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Commentary: The importance of liquid handling quality assurance through the drug discovery process
July 2010
SHARING OPTIONS:
The tug-of-war between quality and productivity is an issue
facing all drug discovery companies, competing in a fierce race
to bring
breakthrough drugs to market before the competition. With the advance of
laboratory automation and the introduction of automated liquid
handlers (ALHs),
high-throughput screening (HTS) processes have provided the ability to rapidly
evaluate a large number of compounds as potential drug
foundations.
However, an increase in throughput does not directly
correlate to decreases in cost or increases in productivity. The drug discovery
process currently comes with an average price tag of $800 million to $1
billion
per new molecular entity and lasts from 10 to 15 years. The continual
mushrooming of drug prices and increased public scrutiny of
pharmaceutical
companies highlight the need for new solutions to streamline drug discovery
operations.
Paying more
attention to liquid handling quality assurance
early in the drug discovery process is one way that companies can enhance
quality and efficiency in
their operations. Liquid handling is an integral part
of the drug discovery process, involved in nearly every stage of drug
development. By
implementing a robust liquid handling quality assurance (LHQA)
program from compound preparation through clinical trials, laboratories can
build
quality into their processes and decrease the overall cost and time
required for drug discovery.
Previously, the lack of adequate technologies for
verification of ALH performance prevented cost-effective quality control
processes. Now, new
technologies are available for rapid and reproducible
assessment of the accuracy and precision of volumes dispensed from automatic
instrumentation.
The cost of false negative and false positive
results
Liquid handling instrumentation is used for a large number
of critical tasks in drug discovery, from dissolving
compounds into DMSO to
adding enzymes to an assay plate and transferring assays from laboratory to
laboratory. Due to the minute volumes typically
handled during HTS and other
drug discovery phases, inaccuracies of just one microliter can affect the
integrity of the process by producing false
positive and false negative
results.
False positive results, while not fatal to the drug
discovery process, can be detrimental to overall efficiency, causing
technicians to waste time and resources chasing dead ends. Today, with the
growing focus on reducing the cost of drugs, pharmaceutical companies are
seeking new ways to streamline R&D in any way feasible. Reducing the
number
of false positives pursued through the drug discovery process can decrease the
number of retests, minimize reagent loss and maximize staff and
equipment
utilization.
To illustrate this point, consider a typical HTS laboratory
testing
approximately one million wells during each screening campaign, with
anywhere from 20 to 25 campaigns conducted annually. While consumable costs
alone
have been reduced to one to five cents per well in recent years, there
are a number of hidden costs to consider during HTS follow-up. First, as false
positive results are pursued through subsequent stages, consumable costs swell
to up to a few dollars per well. Secondly, when reagents are expensive or
in
short supply, the list of compounds for retest is sometimes pared down to a
manageable number, usually through computational chemistry calculations.
These
calculations include data from biologically active (positive) compounds so
false positives make it difficult to produce an accurate computational
model.
Finally, if an HTS group decides to retest all of the hits identified in a
primary 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 these
reasons, each additional screen of a compound passed along due to
false
positive results not only wastes compound, but also leads to delays and inefficient
use of labor and equipment.
While avoidable costs are undesirable in any business,
perhaps the greatest fear of pharmaceutical companies is the missed
opportunity
cost of false negative results.
Dispensing reagents in volumes inaccurate by even miniscule amounts,
especially in complex serial dilution assays, can alter concentration and
prevent the identification of a reaction between compounds, the foundation
for
a new drug. While it is nearly impossible to estimate the value of lost revenue
from this type of error, one could argue that the figure is in the
billions of
dollars. In this case, what you don't know will hurt you.
Where liquid handling error occurs
Identifying and correcting for liquid handling error early
in the drug
discovery process can save time, money, and resources. To design
effective quality control processes, it is essential to understand where liquid
handling equipment is used.
Liquid handling quality control must start as early as
compound
preparation and management, as compound volume errors can introduce
variability into even the most robust assay. In initial compound preparation,
as
powdered compounds are diluted into DMSO or other liquid-based solutions, it
is critical that both volumes be correct for accurate analysis later in the
process.
In the HTS laboratory, automated liquid handlers are used in
screening campaigns to dispense stock solutions
across assay plates containing
volumes of enzymes and substrates. There is potential for volume error whether
dispensing compound solution from the
mother plate directly to the assay plate
or from the mother plate into an intermediary daughter plate with buffer
solution. These errors can propagate
into false positive and false negative
results in the initial screen.
As a progressively smaller
group of compounds is moved
through each stage of the drug discovery process, from 10,000 hits in lead
discovery to 1,000 in lead optimization, to just
a hundred in the lead
profiling stage, liquid handling error inevitably becomes more costly. At each
subsequent stage, compounds are tested more
rigorously to determine their
feasibility for drug manufacture, amplifying the cost of false positives. Lead
profiling assays are the most expensive to
run and control over liquid handling
operations is vital.
