From assay development to assay transfer (Part 1): it’s the little things that matter

Oct 28, 2015

assay development In drug discovery and molecular diagnostics, assay development is only part of the battle, with assay transfer providing its own challenges and complexities. Whether you’re converting a manual assay into an automated one to increase throughput and/or reproducibility, or moving an assay into a QC environment, a few pre-transfer planning steps can go a long way to smoothing out the process. You can read more about three important pre-transfer activities in Lab Report 7: Facilitating Assay Transfer by Controlling Liquid Handling Variables . In this post, we focus on the first of these three activities—understand your assay—with future posts on implementing pipette training and calibration programs, developing effective assay documentation, and the costs of not implementing these three steps during assay transfer.

Little details can have big implications

In assay transfer, little details can be really important, which is why it’s vital to identify the critical points of the assay and understand all the variables that can affect the success of that step. From acceptable tolerances to plate format and composition, you need to really stop and think about the physics, chemistry, biology, and material science involved in the different steps of your assay and how these factors might affect the data quality of the transferred assay.

Here’s an example of a seemingly small change, made during transfer from a manual method to a semi-automated one, that led to an unexpected outcome. The following story was shared with us by Nathaniel Hentz, PhD., Assistant Director of the Analytical Lab at North Carolina State University’s Golden LEAF Biomanufacturing Training and Education Center (BTEC):

When I think of assay transfer problems I immediately think of an ELISA that we now routinely run in our lab. The ELISA was developed entirely by manual pipetting, including the wash steps (in a typical ELISA, there are three to four washing steps). We converted the ELISA to a semi-automated platform, whereby we switched the manual plate washing with a 96-well plate washer and subsequently noticed a tremendous increase in variability. The first thing we did was analyze the data to see if we could detect a pattern. The data appeared random. The next investigation step was to make sure that all the reagents were in fact added in the correct order and for the correct incubation times. Once that was confirmed, attention was shifted to the automated plate washer itself. The aspirate and dispense tips were confirmed to be functional (i.e., no clogging). Then we noticed residual fluid remaining in the well after each aspiration step. During the manual plate washing, all wells were confirmed dry by visual inspection. This comparison and observation led us to optimize the automated plate washer. In fact, we optimized the plate washer with respect to aspirate speed, needle depth/location, number of cycles, dispense speed, and dispense location. After the plate washer was optimized, the assay variability dropped to lower than the manual method.

The root cause of the high variability in this situation was that we thought the plate washer should work with default settings, and didn’t even consider it as a possible issue during our transfer. Generally speaking, it is very helpful to break down an assay to its different liquid handling steps (including plate washing and mixing), visually confirm the assay assembly from the development (originating) lab and then observe at the transfer lab. Sometimes mixing or inappropriate washes can have a much larger effect on the assay than anticipated.

For Dr. Hentz and his group, even the seemingly small step of adding a plate washer had big implications for the robustness of their assay, increasing variability beyond acceptable tolerances. Their experience also highlights the need to evaluate and optimize every liquid handling step, even washing and mixing.

Identifying critical points for assay transfer—recommendations

Because the nature and goals of different assays vary widely, it’s difficult to create a universal set of rules to determine which steps of your assay are critical and which variables around every step—liquid handling, labware, environmental factors—need to be controlled for a successful assay transfer. As Dr. Hentz recommends, liquid handling steps are likely critical points for most assays, and should be evaluated and optimized. Additional parameters to consider include:

  • Plate or tube format (96, 384, etc.)
  • Selected analyte concentration
  • Number and type of dilution steps
  • Number of liquid transfers
  • Number of incubation or centrifugation steps
  • Number of wash steps
  • Dispense order
  • Liquid properties (viscosity, temperature, etc.)

A more comprehensive list of parameters can be found in Lab Report 7: Facilitating Assay Transfer by Controlling Liquid Handling Variables, Table 1.

Conclusion

Assay transfer can be a challenging activity for many biological laboratories. But with a little time spent examining the assay and planning an assay transfer and optimization approach, the process can move more quickly and efficiently forward.

Don’t forget to come back next month for the second of four articles in our assay transfer series: How to Implement Pipette Training and Calibration Programs.


Additional Resources


About the ExpertTanya Knaide

Tanya Knaide

Tanya Knaide is a scientist with over 10 years of experience in leading new product development projects, product launch campaigns and uncovering customer needs to develop innovative new products and services to satisfy them. As Product Manager at Artel, Tanya has led cross-functional and inter-organizational teams that span across R&D, engineering and marketing and ensured that development and marketing projects deliver benefits to the customer in a timely manner.