An automated packed protein G micro-pipette tip assay for rapid quantification of polyclonal antibodies in ovine serum.
The demands on the biopharmaceutical sector to expedite process development have instigated the deployment of micro-biochemical engineering techniques to acquire manufacturing insight with extremely small sample volumes. In conjunction with automated liquid handlers, this permits the simultaneous evaluation of multiple operating conditions and reduces manual intervention. For these benefits to be sustained, novel ways are now required to accelerate analysis and so prevent this becoming a throughput bottleneck. For example, although Protein G HPLC is used to quantify antibody titres in bioprocess feedstocks, it can be time-consuming owing to the serial nature of its application. Although commercial options are available that can process many samples simultaneously, these require separate, potentially expensive instruments. A more integrated approach is desirable wherein the assay is implemented directly on a robot. This article describes a high-throughput alternative to antibody HPLC analysis which uses an eight-channel liquid handler to control pipette tips packed with 40 μL of Protein G affinity matrix. The linearity, range, limit of detection, specificity and precision of the method were established, with results showing that antibody was detected reliably and specifically between 0.10 and 1.00 mg/mL. Subsequently, the technique was used to quantify the antibody titre in ovine serum, which is used as feed material by BTG PLC for manufacturing FDA-approved polyclonal bio-therapeutics. The mean concentration determined by the tips was comparable to that found by HPLC, but the tip method delivered its results in less than 40% of the time and with the potential for further, substantial time-savings possible by using higher capacity robots.
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E7JE, UK. sunil.chhatre@ucl.ac.uk
Sunil
S
Chhatre
Richard
R
Francis
Daniel G
DG
Bracewell
Nigel J
NJ
Titchener-Hooker
D000818:Animals; D000906:Antibodies / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification*; D001331:Automation; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D011485:Protein Binding; D012756:Sheep
D000906:Antibodies; C029152:G-substrate; D009419:Nerve Tissue Proteins