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Biologic drug substance & product · Protein solutions, in-process samples, formulated product

Total Protein by BCA or Bradford Microplate Assay

Samples and a series of protein standards react with a colour reagent in a 96-well plate. Absorbance is read on a microplate reader (562 nm for BCA, 595 nm for Bradford), a standard curve is fitted, and sample protein concentration is read from the curve and multiplied by the dilution factor.

  • Biotech & biologics
  • Microplate colorimetry
  • Plate reader
  • Micropipette
  • Incubator

Reference: Typical method based on USP <1057> and Ph. Eur. 2.5.33 (Bradford and bicinchoninic acid methods), with a bovine serum albumin (BSA) or product-specific standard curve.

PRINCIPLE

Why this method works

In the BCA assay, protein reduces Cu²⁺ to Cu⁺ in alkaline solution (the biuret reaction) and two molecules of bicinchoninic acid bind each Cu⁺ to form a purple complex that absorbs at 562 nm. The colour develops with time and temperature, so standards and samples must be incubated together. In the Bradford assay, Coomassie Brilliant Blue G-250 binds to proteins, mainly to arginine and other basic and aromatic residues; binding shifts the dye's absorbance maximum from about 465 nm to 595 nm. Both assays respond differently to different proteins, so the result is relative to the standard used. A standard of the same protein (or a BSA standard with a correction factor) gives the most accurate results. Absorbance is related to concentration by a standard curve, which is close to linear over a limited range for BCA and is often non-linear for Bradford.

Instruments used

MATERIALS

Reagents & standards

ReagentGrade / specification
BCA working reagentReagent A (BCA, sodium carbonate, sodium tartrate in alkaline buffer) mixed 50:1 with reagent B (4% copper sulphate), prepared fresh
Bradford reagentCoomassie Brilliant Blue G-250 in phosphoric acid and methanol (commercial ready-to-use reagent)
Protein standardBSA reference standard (for example 2 mg/mL) or the product reference standard
DiluentSame buffer as the samples (formulation buffer or water as validated)
SAMPLE PREPARATION

Preparing the sample

  1. 01

    Prepare standards

    Dilute the protein standard to 5–8 levels across the working range.

  2. 02

    Dilute samples

    Dilute samples into the middle of the range with the same buffer.

  3. 03

    Plate

    Pipette blanks, standards, controls and samples into the plate in replicate.

  4. 04

    React

    Add reagent, mix and incubate (BCA: 37 °C for 30 min; Bradford: room temperature about 5–10 min).

  5. 05

    Read

    Cool if needed and read absorbance at 562 nm (BCA) or 595 nm (Bradford).

Procedure

  1. 1Prepare BSA standards at, for example, 0, 125, 250, 500, 750 and 1000 µg/mL in the sample buffer.
  2. 2Dilute each sample so its expected concentration is near the middle of the curve (for example 1 in 5). Prepare at least two independent dilutions.
  3. 3Pipette 25 µL of each blank, standard, control and sample dilution into the plate in duplicate or triplicate.
  4. 4BCA: add 200 µL of working reagent to each well, mix on a plate shaker for 30 s, cover and incubate at 37 °C for 30 min. Cool to room temperature.
  5. 5Bradford: add the reagent volume set in the method (for example 250 µL to 5 µL sample), mix and incubate at room temperature for about 5–10 min, reading within the stable period.
  6. 6Read absorbance at 562 nm (BCA) or 595 nm (Bradford). Subtract the mean blank from every well.
  7. 7Fit the standard curve and calculate sample concentrations.

Standard preparation

  1. 1Stock: BSA 2.0 mg/mL (2000 µg/mL) certified standard.
  2. 21000 µg/mL: 250 µL stock + 250 µL diluent.
  3. 3750 µg/mL: 150 µL stock + 250 µL diluent.
  4. 4500 µg/mL: 100 µL stock + 300 µL diluent.
  5. 5250 µg/mL: 100 µL of the 500 µg/mL standard + 100 µL diluent.
  6. 6125 µg/mL: 100 µL of the 250 µg/mL standard + 100 µL diluent.
  7. 70 µg/mL: diluent only (blank).

