the degradant and preservative 2 was assessed under various analytical conditions. Changing the gradient from the normal condition of 45-75% mobile phase B to 45.9-75% mobile phase B resulted in a minimum resolution of 1.9 for the critical pair. Because the resolution of the active pharmaceutical ingredient and preservative 2 changed in a manner that was parallel to the critical pair as the method conditions changed, this information can be used to determine minimum resolution of the critical pair indirectly. When the critical-pair resolution is 1.9, the resolution of the active pharmaceutical ingredient and preservative 2 peaks is 9.7. As!
long as the chromatographic system maintains the resolution of the active pharmaceutical ingredient and preservative 2 greater than 9.7, the validation data confirm that the critical pair of the separation also will be maintained. Thus, the method resolution standard does not need to be spiked with the degradant to validate separation of the critical pair; the resolution solution simply needs to reflect the critical pair criteria, which it does indirectly using the active pharmaceutical ingredient and preservative 2 standard (the representative pair), and reduces to Figure 3b (Table IV).
[FIGURE 3 OMITTED]
Multiple Critical Pairs
As pharmaceuticals become more complex, so do their associated chromatograms. Separation scientists in the pharmaceutical industry routinely are challenged by chromatographic profiles that contain more than one critical pair. Does this outcome mean that many band pairs all result in the same minimum value? No, multiple band pairs present in the chromatographic profile do not respond in the same representative manner as the traditional critical pair with the lowest calculated resolution. How can this result happen? Usually, it is due to small differences in chemistry between the chromatographic analytes and the target pharmaceutical ingredient. The sources of these components are typically synthesis impurities, fermentation by-products, or intended formulation components.
In the previous section, the critical pair was monitored to qualify the method's robustness. Robustness testing produced a corresponding change in the separation of the chromatographic profile. In Figure 4a, the chromatographic profile of a fermentation product illustrates two critical pairs labeled CP1 and CP2. The pair designated CP1 has the minimum resolution, but the analytes are not considered important to the product and are not monitored for suitability. CP2 is an important band pair and is designated a critical pair for the method because it has the next lowest resolution value. The robustness of this method was challenged at another temperature. The separation profile, illustrated in Figure 4b, shows two additional band pairs that yield resolution at 1.5. The resolution between these additional pairs--CP3 and CP4--was affected by these conditions (Table V) and required surveillance by an analyst to maintain the integrity of the separation. The band pairs at CP3 an!
d CP4 also are labeled as critical pairs for the suitability of the method and need to be monitored during chromatographic analyses. Because the suitability of the separation must be established with two sets of peaks, the separation can be referred to as having multiple critical pairs.
[FIGURE 4 OMITTED]
The Concept of Maximum Resolution
Good manufacturing guidelines require that regulated processes establish and maintain control of the intended process. The establishment of a resolution range using a maximum-resolution parameter in the suitability section of a chromatographic method is an additional attempt to ensure the method maintains its performance integrity and control. Maximum resolution is defined as the greatest value that can exist between the critical pair and still maintain resolution between the remaining components in the chromatographic profile. The resolution range is the established suitability of the method between the minimum and maximum resolution of the critical pair. The need to establish a maximum resolution and corresponding resolution range occurs when a change in a method condition improves the resolution between the critical pair but at the same time decreases resolution between some other band pair in the chromatographic profile. Commonly in pharmaceutical separations, one anal!
yte peak moves to longer retention times at a faster rate than the next peak, which causes potential coelution issues. If the maximum resolution factor was omitted, separation scientists might assume that the chromatography was quite good because the minimum resolution had increased by a large amount; however, in reality the overall separation of the chromatographic profile could have become worse.
Figure 5 shows a simplified example of how a defined maximum resolution determination would be necessary. Table VI lists the resolution values. Figure 5b represents the routine method conditions, and the minimum resolution calculated between the critical pair (peaks 2 and 3) was 1.9. Under a new set of chromatographic conditions, such as a robustness challenge, peak 4 moved to a longer retention time at a faster rate than peak 5. Under these conditions, as Figure 5b shows, a new critical pair was established in peaks 4 and 5. The investigation was further challenged to a point at which the separation between peaks 4 and 5 decreased to baseline resolution ([R.sub.s] = 1.5). In this profile, the resolution factor calculated for the original critical pair (peaks 2 and 3) was found to be 2.9. This resolution is the largest value the original critical pair could have, and it still yields acceptable resolution throughout the chromatographic profile--the maximum resolution. When !
the resolution of the critical pair is kept within the resolution range between the minimum and maximum values, it ensures that all the components of the chromatographic profile are separated.
[FIGURE 5 OMITTED]
The use of maximum resolution parameters has not yet become routine practice in the pharmaceutical industry. We believe the practice should be incorporated into methods in which the robustness of the method warrants such a designation.
Conclusion
Pharmaceutical guidelines, in accordance with current good manufacturing practice regulations, require analytical methods to exhibit and maintain control of the system used. Pharmaceutical scientists are challenged by the need to control each component that responds at 0.1% of the active pharmaceutical ingredient or higher, and this control in terms of specificity and robustness becomes increasingly difficult as the complexity of pharmaceutical matrices evolves. The application of critical-pair principles to a chromatographic separation allows separation scientists to effectively challenge analytical methods and also to maintain and safeguard the integrity of the separation profile.
<pre>
Table 1: Resolution values for Figure 1

Peak Pairs [R.sub.s]

Peaks 1 and 2 7.81
Peaks 2 and 3 2.10

Table II: Resolution values for Figure 2

Peak Pairs [R.sub.s]

Peaks 1 and 2 9.60
Peaks 2 and 3 7.81
Peaks 3 and 4 2.10
Peaks 4 and 5 7.39
Peaks 5 and 6 16.21

Table III: Resolution values for Figure 3

Peak Pairs [R.sub.s]

Preservative 1 and the active
pharmaceutical ingredient 9.60
Active pharmaceutical ingredient
and degradant 7.81
Critical pair: degradant and
preservative 2 2.10
Preservative 2 and impurity 1 7.39
Impurity 1 and impurity 2 16.21

Table IV: Method suitability for Figure 3b

Peak Pairs [R.sub.s]

Preservative 1 and the active 9.62
pharmaceutical ingredient
Active pharmaceutical ingredient
and preservative 2 9.90

Table V: Resolution values for maximum resolution example

Peak Pairs [R.sub.s] [R.sub.s] under Challenged Condition

CP1 0.8 0.5
CP2 1.6 1.4
CP3 2.69 1.5
CP4 2.0 1.5

Table VI: Resolution values for maximum resolution example

[R.sub.s] under
Peak Pairs Original [R.sub.s] Challenged Condition

Peaks 1 and 2 4.1 4.6
Peaks 2 and 3 1.9 2.9
Peaks 3 and 4 5.1 5.5
Peaks 4 and 5 2.6 1.5 </pre>

Acknowledgments
The authors would like to thank Patrick Lukulay and Angel Diaz of Pfizer Corp. for reviewing this manuscript and Larry Thomas and Bill Sanders of Bayer Corp. for their role with the authors in the application of critical-pair modeling.
References
(1) L.R. Snyder and J.J. Kirkland, Introduction to Modern Liquid Chromatography (John Wiley &amp; Sons, New York, 1979).
(2) L.R. Snyder, J.L. Glajch, and J.J. Kirkland, Practical HPLC Method Development (John Wiley &amp; Sons, New York, 2nd ed., 1997).
(3) J.C. Giddings, Unified Separation Science (John Wiley &amp; Sons, New York, 1991).
(4) H.J. Reiger and I. Molnar, J. Chromatogr. A 948, 43-49 (2002).
(5) C.A. Cramers and P.A. Leclercq, J. Chromatogr. A 842, 3-13 (1999).
(6) C.A. Cramers, H.G. Janssen, M.M. van Deursen, and P.A. Leclercq, J. Chromatogr. A 856, 315-329 (1999).
(7) T. Mirza and H.S.I. Tan, J. Pharm. Biomed. Anal. 17, 142-1428 (1998).
(8) V.M. Morris, J.G. Hughes, and P.J. Marriott, J. Chromatogr. A 755, 235-243 (1996).
(9) P. Jandera, J. Chromatogr. A 797, 11-22 (1998).
(10) Guideline on the Validation of Analytical Procedures: Methodology (1) (International Conference on Harmonization of Technical Requirements for the Registration of Drugs for Human Use, Geneva, Switzerland, 9 May 1997).
(11) VICH GL2 (International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products, Brussels, Belgium, October 1998).
(12) Code of Federal Regulations, Title 21, Foods and Drugs (U.S. Government Printing Office, Washington D.C., 1 April 1997), Part 211, pp. 83-103.
(13) USP 24 (United States Pharmacopoeial Convention, Rockville, Maryland, 1999), pp. 2149-2152.
(14) J.M. Green, Anal. Chem. 68, 305A-309A (1996).
(15) M.E. Swartz and I.S. Krull, Pharm. Technol. 22(3), 104-119 (1998).
(16) W.E. Weiser, Analytical Validation, Pharm. Technol. 1998, 20-29 (1998).
(17) U.S. FDA Guidance for Industry: Analytical Procedures and Methods Validation Chemistry, Manufacturing and Controls Documentation (U.S. Food and Drug Administration, Rockville, Maryland, August 2000).
Gregory K. Webster and Christopher L. Basel *
Analytical Research and Development, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, Michigan 48105
* Bayer HealthCare, Animal Health Division, RO. Box 390, Shawnee Mission, Kansas 66201-0390
Address correspondence to G.K. Webster