ALHs are also used when running
compounds through ADMET
assays, which evaluate a compound's absorption, distribution, metabolism,
excretion and toxicity properties. Because ADMET
testing profiles how compounds
will interact with the human body, data produced at this stage is critical in
the decision to pursue or abandon further
development.
It is also important to note that each time compounds are
moved 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 week
to several months. Time
wasted identifying the cause of a failed assay transfer
not only erodes productivity, but can also allow competitors to win the race to
market 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 both
the original
and transferred laboratories. For example, an assay initially
developed in a therapeutic laboratory may require the addition of 50
microliters each of
a compound, substrate, and enzyme to a 96-well plate. In
the HTS laboratory, these volumes will be proportionately reduced to one-tenth
that amount for
screening. If target volumes are incorrect in the HTS
laboratory, the assay may not function and the screening campaign may not
begin, leading to
significant time delays in the drug discovery process.
Verifying the performance of liquid handling equipment can remove one major
source of assay
variability and facilitate a seamless transfer.
Summary
From compound creation to assay development, liquid handling
error can cost time, waste resources and prevent the identification of the next
billion-dollar drug. Regular calibration programs and verification checks help
to reduce liquid handling quality error. The more frequently automated
liquid
handlers are evaluated, the sooner malfunctioning equipment will be detected
and fixed for maximum accuracy and efficiency in the laboratory.
With the help
of advanced quality assurance technology, drug discovery operations can be more
closely controlled for liquid handling accuracy and
precision without
detracting from overall efficiency.
Sources of automated liquid handler errors
When seeking to reduce inaccuracy and imprecision in
automated 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 its
ability to transfer volumes accurately and precision. Although permanent tips
are less costly than
disposable tips, meticulous cleaning protocols are
necessary to avoid contamination in subsequent transfers. This process is time
consuming and may
lead to additional error due to improper washing.
While disposable tips are used only once and
rarely cause
sample contamination, it is important for laboratories using them to realize
the direct connection between tip type and volume transfer
integrity. It is
always recommended that laboratories use vendor-approved tips to optimize
liquid delivery. Tip performance is directly related to
quality because tip
material, shape, fit and wet-ability are important for test repeatability.
Sequential dispensing inaccuracy
Some liquid handling protocols call for a
large volume of
reagent to be aspirated, then systematically dispensed across a microtiter
plate. While sequential dispensing can save time, it can
also lead to
inaccuracies.
To avoid contamination or dilution upon dispensing, users
must ensure that the tips do not touch
any liquid in the wells. It is usually
recommended that liquid be dispensed into a dry well or dispensed using a
non-contact technique above the
buffer-filled wells. When an ALH employs a
sequential transfer, users should confirm that the same volume is dispensed in
each successive transfer, as
it is common for the volume of the first and/or
last dispense to be inaccurate.
Serial dilution transfers
Many laboratories perform dilution testing to determine
toxicity, drug efficacy and
dose response characteristics associated with their
specific assays. In a serial dilution, an important reagent is sequentially
reduced in
concentration using a microtiter plate containing different rows of
lowering amounts of critical reagent. In many applications, a diluted target
reagent will be transferred to a column of wells containing a pre-determined
volume of assay buffer.
Serial dilution
protocols are typically performed by ALHs.
To ensure homogenous transfer of reagents, it is important that users verify
the accuracy of each volume
transfer before the next transfer begins. Any
inconsistencies in the mixture will invalidate the entire experiment.
Pipetting methods and method parameters
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 most common technique is
forward mode, which is suitable for dispensing aqueous reagents. During forward
mode, the full contents of the tip are
released upon discharge. The reverse
mode aspirates more reagent into the tip than is dispensed into the well and is
suitable for viscous or foaming
liquids. While forward-mode pipetting is the
preferred pipetting mode that nearly all manufacturers specify for use, reverse
mode is recommended for
liquids that tend to stick to plastic tips and/or leave
a noticeable film. Along with choice of pipetting technique, automated liquid handling
errors
may occur when variables within the user interface are incorrectly
defined. The user should ensure that all settings are properly defined for each
assay to maintain optimal efficiency.
Dr. Nathaniel Hentz is the associate director of the Biomanufacturing Training and Education Center (BTEC) Analytical Lab at North Carolina State University. Prior to this current role, Hentz served as an independent consultant working with Artel, offering guidance on their efforts toward automated liquid handling quality control within high throughput screening laboratories. Hentz's tenure in the HTS industry includes nearly two years as a senior research investigator at Bristol-Myers Squibb and seven years at Eli Lilly & Co.'s Research Triangle Park Laboratories in North Carolina. Hentz received his Ph.D. in analytical chemistry from the University of Kentucky in 1996 and joined Lilly as a postdoctoral scientist the same year. Back |
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