Amino acid analysis for protein quantification

Early and accurate protein quantification is essential in drug development since other critical assays, including other quantitative analyses, depend on knowing your drug sample's precise total protein concentration. If the concentration determination of your protein standard is incorrect, it will affect your downstream process development and may cause severe delays or even drug candidate failure.

The amino acid analysis is the golden standard for accurately quantifying proteins and peptides according to ICH Q6B guidelines. Therefore, we recommend using it for purified proteins or peptides with known amino acid sequences, complex samples with mixtures of proteins, and protein stock solutions.

Robust amino acid analysis and protein quantification

Determine absolute amount of individual or total amino acids

Amino acid analysis provides highly accurate protein amount and concentration. Compare with the known protein sequence for molar amount

Determine the extinction coefficient

UV measurements combined with amino acid analysis experimentally determine the mass- and molar extinction coefficient of the protein in a given buffer

Determine accurate amount in protein stock solution

Using triplicate measurements and the known amino acid composition of your purified protein, we can determine the exact amount and concentration of the protein stock solution

It is advisable to determine the protein concentration of your sample by amino acid analysis before conducting other quantitative analyses, such as residual HCP quantification or peptide and intact mass analyses. The data ensures the appropriate sample volume and protein amount calculations for protein digestion and mass spectrometry-based workflow preparation. If the protein concentration has been incorrectly measured, the analysis may fail or the results may be less accurate.

The amino acid analysis is the golden standard for accurately quantifying the specific amount of purified proteins and peptides and also for determining the overall protein concentration in complex protein samples. The analysis provides quantitative data for all amino acids except tryptophan and cysteine, which degrade into free amino acids during hydrolysis. The samples can even be run in triplicate for highly accurate quantification.

Traditional protein quantification assays, such as Bradford, Lowry, or BCA, depend on an external reference standard and calibration curve and are prone to interference from chemicals or the presence of other proteins. Furthermore, your purified protein may have a different UV absorption coefficient than the employed calibration standard. Such cases will result in an incorrect concentration determination of the protein standard, which may significantly impact your assay and downstream process development.

Triplicate amino acid analysis (AAA) is the gold standard for protein concentration determination. Generally, it ensures the most accurate quantification of your protein standard, even when the concentration is unknown. The analysis is based on acidic hydrolysis followed by Ion-Exchange HPLC detection and quantifying individual amino acids. The analysis works even with presence of salt and certain detergents.

Knowing the molar extinction coefficient of your purified protein or antibody sample is necessary for accurate protein quantification by UV absorbance and is also required by the ICH Q6B guidelines. The extinction coefficient enables the determination of protein concentrations in laboratory samples using spectrophotometry.

The analysis is a three-step procedure of measuring the exact protein concentration, determining its UV absorbance, and calculating the extinction coefficient using the Beer-Lambert Law. The protein or antibody should be dissolved in the same buffer for subsequent experiments since the molar extinction coefficient depends on the protein's buffer composition and three-dimensional structure.

The method usually requires access to an amino acid analyzer and a spectrophotometer. Alphalyse uses an advanced SoloVPE system with variable-path length measurement, which can identify the linear range of absorbance and allows the calculation of the sample concentration and extinction coefficient using a slope spectroscopy equation derived from the Beer–Lambert Law.

Typical project process

You typically work with
these experts:

Maša Babović, peptide mapping analysis services

Maša Babović

PhD in Biochemistry and Molecular Biology

Vicki Nautrup Nielsen, Biochemical Laboratory Scientist

Vicki Nautrup Nielsen

Laboratory Scientist
  • Facilitate meetings

    Project scope

    We will start with an online meeting to learn more about your project. Based on your needs, you will receive a draft proposal outlining the suggested analyses and expected timeframe.

  • Protein sample


    After signing the final project proposal, we will contact you for details about your samples. An estimated report delivery date will be sent as soon as we receive your samples.

  • SWATH LC-MS analysis and protein quantification using amino acid analysis


    A project leader will oversee the project and send you status updates by email regularly.

    A typical analysis includes:

    Protein quantification / amino acid analysis:

    • Single, duplicate, or triplicate amino acid assay. We recommend analysis in triplicates whenever you need a very accurate protein concentration determination.
    • We can compare the results against a known sequence and perform the analysis when the sequence is unknown or for protein mixtures.

    Molar extinction coefficient

    • Evaluate if the buffer effect the analysis – if yes, the buffer absorbance is subtracted.
    • Determination of mass extinction coefficient at absorbance 280 nm (unless another wavelength is desired).
    • Accurate calculation of the molar extinction coefficient of your protein/antibody based on the molecular weight.
  • 5000 mass spectrometry reports


    A standard report is sent to you by email and includes:

    Protein quantification / amino acid analysis

    • Quantification of 18 amino acids and the total protein content.

    Molar extinction coefficient

    • The mass and molar extinction coefficient at wavelength 280 nm (unless another wavelength is desired).
  • Follow up on reports

    Follow up

    Upon completion of the project, your team is invited to go over the protein quantification results at an online meeting.

