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J Biomol Tech. 2009 December; 20(5): 282–284.
PMCID: PMC2777342

Rapid Screening of Antibody Aggregates and Protein Impurities Using Short Gel Filtration Columns

Abstract

Gel filtration (GF) is an excellent tool to acquire information about sizing (identity), purity, and the multimeric state of a protein of interest. Superdex™ 200 and Superdex 75 are outstanding GF media for such analysis. To speed up analysis and keep sample and buffer consumption at minimum, two prepacked short GF columns have been developed, Superdex™ 200 5/150 GL and Superdex 75 5/150 GL. With lengths of only 15 cm and volume of 3 ml, these columns allow rapid analysis (6–12 min/run) with minimal sample (4–50μl) and buffer consumption.

Purification of antibodies often generates dimers and higher aggregated forms, and during optimization of purification protocols, many samples may need to be analyzed for aggregate content. Gel filtration (GF) provides reliable information about size and the purity of a protein of interest, especially when it is in a multimeric state. GF, however, is often time-consuming, and the analyses become a bottleneck. A new column format—3 ml bed vol and 15 cm long—has been developed, enabling rapid and reliable GF with low sample and buffer consumption, using Superdex™ 75 and Superdex™ 200 media for determination of size and purity status.

RESULTS

Rapid Check of Truncated Recombinant Protein

An affinity-purified recombinant protein of ~17,000 Da MW often contained copurified, truncated protein of ~10,000 Da. A fraction of the purified protein was analyzed on Superdex 75 5/150 GL (Fig. 1). The content of the truncated protein was estimated to be 26%, which was in agreement with the 30% estimated by Superdex 200 10/300 GL (GF).

FIGURE 1
Rapid screening on Superdex 75 5/150 GL of a protein purification fraction containing truncated protein. Column: Superdex 75 5/150 GL; sample: fractions of purified recombinant protein; sample volume: 4 μl; buffer: PBS, pH 7.4; flow rate: 0.3 ...

Rapid Screening of Antibody Purification Conditions

Conditions for a hydrophobic interaction chromatography purification of an antibody were optimized for its dimer and higher aggregate content. Three samples of purified antibody from the optimization work were analyzed on Superdex 200 5/150 GL, and in 45 min, the sample containing the lowest amount of dimer was identified (Fig. 2C). Regulatory demand is a baseline separation of the monomer and the dimer, and thus, the sample with the lowest dimer content was reanalyzed on Superdex 200 10/300 GL, and the dimer content was estimated to be 0.4% (data not shown).

FIGURE 2
Screening dimer content in purified antibody fractions by GF on Superdex 200 5/150 GL. Column: Superdex 200 5/150 GL; sample: fractions of purified, humanized antibody; sample volume: 50 μl; buffer: PBS, pH 7.4, system: ÄKTAexplorer™10. ...

Rapid Purity Check

Two proteins- GAPDH-Strep-tag II and M1Pase-Strep-tag II were purified by affinity chromatography (AC) on Strep Trap™ HP, and the fractions were analyzed on Superdex 75 5/150 GL (GF) and by SDS-PAGE. GAPDH-Strep-tag II was pure and homogenous, according to SDS-PAGE and GF (Fig. 3). The M1Pase-Strep-tag II fraction showed several bands in the SDS-PAGE analysis in nonreducing conditions and was not homogenous in the GF analysis. M1Pase-Strep-tag II is rich in cysteins and probably exists in several forms. Analysis of the protein in reducing conditions resulted in one peak in GF and one band in the SDS-PAGE (data not shown). Similar conclusions could be drawn from the GF and SPS-PAGE analyses, but GF was rapid and more convenient.

FIGURE 3
GF and SDS-PAGE analysis of fractions from purification of GAPDH and M1Pase. The small, late, eluting peaks in the GF chromatograms are buffer components from the StrepTrap™ HP elution buffer. Column: Superdex 75 5/150 GL; sample: fractions of ...

CONCLUSIONS

Superdex 5/150 GL GF columns provided the following:

  • rapid screening of antibody aggregate content in three samples in less than 45 min;
  • estimation of percentage of truncated protein in 12 min;
  • rapid purity check of Strep-tag II fusion proteins giving results similar to SDS-PAGE analysis but more quickly and conveniently.

Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities