In upstream processing, there has been significant progress to maximise titres, cell counts and viability. However, there is little understanding to how changes in the upstream conditions impact product profile in the bioreactor. Currently, analytical techniques require samples to be purified prior to measurement. Although purification is possible, the main issue is that key components (e.g. large aggregates) may be removed as a result, ultimately providing data that may not be a true representation of the cell culture. In addition purification is also time-consuming and costly. Hence, there is a need to develop assays/tools that can help characterise aggregate levels in complex multi-component environment earlier in research and development. This can eventually lead to better decision-making for selecting cell lines and candidate molecules.<br/><br/>Fluorescent dyes have been shown to be able to measure aggregates on purified antibodies. However, dyes solely in the cell culture medium do not provide specificity to monoclonal antibody (mAb) aggregates in a complex mixture. Hence, in this study we evaluated the use of two dyes in fluorescence resonance energy transfer (FRET) to increase specificity in measuring mAb aggregates.<br/><br/>The FRET design utilises a donor dye conjugated to a protein that binds mAbs specifically and an acceptor dye which has specificity towards aggregates. To achieve energy transfer, there are a few requirements which are related the Förster's distance equation: shared overlap between the donor emission spectrum and acceptor excitation spectrum (&gt;30%), distance between the donor and acceptor (1-10 nm), quantum yield of the donor and the relative orientation of the donor and acceptor transition dipoles. Achieving FRET is more challenging as both donors and acceptors are free in solution. In addition, if the acceptor interacts with the inner structure of the aggregate there may be steric hindrance towards it interacting with the donor. To fully understand the FRET design, we investigated the spectral overlap and distance requirements.

Challenges in developing a FRET assay to measure monoclonal antibody aggregates

255th National Meeting (2018)

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