# Determination of the concentration and size-distribution of residual capsular poly(N-acetyl neuraminic acid) from outer membrane vesicle vaccines by micellar electrokinetic capillary chromatography

Laurent Leclercq, Jean-François Cotte, Jérôme Thiebaud, Jean Dubayle, Hervé Cottet

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## To cite this version:

Laurent Leclercq, Jean-François Cotte, Jérôme Thiebaud, Jean Dubayle, Hervé Cottet. Determination of the concentration and size-distribution of residual capsular poly(N-acetyl neuraminic acid) from outer membrane vesicle vaccines by micellar electrokinetic capillary chromatography. Analytica Chimica Acta, 2026, 1398, pp.345295. 10.1016/j.aca.2026.345295. hal-05536270

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# Determination of the concentration and size-distribution of residual capsular poly(N-acetyl neuraminic acid) from outer membrane vesicle vaccines by micellar electrokinetic capillary chromatography

Laurent Leclercq¹*, Jean-François Cotte², Jérôme Thiebaud², Jean Dubayle², Hervé Cottet¹*

¹IBMM, University of Montpellier, CNRS, ENSCM, Montpellier, France

²Sanofi, Analytical Science, 1541 avenue Marcel Mérieux, 69280 Marcy l'Etoile, France

*Correspondence should be addressed to H.C. (herve.cottet@umontpellier.fr) or L.L. (laurent.leclercq@umontpellier.fr).

## ABSTRACT

**Background:** Vaccines based on Outer Membrane Vesicles (OMVs) against *Neisseria meningitidis* require the quantification and characterization of residual capsular polysaccharides (CapsPS) that are not completely removed during the production process. This task is challenging due to the complexity of the CapsPS composition and the presence of OMVs in the samples.

**Results:** Micellar Electrokinetic Chromatography (MEKC) using a negatively charged polyelectrolyte multilayer coating (SMIL) enabled complete separation of CapsPS and OMV residues in approximately 40 min. The use of a 4-layer SMIL coating was crucial for achieving good repeatability of migration times ($t_m$). The method allowed the quantification of CapsPS at concentrations between 80 and 600 mg.L⁻¹. The average number and weight degrees of polymerization were also determined for each batch of OMVs, with polydispersity indexes between 1.2 and 1.5 and degrees of polymerization ($DP_n$) between 30 and 70.

**Significance:** This method can be readily implemented in the pharmaceutical industry for the monitoring and documentation of process development.

**Keywords:** Micellar Electrokinetic Chromatography; Outer Membrane Vesicles; vaccines; capsular polysaccharides; size distribution
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# 1. Introduction

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Outer Membrane Vesicles (OMVs)-based vaccines represent a promising approach in the field of immunization, particularly against bacterial pathogens. These vaccines are derived from the outer membrane of Gram-negative bacteria and contain a variety of antigens capable of stimulating the immune system [1, 2]. OMVs are released naturally during bacterial growth and can be modified to improve their immunogenic properties following various purification steps [3]. OMVs are small spherical nanoparticles, mainly composed of bacterial proteins, lipids, and periplasmic content. In the case of *Neisseria meningitidis* (*Nm*), current vaccines are based on the capsular polysaccharide for several serogroups such as A, C, Y, and W135 [4]. The *Nm* capsular polysaccharide B is poorly immunogenic and prevents the development of an anti *Nm* B vaccine based on capsular polysaccharides [5]. For serogroup B, current commercial vaccines against *Nm* B are made of OMVs and/or recombinant proteins [6]. Therefore, these complex biological products require a thorough analytical understanding, which is essential to ensure consistent vaccine production.

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Among the impurities present in OMV vaccines against *Nm* B is residual capsular polysaccharide (CapsPS). These biomacromolecules are not completely eliminated during the production process and must be monitored [7]. Capsular polysaccharides are complex, bulky carbohydrate structures that form a protective layer around certain bacteria. They also contribute to their virulence by evading the host's immune response. This particular CapsPS from *Nm* serogroup B has been shown to be non-immunogenic in adult humans [8]. Nevertheless, monitoring for residual CapsPS during OMVs production and at the final stage remains important for product characterization and consistency of the production process. The *Nm* B capsule is an (α2→8)-linked poly(*N*-acetyl neuraminic acid) and is anchored in the outer membrane by a 1,2-diacylycerol fragment [9]. CapsPS is quantified using High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) after acid hydrolysis and release of *N*-acetylneuraminic acid (NeuAc). This method measures the total amount of CapsPS amount, but does not provide information on the size of the polysaccharides.

