Analysis of Biologics Molecular Descriptors towards Predictive Modelling for Protein Drug Development Using Time-Gated Raman Spectroscopy

Abstract

Pharmaceutical proteins, compared to small molecular weight drugs, are relatively fragile molecules, thus necessitating monitoring protein unfolding and aggregation during production and post-marketing. Currently, many analytical techniques take offline measurements, which cannot directly assess protein folding during production and unfolding during processing and storage. In addition, several orthogonal techniques are needed during production and market surveillance. In this study, we introduce the use of time-gated Raman spectroscopy to identify molecular descriptors of protein unfolding. Raman spectroscopy can measure the unfolding of proteins in-line and in real-time without labels. Using K-means clustering and PCA analysis, we could correlate local unfolding events with traditional analytical methods. This is the first step toward predictive modeling of unfolding events of proteins during production and storage.

Keywords: CD; DLS; K-means clustering; PCA; Raman spectroscopy; biologics; in-line measurement; pharmaceutical proteins; protein unfolding; tryptophan fluorescence.

Figure 2

Averaged and normalized Time-gated spectra of IgG (glycosylated; blue; N = 11) and IgG (non-glycosylated; red; N = 6) at (A) 25 °C, (B) and at 65 °C. Individual, non-processed, and non-normalized Time-gated spectra (i.e., raw data) are shown in Figures S11 and S12. Regions of significance in respect of the glycosylation status of proteins, as determined by Brewser et al. (2011) [46] are shown in green. Raman values of significance, as presented in Table S5, are depicted in bold and red.

Figure 1

Averaged (N = 11) and normalized time-gated spectra of pepsin at 25 °C (blue) and 65 °C (red). (A,B) correspond to the individual, non-processed, and non-normalized Time-gated spectra as presented in the left and right panes in Figure S10. (i.e., raw data). Raman values of significance, as presented in Table S4 (supplementary data), are depicted in bold and red.

Figure 3

Averaged (N = 11) and normalized Time-gated spectra of (A) ovalbumin at 25 °C (blue) and 50 °C (red), and (B) LmTIM_E65Q at 25 °C (blue) and 86 °C (red) correspond to the individual, non-processed, and non-normalized time-gated spectra as presented in Figures S19 and S20 (i.e., raw data). Raman values of significance, as presented in Tables S6 and S7, are depicted in bold and red.