Protein Determination Procedures - General Chapters - United States Pharmacopeia USP 2025

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

DOWNLOAD PDF HERE

1 INTRODUCTION 

Several procedures exist to determine the total protein in pharmaceutical drug substances and products. Procedures may differ based on the physical characteristic of the protein, which serves as the basis of the measurement principle (e.g., absorbance of ultraviolet light by aromatic amino acid residues or by dye binding). Understanding the protein structure and sample matrix is important when selecting a total protein quantitation procedure. Users must verify that the chosen validated method below is suitable for their specific purposes (see Verication of Compendial Procedures 〈1226〉). 

2 PROCEDURE 

Two options for Method I are presented: Method IA, using denaturing conditions; and Method IB, using native conditions. Depending on the protein structure and the nature of the protein sample (e.g., denaturing conditions may be more suitable for a strong coiled structure with most aromatic amino acids in the interior of the protein), one method may prove superior to the other, and users should select which is the most suitable method based on their protein and verify, as appropriate. At a minimum, both methods are dependent on the following conditions being met: 1) the amino acid sequence and molecular weight of the protein must be known; and 2) the protein must contain tyrosines or tryptophans, and no other chromophores (other than cystines) that absorb near 280 nm (including nucleic acids and diluent components) should be present. 

Method IA. Ultraviolet Light Absorbance Under Denaturing Conditions 

Sample buffer A: Prepare 20 mM sodium phosphate, pH 6.5 by dissolving 0.24 g of sodium dihydrogen phosphate in about 80 mL of water. Adjust the pH with sodium hydroxide solution (10% w/w), and ll with water to 100 mL. Filter. 

Sample buffer B: Prepare 7 M guanidine hydrochloride and 20 mM sodium phosphate, pH 6.5 by dissolving 0.24 g of sodium dihydrogen phosphate and 66.87 g of guanidine hydrochloride in about 50 mL of water. Adjust with sodium hydroxide solution (10% w/w), and ll with water to 100 mL. Filter. 

Reference solution: Prepare 6 M guanidine hydrochloride in Sample buffer A by mixing 330 µL of Sample buffer A and 2000 µL of Sample buffer B. 

Sample preparation A: Dilute the test sample by adding Sample buffer A and Sample buffer B to achieve a nal concentration of 6 M guanidine hydrochloride and an absorbance of about 0.4–0.6. [Note—Gravimetric dilutions can be used and sometimes improve accuracy.] Prepare at least three replicates per test sample. 

Analysis 

Samples: Sample buffer A, Sample buffer B, Reference solution, and Sample preparation A 

Determination of the protein concentration in denaturing buffer: Using a suitable spectrophotometer (see Ultraviolet-Visible Spectroscopy 〈857〉), measure the absorbance of Sample preparation A in 1-cm cuvettes at an optical density of 280 nm against the Reference solution, and subtract the absorbance value at 330 nm to obtain A _corrected .

Calculate the protein concentration (c) in the test sample: 

c = A_corrected  × DF × MW/(ε_{280 nm} × 1 cm) 

corrected

A _corrected = absorbance at 280 nm minus the absorbance at 330 nm 

DF = dilution factor 

MW = calculated molecular weight of the protein 

ε_{280 nm}  = extinction coefficient of the test sample at 280 nm 

ε_{280 nm}  = (n_Trp × 5690 + n_Tyr × 1280 + n_Cys × 120) M⁻¹ × cm⁻¹  

n_Trp = number of tryptophans 

n_Tyr = number of tyrosines 

n_Cys = number of cystines 

System suitability 

The test sample UV spectra must show a maximum between 270 and 285 nm and must show a minimum between 243 and 257 nm. The measured absorbance at 280 nm must be NLT 0.4 and NMT 0.6. The relative standard deviation (RSD) of the calculated concentration of all replicates must be NMT 3.0%. 

Method IB. Ultraviolet Light Absorbance Under Native Conditions 

Sample buffer A: Prepare 20 mM sodium phosphate, pH 6.5 by dissolving 0.24 g of sodium dihydrogen phosphate in about 80 mL of water. Adjust the pH with sodium hydroxide solution (10% w/w), and ll with water to 100 mL. Filter. 

Sample preparation A: Dilute the test sample by adding Sample buffer A to achieve an absorbance of about 0.4–0.6. [Note—Gravimetric dilutions can be used and sometimes improve accuracy.] Prepare at least three replicates per test sample. 

