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Proteins & Peptides Quantitative Analysis

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ICH Guideline Q6B stipulates that quantitative analysis is one of the important testing procedures for biopharmaceutical products. Protein or peptide quantitative analysis helps to understand the total protein or peptide content in a sample or formulated product. CD Formulation's cGMP laboratories are equipped with a variety of advanced instruments and pioneering technologies, dedicated to providing unparalleled protein and peptide quantitative analysis services to the biopharmaceutical industry, as a stand-alone test or as part of a commercial release bio-release test kit or biopharmaceutical stability study test.

Importance of Proteins & Peptides Quantitative Analysis

Protein and peptide drugs are an important class of therapeutics that are increasingly used to treat a variety of diseases, including cancer, autoimmune diseases, and infectious diseases. Accurate protein or quantitative analysis helps generate key data required for a range of other assays, as well as to ensure their safety, efficacy, and quality, including:

Pharmacokinetic studies: Quantitative analysis can provide insights into the absorption, distribution, metabolism, and excretion of protein and peptide drugs in the body. This information is critical for optimizing drug dosing regimens and predicting drug efficacy and toxicity.
Bioequivalence studies: Quantitative analysis is used in bioequivalence studies to compare the pharmacokinetic parameters of generic and branded protein and peptide drugs, ensuring that generic drugs are as effective and safe as branded drugs.
Quality control: Quantitative analysis is essential for quality control to ensure the consistency and purity of protein and peptide drugs. This is essential to maintain product quality during manufacturing and storage and to comply with regulatory standards.

Explore Our Proteins & Peptides Quantitative Analysis Services

As an expert in protein and peptide quantification, CD Formulation has a deep understanding of the physicochemical properties of proteins, peptides, and amino acids. Our protein and peptide quantification services can help you address any potential sources of variation in the accuracy of total protein content determination, determine sample purity, and obtain accurate and reliable data about the protein or peptide being measured.

Thanks to advanced technology platforms and extensive expertise in protein/peptide analysis, we provide you with a range of total protein or peptide quantification methods, including but not limited to:

UV-Vis Spectrophotometry

UV-Vis Spectrophotometry is a simple, direct method for determining protein concentration based on the Beer-Lambert law. The principle of the method is colorimetric, and once the extinction coefficient (molar absorbance) is accurately determined, it can be used to calculate the protein or peptide concentration based on absorbance measurements. The 6 protein quantification methods we commonly use based on UV absorption spectrophotometry include:

Technologies	Procedure & Principle	Concentration Range
280 nm UV Absorption	The method utilizes the maximum absorbance of tyrosine and tryptophan at 280 nm to quantitatively analyze the protein or peptide to be tested.	50 - 2000 µg/mL
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.	150 - 9000 µg/mL
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.	5 - 200 μg/µL
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.	20 - 2000 μg/µL
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.	10 - 2000 g/µL
WST Assay	WST-8 was reduced 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.	50 - 5000 µg/mL

Immunoassays

Immunoassays are highly specific and sensitive and can be used to quantify very low concentrations of proteins or peptides in biological samples. Our scientists often use this technique for pharmacokinetic evaluation of protein or peptide drugs as well as bioequivalence studies.

Available immunoassays include:

Enzyme-linked immunosorbent assay (ELISA)	Fluorescent Immunosorbent assay (FIA)	Chemiluminescent Immunosorbent Assay (CLIA)
ELISA is a powerful method for detecting and quantifying very low concentrations of proteins, peptides, antibodies, and hormones with minimal interference. Detection is achieved by measuring the activity of a reporter enzyme, which produces a measurable product by incubation with an appropriate substrate.	FIA is a variation of ELISA in which the substrate used does not produce color but fluoresces. FIA is more sensitive than ELISA and can be used to measure low concentrations of proteins.	Similar in principle to ELISA, CLIA has higher sensitivity and shorter turnaround time. In addition, it has a wider dynamic range with a linear relationship between the luminescence intensity and the concentration of the substance being measured. Key advantages of CLIA also include the absence of interfering emissions (high specificity), rapid acquisition of analytical signals, and high stability of the reagents and their conjugates.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

LC-MS/MS-based analytical methods are powerful tools for quantitative analysis of biopharmaceuticals. This method is an extremely sensitive and specific analytical technique that can accurately determine the identity and concentration of proteins/peptides in a sample.