Notes for analysts

  • BCA tolerates most detergents but is affected by reducing agents and copper chelators. Bradford tolerates reducing agents but is affected by detergents and strongly basic buffers.
  • Different proteins give different colour yields. When BSA is used as standard, the result is 'BSA equivalent'; a product-specific standard or correction factor gives more accurate values.
  • The BCA reaction keeps developing slowly after incubation, so read all plates at a consistent time after cooling.
  • For Bradford, fit a non-linear (for example quadratic or 4PL) curve if the method allows, because the response flattens at higher concentration.
TEST CONDITIONS

Method parameters (typical)

PlateClear, flat-bottom 96-well plate
Wavelength562 nm (BCA; 540–590 nm filters acceptable if validated) or 595 nm (Bradford)
Sample : reagent25 µL : 200 µL (BCA example)
IncubationBCA 37 °C, 30 min; Bradford room temperature, about 5–10 min
Working rangeBCA about 125–1000 µg/mL (linear fit in this example); Bradford typically about 100–1000 µg/mL, often with a non-linear fit
ReplicatesStandards and samples in duplicate or triplicate
BEFORE YOU REPORT

System suitability & sequence

ParameterLimit
Standard curver ≥ 0.99 (linear) or acceptable goodness-of-fit; back-calculated standards within about ±10–15%
Replicate wellsCV typically ≤ 10%
Control sampleKnown control within its assigned range
Sample absorbanceWithin the range of the standards

Typical sequence

  1. 1Blank (0 µg/mL)
  2. 2Standards 125–1000 µg/mL
  3. 3Control sample
  4. 4Sample dilutions (duplicate)
  5. 5Plate read and blank subtraction
CALCULATION

Calculation with a worked example

Formula
A = m × c + b → c(well) = (A − b) ÷ m; c(sample) = c(well) × DF

Where

A
Blank-corrected absorbance at 562 nm
m
Slope of the standard curve (absorbance per µg/mL)
b
Intercept of the standard curve
c(well)
Protein concentration of the diluted sample (µg/mL)
DF
Sample dilution factor

Given

Standards (µg/mL)
125, 250, 500, 750, 1000
Blank-corrected A562
0.118, 0.231, 0.452, 0.668, 0.880
Sample dilution
1 in 5 (DF = 5), two independent dilutions
Sample A562 (blank-corrected)
0.512 and 0.506

Working

  1. 1

    Linear regression: slope m = 0.0008708 A per µg/mL, intercept b = 0.0126, r = 0.99994

  2. 2
    Dilution 1: c = (0.512 − 0.0126) ÷ 0.0008708 = 573.5 µg/mL; × 5 = 2868 µg/mL
  3. 3
    Dilution 2: c = (0.506 − 0.0126) ÷ 0.0008708 = 566.6 µg/mL; × 5 = 2833 µg/mL
  4. 4
    Mean = (2868 + 2833) ÷ 2 = 2850 µg/mL

Total protein = 2.85 mg/mL (as BSA equivalent)

Try it with your numbers
ACCEPTANCE CRITERIA

What result passes

  • Report against the specification or in-process limit for total protein; state the standard used (for example 'as BSA').
  • Results of independent dilutions agree within the validated limit.
METHOD VALIDATION

Validation parameters (ICH Q2(R2))

ParameterTypical requirement
Specificity / interferenceEffect of buffer components tested; reducing agents, chelators (EDTA) and some sugars interfere with BCA; detergents interfere with Bradford
Linearity and rangeStandard curve over the working range with acceptable fit
AccuracySpike recovery or comparison with A280 or amino acid analysis, typically 90–110%
PrecisionRepeatability and intermediate precision, %RSD typically ≤ 5–10%
RobustnessIncubation time and temperature, reagent lot, read delay
In LimsPulse · Sample Management

Run this method in LimsPulse: specifications and STPs under version control, results calculated and checked against limits, and a signed Certificate of Analysis.

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