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Background: Almost 80 years after implementation of the industrial ethanol fractionation process, based on the pioneering work of Edwin J. Cohn's research group, it has not lost its importance in providing life-saving biotherapies to patients. The focus has shifted from albumin, which was first used to treat intensive care patients, to immunoglobulin G preparations. Nowadays the latter enables effective, life-long treatment of patients suffering from immunodeficiencies. Ethanol concentration, pH, temperature, protein, and salt concentration are diligently varied during the Cohn fractionation process to finally yield fractions in which the main plasma proteins are enriched at adequate purity. Total protein concentration of the intermediates, probably not as important as ethanol levels and pH, has nevertheless an indisputable influence on the process' performance Objectives: Total protein measurement is therefore a valuable in process-control for versatile process performance monitoring. For this purpose, we investigated the application of analytical digital refractometry. Design: For our pilot study, we used six consecutive batches of nine relevant intermediates covering Takeda's whole industrial-scale Cohn Fractionation process. Methods: Total protein concentrations of the intermediates were measured with the method of Kjehldal to obtain reference data and digital analytical refractometry, using 10-kDa ultrafiltration to obtain a protein free permeate serving as a sample-specific blank. Results and Conclusions: Specific refractive index increment dn/dc values, allowing the calculation of the total protein concentrations by refractive index measurement, were obtained for the Cohn Fractionation intermediates investigated. Thus, analytical digital refractometry, a simple, fast, and robust technique, was shown to be fit for this purpose. [ABSTRACT FROM AUTHOR]