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Capillary electrophoresis (CE) and Micellar Electrokinetic Chromatography (MEKC) have been used since the early 1990s for the separation of polysaccharides, either in the absence [10-13] or the presence of micelles [10, 12, 14-16], or in gels [17-21]. Various oligo- and
polysaccharides have been analyzed, including maltose, starch, and dextran [14, 17];
unsaturated disaccharides of hyaluronic acid and chondroitin sulfate [13]; fucoidans [11];
mannuronan [15]; and alginate [18]. The molar mass of polysulfated hyaluronic acid and
NeuAc polymers was determined based on their high-resolution separation by CE in the
presence of a replaceable gel [19-21]. Several reviews have been published on the analysis
of complex polysaccharides [22, 23] and glycoproteins [24] by CE.

CE in different modes has also been used for vaccine characterization [see, e.g., the review in
ref 25], including Human Papillomavirus (HPV) virus-like particles and lipid (squalene)
adjuvant nanoemulsions [26], residual DNA fragments from host cells in the Vero cell rabies
vaccine [27], mRNA vaccines [28, 29], antigenic subunits and isoforms [30], foot-and-mouth
disease virus (FMDV) antigens [31], and the inactivated polio vaccine [32, 33].

With regard to CE applications for the quantification of polysaccharides in vaccines, Lamb et
al. separated and quantified Meningococcal polysaccharides, active components of vaccines
against various *Nm* serogroups, by MEKC in a phosphate/borate buffer using a bare fused
silica capillary [16]. More recently, Li et al. quantified specific oligosaccharides enzymatically
released from *Hericium erinaceus* polysaccharides using CE-LIF [12].

In this work, MEKC using coated capillaries was studied as an advanced analytical method
capable of (i) quantifying and (ii) characterizing CapsPS residues by determining their degree
of polymerization and particle size distribution. This objective is challenging to achieve
because the sample contains not only CapsPS but also OMV residues, which must be
separated to enable quantification and characterization.

## 2. Materials and methods

### Chemicals

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane sulfonic acid (HEPES) and sodium dodecyl sulfate
(SDS) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Ultrapure water
was obtained using a MilliQ system from Millipore (Molsheim, France).
Poly(diallyldimethylammonium chloride) (PDADMAC; high molecular weight, M<sub>w</sub> = 4 × 10<sup>5</sup> –
5 × 10<sup>5</sup> Da) 20% w/w in water was purchased from Aldrich (Lyon, France). Sodium
poly(styrene sulfonate) (PSS; M<sub>w</sub> = 7 × 10<sup>5</sup> Da) was purchased from Acros Organics (Geel,
Belgium). N-acetyl neuraminic acid, borax ($Na_2B_4O_7$, 10 $H_2O$), acetic acid and sodium hydroxide were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Glucuronic acid was purchased from Alfa Aesar (Illkirch, France).

Purified polysaccharide from *Nm B* (PS-MenB, $M_w$ = 3.71 × 10⁵ Da determined by SEC-MALLS) was provided by Sanofi (Marcy-l'Étoile, France). Five lots containing residual capsular polysaccharide issued from OMV vaccine components were also provided by Sanofi (Marcy-l'Étoile, France), numbered from 1 to 5 and stored at 4°C.

## Preparation of the polyelectrolyte coating solutions

PDADMAC and PSS solutions ($3 \text{ g.L}^{-1}$ each) were prepared in 20 mM HEPES buffer (480 mg HEPES and 1 mL of 1 M NaOH, completed to 100 mL with deionized water) at pH 7.4. Both were prepared one night before first use. To prepare the polyelectrolyte coating solution vials, 200 µL of the polyelectrolyte solution were added into a 1 mL polypropylene vial, making sure the liquid level was below the electrode (for Agilent CE equipment using standard electrodes) to avoid cross-contamination between polyelectrolyte vials.

