Page 15 - Shimadzu Journal vol.9 Issue2

Biopharmaceutical

3-2. Impact of Mobile Phase Salt Concentration this monomer peak improved as the sodium chloride concentra-
Sequential analysis was performed to evaluate the impact of mobile tion was increased from 50 to 250 mmol/L. Peak resolution was
phase salt concentration while automatically adjusting salt concentra- also affected; it showed the best value around 50 and 100 mmol/L
tion by the mobile phase blending function, which is the feature of the sodium chloride (Fig. 3, 4), which contributed to accurate peak in-
method scouting system. Fig. 2 shows a chromatogram obtained using tegration. As a conclusions in this case, the better monomer peak
a 100 mmol/L phosphate buffer (pH 7.0) without sodium chloride and shape and appropriate separation from aggregates were achieved
the same chromatogram expanded to show the area around the mon- with a sodium chloride concentration of 100 mmol/L. Hence a
omer peak in more detail. Fig. 3 also shows the same expanded area in mobile phase sodium chloride concentration of 100 mmol/L was
chromatograms obtained using a 100 mmol/L phosphate buffer (pH chosen for this mAb analytical method.
7.0) containing between 50 and 250 mmol/L of sodium chloride.
3-3. Impact of Flow Rate
Fig. 4 shows the relation between sodium chloride concentration It’s known that the flow rate should be optimized based on
in the mobile phase and the peak symmetry factor and resolution. mobile phase composition, packing material particle size, phys-
2
At 0 mmol/L sodium chloride in the mobile phase (Fig. 2), electro- ical and chemical properties of molecules, and other factors .
static interaction between the column packing material and mAb Fig. 5 and Fig. 6 show the impact of mobile phase flow rate on
caused tailing of the monomer peak; but the symmetry factor of separation for this analysis.

mAU
Symmetry factor Aggregates/Monomer
75 2 1. Aggregates 1.5 2.0
50 2. Monomer
3. Fragments 1.2 1.6
25
0 Symmetry Factor 0.9 1.2 Resolution
0.0 2.5 5.0 7.5 10.0 12.5 min
mAU 0.6 0.8
2
NaCl 2.5 1 3 0.3 0.4
0 mmol/L
0.0 0.0
0 50 100 150 200 250
0.0
5.5 6.0 6.5 7.0 7.5 8.0 8.5 min Sodium Chloride Concentration (mmol/L)
Fig. 4. Relationship between Chromatographic Performance and Sodium
Fig. 2. Chromatograms without Sodium Chloride in Mobile Phase
Chloride Concentration
mAU mAU 1. Aggregates
1 2 2 2. Monomer
2.5 1 3
50 mmol/L 3 0.10 mL/min 2.5 3. Fragments
0.0
0.0 2
1 2 2.5 1 3
2.5 0.15 mL/min
100 mmol/L 3 0.0
2
0.0 1 3
1 2 0.20 mL/min 2.5
2.5
150 mmol/L 3 0.0
2
1 3
0.0 2.5
0.25 mL/min
1 2
2.5 0.0
200 mmol/L 3 2 3
0.0 0.30 mL/min 2.5 1
1 2 0.0
2.5
250 mmol/L 3 1 2 3
0.35 mL/min 2.5
0.0
5.5 6.0 6.5 7.0 7.5 8.0 8.5 min 0.0
Fig. 3. Chromatograms with Sodium Chloride in Mobile Phase 1 2 3
0.40 mL/min 2.5
0.0
0.0 5.0 10.0 15.0 20.0 min
Fig. 5. Chromatograms and Flow Rate

Shimadzu Journal vol.9 Issue2 48