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Disposable filtration lightens cleaning and validation load

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Pharmaceutical Technology , 09/01/2003 27 9

Disposable filtration lightens cleaning and validation load: pharmaceutical makers see multiple advantages of single-use systems: single-use filtration technology is becoming increasingly popular as manufacturers seek to cut costs and minimize processing times.(Special Report) Rios, Maribel *~|~*

COPYRIGHT 2003 Advanstar Communications, Inc.

The latest components in sterile filtration technology are not made to last. In fact, pharmaceutical manufacturers are throwing them out after every batch filtered. Tossed out with them, however, are the expense and time of cleaning, cleaning validation, and maintenance as well as the concerns of cross-contamination associated with multi-purpose production lines. Single-use systems are earning double takes from manufacturers impressed with the results from disposable assemblies designed specifically for drug development. But the plastic reconfigurations of pharmaceutical processes are doing more than trimming timelines and fattening revenues. Disposable manufacturing has triggered a new approach to risk management and safety--aspects that have made it an appealing choice for contract service providers and manufacturers of both conventional and biotech pharmaceuticals.
Before manufacturers can consider switching to single-use filtration systems, however, they must have an understanding of their design and performance characteristics as well as their potential effect on manufacturing, testing, and validation le procedures.
Evaluating alternatives
Single-use filter capsules consist of disposable filter cartridges encapsulated in disposable housings typically constructed of gamma-stable polypropylene or polyetherimide. Although several filter capsule models are available, they share many essential characteristics (see sidebar, "Ideal attributes of single-use filter capsules"). And, as explained by Holly Haughney, PhD, vice-president of BioPharmaceuticals Marketing at Pall BioPharmaceuticals (East Hills, NY), "All capsules must provide the same particulate retention and media flow as reusable systems as well as have the same form, fit, and function as a traditional system."
Features such as ease of use, reduced process times, increased personnel safety, and tighter risk--control management have all helped make disposable units an attractive alternative for many applications, and most filtration companies have broadened their product lines to include both stainless steel and disposable designs. As observed by Steve Tingley, director of biopharmaceutical manufacturing at Millipore Corporation (Bedford, MA), "if an application demands a more-retentive, less-expensive, or faster-flowing membrane, that need will have to be met in both reusable and disposable configurations." Choosing the right components then becomes more of a process rather than a selection by rote. Says Tingley, "This provides the end user much more freedom to choose a filter pore size, membrane, and filter device type."
Although the most noticeable impact of switching to disposable systems is the avoidance of the cleaning and cleaning validation requirements associated with reuseable components, the incorporation of a disposable system into an existing fluid path does require a re-evaluation of cleaning procedures, sterilization methods, and connections to other components in the filtration line. Manufacturers must also reconsider their approaches to complying with regulatory testing and validation requirements.
Sterilization
Stainless steel systems are sterilized by following either autoclave or steam-in-place (SIP) procedures. However, most polymer-based housings are not designed for the conditions of in situ steam sterilization because of the high differential pressures (typically 15 psi) that can develop at the elevated temperature for steam sterilization (typically between 121 and 140[degrees]C). Thus, most disposable filter capsules must undergo autoclave sterilization and be connected under aseptic conditions or be purchased as presterilized units or as part of larger configurations.
If filter capsules are to be used with other disposable components such as disposable bags, tubes, and connectors, the entire system can be sent to a contract sterilizer. However, as Tingley observes, "a presterilized component cannot simply be dropped into a stainless steel process." Operators must have a procedure for connecting that presterilized component with other components of the sterilization process, which may or may not be disposable units (e.g., stainless steel tanks). "In situ steaming is not a good option, so people are looking toward other technologies," says Tingley, "including Filters that are validated for irradiation sterilization, gamma irradiation being the method of choice at the moment."
Choosing filtration assemblies that have been sterilized by gamma irradiation eliminates the need for on-site sterilization and sterilization validation. However, manufacturers must ensure that the materials are suitable for gamma sterilization. Such materials include polyvinylidiene fluoride, stabilized polypropylene, nylon, and polyethersulfone. Unstable polypropylene and PTFE cannot undergo sterilization by gamma irradiation. Hydrophobic PTFE filters are used for air or gas filtration and vent filtration applications. For disposable systems, hydrophobic PVDF filters can be used because they can be sterilized by gamma irradiation.
Connections
Regardless of the sterilization method, sterility can only be maintained through the use of appropriate connection devices under aseptic conditions. Because of the range of applications for which disposable systems can be used, there has been an increased demand for complex, specialized configurations customized to particular applications.
According to Pall's Haughney, pharmaceutical manufacturers are increasingly interested in coupled, manifolded systems. "We've seen tremendous interest in terms of the size and the flexibility that these give," says Haughney. "We've also seen more and more companies having multiple steps in their filtration process," she adds.
For example, a polypropylene prefilter can be coupled to a 0.2-[micro]m filter, which is then coupled to a virus filter. These filters can be coupled together in one disposable unit that can also include supplemental equipment such as tubing, aseptic connectors, and bags. The entire assembly can be packaged and then subjected to gamma irradiation so that it is ready for use directly upon receipt. Multiple 10-in. filters also can be coupled together in systems that have integrated disposable polypropylene valves, pressure gauges, and flow detection devices.
In some cases, a connection between a stainless steel unit such as a tank and a disposable filter capsule is required. For example, Millipore has designed a small-valve connector for use between presterilized disposable assemblies such as plastic tubing or filters and stainless steel systems. The Lynx ST connector can be preassembled to the disposable fluid path and gamma sterilized. SIP procedures can then be used to sterilize the stainless steel equipment, piping, and the connector interface before fluid is transferred or sampled. Because the connector is a closed valve, no steam gets into the line, thereby preventing damage to the plastic tubing or filter.
Haughney says disposable connectors are also eliminating the need for bulky laminar-flow hoods where many aseptic connections must currently be made. Pall, for example, recently introduced an aseptic connector that doesn't require a hood or other capital equipment to maintain sterility. The Kleenpak connector can be used in cases in which two pieces of flexible tubing need to be aseptically connected in an uncontrolled environment.
Colder Products Co, (St. Paul, MN) has also introduced a disposable connection device for making sterile connections without the need for a laminar hood. Its Steam Thru device allows sterilization of a biopharmaceutical flow path and initiates connections between a bioreactor and media bag without plumbing connections.
Validation
Validation and compliance remain top concerns for those responsible for ensuring sterile filtration lines. "Disposable manufacturing does not significantly change the science and technology of sterile filtration," says Millipore's Tingley, "but it does change the philosophical approach to the process that you put together and end up assembling and running." For example, Tingley points out that although the use of presterilized disposable systems removes the need to worry about steam sterilization of the filter element, the end user will still have to question the supplier. "Does the supplier have a certificate of sterilization from the irradiator? Can they prove that your product is sterile? That's a shift in the burden from the manufacturer to the supplier."
In addition, although the integrity test requirements for a disposable system are the same as for a traditional system, the user will have to work out a procedure for testing the filter once it's in a disposable format. Similarly, procedures for the validation of extractables will have to be developed. "Again it's a custom validation," explains Tingley. "In disposable manufacturing, more plastics may be involved. In addition to the tubing, you may have the bag, the containment, and perhaps the valves. So the extractables validation is still done. It's done in exactly the same way but instead of being done on the filter, it's done on the entire fluid path" (see sidebar "Validation comparisons").
Filter-capsule qualification, which includes product-material challenge testing as well as extractables and leachables testing, is typically conducted at the filter company. For example, Malk Jornitz, group vice-president of global product management bioprocess at Sartorius Corporation (Edgewood, NY), says validation at Sartorius can be conducted using two methods: "one that includes a choice of using model solvents, depending on the base solvent used within the process, or testing with the client's actual drug product under the client's process conditions."
In either case, the type of filter capsule must be carefully considered. "One of the major problems I see in the industry," says Jornitz, "is that clients don't take into account scalability factors." For example, he points out that when filterability studies are performed, one can use flat filter composites; however, these results can't be used to scale-up linearly because the flow dynamics are different. "If you go through the clinical phases with small-scale volumes, you don't want to revalidate the entire process when you scale up to process batches. That's why we not only scale up these 10-, 20-, and 30-in. filters, but we also scale down from 300 [cm.sup.2] to 150 [cm.sup.2]."
Time and cost
Depending on the complexity and scale of the application, disposable systems can often lead to significant time and cost savings by eliminating the need for preparation and installation of the filter element, cleaning, sterilization, and maintenance. In reuseable configurations, each of these steps must be documented and records must be kept to ensure proper procedures have been followed. But, as pointed out by Pall's Haughney, "With preassembled disposable capsules, the time and labor for assembly, and the documentation associated with that, goes away."
Barry Bardo, director of business development at Meissner Filtration Products (Camarillo, CA), agrees. "Manufacturers want to condense the time requirements associated with the acquisition, installation, and maintenance of more capital-intensive stainless steel filter systems." Because single-use filter capsules are packaged as off-theshelf units, the time required to prepare and set up the filter assemblies is dramatically reduced. Says Millipore's Tingley, "What you gain going to ready-to-use manufacturing is a reduction in validation burden--no CIP validation, minimized sterilization validation burden, and significant time savings. The end user just unpacks and assembles the components."
Although the cost of a single-use filter capsule is typically higher than stainless steel cartridges, Bardo says the operating-expense savings more than overcome the additional per-unit costs. Tingley agrees. "The premium of a high-capacity capsule over a regular cartridge is not that high, and there's clear evidence that this initial cost is offset by the overall savings from implementing disposable systems. End users need to look at their total processes and value all of their costs." Tingley observes that cost considerations should include more than just time saved in cleaning, maintenance, and assembly. "Every time you run that steam clean generator, there's electricity, waste water, waste chemicals, and power. A company must look at all of that."
Risk control and safety
Another prevalent concern for pharmaceutical manufacturers is ensuring minimal contact between personnel and product. "The beauty of disposability," says Sartorius' Jornitz, "is that people don't come into contact with potentially high-potent drug products."
On the flip side, Tingley points out that contact with people is also the single greatest risk associated with final fill and finish. "Companies are going to extraordinary lengths to keep people out of the process, and disposable systems help with this because people don't have to make aseptic connections or run SIP procedures. It's like an extra barrier."
Biotech pharmaceuticals leading the way
Although the time and labor saved by eliminating cleaning validation can be compelling factors in deciding whether to use a disposable system, the major driving force propelling the trend to disposables has clearly been the development of biotechnology-derived drugs.
As described by Jerold M. Martin, senior vice-president and global technical director at Pall BioPharmaceuticals, the manufacture of biological drugs has historically required the use of dedicated facilities and dedicated equipment where it was reasonable to put in a stainless steel system because only a single drug product or vaccine was being handled. "In those days," says Martin, "operators were primarily concerned that cleaning prevent lot-to-lot contamination, and insignificant levels of carryover were allowed."
