Effect of Amino Acid Addition as Stabilizers on Total Protein Content of Intravenous Therapeutic Human Normal Immunoglobulin

Objectives: This study was conducted to show the variation in total protein content of therapeutic immunoglobulin batches after the addition of free amino acids. Methods: A total of 10 different batches of therapeutic human normal immunoglobulin from five different manufacturers were taken. The total protein content in these samples was determined by both Kjeldahl and Biuret methods. The total protein in these samples was also determined by both methods after the addition of 0.3 M glycine, 0.3 M arginine, and 0.3 M histidine separately. Results: In neat samples, both methods quantified similar total protein content. However, both methods showed different protein contents where amino acids such as glycine, arginine, or histidine have been added. The total protein content by the Kjeldahl method in the samples added with glycine, arginine, or histidine was found higher as compared to the Biuret method. Conclusion: Our study suggests that the manufacturers of therapeutic immunoglobulin should replace amino acid stabilizers with nonnitrogenous stabilizer because the Kjeldahl method recommended in various pharmacopeias is based on the detection of total nitrogen. Therefore, the addition of any nitrogenous stabilizer will be quantified as protein and results may be out of specification when minimum and maximum limits are prescribed in the pharmacopeia.