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Minerva Biotecnologica 2017 March;29(1):17-23

DOI: 10.23736/S1120-4826.16.01855-3


language: English

Optimization of large scale production of Haemophilus influenzae type b polyribosyl -ribitol phosphate

Amin ARSANG 1, 2, Akram TABATABAIE 1, Farzam VAZIRI 1, 2, Mehdi NEJATI 1, Mohamad R. ZOLFAGHARI 3, Abolfazl FATEH 1, 2, Fatemeh RAHIMI JAMNANI 1, 2, Ahmad R. BAHRMAND 1, Seyed D. SIADAT 1, 2

1 Department of Mycobacteriology & Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran; 2 Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran; 3 Department of Microbiology, Islamic Azad University, Qom Branch, Qom, Iran


BACKGROUND: Haemophilus influenzae (H. influenzae) type b (Hib) is one of the most common causative agents of meningitis in less than 5-year-old children. The major virulence factor of Hib is its capsular polysaccharide, polyribosyl-ribitol phosphate (PRP), which is used as an active ingredient of Hib vaccines. The purpose of this study was to optimize Hib culture situation to improve large-scale PRP production.
METHODS: H. influenzae type b ATCC10211 was cultivated in 2-liter CY medium with different concentrations of growth limiting factors (Glucose, yeast extract, hemin, nicotinamide adenine dinucleotide [NAD], and level of PRP production and dry cell weight [DCW]) were measured in each experiment. The optimized condition was used for scaling up production of PRP from 2 L to 50 L. PRP was purified according to Joseph S. Kuo’s method using Cetavlon precipitation and Hydroxyl apatite as a contaminant absorbent. Purified PRP was evaluated by in vitro assay such as endotoxin test (Limulus amebocyte lysate test) and biochemical assay such as, proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR).
RESULTS: By using 50-liter fermentor containing modified CY medium with 6g/L Glucose, 2.5 g/L Yeast extract, 0.03 g/L Hemin, 0.015 g/1 NAD and adjusting pH at 7.3 with dissolved oxygen (DO) at 30% about 5.2 g/L DCW, the 1.16 g/L PRP was obtained which was significantly higher than those obtained by normal CY medium. In addition to the high production achieved, the physico chemical analysis of the polysaccharide purified from the supernatant, revealing that PRP structure maintained original chemical composition as well as conformational structure.
CONCLUSIONS: Optimization of CY medium composition and control of medium pH and DO concentration, led to large-scale production of PRP without elevating cost.

KEY WORDS: Haemophilus influenzae type b - Glucose - Yeasts

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