Today, however, the situation is different, with the potency of biotech drugs at such high levels that companies can manufacture a year's supply in a very short period of time. As a result, biotech companies have begun to manufacture multiple products on the same line, which has in turn led FDA to place renewed pressure on them to develop exhaustive cleaning methods, prove the effectiveness of these methods, and prove that any residual cleaning agents are removed. "This became so burdensome," says Martin, "that the industry began to look for ways to avoid it altogether. So, they looked to disposable technology."
Biological entities are usually low-volume, high-value products. Sartorius' Jornitz envisions that "at one point the industry will have a completely disposable facility for these products, which means a disposable upstream, feed stream system, into a disposable bioreactor, then into a disposable cell harvest, a disposable purification column, disposable final filtration, and even a disposable filling system."
Although current applicability is best suited for these low-volume conditions, Jornitz predicts that it's just a matter of time before completely disposable systems are available at the larger scale. "It will happen," he says, "and there are already developments in the pipeline to achieve this goal, including bioreactors, tank linings, and filling systems."
Facing the viral clearance challenge
The threat of contamination from viruses such as West Nile and the virus believed behind severe acute respiratory syndrome as well as organisms such as mycoplasma has placed stringent demands on biotech pharmaceutical manufacturers to prove the purity of their products. As observed by Pall's Martin, the concerns the industry faces about being able to validate their virus filtration systems and whether they have been properly recleaned and resterilized looms so large that many of these companies are abandoning reusable virus filters and incorporating disposable systems instead. To this end, some disposable filtration technology has been specifically designed for the biotech arena. But, there are still several concerns to be resolved.
Hazel Aranha, PhD, manager of bioprocess applications at Pall Corporation (East Hills, NY), observes that virus clearance today is "not an option but a requirement for biopharmaceuticals and biotech-derived products." Any product that is made either directly from human plasma or has any kind of human or animal component in it must be validated for virus clearance.
Examples of products that must be validated for virus clearance include many of the tissue culture cell lines that are often supplemented with serum (e.g., bovine serum) and anything that uses serum during manufacture. According to Aranha, "theoretically you shouldn't have any virus in your product itself. If your product is plasma derived and if the plasma is contaminated with pathogenic viruses like HIV, Hepatitis B and C, the viral contaminants would be detected and the plasma quarantined as current regulations require that all blood be screened for these viruses." However, she adds, "it is possible that plasma may contain very low levels of infectious virus which cannot be detected by current detection systems."
Theoretically, the raw materials, whether plasma-derived or biotech, should not be contaminated with virus. "The primary reason that regulators require viral clearance steps in the process is essentially to document just in case virus has entered your systems," says Aranha, "and that your processes are adequate to provide enough virus clearance." One example, notes Aranha, is the industry's concern last year over West Nile virus. "The industries and blood banks dealing with whole blood considered it as a major concern," she says. "However, manufacturers of plasma-derived products such as clotting factors and enzymes sourced from human plasma were less concerned."
Aranha explains that this was because plasma products are highly processed and their manufacturing operations include viral clearance steps that would clear West Nile virus. "Currently, validation studies conducted to document viral clearance, and thus a level of viral safety assurance, often include a virus belonging to the same family as West Nile virus," she says.
Filters that can completely remove 100% of the bacteria that are in a fluid have been developed. But, in the case of viruses, no test exists that can confirm that no viruses are present. "It is impossible to prove this," says Martin, "but you can remove this question by simply using several different [orthogonal] virus clearance methods."
Many steps in a protein purification process can also either remove or inactivate viruses. For example, notes Martin, "a chromatography column for purifying proteins might also remove viruses, and you could elute the protein off the column in a way that some of the viruses still stay on the column. Or you may be able to stick the protein onto a column and wash viruses through." Martin also points out that FDA considers filtration to be an additional step in the purification process that further enhances the clearance factor and therefore increases the safety of the drug.
Balancing flow and viral clearance. Another challenge associated with viral clearance is finding ways to increase flow while maintaining or even improving filtration efficiency. Several approaches can be used to increase the flow, including modifying the physical characteristics of the filtration system such as increasing the area of membrane within a cartridge or making the membrane thinner. According to Martin, the objective is finding the right balance. "If you make the membrane thinner, then you need to narrow the pore-size distribution to maintain the same virus removal rate." However, as the pore-size distribution narrows, the filter is more liable to plugging. "So," says Martin, "there's a lot of R&amp;D going on right now to develop next generation filters that will have the same or higher titer reductions while increasing the flow."
In addition, Meissner's Bardo says filter companies are increasingly called upon to provide 0.04-[micro]m polyethersulfone systems, usually in combination with other technologies, but also as a virus reduction step. To this end, the company provides a unit that can be used in conjunction with other systems and perform similarly to a prefilter (StyLUX 0.04-[micro]m filter).
"The trick there is that you're really reaching the edge of what so-called microfiltration technology can do," says Bardo. "The pores are getting so tight that it almost risks becoming a solid. You can't get the very smallest viruses, but you can remove some of the larger ones and still have a reasonable amount of flow through such a filter." However, if a membrane gets much tighter, warns Bardo, "the good news is that you remove everything, the bad news is that it flows very slowly. That's the trade-off--viral clearance versus flow. The goal is to improve the flow but maintain or if possible even improve viral clearance."
Mycoplasma removal. Unlike a virus, mycoplasma is a bacteria-like microorganism that has a characteristic called pleomorphic, which means that it will deform and ooze through structures like filters that would otherwise retain more rigid-walled bacteria. A mycoplasma infection can shut down production or development because the small size and absence of a cell wall of mycoplasma enables it to penetrate 0.2/0.22-[micro]m filters.
The presence of mycoplasma in the industry resulted in a new generation of filtration systems (e.g., Pall's Fluorodyne II 0.1 micron DJLP filter and Meissner's SC0.1 version of its StyLUX filter). "Combined with biotech demands on sterility, disposable systems would be best suited for this type of process as opposed to stainless steel systems," says Martin. According to Bardo, a company can have a sterilizing filter that sieves out all the bacteria, but the mycoplasma will still get through many of them. "So as manufacturers, we have to have validated mycoplasma retentive filters," he says.
Future challenges. Although much has been accomplished in the area of viral clearance, Martin recognizes the following three areas of concern yet to be fully resolved.
Need for a rating system for virus filters. Every filter manufacturers qualifies its filters in different ways, and they apply different rating standards. That has created some confusion because there is no standard system by which the filters are rated. There is a standard definition for bacterial removal filter, but there is no such standardized definition for virus filters. According to Martin, a PDA task force is working to develop a standard qualification protocol using a bacteriophage, which is a virus of bacteria. The committee also is writing a technical report that would be an industry-based study about how to select and qualify virus filters. The goal is to address the questions that challenge the users in terms of how best to select a virus filter. Jornitz adds, "viral filtration cannot be compared with bacterial filtration. It is by far more complex and requires evaluation, qualification, and validation of every single application. Often too much emphasis is pu!
t on retention ratings. Nevertheless, the true performance of the filter used can only be measured by appropriate process validation."
Streamlining virus-challenge testing methods. Current FDA guidelines require that a biotech or biological manufacturer generate data with multiple different mammalian viruses to qualify a particular viral clearance step. Several viral clearance steps are involved in the process, which means that a drug manufacturer must challenge several steps, each with multiple different viruses. "Challenges with mammalian viruses are time consuming and expensive," says Martin, "and this can be a large burden on the drug manufacturer; first do to it once and then second if they wanted to change any step in the process, then they have to go through this whole procedure again." Martin explains that researchers have shown that for virus filters--which remove viruses by size exclusion--viruses of bacteria (i.e., a bacteriophage) can be used as models for the mammalian viruses and can then be applied in bacterial virus challenges. These challenges are very rapid and inexpensive to conduct and!
to predict what the filter performance is going to be with the mammalian viruses. In turn, this process enables a drug manufacturer to test out their process development steps quickly and inexpensively.
Understanding the integrity-testing process. The purpose of the integrity test is to confirm that the actual process filter is going to perform at the same level as filters that were used in a validation exercise. No one can take a full-scale process and challenge it with viruses to prove that it works, so filters usually are qualified using very small disks or modules. The integrity test that goes into the production process has to predict the same performance. Martin believes there is a growing need to have these integrity tests be sensitive enough to predict virus removal and to be practical and useful in a production environment. "Many of the integrity tests that have been applied on the lab-bench scale and seem adequate on small-scale filters end up being very problematic on the production floor," he says, "and that is something that actually doesn't get considered when a drug is submitted for approval by FDA. It doesn't come into play until the drug is approved and f!
alls under GMP manufacturing."
A permanent place in the industry
The extent to which disposables will successfully invade the pharmaceutical filtration arena remains to be seen. Millipore's Tingley says he can envision an all-plastic factory or a factory that would contain a manufacturing process all in plastic. "You would just take it out of a box, unroll it, do your processing, and throw it away." However, he adds that "in the shorter term, we're seeing end users with stainless steel in place that want to integrate these disposables with existing stainless steel systems."
Sartorius' Jornitz doesn't see disposable or single-use systems ever completely replacing stainless steel, but says that "they are definitely an alternative that's here to stay. They may perhaps be the solution of choice for biotech applications, and for start-ups, where they can help reduce the tremendous capital investments necessary."
Contract manufacturers, in particular, have seen the advantages offered by single-use filtration systems. "These companies have such a large number of different chemicals in their manufacturing," says Tingley, "that they either have to have a unique set of manufacturing equipment for each drug, or they have to have very clear validation for the cleaning processes, so that they can guarantee that they are not going to have cross contamination from one drug to another." Contract service providers also don't have a lot of time to change or develop new processes. "First and foremost, they are manufacturers," he adds, "so eliminating cleaning and cleaning validation saves them extensive amounts of time."
Whether disposable filtration will remain an alternative or become the predominant methodology, its multiple advantages are clearly already having a major effect on pharmaceutical manufacturing. As observed by Tingley, "anybody who's putting in a new process, anybody who's expanding a manufacturing facility, anybody who's putting in a new manufacturing facility, or anybody who's dealing with biohazard materials is a perfect candidate for this technology."
Ideal attributes of single-use filter capsules
* 2-3 year shelf life
* scalable
* easy hook up to integrity test equipment (convenient placement of vent and drain ports)
* low hold up design
* ease of use (in-line and T-style designs available, multiple inlet/outlet options, various mounting configurations)
* compatible with various filtration media and membranes
* can be used with existing stainless steel components or with other disposable units with appropriate connections.
<pre>
Validation comparison

