title: Strategies to minimize variability and bias associated with manual pipetting in ligand binding assays to assure data quality of protein therapeutic quantification.
abstract: Bioanalytical laboratories require accurate and precise pipetting to assure reproducible and accurate results for reliable data. Two areas where pipetting differences among analysts lead to poor reproducibility are long term stability testing and sample dilution. The purpose of this paper is to illustrate the problems with manual pipetting, describe an automation strategy to mitigate risks associated with manual pipetting, and provide recommendations on a control strategy that properly monitors samples requiring dilutions. We determined differences among various manual pipetting techniques by analysts within a laboratory. To reduce variability in pipetting, a flexible modular liquid handling script was created on the Hamilton Microlab Star (HMS) to perform sample dilution, pre-treatment and plate loading. The script is capable of handling variable dilution factors. Additionally, two dilution controls were prepared and tested at concentrations of high and mid quality controls (QC). These same dilution controls were incorporated into both pre-study validation and in-study QCs to monitor dilution processing and assay performance. Variability of manual pipetting among 11 analysts was more negatively biased with increasing dilution. Forward and reverse pipetting delivering different volumes contributed to the discordance. The dilutional bias with manual pipetting was eliminated using the liquid handler. Total error of dilution controls was less than 20%. The in-study pass rate was 100%. Application of liquid handlers minimizes the variability and bias due to manual pipetting differences among analysts. The incorporation of dilution QCs serves a dual purpose to monitor the dilution process of the samples as well as the binding assay performance.
authors.affiliation: Pharmacokinetics and Drug Metabolism, Amgen Inc, Thousand Oaks, CA, United States.
authors.forename: Kinnari
authors.initials: K
authors.lastname: Pandya
authors.forename: Chad A
authors.initials: CA
authors.lastname: Ray
authors.forename: Laura
authors.initials: L
authors.lastname: Brunner
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Jean W
authors.initials: JW
authors.lastname: Lee
authors.forename: Binodh
authors.initials: B
authors.lastname: DeSilva
mesh_terms: D001331:Automation; D015982:Bias; D019411:Clinical Laboratory Techniques / Q000592:standards*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008024:Ligands; D011506:Proteins / Q000032:analysis*; D011786:Quality Control
chemical_list: D008024:Ligands; D011506:Proteins