Raman spectroscopy characterization of antibody phases in serum

Abstract

When administered in serum, an efficacious therapeutic antibody should be homogeneous to minimize immune reactions or injection site irritation during administration. Monoclonal antibody (mAb) phase separation is one type of inhomogeneity observed in serum, and thus screening potential phase separation of mAbs in serum could guide lead optimization. However, serum contains numerous components, making it difficult to resolve mAb/serum mixtures at a scale amenable to analysis in a discovery setting. To address these challenges, a miniaturized assay was developed that combined confocal microscopy with Raman spectroscopy. The method was examined using CNTO607, a poorly-soluble anti-interleukin-13 human mAb, and CNTO3930, a soluble anti-respiratory syncytial virus humanized mAb. When CNTO607 was diluted into serum above 4.5 mg/mL, phase separation occurred, resulting in droplet formation. Raman spectra of droplet phases in mixtures included bands at 1240 and 1670 cm(-1), which are typical of mAb β-sheets, and lacked bands at 1270 and 1655 cm(-1), which are typical of α-helices. The continuous phases included bands at 1270 and 1655 cm(-1) and lacked those at 1240 and 1670 cm(-1). Therefore, CNTO607 appeared to be sequestered within the droplets, while albumin and other α-helix-forming serum proteins remained within the continuous phases. In contrast, CNTO3930 formed only one phase, and its Raman spectra contained bands at 1240, 1670, 1270 and 1655 cm,(-1) demonstrating homogeneous distribution of components. Our results indicate that this plate-based method utilizing confocal Raman spectroscopy to probe liquid-liquid phases in mAb/serum mixtures can provide a screen for phase separation of mAb candidates in a discovery setting.

Keywords: HC-CDR, heavy chain complementarity-determining region; PBS, phosphate-buffered saline; Raman spectroscopy; circular dichroism; confocal microscopy; droplets; inhomogeneity; mAb, monoclonal antibody; miniature; monoclonal antibody; phase separation; serum; α-helix, alpha helix;CD, circular dichroism; β-sheet, beta-sheet.

Figure 1.

Micrograph images of CNTO607 in serum. CNTO607 titrations at total concentrations in serum at (A) 25 mg/mL, (B) 16.3 mg/mL, (C) 10.6 mg/mL, (D) 6.9 mg/mL, (E) 4.5 mg/mL, (F) 3.0 mg/mL, (G) 1.9 mg/mL, and (H) 0.6 mg/mL.

Figure 2.

Raman spectroscopy images of A: CNTO607 in sodium acetate. B: CNTO3930 in PBS. (i) Spectrum of buffer controls: Sodium acetate (A) and PBS (B), (ii) spectra from the bottom of the wells, (iii) spectra from the top of the wells; (iv, v) corresponding representative images from the bottom and top of the wells. Black circles indicate positions where Raman spectra were collected.

Figure 3.

Raman spectroscopy images of A: CNTO607 in PBS. B: CNTO607 in serum. (i) Spectrum of diluent controls: NaAc/PBS (A) and NaAc/serum (B), (ii) spectra from within the droplet phases at the bottom of the wells, (iii) spectra from the continuous phase at the top of the wells, (iv) representative images from the bottom of the wells, and (v) representative images from the top of the wells. Black circles indicate positions where Raman spectra were collected.

Figure 4.

Raman spectroscopy images of A: CNTO3930 in sodium acetate. B: CNTO3930 in serum. (i) Spectrum of diluent controls: NaAc/PBS (A) and PBS/serum (B), (ii) spectra from the bottom of the wells, (iii) spectra from the top of the wells, (iv, v) representative images from the bottom and top of the corresponding well. Black circles indicate positions where Raman spectra were collected.

Figure 5.

Circular dichroism spectra of A: CNTO607 (solid line) and CNTO3930 (dashed line) both at 0.2 mg/mL. B: Human serum albumin at 0.4 mg/mL (dotted line), and human serum diluted 250 fold in PBS (solid line).