Comment pubs.acs.org/ac
Determination of Meningococcal Serogroups in Formulated Monovalent and Multivalent Polysaccharide and PolysaccharideConjugate Vaccines Matthew C. Cook and Jeremy P. Kunkel* Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Tunney’s Pasture, AL 2201E, Ottawa, Ontario, Canada, K1A 0K9
Anal. Chem. 2014, 86, 5383−5390. DOI: 10.1021/ac5003933
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n their recent publication, Gudlavalleti et al.1 describe a method for the quantitation of antigenic serogroup capsular polysaccharides (A, C, W, Y) in monovalent and quadrivalent polysaccharides, a quadrivalent ACWY-conjugate (diphtheria toxoid) vaccine, and final formulated bulk. The authors applied two acid hydrolysis conditions to obtain monosaccharides representative of the four serogroups, which were separated and quantitated by high-pH anion-exchange chromatographypulsed amperometric detection (HPAEC-PAD) using a multistage gradient and monosaccharide and hydrolyzed polysaccharide standards. The manuscript was received in January 2014 and accepted and published in May 2014. The Discussion section of this paper begins with the sentence: “To our knowledge, there are no published HPAEC-PAD direct methods available for the quantification of meningococcal polysaccharides in conjugate vaccines other than the patent application referred to above.” However, our two papers, “Quantitation of serogroups in multivalent polysaccharide-based meningococcal vaccines: optimisation of hydrolysis conditions and chromatographic methods” by Cook et al.2 and “Serogroup quantitation of multivalent polysaccharide and polysaccharide-conjugate meningococcal vaccines f rom China” by Cook et al.3 were published, indexed, and available online in June 2013 and May 2013, respectively, fully 6 months prior to the submission of Gudlavalleti et al. At the time of submission of their manuscript, a PubMed search using the terms “Meningococcal vaccine chromatography” would have ranked our papers as the no. 2 and no. 3 most recent hits. As of this Comment, they are ranked no. 6 and no. 7 using these same search terms. A reasonable literature search using these or related search terms would not have failed to reveal our work. In addition to the printed content, comprehensive and extensive technical methods and discussion were available online as Supporting Information to these papers (23 pages and 6 pages, respectively). These papers are also well referenced and taken together include a detailed survey of the literature. In Cook et al.2 we systematically assessed six hydrolysis conditions for each of the four (ACWY) meningococcal serogroup polysaccharides (as CRM197 conjugates). The optimum hydrolysis condition for each serogroup was a multivariate of trifluoroacetic acid (TFA) concentration, temperature, and time. The best condition for each serogroup provided maximum yield as a compromise between complete hydrolysis of the polysaccharide and the subsequent degradation of the resulting serogroup-characteristic monosaccharide products. One condition was identified for mannosamine-6Published XXXX by the American Chemical Society
phosphate from MenA, one for neuraminic acid from MenC, and one for glucose and galactose from MenY and MenW, respectively. Gudlavalleti et al. describe the use of only two hydrolysis conditions for the four meningococcal serogroup polysaccharides, one for both MenA and MenC and one for both MenW and MenY. However, we found that the optimal hydrolysis condition for MenA provided only ∼95% yields for MenW and MenY. Conversely, we found that the optimal hydrolysis condition for MenW and MenY provided only ∼95% yield of MenA. These conditions, initially assessed for monovalent solutions, were then confirmed for a ACWY quadrivalent solution. The resulting monosaccharide products were separated, identified, and quantitated using a single HPAEC-PAD protocol, with a customized multistage linear gradient eluent profile and one column setup, for determination of all four serogroup components. Comparison to calibration curves constructed from sets of monosaccharide or hydrolyzed polysaccharide standards allowed for the quantitation of each characteristic serogroup monosaccharide in polysaccharide and polysaccharide-conjugate vaccines (Figure 1). In some instances, saccharide excipients added as stabilizers and bulking agents by various manufacturers will directly interfere with serogroup determinations by having a monosaccharide constituent in common with some serogroup polysaccharides. While Gudlavalleti et al. do not address this complication, we assessed several dialysis and centrifugal filter options from a variety of manufacturers for vaccine deformulation. Those based on cellulose or cellulose ester membranes did not provide consistent results for saccharide excipient removal and/or polysaccharide component retention. However, we demonstrated that reiterative size-exclusion centrifugal filtration prior to sample hydrolysis using a noncellulosic device completely removed all interfering saccharide excipients without loss of serogroup polysaccharides.2 This approach was developed and validated using bivalent AC and quadrivalent ACWY polysaccharide-conjugate “mock” vaccine constructs assembled from monovalent polysaccharideconjugate bulk samples to mimic vaccines with and without saccharide excipient. The mock vaccine samples were either filtered to remove excipient and analyzed by hydrolysis/ HPAEC-PAD or analyzed directly without prior filtration. HPAEC-PAD chromatograms of vaccine hydrolyzates are often complicated by the presence of spurious and unknown peaks, frequently distinct and reproducible in different vaccines,
