Navigating Regulations and Standards for Liquid Delivery Verification

Consider a quality control laboratory that processes 10 to 20 samples per day and releases as many as 20 batches of life-saving vaccines and pharmaceuticals each month. According to FDA stipulations, analytical results from tests performed by these laboratories are used to ensure the safety, purity and effectiveness of these batches. Liquid handling equipment plays a very important role in measuring and analyzing drug samples to determine if they possess these desired properties.

Now, consider the consequences if this liquid delivery instrumentation operates out of specification for one day. One week. One month. How much uncertainty can laboratories legally tolerate? And, even more importantly, how much uncertainty should laboratories tolerate?

While there are federal regulations mandating that laboratories follow liquid delivery quality control processes, each organization has the flexibility to make scientifically valid decisions about methods for and frequency of equipment calibration. Due to the ambiguity of current regulations and the difficulty in keeping up with technological advancements, regulations are incomplete, and a variety of different guides and standards have been brought forward.  This article is intended to help readers navigate current regulations, standards and guidance regarding sufficient calibration procedures, emphasizing the need for more stringent liquid delivery quality assurance.

Liquid Handling and Quality Control

Quality control laboratories routinely prepare samples for testing, producing results that are reviewed, compared to specifications and used to make decisions about whether manufactured products can be released to market. Each step in the QC process is likely to include one or more liquid handling processes. Although some of these steps, such as wash phases, do not rely on accurate volumes, many are highly quantitative.

Some examples of volume-dependent sample preparation processes include:

• Standards preparation: QC laboratories need standard materials in defined concentrations in order to calibrate analytical methods. If standards are inaccurately prepared, calibration will be incorrect and all 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 and inaccurate aliquot volumes can skew results and lead to inaccurate decision making.
• Dilution: Here, a sample solution is mixed with a diluent and a dilution ratio is calculated.  Accurate dilution ratios are dependent on the volumes of both liquids. Because this dilution ratio is used in subsequent calculations, inaccurate dilutions usually propagate to 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 regarding the relationship 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 seeking to 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.  Quantitative measurements seeking to measure levels of impure substances are even more dependent on volume accuracy, and can cause the difference between batch acceptance and an out-of-specification investigation.
• Cell-based assays: These analytical methods can be extremely tedious and expensive to prepare and run. Because of their complexity, cost 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, one of two scenarios ensues. The first is needlessly delaying a good batch of pharmaceuticals. This leads to wasted time, wasted resources and wasted money. And if the biopharmaceutical is in an unstable form, keeping products in limbo for too long can ruin an entire batch.

The second alternative with perhaps even more dire consequences occurs when the quality control laboratory erroneously clears a bad batch for distribution. The results of this error culminate in the biggest fears of most pharmaceutical companies – product recalls, patient injuries and, needless to say, money down the drain.

Consider a manufacturing company that tests pipette performance every six months. After one such test, the company receives notification that one pipette (or several pipettes) failed. By the time the notification is received, one or more batches whose quality was tested with the faulty pipette have been released.  If the product is on the market, this situation would generally require notification of the FDA and the development of a protocol for additional testing. The company would also be at risk of a product recall and potential liability if the drug has already been consumed.

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 very broad and open to interpretation, and 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 CFR 211, Code of Federal Regulations, sets forth 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 set forth by the United States Pharmacopeia (USP).  Unfortunately, USP has little to say about liquid handling.  One general chapter, Chapter 31, deals with glass and plastic volumetric apparatus – specifically volumetric flasks, transfer pipettes, and burettes.  The USP specifies accuracy requirements for these particular pieces of volumetric laboratory equipment, but has not yet issued guidelines about handheld manual or automatic pipettes, or fully 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 handheld manual action pipettes created the need for new standards and the need for preventive maintenance polices in liquid delivery devices.   Similarly, the present trend toward extremely low volumes and higher density formats (e.g., high density microtiter plates) has driven the need for new measurement/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) is an effort to continually improve FDA’s Good Manufacturing Practices, recognizing that static regulations cannot keep pace with the highly dynamic pharmaceutical industry, especially given 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 up-to-date information, cGMP relies on input not only from FDA personnel but also from quality control experts from industry, academia, government and consumer groups as well as a host of independent regulatory bodies.

The International Society for Pharmaceutical Engineering (ISPE), whose mission is to train and educate pharmaceutical manufacturers, is one such organization contributing 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 has a heavy emphasis on criticality assessment and corrective actions, including documentation of non-conformance events.  This guide does not have specific information on liquid handling, but since 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 former National Conference of Standards Laboratories (NCSL), now 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 directing 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 (discussed below), 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/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, Arkansas and Bothell, Washington.

With a long history of importance and responsibility in the world of testing and metrology is the American Society for Testing and Materials (now ASTM International), which 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 since it is one of the few standards that make specific recommendations for both frequency and number of data points in pipetting calibration.

International Guidelines

Due to 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 non-governmental 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 is silent on the particulars 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, including details on accepted metrological requirements and maximum permissible error.

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

Due to 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, ISO added Part 7 to its standard in 2005. Here, ISO formally approved photometry for assessment of liquid delivery equipment performance. Relying on known light absorption properties at specific wavelengths, photometric calibration can provide strong assurance of data integrity, quickly and conveniently.

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^TM, employs 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 0.1μL.

As the industry continues to change and laboratories are faced with new challenges and new solutions, it is likely that ISO will continue to advance its standards. Currently, for example, ISO 8655 is clearly applicable to handheld pipettes. When these standards were prepared, ISO’s focus did not extend to robotic pipettors, explaining 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 very useful in verifying robotic pipettors that dispense to microtiter plates.

Laboratory Trends Emphasizing Greater Process Control

Laboratories are being heavily challenged to evaluate their processes from start to finish by the increasing number of regulations and standards and the industry-wide focus on quality control, evident in the FDA’s Quality System Inspection Technique (QSIT) initiative. This program emphasizes Corrective and Preventive Actions (CAPA), prodding laboratories to identify where problems are occurring or recurring and document corrective actions taken to improve these areas.

The high failure rate of liquid handling instrumentation is cause for concern, especially in light of this 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.

Table 1: Standards Recommending Liquid Delivery Calibration Best Practices

About the Expert

George Rodrigues, Ph.D.

George Rodrigues, Ph.D., is Senior Scientific Manager at Artel, the global leader in liquid delivery quality assurance. Rodrigues is responsible for developing and delivering communications and consulting programs designed to maximize laboratory quality and productivity through science-based management of liquid delivery.  Rodrigues is Artel’s chief representative to key commercial clients, government regulatory bodies and industry organizations.  His speaking and teaching engagements, along with his publications, build awareness of the challenges and solutions for laboratories in maintaining data integrity and confidence in their testing protocols.  He plays a key role in developing the manufacturing and quality assurance processes for Artel products and organizes programs to assist pharmaceutical, biotechnology and clinical laboratories in improving their liquid delivery quality assurance and analytical process control. Rodrigues earned his BS in Chemical Engineering at the U.C. Berkeley, and a PhD in Chemical Engineering at the University of Wisconsin.