Determination of Protein Concentration in the Manufacture

 and Characterization of Biopharmaceuticals

Most biopharmaceuticals are proteins, glycoproteins, or bioconjugates such as biopharmaceuticals which have been modified with poly (ethylene) glycol.   Determination of the concentration of the active pharmaceutical ingredient (API) in final product requires the quantitative measurement of both biological activity and mass (protein concentration).  

I have been interested in the determination of protein concentration for some time. My initial interest resulted from studies on human saliva.  In studying the literature in this area, I discovered that there was great variance between studies and this appeared to be related to the method and protein standard used in individual studies.  I and colleagues published a paper in Analytical Biochemistry in 1989 (1) which provided an understanding of the differences between the various studies.  Methods for the determination of protein concenration such as Coomassie Blue binding (2) and the bicinchoninic acid assay (3) possessed greatly increased sensitivity when compared to the biuret assay, total amino acid analysis or the Kjeldalh method and, in addition, were much easier to perform.   However, these assays were much more dependent on the quality of the protein (1).  The various assays for protein concentration and their limitations have been reviewed by Sapan, Price and Lundblad (4).  Other reviews of methods for the determination of protein concentration have also appeared in recent years.  

Considering the above information, it was with considerable surprise that I discovered that few articles on the development and characterization of biopharmaceuticals which appeared  in two journals surveyed provided significant experimental detail about the the method of protein concentration.  The method used was usually referenced but only in several of the articles was there a mention of the protein standard (usually albumin).   So at the very best, the protein concentration information provided in those articles should have been presented as concentration relative to a standard of albumin.  I searched the literature from January, 2000 to December, 2002 using MedLine(National Library of Medicine, National Institutes of Health; ) for articles referencing methods for the determination of protein concentration.  I found large number of references to both Lowry and bicinchoninic acid.  As I expected, few references were found for the biuret method.  I was somewhat surprised by the small number of references for Coomassie and most of those were concerned with the use of the dye for staining protein bands in gel electrophoresis.  However, when I searched for Bradford and Protein or Bradford and Protein Concentration, a large number of references were obtained.  This is in recognition of the seminal paper on the use of Coomassie Blue G-250 (2).   I have read a large number of the articles obtained through the search by specific method and find that most of them are lacking information sufficient to be of value in biopharmaceutical development and manufacturing.   There is infrequent mention of the standard used and generally reference only to instructions provided by the manufacturer.  There are several recent studies which are worth specific mention.  Zor and Selinger(5) describe a modification of the Coomassie Brilliant Blue G-250 technique which improves the accuracy and sensitivity of the commercial assay.   This modification plots the ratio of absorbance at 590 nm to that at 450 nm versus protein concentration.  While this addresses some issues with respect to possible assay validation(6) such as assay linearity, issues with respect to assay accuracy persist(7).  There have been attempts to apply mathematical corrections to data(8) to allow values obtained by one method to be interchangeable with those obtained with a different method.  While this might prove useful for 'casual' data comparison, it certainly does approach the rigorousness required for a validated assay process to be used within a cGMP environment.  I would further note that my own experience suggests that ease and sensitivity of an assay does not necessarily translate into a validated assay for use in biopharmaceutical manufacturing(6, 9, 10).  As noted in an earlier publication (4), there are ways to improve the sensitivity of accurate methods for total protein analysis.  Additional methods continue to be develop(11) which deserve serious consideration for those concerned with the characterization and manufacture of biopharmaceuticals.  There is also potential for other approaches such as total amino acid analysis.

The above was prepared in 2002. There are some recent artciles which would be quite useful (12-17). I recommend the amido black method (18-20)


1.  Jenzano, J.W., Hogan, S.L., Noyes, C.M., Featherstone, G.L, and Lundblad, R.L.(1989), Comparison of Five Techniques for the Determination of Protein Concentration.  Analytical Biochemistry 159, 370-376.

2.  Bradford, M.M.(1976), Analytical Biochemistry 72, 248-254.

3.  Smith, P.K., Krohn, R.I., Hermansen, G.T., Malia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C.(1985), Analytical Biochemistry 150, 76-85.

4.  Sapan, C.V., Lundblad, R.L. and Price, N.C.(1999), Biotechnology and Applied Biochemistry 29, 99-108.

5.  Zor, T. and Selinger, Z.(1996), Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies.  Analytical Biochemistry 236, 302-308.

6. Lundblad, R.L.(2001), Approach to assay validation for the development of biopharmaceuticals.  Biotechnology and Applied Biochemistry 34, 195-197.

7.  Greive, K.A.,  Balazs, D.H. and Comper, W.D.(2001), Protein Fragments in Urine Have Been Considerably Underestimated by Various Protein Assays.  Clinical Chemistry 47, 1717-1719.

8. Ng, V. and Cho, P.(2000), The relationship between total tear protein concentrations determined by different methods and standards.  Graefes Arch.Clin.Exp.Ophthalmol. 238, 571-576.

9  Guidelines for Industry.  Analytical Procedures and Methods Validation.  Chemistry, Manufacturing, and Control Documentation.  FDA/CDER/CBER, August, 2000,

10. Guidance for Industry.  Bioanalytical Method Validation.  FDA/CDER/CVM, May, 2001,

11.  Zellmer, S., Kaltenborn, G., Rothe, U., Lehnich, H., Lasch, J. and Pauer, H.-D.(1999), Quantiation of Lipids, Liposomes, Amino Acids, and Proteins by Thermal Ultramicrodigestion and an Ultramicrocoulometric Assay, Based on the Reaction of Hypobromite with Ammonia.  Analytical Biochemistry 273, 163-167.

12. Goldring, J.P., Protein quantification methods to determine protein concentration prior to electrophoresis, Methods Mol.Biol. 869, 29-35, 2012.

13. Noble, J.E. and Bailey, M.J., Quantitation of protein, Methods Enzymol. 463, 73-95, 2009.

14. Berkelman, T., Quantification of protein in samples prepared for 2-D electrophoresis, Methods Mol.Biol. 424, 43-49, 2008.

15. Sapan, C.V. and Lundblad, R.L., Review of methods for determination of total protein and peptide concentration in biological samples, Proteomics Clin.Appl. 9. 268-276, 2015.

16. Miranda-Hernandez, M.P., Valle-Gonzalez, E.R., Ferreira-Gomez, D., et al., Theoretrical approximations and experimental extinction coefficients of biopharmaceuticals,Anal.Bioanal.Chem., in press, 2016.

17. Maity, H., Wei, A., Chen, E., et al., Comparison of predicted extinction coefficients of monoclonal antibodies with experimental values, Int.J.Biol.Macromol. 77, 260-265, 2015.

18. Heda, G.D., Kunwar, U., and Heda, R.F., A modified protein assay from microgram to low nanogram levels in dilute samples, Anal Biochem. 445, 67-72, 2014.

19. Goldring, J.P. and Ravaioli, L., Solubilization of protein-dye complexes on nitrocellulose to quantify proteins spectrophotometrically, Anal.Biochem. 242, 197-201, 1996.

20. Schaffner, W. and Weissmann, C., A rapid, sensitive, and specific method for the determination of protein in dilute solution, Anal.Biochem.56, 502-514, 1973.