Fast Quantification of Recombinant Protein Inclusion Bodies within Intact Cells by FT-IR Spectroscopy

Abstract

The accomplishment of the quantification of the recombinant protein content of whole bacterial cells by FT-IR spectroscopy by application of chemometrics is shown. Recombinant Escherichia coli cells expressing an inclusion body forming fusion protein were dried on a 96-well silicon plate for the analysis in a high-throughput FT-IR spectrometer. Acquired spectra of additionally conventionally quantified samples were used to establish a multivariate calibration. The obtained method was tested by predicting inclusion body contents of samples not used for the multivariate model. Results from FT-IR spectra coincided well with the data of universalized electrophoresis analysis. Hence FT-IR spectroscopy could prove as a fast and simple alternative to conventional quantification methods.

Rapid monitoring of recombinant antibody production by mammalian cell cultures using fourier transform infrared spectroscopy and chemometrics

“…Among its advantages are minimal sample preparation time, high throughput automation (usually in batches of 96 or 384), low cost per sample and spectra that can be acquired in less than a minute. IR spectroscopy has previously been used for offline monitoring of cell biomass (Vaidyanathan et al, 1999), recombinant protein production in batch cultures of Escherichia coli (Gross-Selbeck et al, 2007;McGovern et al, 1999), and for quantification of secondary metabolite production (McGovern et al, 2002) as well as nutrient supply (Brimmer and Hall, 1993). FT-IR has also been used as a tool for monitoring temperature-dependent unfolding of purified proteins including an IgG1 antibody (Matheus et al, 2006) and for efficient classification of microorganisms when combined with chemometrics (Timmins et al, 1998;Winder et al, 2004).…”

“…FT-IR spectroscopy has proven to be a particularly valuable tool for the characterization of protein secondary structure (Haris and Chapman, 1992). Specific regions in the IR spectra are indicative of protein secondary structure, and correspond to the amount of protein in the sample (Gross-Selbeck et al, 2007). The region that gives the best correlation for protein accumulation is the amide I band (1,700-1,550 cm À1 ) that belongs predominantly to the C --O stretching vibration of the amide group in the peptide linkage, with some contribution from C-N stretching and N-H bending (Stuart et al, 1997).…”

The Value of UV, Fluorescence, and FTIR Spectroscopy in Biopharmaceutical Development

“…Gross-Selbeck et al [38] applied FT-IR as a high-throughput technique for the analysis of inclusion bodies using dried samples with a 96-well silicon plate. However, instrumentation is now available commercially that enables FT-IR to be run in a high-throughput mode: flow-through cells with a small (w7 mm) fixed pathlength enable a high sample throughput for liquid samples and accessories that enable FT-IR analysis of solids in a microplate format.…”

Analytical Approaches for Assessing Aggregation of Protein Biopharmaceuticals

“…Both procedures have importantly contributed to the understanding of the IB architecture [18,28,49,69,70], that is mainly supported by antiparallel, intermolecular -sheet moieties [48,71] that represent around 20-25% of the total secondary structure [72]. This organization in cross--sheet can be assigned to the band peaking at 1610-1630 cm -1 in the amide I region [15,28,46,67] while native -sheet structures are assigned to the region 1630-1640 cm -1 [18,46,66].…”