HIC
72
Antibody Fragments
Figure 4
shows the separation of Fab and Fc fragments of an antibody
on TSKgel Butyl-NPR. The appearance of additional Fc fragments is due
to the oxidation of methionine residues by 0.10% t-butylhydroperoxide
(tBHP). The numbers above the Fc peaks correspond to the number of
oxidized residues in each fragment.
Antibody Aggregates
The use of a short TSKgel Butyl-NPR column for the separation of a
monoclonal antibody and its high molecular weight aggregates is shown
in
Figure 5
. The total aggregate content of this sample is about 11 %,
which was also confirmed by SEC on TSKgel G3000SW
XL
(5 micron, 7.8
x 300 mm) the current industrial standard for mAb aggregate analysis.
Because of the high efficiency of the nonporous particles of TSKgel
Butyl-NPR only low sample amounts are needed for aggregate analysis.
Antibody Drug Conjugates (ADCs)
ADCs are becoming an increasingly important class of therapeutic
agents for treatment of cancer. One of the most important quality
attributes of an ADC is the drug to antibody ratio (DAR), the average
number of drugs that are conjugated. This determines the amount of
“payload” that can be delivered to the tumor cell.
*
Aditya Wakankar
and others described the analysis of an ADC on TSKgel Butyl NPR that
yielded five predominant peaks that corresponding to mAb containing
zero, two, four, six and eight drugs.
TSKgel Ether-5PW
Antibiotics
The TSKgel Ether-5PW column was used to determine the relative purity of
theantibioticcomponentsC-1027andC-1027-AG(
Figure6
).AntibioticC-1027
is composed of a protein consisting of many hydrophobic and hydroxyamino
acids with a non-protein chromophore. Antibiotic C-1027-AG is composed of
the hydrophobic and hydroxyamino acids without the chromophore.
Applications - TSKgel hic columns
figure 6
Purification of anti-tumor antibiotic
Purificati of anti-tumor antibiotic
Column:
TSKgel Ether-5PW, 7.5mmID x 7.5cm
Sample:
C-1027
C-1027-AG
concentration: 1mg/mL
Injection:
20µL
Elution:
linear gradient from 1.5mol/L to 0mol/L (NH
4
)
2
SO
4
in 0.1mol/L phosphate buffer, pH 7.0
Minutes
0
10
20
5
15
25
0.06
0.04
0.02
0
Absorbance @ 220nm
Column: TSKgel Ether-5PW, 7.5 mm ID x 7.5 cm L; Sample: C-1027, C-1027-AG
concentration: 1 mg/mL; Injection: 20 μL; Elution: linear gradient from
1.5 mol/L to 0 mol/L (NH
4
)
2
SO
4
in 0.1 mol/L phosphate buffer, pH 7.0;
Flow rate: 0.8 mL/min; Detection: UV @ 220 nm
figure 4
Separation of Fab and Fc fragments on TSKgel Butyl-NPR
Separation of F
ab
and F
c
fragments on
TSKgel Butyl-NPR
Column:
Elution:
Gradient:
Flow rate:
Temperature:
TSKgel Butyl-NPR, 4.6mm ID x 3.5cm
Buffer A: 2mol/L (NH
4
)
2
SO
4
, 20mmol/L Tris, pH7
Buffer B: 20mmol/L Tris, pH7
linear from 10%B to 100%B in 34 minutes
1mL/min
30°C
mAU
100
50
0
10 12 14 16
Time (min)
mAU
100
50
0
10 12 14 16
Time (min)
mAU
100
50
0
10 12 14 16
Time (min)
F
ab
peaks
F
c
peaks
1
2
0
C - Incubated with 0.1% tBHP
B - Incubated with 0.01% tBHP
A - Incubated with 0% tBHP (control)
Column: TSKgel Butyl-NPR, 4.6 mm ID x 3.5 cm L; Elution: Buffer A: 2
mol/L (NH
4
)
2
SO
4
, 20 mmol/L Tris, pH 7, Buffer B: 20 mmol/L Tris, pH 7;
Gradient: linear from 10 % B to 100 % B in 34 minutes; Flow rate:1 mL/min;
Temperature: 30°C
figure 5
Analysis of monoclonal antibody and aggregates using a TSKgel
Butyl-NPR column
Column: TSKgel Butyl-NPR, 2.5 µm, 4.6 mm ID × 3.5 cm L,
Mobile phase: A: 3 mol/L NaCl, B: H
2
O, Gradient: 0-100%B in 10 min
Flow rate: 1.0 mL/min, Detection: flourescence, Ex: 280 nm, Em: 348 nm
Injection vol.: 5 µg, Sample: lgG
1
*
Aditya Wakankar et. al. ‘Analytical methods for physicochemical characterization of antibody drug conjugates’, mAbs 3:2, pages 161-172; March/April 2011.
