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Related Concept Videos

Raman Spectroscopy: Overview01:20

Raman Spectroscopy: Overview

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The underlying principle of Raman spectroscopy is based on the interaction between light and matter, specifically molecules' inelastic scattering of photons. When a monochromatic beam of light, typically from a laser source, interacts with a sample, most scattered light has the same frequency as the incident light. This is known as Rayleigh scattering.
However, a small fraction of the scattered light exhibits a frequency shift due to the exchange of energy between the incident photons and...
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Raman Spectroscopy Instrumentation: Overview01:26

Raman Spectroscopy Instrumentation: Overview

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A conventional Raman spectrophotometer includes a laser source, a sample holding system, a wavelength selector, and a detector.
The monochromatic laser source, typically using visible or near-infrared radiation, generates a highly focused beam of light. This light interacts with the molecules of the sample, scattering some of the light. Liquid and gaseous samples are usually tested in ordinary glass capillaries, while solids can be analyzed as powders packed in capillaries or as potassium...
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The Equilibrium Binding Constant and Binding Strength02:18

The Equilibrium Binding Constant and Binding Strength

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The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
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IR Spectroscopy: Hooke's Law Approximation of Molecular Vibration01:16

IR Spectroscopy: Hooke's Law Approximation of Molecular Vibration

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A covalently bonded heteronuclear diatomic molecule can be modeled as two vibrating masses connected by a spring. The vibrational frequency of the bond can be expressed using an equation derived from Hooke's law, which describes how the force applied to stretch or compress a spring is proportional to the displacement of the spring. In this case, the atoms behave like masses, and the bond acts like a spring.
According to Hooke's law, the vibrational frequency is directly proportional to...
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Applications Of NMR In Biology01:25

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Nuclear magnetic resonance (NMR) spectroscopy is a very valuable analytical technique for researchers. It has been used for more than 50 years as an analytical tool. F. Bloch and E. Purcell formulated NMR in 1946 and won the 1952 Nobel Prize in Physics  for their work. Biological macromolecules such as proteins, nucleic acids, lipids, and organic molecules including pharmaceutical compounds, can be studied using this versatile tool that exploits the magnetic properties of certain nuclei.
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Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates
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Application of Raman Spectroscopy to Dynamic Binding Capacity Analysis.

James W Beattie1,2, Ruth C Rowland-Jones3, Monika Farys3

  • 1Department of Life Sciences, Imperial College London, London, UK.

Applied Spectroscopy
|November 1, 2023
PubMed
Summary
This summary is machine-generated.

Raman spectroscopy effectively monitors Protein A resin dynamic binding capacity (DBC) for biotherapeutics. This advanced method offers real-time quality control, surpassing traditional techniques for industrial purification pipelines.

Keywords:
BiotherapeuticHPACRaman spectroscopydynamic binding capacityhigh-performance affinity chromatographymonoclonal antibodyprotein A affinity chromatography

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Area of Science:

  • Biopharmaceutical Manufacturing
  • Analytical Chemistry
  • Process Analytical Technology (PAT)

Background:

  • Protein A affinity chromatography is crucial for isolating biotherapeutics (BTs) like antibodies.
  • Dynamic Binding Capacity (DBC) of chromatography resins decreases with use, impacting product yield and requiring regulatory monitoring.
  • Current DBC assessment methods, such as High-Performance Affinity Chromatography (HPAC), are time-consuming, require specific calibration, and offer limited analytical information.

Purpose of the Study:

  • To evaluate Raman spectroscopy as a novel method for monitoring the DBC of Protein A resins during biotherapeutic purification.
  • To compare the efficacy of Raman spectroscopy against established methods like HPAC and UV chromatography for DBC assessment.
  • To explore the potential of Raman spectroscopy for providing additional quality control information beyond protein concentration.

Main Methods:

  • Utilized Raman spectroscopy to analyze Protein A resins during biotherapeutic loading.
  • Employed partial least square (PLS) analysis in conjunction with Raman spectra for DBC determination without prior calibration.
  • Performed offline analysis in a 96-well plate format, with consideration for inline implementation.

Main Results:

  • Raman spectroscopy demonstrated effectiveness comparable to HPAC and UV methods in monitoring DBC.
  • The technique provided chemical information from spectra, enabling assessment of protein aggregation status and structure.
  • Raman spectroscopy, combined with PLS analysis, allowed for DBC determination of BTs without specific calibration.

Conclusions:

  • Raman spectroscopy is a powerful and improved approach for monitoring DBC in industrial bioprocessing.
  • This method offers enhanced quality control by providing insights into protein concentration, aggregation, and structure.
  • The potential for inline implementation makes Raman spectroscopy a valuable tool for real-time process monitoring and optimization.