John Carroll

The validation of cleaning processes has long played a critical role in pharmaceutical manufacturing. The US Food and Drug Administration (FDA) requires firms to produce written procedures detailing the cleaning processes used for various pieces of equipment, and how those processes will be validated. Typical detection limits mentioned in the pharmaceutical literature are 10 ppm or biological activity levels of 1/1000 of the normal therapeutic dose. Most of the pharmaceutical industry currently uses high performance liquid chromatography (HPLC) to evaluate samples collected from production machines. Highly trained technicians in an analytical laboratory prepare the mobile phase and diluents and set up the HPLC instrument. Each sample collected is then prepared and, typically, through the use of an autoloader, injected and processed consecutively without operator intervention. As cleaning and sample preparation normally occupy a full working day, analysts load the instrument at the end of a one day then, on the following morning, perform any necessary calculations and enter the sample results in the company's laboratory information management system (LIMS). Production usually resumes in the middle of that day, resulting in a minimum loss of one and a half day's production.	

The ion mobility spectrometry (IMS) limit test described in this article defines a pass/fail threshold such that when the IMS signal is below the threshold, it can be proved at a defined confidence level that the measured concentration is truly below the limit. This approach takes advantage of the greatly reduced instrument set up time for IMS and the reduced sample analysis time; typically 5-20 s per sample for IMS compared with 10-20 min for HPLC (Table I). By performing multiple runs with a standard sample at the cleaning action level, the standard deviation of the measurement process is obtained which, combined with the required confidence level, is used to set the IMS signal pass/fail threshold. The result is that pharmaceutical manufacturers have the potential to save millions of dollars by dramatically reducing the time required to verify the cleaning of production equipment before switching over to another product or batch.	

In an effort to reduce the time required for cleaning validation and verification, IMS has been studied as an alternative to HPLC. IMS refers to the principles, practice and instrumentation of characterizing chemical substances based on their gas phase ion mobilities - determined by measuring drift velocities as ions move, under the influence of an electric field, through a gas at ambient pressure.1 	

Table I:  Comparison between a traditional HPLC approach and IMS limit test approach.				

Typical pharmaceutical compounds are thermally desorbed to vaporize the sample. The vaporized sample is then introduced into the IMS via a carrier gas stream before being selectively ionized, typically by an atmospheric chemical ionization (APCI) source. An electronic gate then opens periodically, to admit a finite pulse of product ions into the drift tube. The ions migrate downfield and strike a collector electrode, producing a current. The ion current is amplified and displayed as an ion mobility spectrum or plasmagram, showing ion current versus time. Figure 1 illustrates a cross-sectional view of a drift tube.	

The ratio of the drift velocity of a given ion to the applied electric field strength (ion mobility) depends on the charge, mass and collision cross-section of the ion. The cross-section, in turn, is dictated by molecular size, shape and charge distribution. As size generally scales with mass, correlations exist between the mobility and mass of an ion. Lighter, smaller ions have greater mobility values and therefore, exhibit shorter drift times. Ion mobilities, calculated from the drift times, provide information regarding the chemical identities of observed ions and their neutral precursors. The intensity of each peak in the plasmagram is related to the abundance of the corresponding ion. Thus, quantitative compound information may be obtained by analysing the amplitude or area of an ion peak derived from that compound. 		

Figure 1: A cross-section view of the drift tube.	

Most HPLC-based cleaning validation methods typically implement a one-point calibration based on the assumption that the response is linear and passes through zero (validated during the method development phase). The sample response is then converted to a concentration. The IMS method involves a one-point calibration to define the instrument response based on the assumption that samples with amounts less than the calibration level are clean (validated during the method development phase) and the sample response is converted into a simple clean/dirty response with an associated confidence level. In a sense, the HPLC method could be used as a limit test but the IMS would analyse at least 60 times faster (Table I).				

