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UV–Vis Spectrometers01:14

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The absorbance of UV and visible (UV–visible) radiations is measured using a UV–visible spectrophotometer. Deuterium lamps, which emit UV radiation, and tungsten lamps, which produce radiation in the visible region, are used as light sources in UV–visible spectrophotometers. A monochromator or prism is used for diffraction grating, i.e., to split the incoming radiation into different wavelengths. A system of slits is used to focus the desired wavelength on the sample cell.
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Ultraviolet–visible (UV–visible or UV–Vis) spectroscopy is an analytical technique that investigates the interaction between matter and UV–Vis light within the electromagnetic spectrum. This method is widely used for its versatility, simplicity, and relatively quick data acquisition, making it valuable for both qualitative and quantitative analysis. When UV–Vis radiation passes through a material,  molecules absorb light depending on the energy required for...
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Multi-wavelength UV-based PAT tool for measuring protein concentration.

Anjali Ramakrishna1, Vinay Prathap1, Vijay Maranholkar1

  • 1R & D, Biocon Research Limited, Bommasandra-Jigani Link Road, Bangalore, India.

Journal of Pharmaceutical and Biomedical Analysis
|October 4, 2021
PubMed
Summary

A new multi-wavelength UV method accurately estimates protein concentration during chromatography, overcoming signal saturation issues. This simple, cost-effective process analytical technology (PAT) tool enables dilution-free online measurement for therapeutic protein purification.

Keywords:
ChemometricsContinuous chromatographyPLSProcess analytical technology (PAT)Protein quantificationUltraviolet (UV) spectra

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Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Chemical Engineering

Background:

  • Process chromatography is vital for therapeutic protein purification.
  • Current methods using single UV absorbance saturate at high protein concentrations, hindering accurate quantification.
  • Accurate, real-time protein concentration monitoring is crucial for process control and pooling decisions.

Purpose of the Study:

  • To develop a simple, fast, and cost-effective method for on-line protein concentration estimation.
  • To address the limitations of single UV absorbance, specifically signal saturation and non-linearity.
  • To enable dilution-free online concentration measurement in the high range of 0.8-100 g/L.

Main Methods:

  • A multi-wavelength UV-based approach was developed, utilizing stable absorbance regions from UV spectra.
  • Chemometrics tools, including Principal Component Analysis (PCA) and Partial Least Squares (PLS), were employed for model validation.
  • The method was validated against an established offline UV-based quantification method.

Main Results:

  • The multi-wavelength approach effectively resolved UV signal saturation and non-linearity issues.
  • Model predictions showed high correlation with offline measurements (R² > 98%).
  • The developed process analytical technology (PAT) tool demonstrated <8% variability in online testing for IgG concentration.

Conclusions:

  • The proposed multi-wavelength UV method provides accurate, dilution-free online protein concentration measurement.
  • This simple, low-cost PAT tool is suitable for various stages of downstream processing, from capture to formulation.
  • The technology facilitates integrated and continuous operations in biopharmaceutical manufacturing.