HPAE-PAD method for determination of Hib capsular polysaccharide content

Significance of the Topic

The capsular polysaccharide of Haemophilus influenzae type b (Hib) is a critical virulence factor and a key component of conjugate vaccines that protect infants against serious infections such as meningitis and pneumonia.
Accurate quantification of Hib polysaccharide content is essential for monitoring vaccine stability, ensuring batch-to-batch consistency, and supporting quality control in vaccine production.
High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) offers direct, sensitive carbohydrate analysis without derivatization, reducing analysis time and reagent use.

Study Objectives and Overview

This work aimed to develop and validate an HPAE-PAD method for determining ribitol—the characteristic monosaccharide released from Hib capsular polysaccharide upon acid hydrolysis.
Key objectives included establishing chromatographic conditions, evaluating method performance (linearity, precision, sensitivity, accuracy), and assessing the use of a palladium hydrogen (PdH) reference electrode for improved cost and lifetime.

Methodology and Instrumentation

Sample Preparation:

• Hydrolysis of purified Hib polysaccharide (PRP) standard with 4 N trifluoroacetic acid at 100 °C for 2 h.
• Drying in vacuum concentrator and reconstitution in deionized water, followed by 2- to 32-fold dilution prior to injection.

Chromatographic Conditions:

• System: Thermo Scientific Dionex ICS-5000+ RFIC or ICS-6000.
• Column: Dionex CarboPac MA1, 4 × 250 mm analytical with 4 × 50 mm guard.
• Eluent: 400 mM NaOH (isocratic).
• Flow rate: 0.5 mL/min; Column temperature: 30 °C; Compartment: 25 °C.
• Injection volume: 10 µL (full loop); Total run time: 35 min.
• Electrochemical detection: Carbohydrate 4-potential waveform with Au working electrode and either Ag/AgCl or PdH reference electrode.

Main Results and Discussion

Chromatographic Performance:

• Baseline separation of ribitol from ribose with resolution of 1.7 under isocratic conditions.
• Linear response over 0.1–5 mg/L ribitol, with correlation coefficient (r²) of 0.9993.

Precision:

• Retention time RSD ≤ 0.05%; Peak area RSD ≤ 2.93% at 0.2 and 1 mg/L.

Sensitivity:

• Limit of detection (LOD): 0.017 mg/L (S/N ~3.4).
• Limit of quantification (LOQ): 0.05 mg/L (S/N ~9.3).

Accuracy:

• Spike recovery in hydrolyzed PRP samples ranged 94.0–95.9% for added ribitol levels of 0.2 and 1 mg/L.

Reference Electrode Stability:

• PdH reference provided comparable peak areas to Ag/AgCl over seven days (triplicate injections daily) with no significant drift, suggesting extended electrode life and reduced cost.

Benefits and Practical Applications

The validated HPAE-PAD method enables direct, high-resolution quantification of ribitol in vaccine samples without derivatization, streamlining analysis workflows.
Key advantages include high sensitivity, reproducibility, and minimal sample preparation, making it suitable for vaccine quality control, stability studies, and process monitoring.
The adoption of a PdH reference electrode may lower maintenance costs and downtime in routine carbohydrate assays.

Future Trends and Potential Applications

Integration with automated sample preparation and online dilution systems could further increase throughput and consistency.
Extension of the method to other monosaccharides or oligosaccharides in complex biologics is feasible.
Advances in electrode materials and miniaturized IC systems may enable point-of-use carbohydrate testing in clinical and field settings.
Implementation of data-rich monitoring through real-time detection and machine learning-based peak integration could enhance process analytical technology (PAT) in biopharmaceutical manufacturing.

Conclusion

An HPAE-PAD method using a Dionex CarboPac MA1 column and PdH reference electrode has been successfully developed and validated for the determination of ribitol in Hib capsular polysaccharide hydrolysates.
The assay demonstrates excellent linearity, precision, sensitivity, and accuracy, supporting its utility in vaccine quality control and stability assessment with potential cost savings from extended electrode longevity.

References

1. Lee CJ. Bacterial capsular polysaccharides--biochemistry, immunity and vaccine. Mol Immunol. 1987;24(10):1005–1019.
2. Mangtani P, Mulholland K, Madhi SA, et al. Haemophilus influenzae type b disease in HIV-infected children: a review of the disease epidemiology and effectiveness of Hib conjugate vaccines. Vaccine. 2010;28(7):1677–1683.
3. Schneerson R, Barrera O, Sutton A, Robbins JB. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates. J Exp Med. 1980;152(2):361–376.
4. Put RM, van der Haan AR, van Ijssel J, Hamidi A, Beurret M. HPAEC-PAD quantification of Haemophilus influenzae type b polysaccharide in upstream and downstream samples. Vaccine. 2015;33(48):6908–6913.
5. Mawas F, Bolgiano B, Rigsby P, Crane D, Belgrave D, Corbel M. Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide. Biologicals. 2007;35(4):235–245.
6. Guo Y, Anderson R, McIver J, Gupta RK, Siber GR. A simple and rapid method for measuring unconjugated capsular polysaccharide (PRP) of Haemophilus influenzae type b in PRP-tetanus toxoid conjugate vaccine. Biologicals. 1998;26:33–38.
7. Bardotti A, Ravenscroft N, Ricci S, et al. Quantitative determination of saccharide in Hib glycoconjugate vaccines, alone and in combination with DPT, by HPAEC-PAD. Vaccine. 2000;18:1982–1993.
8. Belfast M, Lu R, Capen R, et al. A practical approach to optimization and validation of a HPLC assay for analysis of polyribosyl-ribitol phosphate in complex combination vaccines. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;832:208–215.
9. Lei QP, Lamb DH, Heller R, Pietrobon P. Quantitation of low level unconjugated polysaccharide in tetanus toxoid-conjugate vaccine by HPAEC/PAD following rapid separation by deoxycholate-HCl. J Pharm Biomed Anal. 2000;21:1087–1091.
10. Sturgess AW, Rush K, Charbonneau RJ, et al. Haemophilus influenzae type b conjugate vaccine stability: catalytic depolymerization of PRP in the presence of aluminum hydroxide. Vaccine. 1999;17:1169–1178.
11. Tsai CM, Gu XX, Byrd RA. Quantification of polysaccharide in Haemophilus influenzae type b conjugate and polysaccharide vaccines by HPAE-PAD. Vaccine. 1994;12:700–706.
12. Thermo Scientific Technical Note 71. Eluent Preparation for High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection. 2021.
13. Thermo Scientific Technical Note 73348. Carbohydrate determinations by HPAE-PAD using a PdH reference electrode. 2021.
14. Thermo Scientific Technical Note 73605. Determination of low- and non-caloric sweeteners in food and beverages by HPAE-PAD. 2021.