The first step is surveying the instrument mass response curve (Figure 2) to select the target mass response point, which provides a compromise between the higher response per unit mass obtained with small masses and the smaller relative standard deviation obtained with larger masses. The limit of linearity is selected because the linear region has the highest sensitivity and generally provides the lowest relative standard deviation. Once the target mass is selected, the swabbing methodology and sample extraction volume is defined using the following factors:				

sample volume used to deliver sample mass to IMS instrument (typically 1-10 mL)

target concentration (determined using target mass and sample volume)

target cleaning level on IMS instrument (typically 100-1000 ng/cm2)

assume worst case swabbing recovery (typically 60-90%)

The above factors are used so that a swab right at the action level will be diluted to the target concentration. 	

For example, if target mass 5 1 ng, sample volume 5 1 mL, target cleaning action level 5 200 ng/cm2, area to be swabbed 5 100 cm2 and swabbing recovery 5 80%, then the target concentration is 1 ng/mL (1 mg/mL) and the action level on the swab is 16 mg. Thus, the volume to extract the swab should be 16 mg/swab/1 mg/mL, which equals 16 mL per swab.	

The next step is determining the signal and uncertainty at the target concentration. This is accomplished by measuring the IMS response for several samples at the target concentration and calculating the mean (x) and standard deviation(s). To determine if the response observed from a test sample is significantly different from this value the Student's t-test can be used, as shown below in Equation 1 where x1 and x2 are the mean values associated with the target and test samples respectively, s is the pooled standard deviation of the target and test samples, n1 and n2 are the samples associated with the target and test samples respectively. 	

The tcalc value is compared with a value obtained from a standard Student's t table for the appropriate confidence level and number of observations. If then the two means are significantly different. The pass/fail threshold can be calculated based on the tabulated value of t for the required confidence level and the number of observations. The threshold value (xt), the maximum instrument response for which it is possible to state with the confidence level in question that the sample is clean, is calculated by rearranging Equation 1 as follows:	

where x1 and x2 are the mean values associated with the target and test samples respectively, s is the pooled standard deviation of the target and test samples, n1 and n2 are the samples associated with the target and test samples respectively. 

The tcalc value is compared with a value obtained from a standard Student's t table for the appropriate confidence level and number of observations. If (the following equation)  then the two means are significantly different. The pass/fail threshold can be calculated based on the tabulated value of t for the required confidence level and the number of observations. 

The threshold value (xt), the maximum instrument response for which it is possible to state with the confidence level in question that the sample is clean, is calculated by rearranging Equation 1 as follows:

Suppose that 10 analyses are run on a standard and the mean IMS response is 100 digital units (du) with a standard deviation of 5 du. Then, it can easily be determined that any solution producing an IMS signal of less than 83 du for one measurement is clean - with at least 99.5% confidence. It is important to note that the short run-time of IMS means that it is sensible to perform extra standard measurements, because they provide better predictions for the standard deviation, increase the limit threshold and, therefore, lower the chances of a false positive.	

In most cases, the swab will test as 'clean' when first measured. In some instances, the swab response will be well above the threshold, indicating the need for further cleaning. In rare cases, the swab response will be slightly above the threshold. IMS generates results so quickly that in this situation it is advisable to analyse a few more aliquots of the sample before deciding to reclean. The swab can be declared clean if the mean of replicate samples is less than the threshold. The pass/fail threshold will be higher when replicate samples are analysed because of the nature of the t-test. 				

Several important precautions should be taken during methods development to protect against false negatives. The first is to verify the consistency of swab recovery, a requirement that also applies to HPLC. The second is to check the instrument response across the range of interest to validate the system response. The third is to evaluate the effect of potential interferences (excipients, for example) which may be present. This is because, occasionally, the analyte signal may be suppressed by the presence of particular compounds, particularly detergents or excipients. 	

Instrumentation. The IonScan-LS (Smiths Detection, Warren, New Jersey, USA) ion mobility spectrometry instrument was used, and the instrumental parameters employed are listed in Table II.				

