A new flow cell and chemometric protocol for implementing in‐line Raman spectroscopy in chromatography

Abstract

On‐line monitoring tools for downstream chromatographic processing (DSP) of biotherapeutics can enable fast actions to correct for disturbances in the upstream, gain process understanding, and eventually lead to process optimization. While UV/Vis spectroscopy is mostly assessing the protein's amino acid composition and the application of Fourier transform infrared spectroscopy is limited due to strong water interactions, Raman spectroscopy is able to assess the secondary and tertiary protein structure without significant water interactions. The aim of this work is to implement the Raman technology in DSP, by designing an in‐line flow cell with a reduced dead volume of 80 μL and a reflector to increase the signal intensity as well as developing a chemometric modeling path. In this context, measurement settings were adjusted and spectra were taken from different chromatographic breakthrough curves of IgG1 in harvest. The resulting models show a small average RMSEP of 0.12 mg/mL, on a broad calibration range from 0 to 2.82 mg/mL IgG1. This work highlights the benefits of model assisted Raman spectroscopy in chromatography with complex backgrounds, lays the fundamentals for in‐line monitoring of IgG1, and enables advanced control strategies. Moreover, the approach might be extended to further critical quality attributes like aggregates or could be transferred to other process steps.

Combining Mechanistic Modeling and Raman Spectroscopy for Monitoring Antibody Chromatographic Purification

“…The acquired Raman spectra of the BT curves are comparable with the spectra described in [10] and are shown in Figure S1A. Due to the high impurity content in the harvest, the spectral features of different species (i.e., target mAb, media components, HCPs, DNA and HMW) overlapped, leading to broad bands and no distinct peak profiles within single spectra.…”

“…Fifteen breakthrough (BT) runs were performed on MabSelect SuRe columns (GE Healthcare, Uppsala, Sweden), prepacked by Repligen GmbH (Ravensburg, Germany, 0.5 × 5 cm), as described in [10]. The feed concentration, flow rate, and the fraction duration were changed between the different BT runs, as described in Table 1.…”

“…The mAb concentrations were determined off-line by HPLC with an analytical standard deviation of 0.01 mg/mL as described in [23]. The HMW, HCP, and DNA content of the harvest were determined as described in [10]. A schematic illustration of the experimental set-up is shown in Figure 2.…”

“…Hence, sets were rotated in order to have every BT run in the external prediction set once, as shown in Table 2. As an example, to build the model for rotation 1 (ROT1), the data of BT#2-6 were used for model calibration and tested on BT#1 (for further details, see [10]).…”

“…Several applications of Raman spectroscopy have been reported in upstream processing [7][8][9], showing the potential of this technology, which often requires specific modeling techniques, such as partial least squares (PLS) regression, to extract the desired information from the measured spectra. Recently, a successful implementation of Raman spectroscopy for the on-line monitoring of monoclonal antibody (mAb) concentrations in downstream processing was reported by Feidl et al [10]. An ad hoc developed flow cell enabled the integration of the Raman technology into the capture (protein A) step of a mAb manufacturing process, providing accurate on-line estimates of mAb concentration.…”