Analysis 

Samples: Sample buffer AandSample preparation A 

Determination of the protein concentration in native buffer: Using a suitable spectrophotometer (see 〈857〉), measure the absorbance of Sample preparation A in 1-cm cuvettes at 280 nm against Sample buffer A and subtract the absorbance value at 330 nm to obtain A_corrected .  

Calculate the protein concentration (c) of the test sample: 

c = A _corrected × DF × MW/(ε_{280 nm-native2}  × 1 cm) 

A_corrected = absorbance at 280 nm minus the absorbance at 330 nm 

DF = dilution factor 

MW = calculated molecular weight of the protein 

ε_{280 nm-native2}  = extinction coefficient of the test sample at 280 nm 

where the extinction coefficient ε_{280 nm-native2}  is calculated as: 

ε_{280 nm-native2} = (n_Trp × 5500 + n_Tyr × 1490 + n_Cys  × 125) M−1 × cm−1 

n_Trp = number of tryptophans 

n_Tyr = number of tyrosines 

n_Cys = number of cystines 

System suitability 

The test sample UV spectra must show a maximum between 270 and 285 nm and must show a minimum between 243 and 257 nm. The measured absorbance at 280 nm must be NLT 0.4 and NMT 0.6. The RSD of the calculated concentration of all replicates must be NMT 3.0%. 

Method II. Bicinchoninic Acid Method 

BCA reagent solution:¹ Prepare a solution of 10 g/L of bicinchoninic acid (BCA), 20 g/L of sodium carbonate monohydrate, 1.6 g/L of sodium tartrate, 4 g/L of sodium hydroxide, and 9.5 g/L of sodium bicarbonate in water to a pH of 11.25. 

Copper sulfate reagent solution: Prepare a solution of 40 g/L of cupric sulfate pentahydrate in water. 

BCA working reagent solution: Mix 50 volumes of BCA reagent solution with 1 volume of Copper sulfate reagent solution. Mix until the green solution is clear. 

Sample dilution buffer: Water or another suitable, non-interfering buffer 

Standard solutions: Ideally, use the USP Reference Standard for the protein under test. If the protein of interest is unknown, is a mixture, or is a specific USP Reference Standard that is unavailable, use USP BSA for Protein Quantitation RS. Dilute portions of this solution with the Sample dilution buffer to obtain NLT ve evenly spaced Standard solutions in triplicate. The selected Standard solution concentrations should result in a linear curve, typically in the range of 25–1000 µg/mL of protein. 

Suitability standard solution: Prepare an independent dilution of the same protein used to make the Standard solutions above using Sample dilution buffer to obtain a Suitability standard solution with a concentration near the midpoint of the standard curve. Prepare the Suitability standard solution in triplicate. 

Sample solution: Prepare a Sample solution with an expected concentration within the standard curve by diluting with the Sample dilution buffer. Prepare triplicate samples. 

Analysis 

Samples: Sample dilution buffer, Standard solutions, Suitability standard solution, and Sample solution 

Accurately transfer 100 µL of Sample dilution buffer, Standard solutions, Suitability standard solution, and Sample solution to individual test tubes. [Note—96-well, plate-based, and automated methods can also be used with suitable volume adjustments to the procedure below.] Add 2.0 mL of BCA working reagent solution to each tube, and mix well. 

Cover and incubate the solutions at 37 ± 2° for 30 ± 1 min, then allow the samples to cool to room temperature for NLT 5 min and NMT 60 min. Determine the absorbances of the solutions at 562 nm with a suitable spectrophotometer (see 〈857〉). Autozero the instrument with the Sample dilution buffer sample. 

Measure the absorbance of all Sample dilution buffer, Standard solutions, Suitability standard solution, and Sample solution samples within 10 min. 

Calculations: Plot the absorbances of the Standard solutions versus the protein concentrations of the Standard solutions, and prepare a standard curve using the linear regression method. Use this standard curve and the absorbance of the Sample solution to determine the concentration of protein in each Suitability standard solution and Sample solution. 

System suitability 

Samples: Standard solutions and Suitability standard solution 

Suitability requirements 

Linearity: The coefficient of determination (r²) for a linear t of all Standard solutions is NLT 0.99. 

