Page 16 - Shimadzu Journal vol.9 Issue2

Biopharmaceutical

As shown in Fig. 6, the resolution between aggregates and mono- mAU
2.5 2 3 1. Aggregates
mer, and between monomer and fragments improved at lower flow pH 5.7 2. Monomer
3. Fragments
rate. Based on these results, a flow rate of 0.25 mL/min was chosen 0.0
as it provides shorter analytical time and appropriate chromato-
2.5 2 3
graphic separation (over 1.5 resolution). pH 6.0 1
0.0
Aggregates/Monomer Monomer/Fragments
2.5 1 2
4.5 3
pH 6.3
0.0
3.6 1 2
Resolution 2.7 pH 6.6 2.5 3
0.0
1.8
0.9 2.5 1 2 3
pH 6.9
0.0 0.0
0.10 0.15 0.20 0.25 0.30 0.35 0.40
Flow rate (mL/min) 2.5 1 2
3
Fig. 6. Relationship between Flow Rate and Peak Resolution pH 7.2
0.0
4.0 5.0 6.0 7.0 8.0 min
Fig. 7. Chromatograms and pH
3-4. Impact of Mobile Phase pH
Due to a large number of dissociable groups on protein molecule,
Symmetry Aggregates/ Monomer/
their charge state and steric structure change depending on pH. Factor Monomer Fragments
Such changes are known to contribute to protein size and the 1.8 3.0
strength of interaction with the column and thereby affect chroma-
1.5 2.5
Symmetry Factor 0.9 1.5
togram peak shapes. 1.2 2.0
Fig. 7 shows chromatograms of mAb obtained with 100 mmol/L 0.6 1.0 Resolution
phosphate buffer containing 100 mmol/L sodium chloride adjusted
0.3 0.5
to different pH. Each mobile phase was automatically prepared
0.0 0.0
with the blending function and provided to the system. The pH 5.7 5.7 6.0 6.3 6.6 6.9 7.2
pH
showed no aggregate and a huge unknown peak after the fragments;
Fig. 8. Relationship between Chroamtographic Performance and pH
meanwhile, the pH 6.0 gave the smaller peaks related to aggregates
and unknown components. Since the isoelectric point (pI) value of
target mAb is larger than the pH level of these two mobile phases,
the impact on the retention deriving from the electrical-charge in- Column
teraction could be more significant in these two conditions.
Shim-pack Bio Diol improves the accuracy of analysis of biophar-
As shown in Fig. 8, mobile phase pH did not significantly affected maceuticals and medium-molecular-weight compounds. Shim-pack
the symmetry factor at pH 6.0 and above, but the resolution be- Bio Diol comes in four pore sizes to accommodate a wide range of
tween aggregates and monomer and between monomer and frag- molecular weights and analysis time can be shortened by the lineup
ments improved with raising pH. The best resolution for the mAb of multiple particle sizes. Refer to the Shimadzu website for details .
3
in this study was obtained at pH 7.2. Proteins normally denature Fig. 9 compares a Shim-pack Bio Diol with another commercially
and recieve damages at highly acidic or alkaline solution. Hence available SEC column installed in the same system (300 mm × 4.6
the effect of pH should be investigated at neutral pH close to the mm I.D., 3 µm, 300 Å). Better separation between monomers and
antibody pI. fragments was achieved using Shim-pack Bio Diol-300.

Shimadzu Journal vol.9 Issue2 49