Optimization of the Lowry Method of Protein Precipitation from the H. influenzae Type b Conjugate Vaccine Using Deoxycholic Acid and Hydrochloric Acid

• Published in: Biotechnology and bioprocess engineering Vol. 11; no. 3; pp. 215 - 222
• Main Authors: Kim, Hyun-Sung, Kim, Sang-Joon, Kim, Hui-Jung, Kim, Han-Uk, Ahn, Sang-Joem, Hur, Byung-Ki
• Format: Journal Article
• Language: Korean
• Published: 한국생물공학회 01.06.2006
• Subjects: 생물공학; method validation; conjugate vaccine; Lowry method; Haemophilus influenzae type b; deoxycholic acid precipitation
• ISSN: 1226-8372; 1976-3816

The Lowry method was used in this study to measure protein in Haemophilus influenzae type b (Hib) conjugate vaccines (polyribosylibitol phosphate-tetanus toxoid; PRP-TT) using deoxycholic acid (DOC) to induce protein precipitation. Trichloroacetic acid (TCA) did not induce precipitation adequately from the Hib conjugate bulk and the freeze-dried Hib conjugate product. Its yield was approximately 50%. The matrix structure of Hib conjugate inhibits precipitation by TCA. Although the Lowry method can be carried out without precipitation in Hib conjugate bulk when no residual impurities (adipic acid dihydrazide [ADH], 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide-HCI [EDAC], phenol and cyanogens bromide [CNBr], etc.) are present, it cannot be used for Hib conjugate products that contain sucrose 8.5%, because 8.5% concentration of sucrose enhanced the protein concentration. DOC- and HCl-induced precipitation is an alternative method for evaluating the protein content of the Hib conjugate bulk and the Hib conjugate product. The precipitation was optimal with a final concentrate of 0.1% for DOC at $4^{\circ}C$ and pH 2. This Lowry method, using DOC/HCI precipitation to induce protein precipitation, was confirmed a consistent, reproducible, and valid test for proteins in Hib conjugate bulk and its freeze-dried product.