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Quantification of residual cetyltrimethylammonium bromide (CTAB) and sodium deoxycholate (DOC) in Haemophilus
1Serum Institute of India Pvt. Ltd., Pune, India.
Insights
Nuclear Magnetic Resonance (NMR) can detect residual reagents, Cetyl trimethylammonium bromide (CTAB) and deoxycholate (DOC), in purified Haemophilus influenzae type b polysaccharide. This method aids regulatory compliance for vaccine manufacturing.
Area of Science:
- Biochemistry and Analytical Chemistry
- Vaccine Manufacturing and Quality Control
Background:
- Hib polysaccharide purification involves Cetyl trimethylammonium bromide (CTAB) precipitation and deoxycholate (DOC) for protein removal.
- Residual reagents like CTAB and DOC must be removed to meet stringent regulatory standards in pharmaceutical products.
Purpose of the Study:
- To establish Nuclear Magnetic Resonance (NMR) as a method for identifying and quantifying residual CTAB and DOC in purified Hib polysaccharide.
- To provide data supporting regulatory compliance for vaccine manufacturers by offering an efficient analytical method.
Main Methods:
- Utilized NMR spectroscopy to detect and quantify residual Cetyl trimethylammonium bromide (CTAB) and deoxycholate (DOC).
- Determined the Limit of Detection (LOD) at 0.1% (10 μg/mL) and Limit of Quantitation (LOQ) at 0.5% (50 μg/mL) for these reagents relative to the polysaccharide.
Main Results:
- NMR successfully identified and quantified residual CTAB and DOC in purified Haemophilus influenzae type b (Hib) polysaccharide.
- Absence of specific NMR peaks confirmed reagent levels below the 0.1% LOD, meeting regulatory thresholds.
Conclusions:
- NMR provides a reliable and efficient method for detecting residual CTAB and DOC in Hib polysaccharide.
- This analytical approach simplifies regulatory validation for vaccine manufacturers, saving time and resources.
Abstract:
CTAB and DOC are used as reagents in the purification of Hib polysaccharide. Polysaccharide is purified by precipitation with CTAB from fermented broth followed by solvent extraction and DOC is used to remove the protein impurities. The reagents used in the purification process should be removed from the product as per regulatory requirements. These two residual reagents can be easily identified and quantified in purified Haemophilus influenzae type b polysaccharide by NMR. The LOD of these residual reagents is 0.1% (10 μg/mL) and LOQ is 0.5% (50 μg/mL) with respect to polysaccharide determined from the spectrum. The absence of the peaks corresponding to CTAB and DOC in the NMR spectrum of purified polysaccharide confirms either they are absent or present at less than 0.1%. The present study provides supporting data from the regulatory viewpoint, which can help in circumventing the time-consuming studies for the vaccine manufacturers to develop different analytical methods for identification and quantification of CTAB and DOC as per regulatory requirements.
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