Relative standard deviation: NMT 5% for triplicate results of the Suitability standard solution 

Accuracy: The calculated concentration of the Suitability standard solution must be within 90%–110% of the theoretical concentration. Acceptance criteria 

Relative standard deviation: NMT 5% for triplicate Sample solution results 

Method III. Bradford Method 

Sample dilution buffer: Water or another suitable, non-interfering buffer 

Standard solutions: Use the USP Reference Standard for the protein under test or USP BSA for Protein Quantitation RS if the protein of interest is unknown, is a mixture, or is a specific USP Reference Standard that is unavailable. Dilute portions of this solution with the Sample dilution buffer to obtain NLT ve evenly spaced Standard solutions in triplicate. Two standard curve ranges are provided for use: either 0.1–1.0 mg/mL of protein (see Analysis procedure A, below) or 5–25 µg/mL of protein (see Analysis procedure B, below). 

Suitability standard solution: Dilute the USP Reference Standard for the protein under test or USP BSA for Protein Quantitation RS with Sample dilution buffer to obtain a Suitability standard solution having a concentration near the midpoint of the standard curve. Prepare the Suitability standard solution in triplicate. 

Sample solution: Prepare a Sample solution with an expected concentration within one of the two concentration ranges in options A or B below by diluting with the Sample dilution buffer. Prepare triplicate samples. 

Coomassie reagent solution: Dissolve 100 mg of brilliant blue G² in 50 mL of alcohol. Add 100 mL of phosphoric acid, dilute with water to 1 L, and mix. Pass the solution through filter paper (Whatman No. 1 or equivalent), and store the filtered reagent in an amber bottle at room temperature. [Note—Slow precipitation of the dye will occur during storage of the reagent. Filter the reagent before use.] 

Analysis: Select one of the two concentration ranges listed below that is suitable for the expected concentrations of the Sample solutions.

• Protein concentration range: 0.1–1.0 mg/mL 

Samples: Sample dilution buffer, Standard solutions, and Sample solution 

Add 1 mL of the Coomassie reagent solution to 20 µL of each Sample dilution buffer, Standard solution, and Sample solution sample. Mix by inversion, avoiding foaming. Incubate 10–30 min. By using a suitable spectrophotometer (see 〈857〉) [Note—Do not use quartz (silica) cuvettes.], use the Sample dilution buffer to set the instrument to zero at 595 nm. Autozero the instrument with the Sample dilution buffer sample. 

Measure the absorbance of all Sample dilution buffer, Standard solutions, Suitability standard solution, and Sample solution samples within 60 min. 

• Protein concentration range: 5–25 µg/mL 

Samples: Sample dilution buffer, Standard solutions, and Sample solution 

Add 0.5 mL of the Coomassie reagent solution to 0.5 mL each of Sample dilution buffer, Standard solution, and Sample solution sample. Mix by inversion, avoiding foaming. Incubate 10–30 min. By using a suitable spectrophotometer (see 〈857〉) [Note—Do not use quartz (silica) cuvettes.], use the Sample dilution buffer to set the instrument to zero at 595 nm. Autozero the instrument with the Sample dilution buffer sample. Measure the absorbance of all Sample dilution buffer, Standard solutions, Suitability standard solution, and Sample solution samples within 60 min. 

Calculations: [Note—The relationship of absorbance to protein concentration is nonlinear; however, if the standard curve concentration range is sufficiently small, it will approach linearity.] Plot the absorbances of the Standard solutions versus the protein concentrations, and determine the standard curve best fitting the plotted points. From this standard curve and the absorbance of the Sample solution, determine the concentration of protein in the Sample solution. 

System suitability 

Samples: Standard solutions and Suitability standard solutions 

Suitability requirements 

Linearity: r2 for a t of all Standard solutions is NLT 0.99. 

Relative standard deviation: NMT 15% for triplicate results of the Suitability standard solution 

Recovery: The calculated concentration of the Suitability standard solution must be within 90%–110% of the theoretical concentration. Acceptance criteria: %CV of triplicates of the Sample solution is NMT 15%. 

Method IV. Lowry Method 

SDS solution: If required, prepare a solution of 50 g/L of sodium dodecyl sulfate (SDS). 

Lowry reagent A: Prepare a solution of 10 g/L of sodium hydroxide in water, and prepare a solution of 50 g/L of sodium carbonate anhydrous in water. Mix equal volumes (2 volumes and 2 volumes) of each solution, and dilute with water or SDS solution, if required, to 5 volumes. [Note—SDS may be added to Lowry reagent A when the test protein contains detergents or lipids or is poorly soluble after treatment for interfering substances.] 