In a typical analysis, a substrate containing the sample of interest is placed on the desorber, which is maintained at a fixed programmed temperature of 290 Â°C. A carrier gas, air, transports thermally volatilized sample material into an ionization/reaction region. Volatilized compounds are selectively ionized using a 63Ni ß-source and a controlled chemical ionization environment to produce molecular ions or ion clusters. The ions are then gated into the drift chamber, at atmospheric pressure, where they are accelerated under an applied electric field toward a collector electrode.	

Identification of compounds is based on the calculation of their characteristic reduced ion mobility K0 (cm2/Vsec) values. K0 is determined using the following equation:	

where tcal is the drift time of the internal calibrant and tobs is that of the observed peak. The K0 of the internal calibrant is a known value and the drift times of the calibrant and observed peak are experimentally measured values. In the external calibration procedure, the internal calibrant K0 value to be used in this equation is set so the K0 observed for the external (primary) calibrant has the value shown in Table II.	

The polarity of the electric field applied to the drift region is either positive or negative, allowing for the analysis of positive or negative ions. Ions of the correct charge are accelerated from the reaction region towards the drift region. Each scan of the IMS spectrum starts when the gating grid opens briefly to admit a burst of ions into the drift tube, and ends just before the gating grid opens again. This interval is the 'scan period.' The data from several scans are co-added together to improve the signal-to-noise ratio and is called a 'segment.' A series of segments with characteristic ion peak patterns for the sample are obtained and can be displayed either as a series of individual segments versus desorption time in seconds (a 3-D plasmagram) or as an average of all segments obtained during the analysis (a 2-D plasmagram).			

Materials. Product X (a novel compound under development) and excipients, provided by GlaxoSmithKline, were used as supplied. Pesticide-grade acetone was used to make up the Product X samples and the excipients were made up in water or ethanol. Teflon substrate (0.45 micron porosity) was obtained from Osmonics (Minnetonka, Minnesota, USA).	

Methods. A 1.0 mL aliquot of the sample was deposited on the Teflon substrate using a 1.0 mL syringe (SGE, Melbourne, Australia) and allowed to evaporate for 15 s prior to analysis. 			

Table III:  IMS response of the target level standard.	

Eight standards containing 1 ng of Product X were analysed to determine the pass/fail threshold. Forty eight test solutions - ranging from 0.25-10 ng - were then analysed. The goal was to verify that the limit test would identify samples containing less than 1.5 ng of Product X as clean and those containing at least 1.5 ng of Product X as dirty. The target level data, listed in Table III, can be summarized as follows:				

Eight measurements of the target level produced a mean response of 398 du. The pass/fail threshold calculated from these eight data points is 339 du. That is, if one analysis of a test sample gives an instrument response of less than 339 du, we can state with at least 99.95% confidence that it contains less than the action level amount of the active pharmaceutical ingredient (API).	

Regarding the test sample data, 24 clean samples (containing less than 1.5 ng of the API) were analysed (Table IV). In 23 of these cases, the sample passed (the instrument response was below the threshold). There was, however, one false positive. For the 24 dirty test samples there were no false negatives. All 'dirty' samples tested as such.		

Figure 3:  IMS plasmagrams of Product X, with and without excipients.	

In the case of the one false positive, the prescribed course of action would be to analyse two additional aliquots of that sample and to apply the limit test to the mean of the three results. The mean would be compared with a revised threshold, which is based on more measurements and is therefore higher. As a worst case example, the three highest responses for 1 ng (329, 330 and 344 du) give a mean response of 334 du, which is less than the revised threshold of 365 du for three test samples. The test sample would therefore be declared clean.	

Articles

A limit test using ion mobility spectrometry (IMS) has the potential to dramatically reduce the time required for cleaning verification and cleaning method development. The traditional approach to cleaning verification, often using HPLC, is relatively resource intensive and can lead to significant delays in reporting results. The main advantage of IMS is that results are seen virtually instantaneously, so any necessary re-measurement can be done very quickly. If the results demonstrate cleanliness, production can resume in a matter of hours not days.