Page 27 - Shimadzu Journal vol.9 Issue2

Biopharmaceutical

Results of SEC Analysis

100 µL of the fraction around the peak top eluted by the affinity
purification as the first step was re-injected and evaluated by the
SEC analysis as the second step. The results are shown in Fig. 5. In
the SEC analysis, a main peak (Peak 2) and a broad peak (Peak 1)
before Peak 2 were detected. To briefly estimate the molecular
weight of the protein from the peaks obtained by SEC analysis,
the mixture of thyroglobulin (669 kDa), aldolase (158 kDa), and
ovalbumin (44 kDa) and the mixture of ferritin (440 kDa) and
conalbumin (75 kDa) were evaluated as standard proteins by SEC
(Fig. 6). The elution times of the protein standard solutions indi- Fig. 5. Chromatogram from SEC Analysis
cate that almost all the purified IgG from human plasma existed
as monomers.

SDS-PAGE Analysis

All fractions from affinity purification and SEC analysis were col-
lected in a 96-well deep well plate. The peak-top fractions were
evaluated by SDS-PAGE analysis (non-reducing and reducing).
The peak-top fractions collected from both affinity purification
and SEC analysis showed bands at the same position as standard
human IgG (Fig. 7). Based on the results, both peaks 1 and 2 in
SEC were IgG. Structurally similar 4 sub-classes of IgG in human
blood could give broadened peak shapes due to the small differ-
ences in molecular size and/or structure. Fig. 6. SEC Chromatograms of Standard Proteins

Conclusion

The liquid handler (LH-40) installed in the LC system provided a
seamless process from purification to analysis and required simply
placing the sample in the liquid handler. For routine work with
prespecified targets, it can be used to analyze only the target frac-
tions. By adding one more column switching valve and increasing
the number of columns, the system can also be used to screen
purification parameters or purify samples in multiple steps. After
fractions are collected in a 96-well plate, they can be used directly
for SDS-PAGE, ELISA, or various other analytical methods. Fig. 7. SDS-PAGE Analysis

Shimadzu Journal vol.9 Issue2 60