Taking Control of Your Quality Control

ABSTRACT

Federal regulations for quality control processes to verify liquid delivery are ambiguous and incomplete, and there are a variety of guides and standards, which add to the confusion. Yet, FDA mandates that laboratories incorporate quality control processes to help ensure the safety, purity, and effectiveness of therapeutics samples. Understanding the processes involved in verifying liquid delivery can help navigate regulations. The processes that depend on volume include standards preparation, aliquoting, and dilution; quantitative tests relying on liquid delivery include yield measurements, purity tests, and cell-based assays. Because liquid-handling processes advance more quickly than corresponding regulations, organizations release standards and guidelines to improve industry operations and promote best practices. Among them are FDA's Current Good Manufacturing Processes initiative and International Society for Pharmaceutical Engineering's Good Automated Manufacturing Practice Guide.

Consider a quality control laboratory that processes 10 to 20 samples of therapeutics per day and releases as many as 20 batches of life-saving vaccines and pharmaceuticals each month. According to FDA requirements, analytical results from laboratory tests—many performed with liquid handling equipment—help to ensure the safety, purity, and effectiveness of these batches.

Federal regulations mandate that laboratories follow liquid delivery quality control processes; however, current regulations are ambiguous and incomplete, and a variety of guides and standards add to the confusion.

Understanding the processes required to verify liquid delivery can help you navigate current regulations, standards, and guidance for calibration procedures.

Table 1. Standards Recommending Liquid Delivery Calibration Best Practices

LIQUID HANDLING AND QUALITY CONTROL

Quality control laboratories routinely prepare samples of therapeutics for testing and produce results that are reviewed, compared with specifications, and used to help decide whether manufactured products are ready for market. Each step in the QC process usually includes one or more liquid-handling processes. Although some of these steps, such as wash phases, do not rely on accurate volumes, many do.

Examples of preparation processes that depend on volume include:

• Standards preparation: QC laboratories need standard materials in defined concentrations to calibrate analytical methods. If standards are inaccurately prepared, calibration will be incorrect and tests that follow will be wrongly reported. This can lead to inaccurate measurement of potency or improper dosage, among other problems.

• Aliquoting: This process, where liquid is taken from a larger sample and dispensed into multiple smaller amounts called aliquots, is highly quantitative. The aliquot volume often figures into future calculations; inaccurate aliquot volumes can skew results and lead to inaccurate decision-making.

• Dilution: A sample solution is mixed with a diluent and a dilution ratio is calculated. Accurate dilution ratios depend on the volumes of both liquids. Because this dilution ratio is used in subsequent calculations, inaccurate dilutions usually create questionable data.

Errors in these and other liquid-handling processes can impact the accuracy of key analytical parameters used to verify the safety, purity, and efficacy of drug batches. Examples of quantitative analytical tests relying on liquid delivery include:

• Yield measurements: Yield measurements provide valuable information about the relation-ship between inputs and outputs of the manufacturing process. When a yield fails to meet defined levels, it often triggers a warning that a manufacturing process may be out of control. However, inaccurate volume measurements can also cause an improper yield measurement, making volume accuracy necessary to avoid false alarms about process yield.

• Purity tests: Qualitative purity tests that detect the presence or absence of foreign or dangerous substances can produce false positive (incorrect identification) or false negative (failure to identify) results if sample volumes are highly inaccurate. Quan-titative measurements that detect levels of impure substances are even more dependent on volume accuracy and can determine the difference between batch acceptance and an out-of-specification investigation.

• Cell-based assays: These analytical methods can be tedious and expensive to prepare and run, because of their complexity and innate variability. Laboratory management should take extra effort to ensure that controllable errors (such as liquid handling error) are eliminated.

If any of these quality tests fail to produce correct results, a good batch of pharmaceuticals could be delayed, leading to wasted time, resources, and money; if the biopharmaceutical is unstable, it becomes unusable. If a quality control laboratory erroneously clears a bad batch for distribution, this could result in a pharmaceutical company's worst fears—product recalls, patient injuries, and wasted money.

Consider what would happen if a manufacturing company that tests pipette performance every six months learns that one pipette, or several, have failed but a batch or product whose quality was tested with the faulty pipette already has been released. If the product is on the market, this situation would require notifying the FDA and developing a protocol for additional testing. If the drug already has been consumed, the company is at risk for a product recall and potential liability.