## CapsPS content determination by HPAEC-PAD

A solution of glucuronic acid at 1 g/L in water was prepared as an internal standard and added to the acetic acid hydrolysis solution to obtain a final concentration of 10 mg/L. The OMV samples were diluted in water by a factor of 250 to 1000 depending on their initial concentration. OMVs samples were then hydrolyzed in acetic acid at high temperature to release NeuAc. HPAEC-PAD was performed using a Dionex ICS6000 system with a CARBOPAC PA10 2×250 mm column (Dionex Corp, Sunnyvale, CA). NeuAc was carried out by isocratic elution at a flow rate of 0.25 mL.min⁻¹ with 60 mM NaOH and 80 mM sodium acetate as the eluent. Components in the eluent were detected by PAD using gold electrode and Ag/AgCl reference electrode. CapsPS content was calculated after external linear calibration with NeuAc standard, from 0.25 to 4 mg/L, and correction with glucuronic acid as an internal standard.

## Polyelectrolyte multilayers coating procedure

Bare fused silica capillaries of 50 µm i.d. × 68.5 cm total length were purchased from Polymicro Technologies (Phoenix, USA). The capillaries were coated with polyelectrolyte multilayers (SMIL), as previously described [34], using the 7100 CE Agilent system
(Waldbronn, Germany). The SMIL coating was prepared by preconditioning the silica capillary with 1 M NaOH for 10 min, then flushing it with water for 5 min and HEPES for 10 min. The coating process consisted of alternating 7-min flushes of polycation (PDADMAC) and polyanion (PSS) solutions, with a 3-min HEPES rinse between each layer. Four layers were applied in total, creating a (PDADMAC/PSS)₂ coating. As previously described [34], low ionic strength HEPES buffer was used as the coating buffer since it provides non-hydrated polyelectrolyte film with extended polyelectrolyte conformation to minimize solute adsorption. After the last HEPES flush, the coating was left still for 5 min, and then flushed with water for 3 min. The final step consisted in rinsing for 10 min with the background electrolyte (BGE) and waiting another 5 min before starting analyses. All flushes were performed at 930 mbar. The coating was found very stable for at least one hundred analyses.

## 134 Micellar Electrokinetic Chromatography

MEKC experiments were carried out on a 7100 CE Agilent system (Waldbronn, Germany). The temperature of the capillary cartridge was set at 25°C. MEKC experiments were performed by applying a voltage of +15 kV. BGE was 50 mM borate buffer (47.67 mg borax in 10 mL deionized water) with various SDS concentrations (up to 250 mM) at pH 9.2. Samples were hydrodynamically injected at 60 mbar for 7 s (0.89 % of total capillary volume). The capillary was flushed with BGE at 1 bar for 5 min between runs. The detection wavelength was 200 nm. All MEKC experiments were performed in triplicates.

Quantification of residual CapsPS was performed by integration of the Abs/t traces relative to a standard. Different standards were tested and compared (see Supporting Information). Since the composition of CapsPS is close to NeuAc structure, NeuAc was finally retained as the standard for CapsPS quantification. The NeuAc peak (DP=1) was detected at 15.5 min.

## 147 3. Results and discussion

Figure 1 shows a schematic representation of the Gram-negative membrane of *Neisseria meningitidis* (*Nm*, also known as meningococcus), which includes various protein antigens, lipooligosaccharides, and a polysaccharide capsule. Purified OMVs targeting *Nm* B have a similar structure, except for the polysaccharide capsule and peptidoglycan, which are
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normally removed during production. The objective of this work is to quantify and
characterize the residual CapsPS present in OMV-based vaccines against Nm B.

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**3.1. BGE optimization on PS standard sample**

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MEKC was selected as a well-adapted separation technique because it allows the separation of biomacromolecules based on differences in size, charge, and hydrophobicity. The SDS concentration was first optimized in the 0-200 mM range, in a 50 mM sodium borate buffer, using a bare fused silica capillary (Figure 2). The PS-MenB sample was used as the standard capsular polysaccharide. Since CapsPS must be negatively charged under alkaline conditions due to carboxyl groups (see the typical chemical structure of PS based on NeuAc repeats in Figure 1) and its interaction with SDS molecules, the electrostatic repulsion between the capillary surface and the solutes was maintained to prevent solute adsorption. PS-MenB polymers were detected at 200 nm due to the presence of amide groups in their structure (Figure 1), appearing after the electroosmotic flow (EOF) peak as anions in counter-electroosmotic mode. As shown in Figure 2, at least 150 mM of SDS was required to achieve better separation between the two modes (see peaks 1 and 2) constituting the PS-MenB sample. These two modes correspond to PS with different molar masses, with migration time increasing as molar mass increases. The selectivity of separation between the two modes was mainly driven by the addition of SDS in the BGE. At higher SDS concentrations (up to 250 mM), better resolution was achieved due to a slight decrease in the EOF magnitude (ionic strength effect), which resulted in increased apparent selectivity on the time scale. It is worth noting that the difference in effective mobility between the two modes was minimal (-36.4 TU for peak 1 vs -37.4 TU for peak 2, where 1 TU = $10^{-9} \text{ cm}^2\text{V}^{-1}\text{s}^{-1}$) due to a similar charge-to-size ratio. Longer PS chains exhibited higher effective mobility, most likely due to greater association with SDS micelles (e.g., via interaction with N-acetyl groups). The voltage was set to +15 kV because multiple spikes, indicating polymer aggregation during electrophoretic separation, were observed at +30 kV (Figure SI1).