A
DOI: 10.1021/acs.analchem.5b00276 Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Comment
but which are not related to the characteristic serogroup peaks of interest. In some cases even the target analyte peaks are not of ideal shape and resolution, despite the considerable efforts taken to refine the chromatography program. This is especially true of vaccine samples which have not been deformulated by reiterative centrifugal filtration. Excellent results for all four serogroups were obtained using our method with careful and consistent manual integration for each product/serogroup chromatogram. However, it was noted that deformulation decreased the complexity and improved the consistency of all chromatograms, including those in which the presence of saccharide excipients (e.g., lactose, sucrose) did not directly interfere with determination of the target analyte. Therefore, as suggested in our papers,2,3 since publication of these methods we now routinely remove saccharide excipients from meningococcal polysaccharide-based vaccines in which they occur for the determination of all serogroup polysaccharides regardless of direct interference, despite the additional effort that this entails. The “total polysaccharide equivalent” content for each serogroup in each vaccine was calculated based on repeating unit concentrations, with conversion of molar concentrations to mass concentrations. One mole of each serogroup-characteristic monosaccharide was considered equal to 1 mol of the respective repeating units. The levels of O-acetylation and types/ratios of counterions are highly dependent on the distinct bacterial strains and growth conditions used and the purification and processing techniques employed. Because we intended our methods for use with a wide spectrum of meningococcal vaccines, and this information is not always available or forthcoming, the molar masses of repeating units were not adjusted to account for the contributions from counterions or from variable O-acetylation of N-acetylmannosamine-6-phosphate (MenA) or N-acetylneuraminic acid (MenC, MenY, MenW). As a result, the actual concentrations of serogroup polysaccharides in all vaccines complete with counterions and O-acetylation will be variably but only marginally higher than measured. Curiously, Gudlavalleti et al. employ a mixed approach for quantitation. They use literature values for the molar masses of the repeating units of MenC and MenA (both of which will have different levels of Oacetylation and types/ratios of counterions than the ones in their study). However, for MenW and MenY, they do not address counterion content, and based on “low” and “high” Oacetylation levels, as measured by NMR, they assume nonacetylated MenW and monoacetylated MenY. Thus, they treat MenW and MenY differently with respect to quantitation despite their otherwise overall structural similarity and identical nonacetylated molar masses. Our method, including deformulation, acid-hydrolysis, and chromatography, was demonstrated to be highly reproducible and very useful for the evaluation of antigen content and lot-tolot consistency of manufacture. It has also proven to be robust and widely applicable for routine analysis and serogroup quantitation. Gudlavalleti et al. demonstrate their method with only one polysaccharide-conjugate vaccine. To date, we have applied our serogroup polysaccharide determination method to test a dozen marketed polysaccharide or polysaccharideconjugate (tetanus toxoid, TT; diphtheria toxoid, DT; mutant diphtheria toxin, CRM197 protein) vaccines, with at least two lots, and in some cases more than 15 lots, of each vaccine tested (>70 product lots total) (Cook et al.2,3 and unpublished data). Aggregate multioperator data from the evaluation of these
Figure 1. Comparison of HPAEC-PAD chromatograms of standards, “mock” quadrivalent polysaccharide-conjugate vaccine, and real quadrivalent polysaccharide-conjugate vaccine chromatograms for determination of serogroups A, C, W, and Y. MenA polysaccharide standard, “mock” polysaccharide-conjugate vaccine, and real polysaccharide-conjugate vaccine were hydrolyzed under optimal conditions for each respective serogroup: MenA (1.0 M TFA/90 °C/2 h), MenC (2.0 M TFA/90 °C/3 h), MenW and MenY (0.1 M TFA/80 °C/2 h). (Traces offset by 5 nC from one another for better clarity.) Adapted and reprinted with permission from the Supporting Information of ref 2. Copyright 2013 Elsevier. (a) MenA: MenA polysaccharide hydrolyzate standard (blue trace), containing 2 nmol of ManN-6-P; “mock” quadrivalent ACWY polysaccharide-conjugate vaccine (red trace); real quadrivalent ACWY polysaccharide-conjugate vaccine (green trace, no saccharide excipient). (b) MenC: monosaccharide standard mix (blue trace), containing 0.8 nmol of Neu5Ac; “mock” quadrivalent ACWY polysaccharide-conjugate vaccine (red trace); real quadrivalent ACWY polysaccharide-conjugate vaccine (green trace, no saccharide excipient). (c) MenW and MenY: monosaccharide standard mix (blue trace), containing 1.2 nmol each of Gal (for MenW) and Glc (for MenY); “mock” quadrivalent ACWY polysaccharide-conjugate vaccine (red trace); real quadrivalent ACWY polysaccharide-conjugate vaccine (green trace, no saccharide excipient). B
DOI: 10.1021/acs.analchem.5b00276 Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Comment
monovalent and multivalent bulks and vaccines, in liquid and lyophilized formulations with various buffers, salts, and saccharide excipients, continue to demonstrate a method CV of