Stainless steel system Disposable system

* validate sterilization process No SIP. Can be autoclaved and
aseptically connected or
purchased presterilized (gamma
irradiated)
* validate cleaning system No CIP validation necessary
* integrity test before and after use Same requirements, commonly
available as a service from
supplier
* bacterial retention validation Same requirements
* validation of the extractables Required, though may be done
on the entire assembly,
including bags, tubings,
connections, and valves. </pre>

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Advanced Computer Systems Validation For Pharmaceutical, Biotech & Medical Device

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M2 Presswire , 09/09/2003

Advanced Computer Systems Validation For Pharmaceutical, Biotech &amp; Medical Device Companies 17th &amp; 18th November 2003, The Hatton, London -- Supported by: ABB Eutech -- Visit us at http://www.smi-online.co.uk/csvadvanced8.asp.

COPYRIGHT 2003 Ingram Investment Ltd.

M2 PRESSWIRE-9 September 2003-SMI: Advanced Computer Systems Validation For Pharmaceutical, Biotech &amp; Medical Device Companies 17th &amp; 18th November 2003, The Hatton, London -- Supported by: ABB Eutech -- Visit us at http://www.smi-online.co.uk/csvadvanced8.asp(C)1994-2003 M2 COMMUNICATIONS LTD RDATE:09092003
PLUS A HALF-DAY POST-CONFERENCE EXECUTIVE BRIEFING Documentation Requirements for CSV - The 'How-To's' of Computer System Validation Documentation 19th November 2003, The Hatton, London
PLUS A FULL-DAY POST-CONFERENCE EXECUTIVE BRIEFING Technical Aspects of Implementing 21 CFR Part 11 19th November 2003, The Hatton, London In association with Systonomy
This Conference will provide a forum for validation professionals to hear about the technical implementation of legislation governing the validation of computer systems. It will provide the very latest update on 21 CFR Part 11, GAMP regulations and compliance issues, including audit trends, common pitfalls and an evaluation of FDA warning letters. It will also look at the tools and concepts of Six Sigma.
SMi would also like to draw your attention to our corresponding event: Computer Systems Validation: Key focus: Laboratory Computer Systems - For Pharmaceutical, Biotech and Medical Device Companies, being held on 22nd &amp; 23rd September 2003.
This essential meeting will help you:
* UNDERSTAND technical aspects of complying with processes and procedures
* OPTIMISE system prioritisation - GAP analysis &amp; Part 11 assessment
* MAXIMISE benefits of legacy systems validation
* MITIGATE enforcement actions in the event your organisation is cited for process control deficiencies
* EVALUATE the new FDA Warning Letter review process
* ASSESS the latest inspection and enforcement practices
* LEARN about the risk-based approach to Part 11 compliance
* DISCOVER the process and benefits of using Six Sigma for computerised systems validation
Gain an insight from the key industry experts in the field:
* KLAUS KRAUSE, Corporate Manager, Computer Systems Compliance, AMGEN
* RON ARMSTRONG, Manager, R&amp;D QA, Computer Systems Compliance, BOEHRINGER INGELHEIM
* MICHAEL BRITT, Site Manager, Systems Validation Services, ROCHE
* DR ROBERT STEPHENSON, Regulatory Systems Team Leader, PGMIT, PFIZER
* DR LUDWIG HUBER, Product Marketing Manager, AGILENT TECHNOLOGIES
* Dr TERI STOKES, Director, GXP International
* KATE SAMWAYS, Director, KAS Associates &amp; Secretary, EUROPEAN STEERING COMMITTEE (GAMP)
* RON JOHNSON, Executive Vice President, QUINTILES (FORMERLY FDA)
* CAROLYN STOCKDALE, Manager, Quality &amp; Regulatory Compliance - Validation, PHASE FORWARD
The SMi Group is a world leader in business to business information with products spanning over 250 countries - within the past year over 30,000 senior executives from blue chip companies have benefited from SMi's highly targeted conferences, executive briefings, newsletters and management reports.
Website: http://www.smi-online.co.uk/csvadvanced8.asp

