Total Protein Determination in Pharmaceutical Products (Vaccines) by TNb Analysis on multi N/C 2100S pharma Application Note · multi N/C 2100S pharma

Introduction

In pharmaceutical vaccine production, starting, intermediate and end products need to be controlled for the level of antigens. They are tested for the quantity of attenuated or devitalized viruses or bacteria. Since these antigens typically consist of proteins, the analytical quantification of total protein is a method of choice. Pharmacopoeia regulations list a number of relevant methods, among them are several UV/Vis assays (e.g. Lowry, Bradford, BCA assay) and two total nitrogen methods, which is the Kjeldahl and the catalytic combustion method. This application note focuses on the catalytic combustion method which is described in Pharm. Eur. Monograph 2.5.33, Method 7 B: high temperature pyrolysis of the nitrogen compounds in an oxygen atmosphere to nitric oxide (NO) followed by chemiluminescence detection (CLD). This method description almost coincides with the EN 12260, which describes TNb determination in environmental water samples by catalytic high temperature combustion and CLD detection of the formed NO molecules. As this is what multi N/C analyzers are designed for they can be applied for TNb determination in pharmaceutical products as well. The TNb can than be converted into total protein to determine the level of antigens.

Since the nitrogen content of proteins varies, it is widely accepted to convert total nitrogen concentrations into total protein concentrations by multiplying a factor of 6.25 according to the following formula:
C [Total Protein] = c [total nitrogen] x 6.25