Commentary: The importance of liquid handling quality assurance through the drug discovery process

The choice of reverse- or forward-mode pipetting is often based on operator preference, but choosing the right technique can help to minimize error in automated liquid handling.

The tug-of-war between quality and productivity is an issuefacing all drug discovery companies, competing in a fierce race to bringbreakthrough drugs to market before the competition. With the advance oflaboratory automation and the introduction of automated liquid handlers (ALHs),high-throughput screening (HTS) processes have provided the ability to rapidlyevaluate a large number of compounds as potential drug foundations.  
However, an increase in throughput does not directlycorrelate to decreases in cost or increases in productivity. The drug discoveryprocess currently comes with an average price tag of $800 million to $1 billionper new molecular entity and lasts from 10 to 15 years. The continualmushrooming of drug prices and increased public scrutiny of pharmaceuticalcompanies highlight the need for new solutions to streamline drug discoveryoperations.

Paying more attention to liquid handling quality assuranceearly in the drug discovery process is one way that companies can enhancequality and efficiency in their operations. Liquid handling is an integral partof the drug discovery process, involved in nearly every stage of drugdevelopment. By implementing a robust liquid handling quality assurance (LHQA)program from compound preparation through clinical trials, laboratories canbuild quality into their processes and decrease the overall cost and timerequired for drug discovery. 
Previously, the lack of adequate technologies forverification of ALH performance prevented cost-effective quality controlprocesses. Now, new technologies are available for rapid and reproducibleassessment of the accuracy and precision of volumes dispensed from automaticinstrumentation. 
The cost of false negative and false positive results
Liquid handling instrumentation is used for a large numberof critical tasks in drug discovery, from dissolving compounds into DMSO toadding enzymes to an assay plate and transferring assays from laboratory tolaboratory. Due to the minute volumes typically handled during HTS and otherdrug discovery phases, inaccuracies of just one microliter can affect theintegrity of the process by producing false positive and false negativeresults.
False positive results, while not fatal to the drugdiscovery process, can be detrimental to overall efficiency, causingtechnicians to waste time and resources chasing dead ends. Today, with thegrowing focus on reducing the cost of drugs, pharmaceutical companies areseeking new ways to streamline R&D in any way feasible. Reducing the numberof false positives pursued through the drug discovery process can decrease thenumber of retests, minimize reagent loss and maximize staff and equipmentutilization.
To illustrate this point, consider a typical HTS laboratorytesting approximately one million wells during each screening campaign, withanywhere from 20 to 25 campaigns conducted annually. While consumable costsalone have been reduced to one to five cents per well in recent years, thereare a number of hidden costs to consider during HTS follow-up. First, as falsepositive results are pursued through subsequent stages, consumable costs swellto up to a few dollars per well. Secondly, when reagents are expensive or inshort supply, the list of compounds for retest is sometimes pared down to amanageable number, usually through computational chemistry calculations. Thesecalculations include data from biologically active (positive) compounds sofalse positives make it difficult to produce an accurate computational model.Finally, if an HTS group decides to retest all of the hits identified in aprimary screen, which can be as high as 1 percent of the compound collection,it can take weeks to produce enough of a recombinant protein. For thesereasons, each additional screen of a compound passed along due to falsepositive results not only wastes compound, but also leads to delays and inefficientuse of labor and equipment.
While avoidable costs are undesirable in any business,perhaps the greatest fear of pharmaceutical companies is the missed opportunitycost of false negative results. 
Dispensing reagents in volumes inaccurate by even miniscule amounts,especially in complex serial dilution assays, can alter concentration andprevent the identification of a reaction between compounds, the foundation fora new drug. While it is nearly impossible to estimate the value of lost revenuefrom this type of error, one could argue that the figure is in the billions ofdollars. In this case, what you don't know will hurt you.
Where liquid handling error occurs
Identifying and correcting for liquid handling error earlyin the drug discovery process can save time, money, and resources. To designeffective quality control processes, it is essential to understand where liquidhandling equipment is used.
Liquid handling quality control must start as early ascompound preparation and management, as compound volume errors can introducevariability into even the most robust assay. In initial compound preparation,as powdered compounds are diluted into DMSO or other liquid-based solutions, itis critical that both volumes be correct for accurate analysis later in theprocess.

