Minibodies and Multimodal Chromatography Methods: A Convergence of Challenge and Opportunity

Abstract

This case study describes early phase purification process development for a recombinant anticancer minibody produced in mammalian cell culture. The minibody did not bind to protein A. Cation-exchange, anion-exchange, hydrophobic-interaction, and hydroxyapatite (eluted by phosphate gradient) chromatographic methods were scouted, but the minibody coeluted with BSA to a substantial degree on each. Hydroxyapatite eluted with a sodium chloride gradient separated BSA and also removed a dimeric contaminant, but BSA consumed so much binding capacity that this proved impractical as a capture tool. Capto MMC media proved capable of supporting adequate capture and significant dimer removal, although both loading and elution selectivity varied dramatically with the amount of supernatant applied to the column. An anion-exchange step was included to fortify overall virus and DNA removal. These results illustrate the value of multimodal chromatography methods when affinity chromatography methods are lacking and conventional alternatives prove inadequate.

Figure 1

Coregistered microPET/CT scans of mice bearing LAPC-9 or Capan-1 xenografts, 21 hours after injection of 124I-labeled A11 minibody (100–150 μCi); LAPC-9 is an androgen-dependent human prostate cancer cell line, and Capan-1 is a human pancreatic cancer cell line. Tumor locations are indicated by yellow arrows. Response scale is red (high) to blue (low).

Figure 2

Structural comparison of a human IgG1 and an anti-PSCA minibody; references 2, 28, and 29 provide more information about the minibody's development, structure, characterization, and applications.

Figure 3

Nonreduced SDS-PAGE of HA scouting with a phosphate gradient; AG indicates supernatant after conditioning with AG1×8, FT flow-through, TRF transferrin, and numbers are elution fractions throughout the gradient.

Figure 4

HA fractionation of minibody supernatant with NaCl gradient; chromatogram and nonreduced SDS-PAGE; gray area approximates minibody distribution; abbreviations are as in Figure 2. Most of the minibody elutes in fraction 23 relatively free of BSA and TRF.

Figure 5

HA fractionation of minibody and dimer following MMC and AX

Figure 6

Column loading's effect on purification performance of MMC; nonreduced SDS-PAGE; note the difference between the ratio of minibody to BSA in EL1 with the larger load (left) and the ratio with the smaller load (right, EL). FT refers to flow-through fractions, EL to elution fractions; other abbreviations are as in Figure 3.

Figure 7

Dimer and aggregate distribution in MMC fractions; (left) a Superdex 75 profile of the MMC eluate with dimer peak at 16.51 minutes, higher aggregates at 14.59 minutes; (right) the SEC profile of a 1 M NaCl strip dominated by dimer and aggregates. Note that the minibody apparently coelutes on center with contaminating BSA at about 18.2 minutes. Compare with Figure 9.

Figure 8

SDS-PAGE of purification process stages

Figure 9

Dimer and aggregate removal by HA (chromatogram in Figure 4); the first HA peak is essentially all nonaggregated minibody; the second is dominated by dimers and aggregates. Peak 2 was concentrated before application to SEC. Compare with Figure 7; refer to Table 2 for relative amounts of minibody in the respective fractions.