HPLC-SEC can also be used to estimate the amount of unconjugated protein in conjugate samples.
For example, the area of unreacted CRM 197 can be quantified
with respect to a calibration curve built with CRM 197 samples in the
range 5–50 μg/mL. The percentage of unconjugated CRM 197 is
calculated dividing the amount of free CRM 197 detected by HPLCSEC by the total amount of protein quantified in the sample by
micro BCA.
Table 4
Dilutions for preparing the KDO calibration curve for semicarbazide/HPLC-SEC method
KDO
KDONH 4 40 μg/mL (μL)
Water (μL)
μg/mL
nmol/mL
4
15.7
10
90
8
31.4
20
80
14
54.9
35
65
20
78.4
50
50
40
156.8
100
0
Fig. 5 HPLC-SEC profiles (ABS 252 nm) of OAg and KDO standard after derivatization with semicarbazide (SCA)
and of underivatized OAg (blank). Each sample (80 μL) run on a Tosoh TSK gel 3000 PWXL column; eluent
0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN, pH 7.2; flow rate 0.5 mL/min
288
Francesca Micoli et al.
For example, the area of unreacted CRM 197 can be quantified
with respect to a calibration curve built with CRM 197 samples in the
range 5–50 μg/mL. The percentage of unconjugated CRM 197 is
calculated dividing the amount of free CRM 197 detected by HPLCSEC by the total amount of protein quantified in the sample by
micro BCA.
Table 4
Dilutions for preparing the KDO calibration curve for semicarbazide/HPLC-SEC method
KDO
KDONH 4 40 μg/mL (μL)
Water (μL)
μg/mL
nmol/mL
4
15.7
10
90
8
31.4
20
80
14
54.9
35
65
20
78.4
50
50
40
156.8
100
0
Fig. 5 HPLC-SEC profiles (ABS 252 nm) of OAg and KDO standard after derivatization with semicarbazide (SCA)
and of underivatized OAg (blank). Each sample (80 μL) run on a Tosoh TSK gel 3000 PWXL column; eluent
0.1 M NaCl, 0.1 M NaH 2 PO 4 , 5% ACN, pH 7.2; flow rate 0.5 mL/min
288
Francesca Micoli et al.