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**3.2. PS quantification in different OMV lots using SMIL coating**

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The optimized BGE was used to analyze an OMV (lot #1) using a (PDADMAC/PSS)₂ SMIL coating. Bare fused silica capillaries showed relatively low migration time repeatability (RSD ~ 13.2 %), as shown by the shift towards higher migration times in Figure SI2 for three successive runs. In contrast, SMIL-coated capillaries showed much better repeatability (see
Figure SI3, RSD ~ 2.6 %). This result confirms that SMIL coatings offer excellent repeatability compared to bare fused silica [34]. The electropherogram of OMVs from lot #1 under these optimized conditions is shown in Figure 3. Residual CapsPS was detected between 18 and 37 min, clearly separated from the broad signal (45-75 min) corresponding to OMV residues. This migration order can be explained by the chemical nature of OMVs, which are expected to be more hydrophobic than CapsPS and therefore interact more strongly with SDS micelles. Indeed, OMVs are mainly composed of proteins, lipids, and lipopolysaccharides [35]. This remarkable separation demonstrates the great potential of MEKC for analyzing residual CapsPS in highly complex sample containing OMV components. In OMVs lot #1, the degree of polymerization of CapsPS was much lower than in the PS-MenB sample. Consequently, the CapsPS profile in lot #1 appears on the electropherogram as well-separated PS oligomers, with a typical "hedgehog" shape, generally observed for oligomers with the lowest molar mass. The peak numbering was easily determined by spiking the sample with the monomer NeuAc (DP = 1) and successively counting the peaks at higher migration times.

The optimized conditions were also applied to quantify the residual CapsPS in 5 different lots of OMV. Figure 4 shows $h(t) = S(t)/t$ as a function of migration time $t$, where $S(t)$ is the absorbance signal. This transformation of the y-axis from $S(t)$ to $h(t)$ is a convenient way to correct for differences in the migration velocities of PS oligomers. Quantification by integrating the peaks in Figure 4 is more accurate than interpreting the raw electropherogram (i.e. $S(t)$ vs $t$) and then dividing each peak area by its migration time. For more details on this topic, see previous work explaining this approach [36]. Prior to integration, the baselines were corrected for drift, as indicated by the dotted lines in Figure 4. The electropherograms with the corrected baselines are shown in Figure 5A. The residual concentrations of CapsPS in the different lots were obtained by integrating the traces in Figure 5A and using NeuAc as a standard (see calibration curve shown in Figure SI4-D). The residual CapsPS concentrations are reported in Table 1 and are consistent with the values obtained using an orthogonal method (i.e., HPAEC-PAD), shown in the last column. Different standards were compared (see calibration plots in Figure SI4, A-D), including PS-MenB, colominic acid, and N-acetylmuramic acid. However, PS MenB is not a pure sample (80 % considering the salt counter ions), and the composition of the residual CapsPS is expected to
be closer to the structure of NeuAc than to that of colominic acid or N-acetylmuramic acid. Depending on the OMV lot and production processes (including the various purification steps), CapsPS residues ranged from 70 and 600 mg.L⁻¹, in agreement with process optimizations aimed at removing CapsPS. Relative standard deviations of less than 6% were obtained by MEKC for the residual concentration of CapsPS.