Curious to know more about protein quantification?

Leading edge technologies

Whatever challenge or question you may have, be it related to protein quantification or something else, we are here to help you solve it. One of our protein analysis experts will discuss the best analysis approach or method for your project by email or online meeting – without obligation.

Client stories

GMP validated LC-MS-based HCP analysis

Video: GMP-validated HCP analysis based on LC-MS

The world’s first mass-spectrometry-based analysis of HCP impurities under GMP conditions
19 Characterization of HPCL peaks

Video: Analyzing unexplainable HPLC peaks or shoulders

Using an IEX-MS setup as an essential part of quality control and documentation, especially when experiencing new peaks or shoulders
forced degradation study

Stability study – an important part of biologics license application

Our client needed a stability study of protein degradation products for regulatory documentation
mAb stability study using LC-MS

Characterization program for therapeutic mAbs

“We identified a scale-up problem and now use the analysis to test the quality of all our batches”
Antibody characterization for cancer drug

High-throughput antibody characterization service

GTP Bioways develops a massive number of high-quality mAbs with Alphalyse’s LC-MS analysis for characterization
Optimized HPLC analysis method

Optimized HPLC analysis of peptides for clinical trials

DTA Consulting wanted to improve their client's HPLC setup for GMP production of a peptide with high stability.

Benefits of amino acid analysis and extinction coefficient analysis

  • Compare the measured amino acid composition with the expected composition calculated from the protein sequence.

  • Accurate quantification of protein concentration in reference samples.

  • Get the extinction coefficient for your protein in your preferred buffer system.

  • Use the information for fast protein concentration measurements in your lab using UV

What clients say

Testimonial Alphalyse
"The analysis showed that the new ELISA standard made the calibration slope shift, resulting in a misinterpretation of the total amount of HCPs in the drug sample"

Director QC
EU-based biotech
Testimonial Alphalyse
“I found the group at Alphalyse knowledgeable, easy to work with, and helpful in
planning the study”

VP CMC and Quality
US Biotech
Testimonial Alphalyse
“Extremely professional service, high-level quality of the results, and excellent communication”

Program Manager
European Biotech
Testimonial Alphalyse
"The mass spec results were extremely detailed and almost identical between replicates and can probably replace ELISA as the HCP analysis approach for our future C&GT projects"

Chief Scientific Officer
European biopharma company
Testimonial Alphalyse
"We hoped our biosimilar product was cleaner, more stable, and safer than the originator. However, the results were so favorable that not only can we use them as documentation for regulatory filing, but also as part of our marketing"

Head of Analytical Development
Developer of a mAb-biosimilar
Testimonial Alphalyse
"Thanks to the ELISA-MS data, we knew which kit to use for the setup and validation of our HCP ELISA assay! Furthermore, we knew exactly which individual HCPs were covered by
the ELISA antibodies"

Director CMC
US Biotech
Testimonial Alphalyse
"Based on the data provided by Alphalyse, we settled on a kit that rovided both excellent coverage of the HCPs overall and the individual HCPs of concern"

Senior CMC specialist

US-based Biotech
Aicuris logo
"Alphalyse provided a very well-designed and executed HCP analysis, fruitful technical discussions, and flexibility in terms of writing the report."

Thore Schmedt, Associate Director
AiCuris Anti-infective Cures AG, Germany
SSI logo
"Alphalyse's HCP analysis saved us the development of an ELISA assay that may not have worked anyway. The HCP team explained test results competently and was very open to discussing the method capabilities."

Max Kristiansen, MSc, Special Consultant Assay Development
Statens Serum Institut (SSI), Denmark
Savara logo
"Using the Alphalyse LC-MS/MS coverage method in HCP ELISA selection, we estimate a savings of approximately $1M and, likely, one year of development time."

Lars Skriver
Senior Science Officer

SAVARA Aps, Denmark
Actinobac Biomed Inc logo
"The FDA approved our IND! They accepted the MS data without also requesting the standard HCP-ELISA immunoassay."

Scott Kachlany, founder
Actinobac Biomed Inc.

Knowledge center

When should I use amino acid analysis instead of standard Bradford, Lowry, or BCA?

The results from these analyses can be adequately accurate but are all based on protein quantification relative to an external calibration curve on a standard. There is a risk that interference from chemicals or the presence of other proteins may significantly affect these analyses and provide incorrect results. We recommend using amino acid analysis as it is a direct quantitative analysis of each individual amino acid, using calibration curves in each individual amino acid.

Will high salt levels in my buffer impact the amino acid analysis results?

No, a saline buffer will not affect the results. Our HPLC analysis uses Ion-Exchange HPLC detection and quantification of each amino acid, which works well even at high salt levels.

Talk to us

Whatever protein-related challenge or question you may have, we would love to help. Our experts can help you decide on the best analytical approach for your project by email or online meeting - providing advice without obligation.

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