Cleaning verification represents the start and finish of each batch in pharmaceutical manufacturing. Two commonly applied analytical approaches to verify pharmacological cleanliness are: total organic carbon (TOC) and high-performance liquid chromatography (HPLC). TOC is a non-specific technique with analysis times ranging from 2 to 10 minutes, depending on the analytical instrument. HPLC has the advantages that methods can be developed for specific analytes and the limit of detection can be one to two orders of magnitude lower than TOC. Due to this, HPLC-UV is a popular cleaning verification (CV) method.
The potential downside of an HPLC cleaning verification approach is analysis time. The number of analytical swab samples during batch manufacturing may range from 50 to 100. With an analytical run time between 5 and 10 minutes per sample depending on the method, analysis time may range from 6 to 20 hours including system suitability samples. This is a potentially rate limiting factor for manufacturing activities. Piramal Pharma Solutions – Lexington applies fundamental analytical principles to potentially reduce sample analysis time to durations approaching 2 minutes, equivalent to best in class TOC analysis but with greater sensitivity, specificity, and compatibility with organic cleaning solvents for water insoluble molecules.
CV by HPLC-UV
The following equation dominates resolution in a chromatographic method.
Where N is the column efficiency, α is the selectivity, and k is the retention factor. Chromatographers usually recommend to keep k (retention factor) around 5 to maximize the resolution within an acceptable time frame. The following chromatographic conditions are popular choices for small molecules in the current pharmaceutical industry (Table 1).
Table 1. Typical chromatographic conditions for Assay and Impurity HPLC method
As a CV method, the method usually only needs to separate the active from placebo and swab extraction. The separation between the active and its impurities is usually not required. The retention factor, k and column length (proportional to N) can be reduced to minimize sample analysis duration. Therefore, the following chromatographic conditions are recommended for a CV method (Table 2).
Table 2. Recommended chromatographic conditions for CV HPLC method (Isocratic)
Organo silicon stationary phase columns are selected due to their wide pH range which provides more choices for mobile phases.
If we assume the run time is eight times of the unretained time [T0=0.35mL/(2mL/min)=0.175 min], it will be about 1.4 min.
Sample injections, including needle washing and vial/tray moving, may take about one or two min for an HPLC system. A high throughput setting in an autosampler is necessary to reduce the overall run time.
In some cases (e.g. some large peptides), an isocratic method may be not practically achievable. An example gradient method is shown below (Table 3). A small system dwell volume is desired to reduce the run time.
Table 3. One example chromatographic conditions for CV HPLC method (Gradient)
* May reduce to achieve a better efficiency, which may increase the run time.
# may change due to the different dwell volume in an HPLC system to keep the same equilibrium volume.
CV by UHPLC-UV
A UPLC system usually has less dwell volume, extra column volume and sample injection time compared with an HPLC system, which can lead to less run time. However, the UPLC system also has the following shortcomings:
- Higher cost for instrument and column with less robustness (easier clogged).
- Few UPLC column with length less than 30 mm available currently.
- CV sample usually require filtration before injection, which increase the sample preparation time and method validation time.
Therefore, additional cautions are needed when a UPLC system is applied for CV work.
An HPLC-UV CV method with run time of 1.4 to 3 min is achievable if an appropriate column and chromatographic conditions are applied. Using methods with this duration, even with approximately 50 to 100 swab samples for aseptic manufacturing using isolators, analytical duration can be reduced to a brief as just over 2 hours.
Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan, Third Edition, 2010, “Introduction to Modern Liquid Chromatography”