Katy West Marketing Department SMi Conferences Ltd Tel: +44 (0) 20 7827 6108 Fax: +44 (0) 20 7827 6109 Email: mailto:kwest@smi-online.co.uk
Please scroll down to view the full Conference programme.
Day One - 17th November 2003
8.30 Registration &amp; Coffee
9.00 Chairperson's Opening Remarks Dr Teri Stokes, Director, GXP International
KEYNOTE ADDRESS: CSV MODELS 9.10 IQ, QQ, PQ in computer validation - the who, what and why
* Who does what in CSV and why?
* What is computerised system validation (CSV) about?
* Why have three types of qualification?
* How is a validation plan different from a test plan?
* What is the real reason for using summary reports? Dr Teri Stokes, Director, GXP International
GOVERNING PROCESSES AND PROCEDURES FOR CSV 9.40 Creating useful tools for compliance
* Corporate and global procedures
* Local procedures
* Practical SOPs
* Change control
* Fitting the needs of the business Ron Armstrong, Manager, R&amp;D QA, Computer Systems Compliance, Boehringer Ingelheim
TECHNICAL ASPECTS OF COMPLYING WITH PROCESSES AND PROCEDURES 10.20 Taking the pain out of computer systems validation training and communication
* Auditing against procedures
* Documenting variances and justifications for deviations
* Procedures for systems involving equipment and databases Judi Boyle, Associate Director, R&amp;D QA, External GMP &amp; Part 11 Computer Compliance, Boehringer Ingelheim
11.00 Morning Coffee
TYPICAL DOCUMENTATION REQUIREMENTS FOR CSV 11.20 Overview of computer system validation methodologies
* General validation concepts and definitions
* Governing processes and procedures - The FAQs of documentation requirements
* Why is documentation required?
* Who is responsible for documentation?
* What should be documented?
* How should we prepare documentation? Carolyn Stockdale, Manager, Quality &amp; Regulatory Compliance - Validation, Phase Forward
NEW REGULATIONS AND GUIDANCE FOR COMPUTER SYSTEMS 12.00 Computer validation and E-records/signatures
* EU regulations and guidelines, eg PIC/S
* FDA's current thinking and implementation of part 11
* Inspection and enforcement practices
* Recent Warning Letters
* Impact of risk-based system inspections
* New focus: IT infrastructure and networked systems
* Recommendations from the industry Dr Ludwig Huber, Product Marketing Manager, Agilent Technologies
12.40 Networking Lunch
COMPLIANCE WITH 21 CFR PART 11 2.00 Issues and enforcement strategy - what is the FDA really looking for?
* Clear at last? - FDA's latest guidance on Part 11
* A risk-based approach to Part 11 compliance
* Part 11 - business as usual? Dr Robert Stephenson, Regulatory Systems Team Leader, PGMIT, Pfizer
RISK BASED PART 11 COMPLIANCE STRATEGIES 2.40 Developing a rigorous and systematic approach for cGMP automated systems
* Scope of Part 11 and the current FDA thinking
* Predicate rules - the new essence
* New &amp; legacy systems - what is FDA saying?
* Risk-based approach - the central point
* GAP analysis &amp; Part 11 assessment - system prioritisation
* Compliance strategy - a practical approach Dr Rachita Sharma, President, FDA Computer Validation (contracting for Bayer &amp; Baxter)
3.20 Afternoon Tea
IS 21 CFR 11 STILL THE GOLD STANDARD? 3.40 Outlook in an uncertain future
* Traditional position of FDA as 'strictest' regulator
* Strengthening European regulatory framework
* Changes in FDA guidance and enforcement
* Trends in European regulatory enforcement
* Role of externally published good practice John Farrell, Principal Consultant, The Synapse Partnership
LEGACY SYSTEMS 4.20 Bringing existing systems into compliance
* Understanding the legacy system problem
* Establishing a programme for legacy system validation
* Benefits of doing legacy systems validation
* Defending legacy systems during inspections Kate Samways, Director, KAS Associates &amp; Secretary, European Steering Committee, (GAMP)
5.00 Chairperson's Closing Remarks and Close of Day One
Day Two - 18th November 2003
8.30 Re-registration &amp; Coffee
9.00 Chairman's Opening Remarks John Farrell, Principal Consultant, The Synapse Partnership
THE LATEST FDA GUIDANCE REVIEW ON PART 11 AND PROCEDURAL CHECKLISTS 9.10 What is the FDA looking for in an inspection?
* History of guidance document
* FDA intention vs industry reaction
* Pure vs hybrid systems
* Current validation practices
* Audit trail - the most confused topic
* Archival, copies of records &amp; record retention
* Procedural vs technical controls
* A checklist for Part 11 success
* Current FDA inspection trends Dr Bhanu Sharma, Manager, Computer Systems Validation &amp; Compliance for a leading Pharmaceutical company
THE FDA-483 AND THE WARNING LETTER 9.40 How to handle 483 observations
* What is FDA's foreign inspection programme?
* Can you get a Warning Letter even if you did not get a 483?
* If no 483 is issued, should you respond to observations in the EIR?
* Who writes FDA Warning Letters?
* Can you avoid getting a Warning Letter?
* What does the new FDA Warning Letter review process mean to you?
* What regulatory action can result following a Warning Letter?
* How does a Warning Letter affect a foreign company?
* What can you do if FDA puts you on import alert? Ron Johnson, Executive Vice President, Quintiles (formerly FDA)
GAMP 10.20 A framework for risk based validation
* Influence of different risks on your computer validation programme
* When and how to evaluate risk throughout the life cycle
* Expectations for validation of different types of application (eg LIMS, custom development, spreadsheets)
* Using GAMP to validate your computerised system Chris Reid, Director &amp; Principal Consultant, Integrity Solutions &amp; Member, European Steering Committee, GAMP
11.00 Morning Coffee
APPLICABILITY OF SOPS TO IT DEPARTMENTS 11.20 What does it mean to them?
* What is achieved by SOPs?
* What are they written for?
* How does this affect the IT team?
* Existing technical activities are performed accordingly David Stephenson, Consultant, ABB Eutech (formerly GlaxoSmithKline)
QUALIFICATION OF NETWORK INFRASTRUCTURE AND SYSTEMS VALIDATION 12.00 Critical considerations
* Definitions - the 4 Qs
* How does infrastructure align with the business model?
* The elements of infrastructure
* Qualification vs validation
* Steps to qualification of infrastructure
* Supporting your customers/users validation needs Michael Britt, Site Manager, Systems Validation Services, Roche
12.40 Networking Lunch
SECURING YOUR NETWORK 2.00 Is your data really safe? Find out how to protect it
* Who owns the network? - get it under control
* Remember 85% of attacks are from inside your company
* Secure GXP data with at least a router and firewall
* Use all the security features of the operating system
* Explain very clearly to staff the reasons for network security and then act on security breaches
* Data is the beginning of corporate intellectual property - guard it well Bob Taylor, Technical Director, Smart Tech Worldwide (formerly GlaxoSmithKline)
QUALITY MANAGEMENT SYSTEM FOR COMPUTER SYSTEMS COMPLIANCE 2.40 Achieve and maintain compliance
* System structure
* Components and controls
* Global computer system registry as a quality management tool Klaus Krause, Corporate Manager, Computer Systems Compliance, Amgen
3.20 Afternoon Tea
EFFECTIVE GAP ANALYSIS AS A TOOL FOR COMPLIANCE 3.40 How to fit GAP analysis into compliance initiatives
* Effective GAP analysis techniques
* Preparing for a GAP analysis: developing worksheets and checklists
* Conducting a computer systems GAP analysis
* Conducting a 21 CFR Part 11 GAP analysis
* Risk analysis techniques
* Practical and impractical corrective methods
* How to mitigate enforcement actions in the event your organisation is cited for deficiencies Dr Jennifer Methfessel, Senior Consultant, Life Sciences, ABB Eutech
TOOLS AND CONCEPTS OF SIX SIGMA 4.20 The process and benefits using Six Sigma for computerised systems validation
* The economics of computerised systems compliance and validation
* How Six Sigma and software process improvement fit into quality regulation systems
* What Six Sigma tools are usable in computerised system validation and how process improvement methodologies operate
* The use of measurement to prove objective success and achievement
* Challenges for deploying such initiatives within software/IT communities Dr Radouane Oudrhiri, Chief Technology Officer, Systonomy
5.00 Chairman's Closing Remarks and Close of Conference
CONTACT: Katy West, Marketing Department, SMi Conferences LtdTel: +44 (0)20 7827 6108Fax: +44 (0)20 7827 6109e-mail: kwest@smi-online.co.uk
((M2 Communications Ltd disclaims all liability for information provided within M2 PressWIRE. Data prepared by named party/parties. Further information on M2 PressWIRE can be obtained at http://www.presswire.net on the world wide web. Inquiries to info@m2.com)).