### 3.3. Molar mass characterization of residual CapsPS in different OMV lots by MEKC using SMIL coating

After quantification of residual CapsPS in the OMV samples, size-based characterization of the CapsPS samples was investigated by converting the electropherogram into an effective mobility scale distribution (Figure 5B) and, finally, into a weight-distribution of the degree of polymerization (DP). Starting from the baseline-corrected electropherograms h(t) vs t presented in Figure 5A, the x-axis was first transformed into an effective mobility (μep) scale by correcting the apparent mobilities from the electroosmotic mobility of each run according to:

$$ \mu_{ep} = \frac{L_d}{E} \left( \frac{1}{t} - \frac{1}{t_{eof}} \right) \qquad (1) $$

where E is the applied electric field, t_{eof} is the EOF migration time and L_d is the capillary length to the detector. To keep a weight distribution of effective mobilities, the y-axis must be transformed according to mathematical rules of variable changes, leading to [36]:

$$ P(\mu) = \frac{S(t)}{t} \times \frac{1}{\left| \frac{d\mu_{ep}}{dt} \right|} = S(t) \times t \qquad (2) $$

These P(μ) distributions are shown in Figure 5B together with the integration limits (see vertical dotted lines). As discussed earlier, higher CapsPS DPs lead to higher effective mobilities (in absolute value). The transformation into a weight-based distribution of the DP was next obtained using a μep vs DP calibration curve shown in Figure 5 insert. The semi-empirical equation obtained by curve fitting on the 25 first oligomers (i.e. DP≤25) is given in Figure 5 caption. This equation allowed the conversion of the effective mobility scale into a DP scale. The y-axis corresponding to the weight-based distribution in DP is given by [36]:

$$ P(DP) = \frac{P(\mu)}{\left| \frac{dDP}{d\mu_{ep}} \right|} \qquad (3) $$

Calculations were performed in an Excel sheet using the previously mentioned μep vs DP
calibration curve. Using this approach, the final DP distributions for the 5 OMV samples are presented in Figure 5. The corresponding average DPn and weight-average DPw were determined by integration of the aforementioned distributions between DP = 2 and DP = 190 according to:

$$246 \qquad DP_n = \frac{\sum_{i=2}^{190} A_i}{\sum_{i=2}^{190} \frac{A_i}{DP_i}} \qquad (4)$$

$$247 \qquad DP_w = \frac{\sum_{i=2}^{190} A_i DP_i}{\sum_{i=2}^{190} A_i} \qquad (5)$$

The numerical values are summarized in Table 1 along with the corresponding dispersity index. Lot #1 was the most polydisperse sample (PDI=1.5), whereas the others (lot #2 to lot #5) exhibited a PDI of 1.2. The number-average DPn ranged from 30 for lot #1 to 69 for lot #5. Relative standard deviations of less than 4% on average were obtained on DPn.

With regard to size-based resolution, it is clear from Figure 5 that MEKC allows high-resolution separations with monomeric distributions up to about DP=25. For higher DP values, separation by MEKC remains effective even if oligomeric resolution is not achieved. In this regard, the counter-electroosmotic mode using a SMIL coating is favorable because it increases the apparent selectivity for higher molar mass compared to a neutral-coated capillary. It is clear that the peak shapes and lots differences observed in Figure 5C demonstrate that MEKC is able to discriminate differences in molar mass distribution up to DP~125 (for lot #5). It is difficult to determine the exact limit of this method in terms of maximal molar mass as we did not have samples with higher molar masses. The trueness of the DP determination is directly related to the extrapolation of the empirical calibration curve to a high DP (see inset in Figure 5C). The calibration curve was established based on the electrophoretic mobility of the first 25 oligomers present in lot #1 with an excellent correlation coefficient R²=0.9999. The average DPn of the 5 samples varies between 30 and 70 with typical DP distributions ranging between 1 and 125 monomers.

So far, this MEKC method dedicated to residual CapsPS from OMVs had not been described in the literature. In the absence of an official method from the pharmacopoeia for CapsPS, the commonly used and published alternative method is HPAEC-PAD [37, 38]. While both methods MEKC and HPAEC-PAD allow quantifying residual CapsPS (see Table 2 for a matter of comparisons), an in-depth characterization of residual CapsPS was performed by determining their molar mass distribution using MEKC. This information is valuable for
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monitoring the elimination of CapsPS during OMV production and for documenting product knowledge. Moreover, MEKC presents the advantage of minimal sample preparation with a high matrix tolerance regarding the OMV content.