Lowry reagent B: Prepare a fresh solution of 29.8 g/L of disodium tartrate dihydrate in water. Prepare a solution of 12.5 g/L of cupric sulfate pentahydrate in water. Mix equal volumes (2 volumes and 2 volumes) of each solution, and dilute with water to 5 volumes. Lowry reagent C: Mix 50 volumes of Lowry reagent A with 1 volume of Lowry reagent B. [Note—Prepare fresh and protect from light.] Sample dilution buffer: Purified water or other non-interfering buffer 

Standard solutions: Use the USP Reference Standard for the protein under test or USP BSA for Protein Quantitation RS if the protein of interest is unknown, is a mixture, or is a specific USP Reference Standard that is unavailable. Dilute portions of this solution with the Sample dilution buffer to obtain NLT ve evenly spaced Standard solutions. The standard concentrations should be selected to result in a linear curve, typically in the range of 10–200 µg/mL of protein. 

Suitability standard solution: Dilute the USP Reference Standard for the protein under test or USP BSA for Protein Quantitation RS with Sample dilution buffer to obtain a Suitability standard solution having a concentration near the midpoint of the standard curve. Prepare and analyze the Suitability standard solution in triplicate. 

Sample solution: Prepare a Sample solution with an expected concentration within the standard curve by diluting with the Sample dilution buffer. Prepare and analyze samples in triplicate. 

Diluted Folin–Ciocalteu's phenol reagent: Dilute Folin–Ciocalteu's phenol reagent with water (1:1). Alternative dilutions may be used provided that the pH of the samples (i.e., Standard solutions and Sample solutions after addition of Lowry reagent C and the Diluted Folin– Ciocalteu's phenol reagent) is 10.0–10.6. 

Analysis 

Samples: Sample dilution buffer, Standard solutions, Suitability standard solution, and Sample solution 

Accurately transfer 1.0 mL of Sample dilution buffer, Standard solutions, Suitability standard solution, and Sample solution samples to individual test tubes. To each test tube add 5.0 mL of Lowry reagent C. Allow to stand at room temperature for 10 min. Add 0.5 mL of Diluted Folin–Ciocalteu's phenol reagent to each test tube, mix immediately, and incubate at room temperature for 30 min. Determine the absorbance of each solution at 750 nm with a suitable spectrophotometer (see 〈857〉). Autozero the instrument with the Sample dilution buffer. 

Calculations: Plot the absorbances of the Standard solutions versus the protein concentrations of the Standard solutions, and prepare a standard curve using the linear regression method. Use this standard curve and the absorbances of the Sample solutions to determine the concentration of protein in each of the Sample solution samples and Suitability standard solution. 

System suitability 

Sample: Suitability standard solution 

Suitability requirements 

Linearity: r2 for a linear t of all standards is NLT 0.995. 

Relative standard deviation: NMT 5% for triplicate results of the Suitability standard solution 

Recovery: The calculated concentration of the Suitability standard solution must be within 90%–110% of the theoretical concentration. Acceptance criteria 

Relative standard deviation: NMT 5% for triplicate sample results 

Method V. Amino acid Analysis 

Separation and determination of hydrolyzed amino acids by ion-exchange chromatography, followed by postcolumn ninhydrin derivatization, is described for protein samples containing a protein of known molecular weight and amino acid composition. [Note—For additional information, see Biotechnology-Derived Articles—Amino Acid Analysis 〈1052〉, which may be a helpful but not mandatory resource.] 

Hydrolysis solution: 6 N hydrochloric acid 

Sample hydrolysate preparations: Prepare a protein sample such that the content of amino acids is within the established linear working range of the procedure. [Note—Glassware used for hydrolysis must be clean.] Place suitable volumes of the sample and Hydrolysis solution in a hydrolysis tube. [Note—Common volumes range from 100 to 500 µL for the sample and from 1 to 2 mL for the Hydrolysis solution such that complete protein digestion is demonstrated within the hydrolysis incubation time.] Cool the mixture until solidification, and evacuate until the pressure is <0.2 bar. Flame-seal the hydrolysis tube in vacuum by melting the neck of the tube. [Note—Alternative commercially available hydrolysis vials also can be used.] Hydrolyze at 110° for 22 h. Allow the tube to cool, and remove the solvent by centrifugal vacuum concentrator. 