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HPLC method development and validation for pharmaceutical analysis

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Pharmaceutical Technology Europe , 03/01/2004 16 3
HPLC method development and validation for pharmaceutical analysis: this article presents a simple and systematic approach to HPLC method development, beginning with sample preparation and finishing with practical analytical method validation.(high performance liquid chromatography ) Shabir, Ghulam A. *~|~*
COPYRIGHT 2004 Advanstar Communications, Inc.

The wide variety of equipment, columns, eluent and operational parameters involved makes high performance liquid chromatography (HPLC) method development seem complex. The process is influenced by the nature of the analytes and generally follows the following steps: * step 1 -- selection of the HPLC method and initial system
* step 2 -- selection of initial conditions
* step 3 -- selectivity optimization
* step 4 -- system optimization
* step 5 -- method validation.
Depending on the overall requirements and nature of the sample and analytes, some of these steps will not be necessary during HPLC analysis. For example, a satisfactory separation may be found during step 2, thus steps 3 and 4 may not be required. The extent to which method validation (step 5) is investigated will depend on the use of the end analysis; for example, a method required for quality control will require more validation than one developed for a one-off analysis. The following must be considered when developing an HPLC method:
* keep it simple
* try the most common columns and stationary phases first
* thoroughly investigate binary mobile phases before going on to ternary
* think of the factors that are likely to be significant in achieving the desired resolution.
Mobile phase composition, for example, is the most powerful way of optimizing selectivity whereas temperature has a minor effect and would only achieve small selectivity changes. pH will only significantly affect the retention of weak acids and bases. A flow diagram of an HPLC system is illustrated in Figure 1.
HPLC method development
Step 1 -- selection of the HPLC method and initial system. When developing an HPLC method, the first step is always to consult the literature to ascertain whether the separation has been previously performed and if so, under what conditions--this will save time doing unnecessary experimental work. When selecting an HPLC system, it must have a high probability of actually being able to analyse the sample; for example, if the sample includes polar analytes then reverse phase HPLC would offer both adequate retention and resolution, whereas normal phase HPLC would be much less feasible. Consideration must be given to the following:
Sample preparation. Does the sample require dissolution, filtration, extraction, preconcentration or clean up? Is chemical derivatization required to assist detection sensitivity or selectivity?
Types of chromatography. Reverse phase is the choice for the majority of samples, but if acidic or basic analytes are present then reverse phase ion suppression (for weak acids or bases) or reverse phase ion pairing (for strong acids or bases) should be used. The stationary phase should be [C.sub.18] bonded. For low/medium polarity analytes, normal phase HPLC is a potential candidate, particularly if the separation of isomers is required. Cyano-bonded phases are easier to work with than plain silica for normal phase separations. For inorganic anion/cation analysis, ion exchange chromatography is best. Size exclusion chromatography would normally be considered for analysing high molecular weight compounds (>2000).
Gradient HPLC. This is only a requirement for complex samples with a large number of components (>20-30) because the maximum number of peaks that can be resolved with a given resolution is much higher than in isocratic HPLC. This is a result of the constant peak width that is observed in gradient HPLC (in isocratic HPLC peak width increases in proportion to retention time). The method can also be used for samples containing analytes with a wide range of retentivities that would, under isocratic conditions, provide chromatograms with capacity factors outside of the normally acceptable range of 0.5-15.
Gradient HPLC will also give greater sensitivity, particularly for analytes with longer retention times, because of the more constant peak width (for a given peak area, peak height is inversely proportional to peak width). Reverse phase gradient HPLC is commonly used in peptide and small protein analysis using an acetonitrile-water mobile phase containing 1% trifluoroethanoic acid. Gradient HPLC is an excellent method for initial sample analysis. Column dimensions. For most samples (unless they are very complex), short columns (10-15 cm) are recommended to reduce method development time. Such columns afford shorter retention and equilibration times. A flow rate of 1-1.5 mL/min should be used initially. Packing particle size should be 3 or 5 [micro]m.
Detectors. Consideration must be given to the following:
* Do the analytes have chromophores to enable UV detection?
* Is more selective/sensitive detection required (Table I)?
* What detection limits are necessary?
* Will the sample require chemical derivatization to enhance detectability and/or improve the chromatography?
Fluorescence or electrochemical detectors should be used for trace analysis. For preparative HPLC, refractive index is preferred because it can handle high concentrations without overloading the detector.
UV wavelength. For the greatest sensitivity [[lambda].sub.max] should be used, which detects all sample components that contain chromophores. UV wavelengths below 200 nm should be avoided because detector noise increases in this region. Higher wavelengths give greater selectivity.
Fluorescence wavelength. The excitation wavelength locates the excitation maximum; that is, the wavelength that gives the maximum emission intensity. The excitation is set to the maximum value then the emission is scanned to locate the emission intensity. Selection of the initial system could, therefore, be based on assessment of the nature of sample and analytes together with literature data, experience, expert system software and empirical approaches.
Step 2 -- selection of initial conditions. This step determines the optimum conditions to adequately retain all analytes; that is, ensures no analyte has a capacity factor of less than 0.5 (poor retention could result in peak overlapping) and no analyte has a capacity factor greater than 10-15 (excessive retention leads to long analysis time and broad peaks with poor detectability). Selection of the following is then required.
Mobile phase solvent strength. The solvent strength is a measure of its ability to pull analytes from the column. It is generally controlled by the concentration of the solvent with the highest strength; for example, in reverse phase HPLC with aqueous mobile phases, the strong solvent would be the organic modifier; in normal phase HPLC, it would be the most polar one. The aim is to find the correct concentration of the strong solvent. With many samples, there will be a range of solvent strengths that can be used within the aforementioned capacity limits. Other factors (such as pH and the presence of ion pairing reagents) may also affect the overall retention of analytes.
Gradient HPLC. With samples containing a large number of analytes (>20-30) or with a wide range of analyte retentivities, gradient elution will be necessary to avoid excessive retention.
Determination of initial conditions. The recommended method involves performing two gradient runs differing only in the run time. A binary system based on either acetonitrile/water (or aqueous buffer) or methanol/water (or aqueous buffer) should be used.
Step 3 -- selectivity optimization. The aim of this step is to achieve adequate selectivity (peak spacing). The mobile phase and stationary phase compositions need to be taken into account. To minimize the number of trial chromatograms involved, only the parameters that are likely to have a significant effect on selectivity in the optimization must be examined. To select these, the nature of the analytes must be considered. For this, it is useful to categorize analytes into a few basic types (Table II).
Once the analyte types are identified, the relevant optimization parameters may be selected (Table III). Note that the optimization of mobile phase parameters is always considered first as this is much easier and convenient than stationary phase optimization.
Selectivity optimization in gradient HPLC. Initially, gradient conditions should be optimized using a binary system based on either acetonitrile/water (or aqueous buffer) or methanol/water (or aqueous buffer). If there is a serious lack of selectivity, a different organic modifier should be considered.
Step 4 -- system parameter optimization. This is used to find the desired balance between resolution and analysis time after satisfactory selectivity has been achieved. The parameters involved include column dimensions, column-packing particle size and flow rate. These parameters may be changed without affecting capacity factors or selectivity.
Step 5 -- method validation. Proper validation of analytical methods is important for pharmaceutical analysis when ensurance of the continuing efficacy and safety of each batch manufactured relies solely on the determination of quality. The ability to control this quality is dependent upon the ability of the analytical methods, as applied under well-defined conditions and at an established level of sensitivity, to give a reliable demonstration of all deviation from target criteria.
Analytical method validation is now required by regulatory authorities for marketing authorizations and guidelines have been published. It is important to isolate analytical method validation from the selection and development of the method. Method selection is the first step in establishing an analytical method and consideration must be given to what is to be measured, and with what accuracy and precision.
Method development and validation can be simultaneous, but they are two different processes, both downstream of method selection. Analytical methods used in quality control should ensure an acceptable degree of confidence that results of the analyses of raw materials, excipients, intermediates, bulk products or finished products are viable. Before a test procedure is validated, the criteria to be used must be determined.
Analytical methods should be used within good manufacturing practice (GMP) and good laboratory practice (GLP) environments, and must be developed using the protocols set out in the International Conference on Harmonization (ICH) guidelines (Q2A and Q2B). (1,2) The US Food and Drug Administration (FDA) (3,4) and US Pharmacopoeia (USP) (5) both refer to ICH guidelines. The most widely applied validation characteristics are accuracy, precision (repeatability and intermediate precision), specificity, detection limit, quantitation limit, linearity, range, robustness and stability of analytical solutions. Method validation must have a written and approved protocol prior to use. (6)
This article reviews and demonstrates practical approaches to analytical method validation with reference to an HPLC assay of progesterone (Figure 2) in a gel formulation. Progesterone is widely used for dysfunctional uterine bleeding or amenorrhoea, (7,8) for contraception (either alone or with, for example, oestradiol or mestranol in oral contraceptives) and in combination with oestrogens for hormone replacement therapy in postmenopausal women. (9,10)
Experimental Chemicals and reagents
All chemicals and reagents were of the highest purity. HPLC-grade methanol was obtained from Merck (Darmstadt, Germany). Progesterone reference standard was purchased from Sigma Chemicals (St Louis, Missouri, USA). Deionized distilled water was used throughout the experiments.
HPLC instrumentation
The HPLC systems used for the validation studies consisted of Series 200 UV/Visible Detector, Series 200 LC Pump, Series 200 Autosampler and Series 200 Peltier LC Column Oven (all Perkin Elmer, Boston, Massachusetts, USA). The data were acquired via TotalChrom Workstation (Version 6.2.0) data acquisition software (Perkin Elmer), using Nelson Series 600 LINK interfaces (Perkin Elmer).
All chromatographic experiments were performed in the isocratic mode. The mobile phase was a methanol/water solution (75:25 v/v). The flow rate was 1.5 mL/min and the oven temperature was 40[degrees]C. The injection volume was 20 [micro]L and the detection wavelength was set at 254 nm. The chromatographic separation was on a 250 X 4.6 mm ID, 10 [micro]m [C.sub.18] [mu]-Bondapak column (Waters, Milford, Massachusetts, USA).
Results and discussion Linearity and range