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## 4. Conclusion

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In this work, we demonstrated that MEKC is a powerful separation method for the quantification and size characterization of residual CapsPS in OMVs lots. The quantitative results are consistent with those obtained using the orthogonal HPAEC-PAD method, and additional size information was successfully extracted. This achievement was challenging due to the complexity of PS polydispersity and the presence of OMV residues in the samples.

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A concentration of 250 mM SDS allowed complete separation between CapsPS residues and OMV residues in approximately 40 minutes. The use of a 4-layer SMIL coating was crucial to achieve good repeatability of the migration time. The separation voltage was limited to +15 kV to avoid the formation of aggregates (indicated by spikes) in the capillary. After baseline correction, it has been possible to quantify residual CapsPS at levels ranging from 80 to 600 mg.L⁻¹. Thanks to the effective separation of the PS molar mass distributions, it was possible to determine the average number and weight *DP* for each lot of OMV, with PDI values between 1.2 and 1.5 and *DPn* ranging from 30 to 70. This was achieved by transforming the migration time into an effective mobility scale and, finally, into a *DP* scale. We believe that this method can be easily implemented in the pharmaceutical industry for monitoring and documenting process development.

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## Credit authorship contribution statement

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Laurent Leclercq: Investigation, Data curation, Formal analysis, Writing – original draft. Jean-François Cotte: Writing – review & editing, Methodology. Jérôme Thiebaud: Methodology.

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Jean Dubayle: Writing – review & editing, Methodology, Investigation. Hervé Cottet: Writing – review & editing, Methodology, Formal analysis, Supervision, Funding acquisition, Conceptualization.

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## Acknowledgement

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Marina Brossaud (Sanofi) is gratefully acknowledged for providing *Neisseria meningitidis* polysaccharides sample. Denis Bouchard and Alain Médard (Sanofi) are gratefully

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acknowledged for HPAEC-PAD experiments. Christelle Fontaine (Sanofi) is gratefully acknowledged for supporting this study. The authors thank Jean-Sébastien Bolduc (Sanofi) for proof-reading the manuscript.

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**Declaration of competing interest**

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Jean-François Cotte, Jérôme Thiebaud, and Jean Dubayle are Sanofi employees and may hold shares and/or stock options in the company. Laurent Leclercq and Hervé Cottet declare no competing interests.

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**Disclosure**

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This work was partly funded by Sanofi under a Cooperative Research and Development Agreement with the University of Montpellier and the CNRS.

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**Appendix A. Supplementary data**

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Supplementary data to this article can be found online.

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**Data availability**

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Data will be made available on request.

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<figure><img src="image_3.png" /></figure>

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Figure 1. Schematic representation of type B *Neisseria meningitidis* comprising various protein antigens, lipooligosaccharide, and the polysaccharide capsule with its chemical structure given at the top.
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Comparative electropherograms of PS-MenB on a fused silica capillary in BGE at various SDS concentrations. Experimental conditions: 68.5 cm total length (60 cm to the detector) x 50 µm i.d. Background electrolyte: 50 mM borate buffer + SDS, pH 9.2. Applied voltage: +15kV. Hydrodynamic injection: 60 mbar, 6 s (0.77% of total capillary volume). Sample concentration: 10 g.L⁻¹ in BGE without SDS. Temperature: 25°C. Detection wavelength: 200 nm.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Time-corrected electropherogram showing the separation of residual CapsPS from OMV residues in sample 1 on a 4-layers SMIL-coated capillary. Experimental conditions: (PDADMAC/PSS)₂ coated fused silica capillary, 68.5 cm total length (60 cm to the detector) x 50 µm i.d. Background electrolyte: 50 mM borate buffer + 250 mM SDS, pH 9.2. Applied voltage: +15kV. Hydrodynamic injection: 60 mbar, 7 s (0.89 % of total capillary volume). Temperature: 25°C. Detection wavelength: 200 nm.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4. Comparative time-corrected electropherograms of sample #1 (–), sample #2 (–), sample #3 (–), sample #4 (–) and sample #5 (–). Experimental conditions: (PDADMAC/PSS)₂ coated fused silica capillary, 68.5 cm total length (60 cm to the detector) x 50 µm i.d. Background electrolyte: 50 mM borate buffer + 250 mM SDS, pH 9.2. Applied voltage: +15kV. Hydrodynamic injection: 60 mbar, 7 s (0.89 % of total capillary volume). Temperature: 25°C. Detection wavelength: 200 nm. Each sample was injected as is. Dotted lines correspond to the baseline used for the quantification of total residual CapsPS. Lower limit of integration was 18 min and upper limit was 38 min. The NeuAc peak (DP=1) was detected at 15.5 min.</figcaption></figure>

