The challenge of applying Raman spectroscopy to monitor recombinant antibody production

Abstract

UV resonance Raman (UVRR) spectroscopy combined with chemometric techniques was investigated as a physiochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) cells. Due to the enhanced selectivity of the UVRR, spectral variations arising from protein, small molecule substrates, and nucleic acid medium components could be measured simultaneously and we have successfully determined antibody titre. Medium samples were taken during culture of three CHO cell lines: two antibody-producing cell lines and a non-producing cell line, and analysed by UVRR spectroscopy using an excitation laser of 244 nm. Principal component analysis (PCA) was applied to the spectral sets and showed a linear trend over time for the antibody-producing cell lines that was not observed in the non-producing cell line. Partial least squares regression (PLSR) was used to predict antibody titres, glucose utilization and lactate accumulation, and compared very favourably with gold standard data acquired with the much slower techniques of ELISA and liquid chromatography. Further analysis of the UVRR spectral sets using two-dimensional correlation moving windows also revealed that spectral variations due to protein and nucleic acid concentrations in the medium during cell culture varied between each of the three cell lines investigated.

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

“…The spectra showed contributions from glucose, lactate, and the antibody product. 89 Using chemometric methods, they were able to build PLS models with this UVRRS data for antibody, glucose, and lactic acid concentrations. The antibody concentration models spanned a range of $50-600 mg/L for the two cell lines, and reference measurements (ELISA and HPLC) showed they had errors of 11% and 8%.…”

“…This means that the spectra from different bioprocesses can look very similar (e.g., the UVRR spectra of supernatant liquid collected from the different cell lines in Figure 5d). 89 It is often difficult to unscramble the data from the various chemical components (metabolites, lipids, product protein, HCP, nucleotides, etc.) and the protein bands (if present) will be extensively overlapped with amino acid bands from the media; this all increase the difficulty of the analysis.…”

Insights into Protein Structure and Dynamics by Ultraviolet and Visible Resonance Raman Spectroscopy

“…81 Histidine protonation states are easily determined by UVRR studies of protein in D 2 O buffer, and such experiments have been performed on superoxide dismutase and galectins. 112–114 An area with growing use of UVRR is the pharmaceutical industry, in which UVRR experiments have been reported for recombinant therapeutic proteins 115 and antibodies 116 as well as drug binding. 117,118 Time-resolved UVRR experiments have also probed dynamics of heme proteins and photoactive yellow protein, 119–122 and a variety of UVRR experiments have investigated intermediates in protein folding.…”

Closed loop control of lactate concentration in mammalian cell culture by Raman spectroscopy leads to improved cell density, viability, and biopharmaceutical protein production

“…We chose Raman spectroscopy for its ability to perform direct, online measurements of solution analytes, including glucose, and lactate. Because of its many advantages, Raman spectroscopy has increasingly been used as a tool for the online monitoring of cell culture metabolites (Abu‐Absi et al, ; André et al, ; Ashton et al, ; Avila et al, ; Iversen et al, ; Mehdizadeh et al, ; Singh et al, ; Whelan et al, ), and even control a CHO cell culture glucose feeding (Berry et al, ; Craven et al, ).…”