The linearity of a test procedure is its ability (within a given range) to produce results that are directly proportional to the concentration of analyte in the sample. The range is the interval between the upper and lower levels of the analyte that have been determined with precision, accuracy and linearity using the method as written. ICH guidelines specify a minimum of five concentration levels, along with certain minimum specified ranges. For assay, the minimum specified range is 80-120% of the theoretical content of active. Acceptability of linearity data is often judged by examining the correlation coefficient and y-intercept of the linear regression line for the response versus concentration plot. The regression coefficient ([r.sup.2]) is >0.998 and is generally considered as evidence of acceptable fit of the data (Figure 3) to the regression line. The per cent relative standard deviation (RSD), intercept and slope should be calculated.
In the present study, linearity was studied in the concentration range 0.025-0.15 mg/mL (25-150% of the theoretical concentration in the test preparation, n=3) and the following regression equation was found by plotting the peak area (y) versus the progesterone concentration (x) expressed in mg/mL: y=3007.2x + 4250.1 ([r.sup.2]=1.000). The demonstration coefficient ([r.sup.2]) obtained for the regression line demonstrates the excellent relationship between peak area and concentration of progesterone. The analyte response is linear across 80-120% of the target progesterone concentration.
Accuracy
A method is said to be accurate if it gives the correct numerical answer for the analyte. The method should be able to determine whether the material in question conforms to its specification (for example, it should be able to supply the exact amount of substance present). However, the exact amount present is unknown, which is why a test method is used to estimate the accuracy. Furthermore, it is rare that the results of several replicate tests all give the same answer, so the mean or average value is taken as the estimate of the accurate answer.
Some analysts adopt a more practical attitude to accuracy, which is expressed in terms of error. The absolute error is the difference between the observed and the expected concentrations of the analyte. Percentage accuracy can be defined in terms of the percentage difference between the expected and the observed concentrations (Equation 1).
Percentage accuracy tends to be lower at the lower end of the calibration curve. The term accuracy is usually applied to quantitative methods but it may also be applied to methods such as limit tests. Accuracy is usually determined by measuring a known amount of standard material under a variety of conditions but preferably in the formulation, bulk material or intermediate product to ensure that other components do not interfere with the analytical method. For assay methods, spiked samples are prepared in triplicate at three levels across a range of 50-150% of the target concentration. The per cent recovery should then be calculated. The accuracy criterion for an assay method is that the mean recovery will be 100[+ or -]2% at each concentration across the range of 80-120% of the target concentration. To document accuracy, ICH guidelines regarding methodology recommend collecting data from a minimum of nine determinations across a minimum of three concentration levels cover!
ing the specified range (for example, three concentrations, three replicates each).
In the present study, the accuracy of the method was evaluated by recovery assay, adding known amounts of progesterone reference standard to a known amount of gel formulation, to obtain three different levels (50, 100 and 150%) of addition. The samples were analysed, and mean recovery and %RSDs calculated. The data presented in Table IV show that the recovery of progesterone in spiked samples met the evaluation criterion for accuracy (100 [+ or -] 2.0% across 80-120% of target concentrations).
Specificity
Developing a separation method for HPLC involves demonstrating specificity, which is the ability of the method to accurately measure the analyte response in the presence of all potential sample components. The response of the analyte in test mixtures containing the analyte and all potential sample components (placebo formulation, synthesis intermediates, excipients, degradation products and process impurities) is compared with the response of a solution containing only the analyte. Other potential sample components are generated by exposing the analyte to stress conditions sufficient to degrade it to 80-90% purity. For bulk pharmaceuticals, stress conditions such as heat (50-60[degrees]C), light (600 FC of UV), acid (0.1 M HCl), base (0.1 M NaOH) and oxidant (3% [H.sub.2][O.sub.2]) are typical. For formulated products, heat, light and humidity (70-80% RH) are often used. The resulting mixtures are then analysed, and the analyte peak is evaluated for peak purity and resolut!
ion from the nearest eluting peak.
Once acceptable resolution is obtained for the analyte and potential sample components, the chromatographic parameters, such as column type, mobile phase composition, flow rate and detection mode, are considered set. An example of specificity criterion for an assay method is that the analyte peak will have baseline chromatographic resolution of at least 2.0 from all other sample components. In this study, a weight of sample placebo equivalent to the amount present in a sample solution preparation was injected to demonstrate the absence of interference with progesterone elution (Figure 4).
Precision
Precision means that all measurements of an analyte should be very close together. All quantitative results should be of high precision--there should be no more than a [+ or -]2% variation in the assay system. A useful criterion is the relative standard deviation (RSD) or coefficient of variation (CV), which is an indication of the imprecision of the system (Equation 2).
According to the ICH, (2) precision should be performed at two different levels--repeatability and intermediate precision. Repeatability is an indication of how easy it is for an operator in a laboratory to obtain the same result for the same batch of material using the same method at different times using the same equipment and reagents. It should be determined from a minimum of nine determinations covering the specified range of the procedure (for example, three levels, three repetitions each) or from a minimum of six determinations at 100% of the test or target concentration.
Intermediate precision results from variations such as different days, analysts and equipment. In determining intermediate precision, experimental design should be employed so that the effects (if any) of the individual variables can be monitored. Precision criteria for an assay method are that the instrument precision and the intra-assay precision (RSD) will be [less than or equal to]2%.
In this study, the precision of the method (repeatability) was investigated by performing six determinations of the same batch of product. The resulting data are provided in Table V, which show that the repeatability precision obtained by one operator in one laboratory was 0.28% RSD for progesterone peak area and, therefore, meets the evaluation criterion.
The intermediate precision was demonstrated by two analysts, using two HPLC systems and who evaluated the relative per cent purity data across the two HPLC systems at three concentration levels (50%, 100%, 150%) that covered the assay method range (0.025-0.15 mg/mL). The mean and RSD across the systems and analysts were calculated from the individual relative per cent purity mean values at 50%, 100% and 150% of the test concentration. The data are presented in Table VI, and show [less than or equal to]2.0% RSD, therefore, meeting the evaluation criterion.
Limits of detection and quantitation
The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected, not quantified. It is expressed as a concentration at a specified signal:noise ratio, (2) usually 3:1. The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte in a sample that can be determined with acceptable a signal:noise ratio 10:1. LOD and LOQ may also be calculated based on the standard deviation of the response (SD) and the slope of the calibration curve(s) at levels approximating the LOD according to the formulae: LOD = 3.3(SD/S) and LOQ = 10(SD/S).
The standard deviation of the response can be determined based on the standard deviation of the blank, on the residual standard deviation of the regression line, or the standard deviation of y-intercepts of regression lines. The method used to determine LOD and LOQ should be documented and supported, and an appropriate number of samples should be analysed at the limit to validate the level. In this study, the LOD was determined to be 10 ng/mL with a signal:noise ratio of 2.9. The LOQ was 20 ng/mL with a signal:noise ratio of 10.2. The RSD for six injections of the LOQ solution was [less than or equal to]2%.
Analytical solution stability
Validation of sample and standard solution preparation may be divided into sections, each of which can be validated. These include extraction; recovery efficiency; dilution process when appropriate; and addition of internal standards when appropriate. Although extraction processes do not actually affect the measuring stage they are of critical importance to the analytical test method as a whole. The extraction process must be able to recover the analyte from the product; it must not lose (for example, by oxidation or hydrolysis) any of the analyte in subsequent stages, and must produce extraction replicates with high precision. For example, during analysis of an ester prodrug the extraction process involves the use of strongly alkaline or acid solutions, it may cause some of the prodrug to be hydrolysed and, therefore, give false results.
Reference substances should be prepared so that they do not lose any of their potency. Thus it is necessary to validate that the method will give reliable reference solutions that have not been deactivated by weighing so little that an error is produced; adsorption onto containers; decomposition by light; and decomposition by the solvent. If the reference is to be made up from a stock solution then it must be validated that the stock solution does not degrade during storage. Reagent preparation should be validated to ensure that the method is reliable and will not give rise to incorrect solutions, concentrations and pH values.
Samples and standards should be tested during a period of at least 24 h (depending on intended use), and component quantitation should be determined by comparison with freshly prepared standards. For the assay method, the sample solutions, standard solutions and HPLC mobile phase should be stable for 24 h under defined storage conditions. Acceptable stability is [less than or equal to]2% change in standard or sample response, relative to freshly prepared standards. The mobile phase is considered to have acceptable stability if aged mobile phase produces equivalent chromatography (capacity factors, resolution or tailing factor) and the assay results are within 2% of the value obtained with fresh mobile phase.
In the present study, the stabilities of progesterone sample and standard solutions were investigated. Test solutions of progesterone were prepared and chromatographed initially and after 24 h. The stability of progesterone and the mobile phase were calculated by comparing area response and area per cent of two standards with time. Standard and sample solutions stored in a capped volumetric flask on a lab bench under normal lighting conditions for 24 h were shown to be stable with no significant change in progesterone concentration during this period (Table VII).
Robustness
Robustness measures the capacity of an analytical method to remain unaffected by small but deliberate variations in method parameters. It also provides some indication of the reliability of an analytical method during normal usage. Parameters that should be investigated are per cent organic content in the mobile phase or gradient ramp; pH of the mobile phase; buffer concentration; temperature; and injection volume. These parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment. The chromatography obtained for a sample containing representative impurities when using modified parameter(s) should be compared with the chromatography obtained using the target parameters.
Conclusion
Method development involves a series of sample steps; based on what is known about the sample, a column and detector are chosen; the sample is dissolved, extracted, purified and filtered as required; an eluent survey (isocratic or gradient) is run; the type of final separation (isocratic or gradient) is determined from the survey; preliminary conditions are determined for the final separation; retention efficiency and selectivity are optimized as required for the purpose of the separation (quantitative, qualitative or preparation); the method is validated using ICH guidelines. The validated method and data can then be documented.
<pre>
Table I HPLC detector comparison.

