With fVi avMW 43 kDa, the following procedure was used to prepare conjugates. Polysaccharide was solubilized in 100 mM MES pH 6 at a concentration of 50 mg/mL. NHS and then EDAC were added to have 0.33 M NHS and EDAC/Vi repeating units molar ratio of 5. After the reaction was mixed at room temperature for 1h, the protein previously derivatized with ADH [10 (link), 21 (link)], was added to give a Vi concentration of 7.8 mg/mL in 20 mM MES, pH 6 and mixed at room temperature for 2h. For full length Vi, the PS concentration in the EDAC/NHS activation step was reduced to 4.2 mg/mL and to 1.7–3.5 mg/mL in the conjugation step in order to avoid gel formation. Different ratios of Vi to protein were used: 1:1, 2:1 or 1:2 in weight.
Full-length Vi-CRM197 conjugates were purified by tangential flow filtration by using a 300k membrane (Sartocon Slice Cassette 200 cm2 PES). Twenty cycles of diafiltration against 1M NaCl 20 mM NaH2PO4 pH 7.2 and subsequently twenty cycles of diafiltration against 20 mM NaH2PO4 pH 7.2 (Pin 2.0 bar; Pout 0.2 bar; permeate flow rate = 30–33 mL/min) were performed. For full-length Vi-DT conjugate purification was performed with a 100k membrane (Hydrosart 200 cm2 in stabilized cellulose). Full-length Vi-TT conjugate and fVi conjugates were purified by size exclusion chromatography on a 1.6 cm x 60 cm Sephacryl S300 column or 1.6 cm x 60 cm Sephacryl S100 HR column respectively [GE Healthcare] eluted at 0.5 mL/min in PBS. Fractions at higher MW that did not overlap free PS and free protein run on the same column in the same conditions were collected.
Activated Vi (with EDAC/NHS) was not isolated before protein addition, but a fraction of the mixture was sampled in process and characterized for quantifying the % of activated Vi repeating units (molar ratio % of NHS/Vi repeating units). The sample was desalted by PD10 column (SephadexTM G-25M, GE Healthcare) against HCl 55 ppm and analyzed by ion pair HPLC-RP for NHS quantification and by HPAEC-PAD for Vi PS quantification. For quantification of NHS ester groups introduced on Vi PS, samples were eluted on a C18 column (Phenomenex, Gemini-NX 5 μ) with 80% 10 mM TBABr, 0.17% NH4OH, 20% ACN in isocratic condition with a flow rate of 1 mL/min. Eluent pH allowed ester-NHS groups hydrolysis and formation of N-hydroxysuccinimidate anion that was detected at 260 nm eluted as ion pair with TBA. Calibration curve was built using NHS as standard in the range 3–50 nmol/mL.
Full-length Vi-CRM197 conjugates were purified by tangential flow filtration by using a 300k membrane (Sartocon Slice Cassette 200 cm2 PES). Twenty cycles of diafiltration against 1M NaCl 20 mM NaH2PO4 pH 7.2 and subsequently twenty cycles of diafiltration against 20 mM NaH2PO4 pH 7.2 (Pin 2.0 bar; Pout 0.2 bar; permeate flow rate = 30–33 mL/min) were performed. For full-length Vi-DT conjugate purification was performed with a 100k membrane (Hydrosart 200 cm2 in stabilized cellulose). Full-length Vi-TT conjugate and fVi conjugates were purified by size exclusion chromatography on a 1.6 cm x 60 cm Sephacryl S300 column or 1.6 cm x 60 cm Sephacryl S100 HR column respectively [GE Healthcare] eluted at 0.5 mL/min in PBS. Fractions at higher MW that did not overlap free PS and free protein run on the same column in the same conditions were collected.
Activated Vi (with EDAC/NHS) was not isolated before protein addition, but a fraction of the mixture was sampled in process and characterized for quantifying the % of activated Vi repeating units (molar ratio % of NHS/Vi repeating units). The sample was desalted by PD10 column (SephadexTM G-25M, GE Healthcare) against HCl 55 ppm and analyzed by ion pair HPLC-RP for NHS quantification and by HPAEC-PAD for Vi PS quantification. For quantification of NHS ester groups introduced on Vi PS, samples were eluted on a C18 column (Phenomenex, Gemini-NX 5 μ) with 80% 10 mM TBABr, 0.17% NH4OH, 20% ACN in isocratic condition with a flow rate of 1 mL/min. Eluent pH allowed ester-NHS groups hydrolysis and formation of N-hydroxysuccinimidate anion that was detected at 260 nm eluted as ion pair with TBA. Calibration curve was built using NHS as standard in the range 3–50 nmol/mL.
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