UV-Vis Spectrophotometry Technology

UV-Vis spectroscopy is a well-established method in biomolecule research. ICH Guideline Q6B states that spectrophotometric protein quantification is one of the typical procedures for protein quantitative analytical testing. CD Formulation's cGMP laboratory is equipped with a variety of advanced instruments and groundbreaking technologies, dedicated to providing protein quantification analysis services based on UV-Vis spectroscopy technology to the biopharmaceutical industry, as a stand-alone test or as a commercial release bio-release test kit or biopharmaceutical-stability-study-test.

What is UV-Vis Spectrophotometry Technology?

UV-Vis spectrophotometry is a technique used to measure the concentration of proteins in a solution by observing how they absorb and transmit light in the ultraviolet-visible region of the electromagnetic spectrum. UV absorption is the process by which a molecule absorbs ultraviolet light and excites electrons (giving them high energy). This energy causes the electrons to jump from the ground state to an excited state.

In this technique, a protein/peptide sample is exposed to a range of wavelengths of light and then the protein is detected and quantified by measuring the UV absorbance. This technique is the simplest of the various protein drug testing methods and can quantify the protein concentration by measuring the UV absorbance of the protein based on its specific amino acid residues.

Fig. 1 A simplified schematic of a UV-Vis spectrophotometer. (CD Formulation)

Basic Principles of UV-Vis Spectrophotometry Technology

UV-Vis spectrophotometry is a simple and direct method for determining protein concentration based on the Beer-Lambert law. The method is based on colorimetry and once the extinction coefficient (molar absorbance) is accurately determined, the concentration of the protein or peptide can be calculated from the absorbance measurement.

Beer–Lambert Law：

A =εbc

• A = the absorbance.
• ε = the molar extinction coefficient.
• c = the molar concentration.
• b = the optical path length in centimeters (e.g. the length of the cuvette).

In UV-Vis Spectrophotometry, the protein content in the sample can be calculated by measuring the absorbance of ultraviolet light at different wavelengths and then using the known molar absorption coefficient. This method is simple and fast, and is also applicable to protein samples containing multiple amino acids.

Our Services for Protein Quantification Based on UV-Vis Spectrophotometry Technology

With decades of experience supporting protein/peptide biopharmaceutical development and manufacturing, our team of highly qualified experts can provide a full range of UV-Vis Spectrophotometry-based protein quantification services to accelerate the implementation and success of your project.

Our UV-Vis Spectrophotometry-based protein quantification services support you at all stages of protein/peptide drug development and manufacturing - from early research to post-release testing under GMP.

At CD Formulation, we offer the following 6 UV-Vis Spectrophotometry-based protein quantification methods:

280nm UV Absorption

The method utilizes the maximum absorbance of proteins containing aromatic amino acids (such as tyrosine and tryptophan) at 280 nm to quantify the proteins or peptides to be detected.

Concentration Range: 50 - 2000µg/mL

Advantages:

• Simple method.
• The assay does not affect protein activity.
• Small amounts of sample.
• Highly sensitive.
• Doesn't require any additional reagents.

Disadvantages:

• The presence of interfering compounds that absorb light at 280 nm, such as nucleic acids, can obscure the measurement.
• Each protein absorbs differently and a suitable reference protein is needed.
• Some proteins that don't absorb at 280 nm can't be measured, such as collagen and gelatin.

Lowry Assay

An alkaline copper solution is added to the protein or peptide solution to be tested, in which tyrosine, tryptophan, and cysteine reduce molybdic acid and phosphotungstic acid in the phenol reagent, making the solution blue, and the maximum absorbance at 750nm is used for quantitative analysis.

Concentration Range: 5 - 200µg/µL

Advantages

• High sensitivity.

Disadvantages

• The process is complex and the preparation time is long.
• Reducing substances will interfere with the measurement results.
• The color development speed of each protein is different.

BCA Assay

It is formed by combining the biuret method with bicinchoninic acid (BCA). The copper ions generated by the reaction of the protein or peptide solution to be tested with biuret react with 2 molecules of BCA, and the solution turns purple, and the maximum absorbance at 560nm is used for quantitative analysis.

Concentration Range: 20 - 2000µg/µL

Advantages:

• Simple operation.
• High sensitivity.
• Wide detection range.

Disadvantages:

• The reaction is easily affected by reducing agents and chelating agents (such as thiols, phospholipids, and ammonium sulfate).

Biuret Assay

An alkaline solution of biuret reagent containing copper sulfate and Rochelle salt is added to the protein or peptide solution to be tested, and the maximum absorbance at 540nm is used for quantitative analysis, in which the peptide chain in the protein or peptide solution chelates with copper ions to generate a purple compound.

Concentration Range: 150 - 9000 µg/mL

Advantages

• Simple procedure.
• Constant color development rate.

Disadvantages

• Low sensitivity.
• It can't measure samples with low protein concentrations.
• The color reaction is easily affected by high concentrations of trisaminomethane, amino acids, and ammonium ions.

Bradford Assay

When the protein or peptide solution to be tested is combined with Coomassie Brilliant Blue G250, its maximum absorbance moves from 465 nm to 600 nm which was used for quantitative analysis.

Concentration Range: 10 - 2000 g/µL

Advantages:

• Very simple to use.
• The one-step method with less than 10 minutes of detection time.

Disadvantages:

• Requires large sample volumes.
• Contamination with common protein surfactants (e.g., Triton X, SDS) may interfere with the color development reaction.
• It isn't useful for proteins with above- or below-average levels of basic amino acids.

WST Assay

WST-8 was used with a high pH solution of the protein or peptide to be tested, causing the sample to turn blue. Quantitative analysis was performed using the maximum absorbance at 650 nm.

Concentration Range: 50 - 5000µg/mL

Advantages:

• Simple method
• Not affected by protein surfactants.

Disadvantages:

• Each protein has a different color development rate.

Note*: CD Formulation also provides protein quantification services based on LC-MS/MS and immunoassays.

Fig. 1 Workflow of protein/peptide quantitative analysis. (CD Formulation)

Custom UV-Vis Spectrophotometry Services

Why Choose Our UV-Vis Spectrophotometry Technology?

• We have a team of experts with rich experience in performing UV-vis analytical method development and validation.
• We have accumulated decades of expertise and successful project experience using UV-vis technology to support protein/peptide biopharmaceutical development.
• Our laboratories are equipped with state-of-the-art equipment and technologies to support any type of protein characterization.
• We provide flexible experimental design and customized solutions.
• Fast turnaround time: detailed technical reports are provided within 5-7 days.

Publication

CD Formulation aims to provide a powerful analytical tool for quantitative analysis of protein/peptide concentration. Please feel free to contact us if you are interested in our services. Learn how ourr UV-vis spectrophotometry technology can support the smooth implementation of your protein/peptide biopharmaceutical program.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.