Parameter Refractive index UV/Vis Fluorescence

Detection [10.sup.-6] [10.sup.-9] [10.sup.-12]
sensitivity (g)
Linear range [10.sup.4] [10.sup.5] [10.sup.3]
Flow sensitivity Yes No No
Temperature Yes No No
sensitivity

Parameter Electrochemical

Detection [10.sup.-12]
sensitivity (g)
Linear range [10.sup.8]
Flow sensitivity Yes
Temperature Yes
sensitivity

Table II The basic types of analytes used in HPLC.

Analyte Characteristics

Neutral No significantly acidic or basic functional groups
Weak acid Has carboxylic acid function or phenolic -OH
Weak base Aromatic amine
Strong base Non-aromatic amine

Table III HPLC optimization parameters.

Analytes HPLC method Optimize

Neutral Reverse phase Solvent strength,
solvent type
Weak acids and/or weak bases Ion suppression pH, solvent strength,
solvent type
Strong acid and/or strong bases Ion pairing Ion pairing reagent
concentration, pH
solvent strength,
solvent type
Inorganic anions/cations Ion exchange Eluting ion
concentration

Table IV Accuracy/recovery of progesterone from samples of known
concentration.

Amount of
progesterone (mg) % Recovery
Sample Per cent of nominal Added Recovered (n = 3) %RSD

1 50 3.179 3.156 99.28 0.62
2 100 4.258 4.240 99.58 0.43
3 150 5.253 1225.195 98.90 0.71
Mean 99.25 0.59

Table V Demonstration of the repeatability of the HPLC assay for
progesterone.

Sample Peak area Progesterone (% w/w)

1 15951 4.221
2 15895 4.140
3 15877 4.073
4 15876 4.127
5 15848 4.098
6 15822 4.102
Mean 15878 4.127
SD 43.95 0.051
%RSD 0.28 1.250

Table VI Demonstration of the intermediate precision of the HPLC assay
results for progesterone.

HPLC system 1 HPLC system 2
S1 S2 S3 S1 S2 S3
Sample (50%) (100%) (150%) (50%) (100%) (150%)

Analyst 1 100.54 100.08 99.89 99.62 99.82 99.94
Analyst 2 100.19 100.07 99.73 99.94 100.13 99.97
Mean (HPLC) 100.37 100.08 99.81 99.78 99.98 99.96
Mean (Analyst 1 + 2) 100.07 100.03 99.88
RSD (criteria S1 S2 S3
[less than or equal to]
2%)
HPLC system 1 + 2 0.42 0.07 0.11
Analyst 1 + 2 0.39 0.14 0.11

Table VII Stability results of progesterone samples and standard
solutions (n = 3).

Progesterone recovery
Time (h) (mg/100 mL) Per cent of initial

Standard 0 3.955 100.0
24 3.952 99.9
Sample 0 5.503 100.0
24 5.478 99.5 </pre>
Equation 1
%Accuracy = [[Mean observed concentration]/[Expected concentration]] X 100
Equation 2
%RSD [[Standard deviation]/[Mean value]] X 100
Article reprints may be purchased from Steve McQuair.
Tel. +44 1244 393 419
Fax +44 1244 383 356
smcquair@advanstar.com
References
1. International Conference on Harmonization, "Q2A: Text on Validation of Analytical Procedures," Federal Register 60(40), 11260-11262 (1995).
2. International Conference on Harmonization, "Q2B: Validation of Analytical Procedures: Methodology; Availability," Federal Register 62(96), 27463-27467 (1997).
3. FDA, "Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls Documentation; Availability," Federal Register (Notices) 65(169), 52776-52777 (2000).
4. www.fda.gov/cder/guidance/cmc3.pdf
5. USP 25-NF 20, Validation of Compendial Methods Section (1225) (United States Pharmacopeal Convention, Rockville, Maryland, USA, 2002) p 2256.
6. G.A. Shabir, "Validation of HPLC Chromatography Methods for Pharmaceutical Analysis. Understanding the Differences and Similarities Between Validation Requirements of FDA, the US Pharmacopeia and the ICH," J. Chromatogr. A. 987(1-2), 57-66 (2003).
7. C.E. Wood, "Medicare Program; Changes to the Hospital Outpatient Prospective," Med. J. Aust. 165, 510-514 (1996).
8. A. Prentice, "Medical Management of Menorrhagia," Br. Med. J. 319, 1343-1345 (1999).
9. D.T. Baired and A.F. Glasier, "Hormonal Contraception," New Engl. J. Med. 328, 1543-1549 (1993).
10. P.E. Belchetz, "Hormonal Treatment of Postmenopausal Women," New Engl. J. Med. 330, 1062-1071 (1994).
Ghulam A. Shabir is a senior scientist at Abbott Laboratories, MediSense Products, R &amp; D, Range Road, Witney, Oxon OX29 0YL, UK.
Tel. +44 1993 863 099
Fax + 44 1235 467 737
ghulam.shabir@abbott.com

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