LIQUID DELIVERY QUALITY REQUIREMENTS

In light of the severe consequences that can arise from improperly functioning liquid-handling instrumentation, most would conclude that federal regulations must tightly govern equipment performance verification and quality control systems. Unfortunately, this is not the case. Although regulations do exist, they are broad and open to interpretation; most have not caught up with advancements in liquid-handling technology.

For example, the FDA's Food, Drug and Cosmetics Act of 1938 simply states that drugs must be safe, pure, and effective, and that to achieve these goals, manufacturing processes must be controlled.

21 Code of Federal Regulations (CFR) 211, establishes Good Manufacturing Practices. Two sections of this ruling are relevant to liquid handling. Subpart D states that equipment used to manufacture drugs should "be routinely calibrated, inspected, or checked." Subpart I emphasizes laboratory controls and requires calibration of all instruments, including liquid-handling devices, "at suitable intervals in accordance with an established written program." It is left up to individual laboratories to define and defend their choices.

Laboratories are also required to follow standards established by the United States Pharmacopeia (USP), but USP has little to say about liquid handling. Chapter 31 specifies accuracy requirements for volumetric flasks, transfer pipettes, and burettes, but has not yet issued guidelines about hand-held manual or automatic pipettes, or automated liquid handling equipment.

GUIDELINES EMERGE TO OUTLINE BEST PRACTICES

It is evident that liquid-handling processes advance more quickly than corresponding regulations. For example, the transition from glass to hand-held manual action pipettes created the need for new standards and for preventive maintenance policies in liquid delivery devices. Similarly, the current trend toward extremely low volumes and higher density formats (e.g., high-density microtiter plates) has driven the need for new measurement and calibration methods, as well as the need to select from and standardize the best of these new methods. Because regulations provide inadequate guidance for modern laboratories, independent organizations release standards and guidelines to improve industry operations and promote best practices.

Current Good Manufacturing Processes (cGMP), FDA's initiative to continually improve Good Manufacturing Practices, recognizes that static regulations cannot keep pace with the highly dynamic pharmaceutical industry—especially when considering the often lengthy regulation revision process. This initiative provides laboratories with current best practices to avoid the need to continually amend federal requirements. For the most current information, cGMP relies on input from FDA personnel and quality control experts from industry, academia, government, and consumer groups, as well as from a number of independent regulatory bodies.

The International Society for Pharmaceutical Engineering (ISPE), whose mission is to train and educate pharmaceutical manufacturers, is one such organization that contributes to cGMP. ISPE produces Good Automated Manufacturing Processes (GAMP) Good Practice Guide: Calibration Management. This guide takes a structured approach to setting up a calibration management system that follows the validation life cycle and is oriented towards engineering process control. It emphasizes criticality assessment and corrective actions, including documentation of non-conformance events. This guide does not have specific information on liquid handling; but because liquid handling is a frequent source of equipment non-conformance events, the principles here are worth noting, particularly in light of present FDA enforcement focus on corrective and preventive actions.

Several other organizations issue recommendations, such as the National Conference of Standards Laboratories (NCSL) International, which issued Recommended Practice 6 (RP-6) to help biomedical and pharmaceutical laboratories establish effective calibration control systems. In many ways, RP-6 is a complement to GAMP. Written from a metrology and equipment management perspective, RP-6 emphasizes the nuts and bolts, such as calibration and measurement traceability, calibration history records and good labeling practices. The recommendations in RP-6 help laboratories move towards establishing calibration programs that are in compliance with 21 CFR 211.

The Association of Analytical Communities (AOAC), whose vision is "worldwide confidence in analytical results," is another agency that directs quality assurance initiatives. The organization's standard, entitled "Accreditation Criteria for Laboratories Performing Microbiological and Chemical Analyses in Foods, Feeds, and Pharmaceutical Testing," includes the full text of ISO 17025, with additional appended information to benefit the target laboratories.

Appendix A establishes a minimum calibration frequency of every three months for "volumetric delivery devices" (including mechanical action pipettes and mechanical burettes) and notes that "all data acquired on instruments that fail a parameter are suspect between the failing assessment date and the last successful calibration or verification date."

Laboratories subject to these requirements include those testing foods for international export or those bound by contract to their customers. In addition, FDA has adopted this particular AOAC extension to ISO 17025 in its own voluntary accreditation program that now includes a number of district laboratories and also the FDA regional laboratories in Jefferson, AR, and Bothell, WA.

