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Raman spectroscopy characterization of antibody phases in serum.

Baker AE, Mantz AR, Chiu ML - MAbs (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: a Janssen R&D; Biotech CoE ; Spring House , PA USA.

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.

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Related in: MedlinePlus

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).
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f0005: 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).

Mentions: The CD spectra of each mAb, human serum albumin, and human serum were collected to determine their secondary structures and to help validate Raman secondary structure results. Figure 5a shows the CD spectra of CNTO607 and CNTO3930 evaluated in their respective formulation buffers.Figure 5.


Raman spectroscopy characterization of antibody phases in serum.

Baker AE, Mantz AR, Chiu ML - MAbs (2014)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4622053&req=5

f0005: 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).
Mentions: The CD spectra of each mAb, human serum albumin, and human serum were collected to determine their secondary structures and to help validate Raman secondary structure results. Figure 5a shows the CD spectra of CNTO607 and CNTO3930 evaluated in their respective formulation buffers.Figure 5.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: a Janssen R&D; Biotech CoE ; Spring House , PA USA.

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.

Show MeSH
Related in: MedlinePlus