A Flexible Antibody Purification Process Based on ReadyToProcess Products with Capto Adhere

Extracted from Multimodal Chromatography (PDF), GE Healthcare, 2013

In this study, a series of experiments was undertaken to determine whether shorter time-to-clinic and cost savings could be achieved using ReadyToProcess products. The work involved scaling up a two-step chromatography purification process from 4.7 mL HiScreen columns (diameter 7.7 mm) to pilot scale using 1 L ReadyToProcess columns (diameter 80 mm). Chromatography was run using ReadyToProcess columns on an ÄKTA ready system, and filtration was performed using ReadyToProcess filters and a fully disposable cross-flow filtration system for ReadyToProcess hollow fiber cartridges. The chromatography steps were performed on the same ÄKTA ready system; only the flow kit was changed between the runs. The process consisted of a capture step on MabSelect SuRe and a polishing step on Capto adhere with a buffer exchange step in between and a formulation step at the end. The buffer-exchanged sample was loaded in one cycle onto a 1 L ReadyToProcess Capto adhere column. The load was 60 g/L. The flowthrough, wash, and elution fractions were collected in one pool. The starting aggregate concentration of 10% was reduced to 0.4% in this single step (Fig 4.28). The monomer yield was 89%, which was judged to be good considering the high aggregate content at the start.

The full series of experiments was able to reduce the HCP concentration from 37 500 ppm to 1.0 ppm (Table 4.4). In addition, the Capto adhere step removed aggregates from a concentration of 10% down to 0.4%, and the protein A ligand leakage was reduced to below the limit of quantitation (LOQ) from 9 ppm. The total yield of the downstream process, including all filtration steps, was 81%.

GF analysis of the MAb in the Capto adhere step

Fig 4.28. GF analysis of the MAb in the Capto adhere step — sample before purification (green), purified fraction (purple), and strip fraction (blue). The curves were normalized with respect to the monomer peak of the purified fraction.

Table 4.4. Summary of monomer yield, aggregate content, and HCP reduction in the scale-up

Process step HCP (ppm) Ligand (ppm) Aggregate content (%) Yield (%)
Fermentation 37 000 Not done 10  
Harvest 37 000 Not done 10 100
MabSelect SuRe (2 cycles) 19 8.8 10 96.01
UF/DF 1 12 9.1 10 97.7
Polishing, Capto adhere < LOQ2 < LOQ2 0.4 89.0
UF/DF 2 & sterile filtration 1.0 0.1 0.4 97.4
Total yield:       81.3

1 Average of 2 cycles.
2
LOQ = level of quantification (4.6 ng/mL for HCP, 3 ng/mL for ligand).

For more information on this example, see application note 28-9403-48, “A flexible antibody purification process based on ReadyToProcess products.

Materials

     
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