Fig. 1 Workflow in LC-MS/MS analysis of protein samples. Fig. 1 Workflow and critical factors in LC-MS/MS analysis of protein samples.(Song JG, et al.,2023)

Reversed-phase chromatograms can be used to determine the purity of proteins or peptides. The purity of a protein or peptide is quantified by the peak area, which is equal to the ratio of the total area of all peaks detected in the chromatogram to the area of the main peak.
In addition to reversed-phase HPLC, our protein analysis experts can also determine the molecular weight information of proteins/peptides by mass spectrometry.
We also provide amino acid analysis (AAA) by high-performance liquid chromatography (HPLC) or ion chromatography (IC) to determine the amino acid composition of peptides or protein products, thereby ensuring the accuracy and consistency of experiments.

General Workflow of Protein & Peptide Quantitative Analysis

Fig. 2 Workflow of protein/peptide quantitative analysis. (CD Formulation)

Sample preparation: Protein extraction, digestion, and purification techniques are used to isolate the proteins and peptides of interest from complex biological samples.
Quantitative analysis: Once the samples are prepared, they are analyzed using mass spectrometry, UV-Vis spectrophotometry, or other quantitative techniques to determine the abundance of different proteins and peptides in the sample. This may involve comparing the abundance of target proteins or peptides to internal standards or reference samples to quantify their concentration.
Data analysis: The quantitative data obtained from the analysis is processed and analyzed using bioinformatics tools and software to identify and quantify the proteins and peptides present in the sample. This may involve statistical analysis, protein identification, and quantification, as well as data visualization and interpretation.
Reporting and interpretation: The results of the quantitative analysis are compiled into a report that includes tables and graphs showing the abundance of different proteins and peptides, as well as comparisons to reference samples or controls.
Validation and quality control: This involves running validation experiments, checking for reproducibility and consistency, and verifying the results against known standards or controls.
Data submission: Once the results have been validated and confirmed, the final data and report are submitted to you for further analysis or publication.

Why Choose Us for Proteins & Peptides Quantitative Analysis?

Our team of scientists and researchers have specialized expertise in protein and peptide quantitative analysis.
We utilize state-of-the-art technology and techniques for protein and peptide analysis, allowing for precise quantification and detection of even low abundance proteins.
We offer customized solutions to meet the specific needs of each client, including method development, validation, and data interpretation.
We adhere to stringent quality control measures to ensure the highest level of accuracy and reliability in our analysis.
We provide flexible experimental design and testing options can meet any customer's specific needs.

Publication

Published Data

Technology: LC-MS bioanalysis of intact proteins and peptides

Journal: Biomed Chromatogr.

IF: 1.8

Published: 2020

Results:

The authors review the latest development in LC-MS bioanalysis of intact proteins and peptides by summarizing recent publications and discussing the important topics such as the comparison between top-down intact analysis and bottom-up surrogate peptide approach, as well as simultaneous quantitation and catabolite identification. Key bioanalytical issues around intact protein bioanalysis such as sensitivity, data processing strategies, specificity, sample preparation and LC condition are elaborated.

At CD Formulation, our experienced protein scientists use a wide range of analytical techniques to accurately and reliably quantify therapeutic proteins or peptides. Please feel free to contact us if you are interested in our services. We will provide you with the most professional advice and support to ensure the smooth launch and implementation of your project.

References

Gillette MA, Carr SA. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat Methods. 2013, 10(1):28-34.
Song JG, Baral KC, Kim GL, et al. Quantitative analysis of therapeutic proteins in biological fluids: recent advancement in analytical techniques. Drug Deliv. 2023, 30(1):2183816.
Kang L, Weng N, Jian W. LC-MS bioanalysis of intact proteins and peptides. Biomed Chromatogr. 2020, 34(1):e4633.

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