Measurement of total nitrogen (TN) for protein estimation in pharmaceutical vaccines on the Shimadzu TOC-L with TNM-L

Introduction

This article introduces the application of Total Nitrogen Measurement (TNM) as a powerful analytical technique for protein estimation in biopharmaceutical products such as vaccines. The study was performed at the Serum Institute of India Pvt. Ltd., Pune. Nitrogen is a fundamental component of amino acids, which are the building blocks of proteins. Measurement of nitrogen content is widely being used for the estimation of proteins and its metabolites in pharmaceuticals, foods, beverages, and plant materials. Nitrogen commonly occurs in two forms-organic and inorganic. The inorganic forms include nitrate (NO₃^-), nitrite (NO₂^-), and ammonium (NH₄⁺). In pharmaceutical vaccine production, the quantity of the antigen needs to be controlled at the initial-, the intermediate-, and the final stages of the production cycle. Thus, viral vaccines or bacterial vaccines are tested for the quantity of respective attenuated or inactivated viruses or bacteria. Since these antigens typically consist of proteins, analytical quantification of total protein becomes crucial. Diphtheria and tetanus toxoids combined with pertussis antigens are used as a combination DTP vaccine. It confers immunity in children against diphtheria, tetanus, and pertussis. A conventional Kjeldahl method is used for protein nitrogen estimation in vaccines. Its major limitation is the need of different correction factors for different proteins as proteins have variable amino acid sequences. In addition, it is time-consuming and laborious. Further, the use of concentrated sulfuric acid at high temperatures can pose a considerable hazard to safety and health. In this application, high temperature combustion coupled with chemiluminescence detection technique was explored for the estimation of organic nitrogen and inorganic nitrogen. This method provides a fast and an efficient way to monitor nitrogen loading by Total Nitrogen (TN) analysis. The results were compared with both experimental values (Kjeldahl method) and theoretical values.

February 4, 2020 GMT