The American Society for Testing and Materials (ASTM International), develops market-relevant standards on a global scale. ASTM International standard E1154 states that liquid-delivery device calibration should be performed every three months with 10 data points, while a "quick check" verification should be performed every month with four data points. This standard is not required practice in the pharmaceutical industry, but it does provide a point of reference for laboratories evaluating internal programs. It is one of the few standards that make specific recommendations for both frequency and number of data points in pipette calibration.

INTERNATIONAL GUIDELINES

Because of the globalization of the pharmaceutical industry, international organizations are emerging to provide border-spanning guidance to the vast number of companies operating in multiple countries. For example, the International Organization for Standardization (ISO) emerged as a key global guiding body for a range of industries, particularly laboratories. A nongovernmental organization, this network identifies and adopts relevant standards that can improve practices and ensure quality in products and services. These standards are highly useful for international companies to coordinate laboratory operations and maintain consistent quality programs worldwide.

ISO 17025 presents general requirements for the competence of testing and calibration laboratories, and includes both quality system and technical requirements. This standard does not address the specifics of liquid handling, but does state that all equipment that can contribute significant uncertainty must be calibrated using traceable means and with a stated uncertainty. For nearly all analytical methods, liquid handling undoubtedly falls into this equipment category. Also in ISO 17025 is the recommendation that standard calibration and check methods be used as they are more easily validated and less expensively defended in audits. A growing list of FDA laboratories are ISO 17025 accredited, including district and regional laboratories in Arkansas, California, Colorado, Pennsylvania, and Washington, providing evidence of FDA's support of this standard.

To provide further guidance to laboratories, ISO Technical Committee 48 released a seven-part series, ISO 8655, defining accepted liquid delivery performance and calibration practices. Parts one through five define and specify minimum performance requirements for accuracy and precision in liquid handling.

The next two sections of ISO 8655 provide guidance regarding accepted methods for verifying performance of liquid delivery devices. Part 6, released in 2002, discusses gravimetric calibration, which verifies liquid volumes by measuring weight on a balance.

NEW GUIDELINES RECOMMEND PHOTOMETRY

In 2005, ISO added Part 7 to Standard 8655 because of advancing technologies that overcome several limitations of gravimetry—such as susceptibility to evaporation errors, difficulty in verifying the performance of individual channels in multi-channel devices, and the requirement of a temperature and humidity controlled environment for accurate results. In this section, ISO formally approves photometry for assessment of liquid delivery equipment performance. By relying on known light absorption properties at specific wave lengths, photometric calibration can quickly and conveniently provide strong assurance of data integrity .

Two specific variants of photometric calibration are highlighted by the standard: single-dye and dual dye. As its name implies, single-dye photometry measures light absorption in one colorimetric solution to verify volume. The dual-dye approach to calibration, called Ratiometric Photometry, uses two highly characterized solutions to combat accuracy problems typically associated with single-dye absorbance measurements, and yields results with uncertainty of less than one percent for volumes as low as 0.1 μL.

As the industry changes and laboratories encounter new challenges and solutions, it is likely that ISO will continue to advance its standards. Currently, for example, ISO 8655 clearly applies to handheld pipettes. When these standards were prepared, ISO's focus did not extend to robotic pipettors, which explains the exclusion of these liquid handling devices. The scope of the committee now includes a broader range of laboratory equipment and is likely to move in one of two directions: write additional standards to guide calibration of automated liquid handlers or revise existing standards to include them. It is important to note that 8655 Part 7 does approve "vertical beam photometry," which is useful in verifying robotic pipettors that dispense to microtiter plates.

SUMMARY

Laboratories today must evaluate their processes from start to finish, because of the increasing number of regulations and standards and the industry-wide focus on quality control. This priority is evident in the FDA's Quality System Inspection Technique (QSIT) initiative. The program emphasizes corrective and preventive actions and requires laboratories to identify where problems occur or recur, and to document corrective actions taken.

The high failure rate of liquid handling instrumentation is cause for concern, especially in light of the growing focus on quality and the drastic consequences of failure. Forward-looking laboratories are taking measures to verify accuracy and precision and maintain liquid handling quality control to facilitate compliance and produce quality products.

George Rodrigues, PhD, is senior scientific manager at ARTEL, 25 Bradley Drive, Westbrook ME 04092, 207.854.0860, fax 207.854.0867 grodrigues@artel-usa.com