In the HTS laboratory, automated liquid handlers are used inscreening campaigns to dispense stock solutions across assay plates containingvolumes of enzymes and substrates. There is potential for volume error whetherdispensing compound solution from the mother plate directly to the assay plateor from the mother plate into an intermediary daughter plate with buffersolution. These errors can propagate into false positive and false negativeresults in the initial screen.
As a progressively smaller group of compounds is movedthrough each stage of the drug discovery process, from 10,000 hits in leaddiscovery to 1,000 in lead optimization, to just a hundred in the leadprofiling stage, liquid handling error inevitably becomes more costly. At eachsubsequent stage, compounds are tested more rigorously to determine theirfeasibility for drug manufacture, amplifying the cost of false positives. Leadprofiling assays are the most expensive to run and control over liquid handlingoperations is vital. 
ALHs are also used when running compounds through ADMETassays, which evaluate a compound's absorption, distribution, metabolism,excretion and toxicity properties. Because ADMET testing profiles how compoundswill interact with the human body, data produced at this stage is critical inthe decision to pursue or abandon further development.
It is also important to note that each time compounds aremoved from one drug discovery stage to the next, an assay transfer takes place.Assay transfers are rarely seamless, and can cause process delays of one weekto several months. Time wasted identifying the cause of a failed assay transfernot only erodes productivity, but can also allow competitors to win the race tomarket with the next blockbuster drug.
While there are many causes of failed assay transfers,inaccuracy in the volume transfer process can be a major source of error,leading to a lengthy problem identification process involving parties from boththe original and transferred laboratories. For example, an assay initiallydeveloped in a therapeutic laboratory may require the addition of 50microliters each of a compound, substrate, and enzyme to a 96-well plate. Inthe HTS laboratory, these volumes will be proportionately reduced to one-tenththat amount for screening. If target volumes are incorrect in the HTSlaboratory, the assay may not function and the screening campaign may notbegin, leading to significant time delays in the drug discovery process.Verifying the performance of liquid handling equipment can remove one majorsource of assay variability and facilitate a seamless transfer.
From compound creation to assay development, liquid handlingerror can cost time, waste resources and prevent the identification of the nextbillion-dollar drug. Regular calibration programs and verification checks helpto reduce liquid handling quality error. The more frequently automated liquidhandlers are evaluated, the sooner malfunctioning equipment will be detectedand fixed for maximum accuracy and efficiency in the laboratory. With the helpof advanced quality assurance technology, drug discovery operations can be moreclosely controlled for liquid handling accuracy and precision withoutdetracting from overall efficiency. 

Sources of automated liquid handler errors
When seeking to reduce inaccuracy and imprecision inautomated liquid handling, the following common errors may be to blame:
Tip type and contamination   
The type of tip used on an ALH is a critical factor in itsability to transfer volumes accurately and precision. Although permanent tipsare less costly than disposable tips, meticulous cleaning protocols arenecessary to avoid contamination in subsequent transfers. This process is timeconsuming and may lead to additional error due to improper washing.
While disposable tips are used only once and rarely causesample contamination, it is important for laboratories using them to realizethe direct connection between tip type and volume transfer integrity. It isalways recommended that laboratories use vendor-approved tips to optimizeliquid delivery. Tip performance is directly related to quality because tipmaterial, shape, fit and wet-ability are important for test repeatability.
Sequential dispensing inaccuracy
Some liquid handling protocols call for a large volume ofreagent to be aspirated, then systematically dispensed across a microtiterplate. While sequential dispensing can save time, it can also lead toinaccuracies.
To avoid contamination or dilution upon dispensing, usersmust ensure that the tips do not touch any liquid in the wells. It is usuallyrecommended that liquid be dispensed into a dry well or dispensed using anon-contact technique above the buffer-filled wells. When an ALH employs asequential transfer, users should confirm that the same volume is dispensed ineach successive transfer, as it is common for the volume of the first and/orlast dispense to be inaccurate.
Serial dilution transfers
Many laboratories perform dilution testing to determinetoxicity, drug efficacy and dose response characteristics associated with theirspecific assays. In a serial dilution, an important reagent is sequentiallyreduced in concentration using a microtiter plate containing different rows oflowering amounts of critical reagent. In many applications, a diluted targetreagent will be transferred to a column of wells containing a pre-determinedvolume of assay buffer.
Serial dilution protocols are typically performed by ALHs.To ensure homogenous transfer of reagents, it is important that users verifythe accuracy of each volume transfer before the next transfer begins. Anyinconsistencies in the mixture will invalidate the entire experiment.
Pipetting methods and method parameters
The choice of reverse- or forward-mode pipetting is oftenbased on operator preference, but choosing the right technique can help tominimize error in automated liquid handling. The most common technique isforward mode, which is suitable for dispensing aqueous reagents. During forwardmode, the full contents of the tip are released upon discharge. The reversemode aspirates more reagent into the tip than is dispensed into the well and issuitable for viscous or foaming liquids. While forward-mode pipetting is thepreferred pipetting mode that nearly all manufacturers specify for use, reversemode is recommended for liquids that tend to stick to plastic tips and/or leavea noticeable film. Along with choice of pipetting technique, automated liquid handlingerrors may occur when variables within the user interface are incorrectlydefined. The user should ensure that all settings are properly defined for eachassay to maintain optimal efficiency.

Dr. Nathaniel Hentz is the associate director of theBiomanufacturing Training and Education Center (BTEC) Analytical Lab at NorthCarolina State University. Prior to this current role, Hentz served as anindependent consultant working with Artel, offering guidance on their effortstoward automated liquid handling quality control within high throughputscreening laboratories. Hentz's tenure in the HTS industry includesnearly twoyears as a senior research investigator at Bristol-Myers Squibb and seven yearsat Eli Lilly & Co.'s Research Triangle Park Laboratories in NorthCarolina.Hentz received his Ph.D. in analytical chemistry from the University ofKentucky in 1996 and joined Lilly as a postdoctoral scientist thesame year. 

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