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>Figure 5. Baseline-corrected electropherograms in timescale (A), effective mobility-scale (B), and weight distributions in degree of polymerization (C) for sample #1 (--), sample #2 (--), sample #3 (--), sample #4 (--) and sample #5 (--). Experimental conditions: (PDADMAC/PSS)₂ coated fused silica capillary, 68.5 cm total length (60 cm to the detector) x 50 µm i.d. Background electrolyte: 50 mM borate buffer + 250 mM SDS, pH 9.2. Applied voltage: +15kV. Hydrodynamic injection: 60 mbar, 7 s (0.89 % of total capillary volume). Temperature: 25°C. Detection wavelength: 200 nm. Each sample was injected as is. Baselines were corrected according to the dashed traces shown in Fig. 4. Lower limit of integration was 18 min (i.e. at $\mu_{ep}$ = -24.8 TU corresponding to DP between 1 and 2) and upper limit was 38 min (i.e. $\mu_{ep}$ = -38.5 TU corresponding to DP = 190). Insert: Calibration curve in DP.</figcaption></figure>

For $DP>3$, equation is $DP = 1.4937830628 \cdot 10^{51} \times \mu_{ep}^{6} + 2.7630476617 \cdot 10^{44} \times \mu_{ep}^{5} + 2.1264532838 \cdot 10^{37} \times \mu_{ep}^{4} + 8.7149695124 \cdot 10^{29} \times \mu_{ep}^{3} + 2.0059180132 \cdot 10^{22} \times \mu_{ep}^{2} + 2.4583723562 \cdot 10^{14} \times \mu_{ep} + 1.2532431350 \cdot 10^{6}$. For $2<DP<3$, equation is $DP = -4.5328860886 \cdot 10^{8} \times \mu_{ep} - 9.9265219165$.

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**Table 1.** Physicochemical characteristics of oligosaccharides, in terms of DP<sub>w</sub>, DP<sub>n</sub>, polydispersity index PDI, and concentration in the 5 lots. See Figure 4 for experimental conditions. * N-acetyl neuraminic acid (NeuAc) used as standard (RSD on n=3 experiments). § HPAEC-PAD determination (RSD on n=2 experiments).

<table><thead><tr><th>Lot</th><th>DP<sub>w</sub></th><th>DP<sub>n</sub></th><th>&lt;PDI&gt;</th><th>[oligoS]<sup>*</sup> (mg.L<sup>-1</sup>)</th><th>[oligoS]<sup>§</sup> (mg.L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>#1</td><td>45 ± 3</td><td>30 ± 1</td><td>1.5</td><td>602 ± 23</td><td>624 ± 5</td></tr><tr><td>#2</td><td>66 ± 4</td><td>56 ± 2</td><td>1.2</td><td>173 ± 9</td><td>139 ± 20</td></tr><tr><td>#3</td><td>71 ± 5</td><td>59 ± 2</td><td>1.2</td><td>83 ± 6</td><td>109 ± 1</td></tr><tr><td>#4</td><td>72 ± 5</td><td>62 ± 2</td><td>1.2</td><td>92 ± 6</td><td>100 ± 14</td></tr><tr><td>#5</td><td>82 ± 6</td><td>69 ± 3</td><td>1.2</td><td>78 ± 5</td><td>64 ± 2</td></tr></tbody></table>

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**Table 2.** Comparison of the proposed MEKC method with the HPAEC-PAD technique described in the literature for the quantification of CapsPS including the limit of quantification (LOQ).

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<table><thead><tr><th>Parameter</th><th>MEKC (this work)</th><th>HPAEC-PAD</th></tr></thead><tbody><tr><td>Quantifies total NeuAc</td><td>Yes</td><td>Yes</td></tr><tr><td>Resolves size distribution</td><td>Yes</td><td>No</td></tr><tr><td>Matrix tolerance (OMV)</td><td>High</td><td>Moderate</td></tr><tr><td>LOQ (mg/L)</td><td>16</td><td>0.25</td></tr><tr><td>Analysis time</td><td>~ 40 min</td><td>~ 45 min</td></tr><tr><td>Sample preparation</td><td>Minimal</td><td>Hydrolysis required</td></tr></tbody></table>

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