Chromatography 

Solution A: Sodium citrate buffer (0.2 N in sodium), containing 2.0% trisodium citrate dihydrate, 0.1% phenol, 0.2% thiodiglycol, and 2.0% propan-2-ol. Adjust with concentrated hydrochloric acid to a pH of 3.2. 

Solution B: Sodium citrate buffer (0.2 N in sodium), containing 2.0% trisodium citrate dihydrate and 0.1% phenol. Adjust with concentrated hydrochloric acid to a pH of 4.25. 

Solution C: Sodium citrate buffer (1.2 N in sodium), containing 2.0% trisodium citrate dihydrate, 0.1% phenol, and 5.8% sodium chloride. Adjust with concentrated hydrochloric acid to a pH of 6.45. 

Solution D: 0.4 M sodium hydroxide containing 0.1% ethylenediaminetetraacetic acid 

Mobile phase: See Table 1. Equilibrate the system before each run. 

Table 1 

Loading buffer: Sodium citrate buffer (0.2 N in sodium), containing 2.0% trisodium citrate dihydrate, 0.1% phenol, and 2.0% thiodiglycol. Adjust with concentrated hydrochloric acid to a pH of 2.2. 

Post Column reagent 

Solution 1: Solution containing 7.6% acetic acid, 47.7% ethylene glycol, 28.7% water, and 16.0% potassium acetate Solution 2: Solution containing 9% methanol, 8% ninhydrin, 46.4% diethylene glycolmethyl ether, 36% ethylene glycol, and 0.6% hydrindantin. Prepare Postcolumn reagent by mixing 1.75 L of Solution 1 and 0.25 L of Solution 2. Use nitrogen sparging during preparation and mixing. Keep the solution under nitrogen pressure. 

Standard solution: Prepare a solution having known equimolar amounts of L-alanine, L-arginine, l-aspartic acid, l-glutamic acid, glycine, l histidine, l-isoleucine, l-leucine, l-lysine, l-methionine, l-phenylalanine, l-proline, l-serine, l-threonine, l-tyrosine, and l-valine with half the equimolar amount of l-cystine. [Note—Suitable concentrations are 250 and 125 nmol/mL, respectively.] 

Sample solution: Reconstitute the Sample hydrolysate preparations in a suitable volume of Loading buffer. 

Blank: Hydrolysis solution that has been taken through analytical steps including hydrolysis 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: 440 and 570 nm 

Columns 

Prewash: 4.6-mm × 10-cm; 20-µm sodium form, packing L58 

Analytical: 4.6-mm × 20-cm; 8-µm sodium form, packing L58 

Injection volume: 40 µL, at 1 min with baseline reset 

Postcolumn reaction: As the amino acids are eluted from the column, Postcolumn reagent is added at a rate of 25 mL/h. After mixing, the column effluent and the Postcolumn reagent are passed through a reaction coil of approximately 10-m × 0.3-mm PTFE tubing at a temperature of approximately 135°, where a purple color is developed. 

Reaction coil temperature: 135° 

System suitability 

Sample: Standard solution 

Suitability requirements: All 17 amino acid peaks must be visible in the Standard solution. 

Resolution: NLT 1.2 between the pairs of l-threonine and l-serine, L-Cystine and l-valine, and l-isoleucine and l-leucine Tailing factor: 0.8–1.5 for the peak of l-aspartic acid 

Relative standard deviation: NMT 2.0% determined from the l-aspartic acid peak 

Analysis 

Samples: Blank, Standard solution, and Sample solution 

Record and measure the responses for each amino acid peak in the Standard solution and Sample solution. Use the responses obtained from the Standard solution to calculate the content of each amino acid in the sample. Analyze the Blank to confirm purity of the reagent and diluent. 

Calculations: Use well-recovered amino acids to quantify the protein. [Note—For additional information, see Biotechnology-Derived Articles —Amino Acid Analysis 〈1052〉, Protein Hydrolysis, which may be a helpful but not mandatory resource.] Divide the quantity, in nmol, of each of the well-recovered amino acids by the expected number of residues for that amino acid to obtain the protein content based on each well-recovered amino acid. Average the protein content results calculated. Discard protein content values that deviate >5% from the mean. Recalculate the mean protein content from the remaining values to obtain the protein content of the sample. 

USP Reference Standards 〈11〉 

USP BSA for Protein Quantitation RS 

¹ Suitable BCA reagents are available from Thermo Scientific catalog #23225 or equivalent. 

² Suitable brilliant blue G dye is available from Sigma–Aldrich, catalog #B0770.