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. 2022 Mar 17;8(4):449–460. doi: 10.1021/acscentsci.1c01479

Table 1. Impurity Considerations during Scale-up of the TT Modification and Conjugation Steps in the Absence of Oligosaccharide 2 and TT.

impurity guideline for GMP maximum allowed quantity in the formulated vaccine (μg/dose or μg/0.5 mL unless indicated otherwise) maximum allowed quantity in the bulk vaccinea (mM) estimated maximum concentration in the bulk vaccine (mM)
GMBS/GMBA ICH-M7 12 <9 15.2b
N-hydroxysuccinimide ICH-M7 12 <9 15.2b
DMSO ICH-Q3D 5000 ppm <6400c 761d
hydroxylamine·HCl ICH-M7draft status 2 <12 37.4e
acetohydroxamic acidf n.a. 72 <73 2.5g
EDTAh ICH-M7 12 <6 5h
cysteamine·HCli n.a. 200 <518 14.4j
a

The bulk SF2a-TT15 is ≥100 times more concentrated than the formulated vaccine candidate.

b

N-Hydroxysuccinimide is a byproduct resulting from GMBS coupling to TT or from its hydrolysis into GMBA. Here, we assume 100% formation of byproduct.

c

2019 specification for DMSO is 6400 mM, which corresponds to 5000 ppm per vaccine dose.

d

[(volume DMSO mL/total volume mL) × 1000 = g/L]/Mw (DMSO) g/mol thus (6.33/106.33) × 1000/78.13.

e

(0.26 g/100 mL) × 1000 mL/Mw: 69.49 g/mol.

f

Acetohydroxamic acid is used as a treatment for bladder infections. A normal dose is 12 mg/kg/day, which translates to 720 mg/day for a 60 kg adult. When applying a minimum 1000-fold decline with respect to process performance and an additional 10-fold safety margin, a maximum amount of 1/10000 of the starting concentration of thiol 3 is acceptable.

g

6.47 mg/mL: same concentration as thiol 3.

h

The concentration of EDTA in conjugation buffer is 5 mM.

i

Cysteamine·HCl is administered as a treatment for nephropathic cystinosis. A daily dose of 2 g/day is accepted. When applying a minimum 1000-fold decline with respect to process performance and an additional 10-fold safety margin, a maximum amount of 1/10000 of initial concentration is acceptable or 200 μg/dose.

j

(180 mg/110 mL total) *1000 mL/Mw: 113.61 g/mol. ICH: International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use by their flow paths, LP screen and open channel, respectively. GMBA: 4-maleimidobutyric acid.