3.5 A Case Study:
Nuvia™ cPrime™
and CMM Hypercel™
Resins Nuvia cPrime and CMM Hypercel were superior compared
to the other commercial multimodal resins in terms of achieved
product yield and purity. The chemistry of ligands in both resins
includes hydrophobic (benzene) and cation exchange (carboxylic
acid) groups (Fig. 4). Multiple conditions were evaluated rapidly
(pH, salt, and buffer) and these two resins were selected for scaleup using the A ¨ KTA™ pure (GE Healthcare, Uppsala Sweden).
Initial experiments on the Tecan at 0.6 mL scale resulted in
Nuvia cPrime outperforming CMM Hypercel (Table 7) under
multimodal buffered conditions to purify rotavirus antigen
A. Nevertheless, both antigens (A and B) were purified at smaller
(0.6 mL) and larger scale (>1 mL) in order to assess the reproducibility of the HT method. The deployment of CMM Hypercel at
HT scale is shown in Fig. 5 as an example of method application
(Columns 7 and 8 MMC conditions in Table 3). Further work was
undertaken using rotavirus antigen B and the evaluation of both
resins in order to develop a purification process.
The chromatogram in Fig. 5 shows the purification of rotavirus
antigen A using CMM Hypercel. The employed conditions exploit
the multimodal capability of the resin as the equilibration buffer has
0.8 M ammonium sulfate for hydrophobic binding and a pH of 4 to
induce attraction between negatively charged ligand and positively
charged product. Elution is achieved by reducing the ammonium
sulfate concentration and increasing the pH. This sets a dual gradient (Fig. 5) and resulted in 97% purity and a yield of 49% based on
densitometry (Table 7). The separation between the impurities
Fig. 4 The structure of multimodal resins Nuvia cPrime and CMM Hypercel
Table 7
Purification of rotavirus antigen A using 0.6 mL Nuvia™ cPrime™ and CMM Hypercel™ under MMC
buffered conditions performed on the Tecan EVO
® 200
Resin
Scale (mL) Conditions used to purify Purity (%) Yield (%) OD 260/280
CMM Hypercel™
0.6
MM
97
49
0.59
Nuvia™ cPrime™
0.6
MM
97
56
0.59
HTPD Purification of Viral Antigens
129
Nuvia™ cPrime™
and CMM Hypercel™
Resins Nuvia cPrime and CMM Hypercel were superior compared
to the other commercial multimodal resins in terms of achieved
product yield and purity. The chemistry of ligands in both resins
includes hydrophobic (benzene) and cation exchange (carboxylic
acid) groups (Fig. 4). Multiple conditions were evaluated rapidly
(pH, salt, and buffer) and these two resins were selected for scaleup using the A ¨ KTA™ pure (GE Healthcare, Uppsala Sweden).
Initial experiments on the Tecan at 0.6 mL scale resulted in
Nuvia cPrime outperforming CMM Hypercel (Table 7) under
multimodal buffered conditions to purify rotavirus antigen
A. Nevertheless, both antigens (A and B) were purified at smaller
(0.6 mL) and larger scale (>1 mL) in order to assess the reproducibility of the HT method. The deployment of CMM Hypercel at
HT scale is shown in Fig. 5 as an example of method application
(Columns 7 and 8 MMC conditions in Table 3). Further work was
undertaken using rotavirus antigen B and the evaluation of both
resins in order to develop a purification process.
The chromatogram in Fig. 5 shows the purification of rotavirus
antigen A using CMM Hypercel. The employed conditions exploit
the multimodal capability of the resin as the equilibration buffer has
0.8 M ammonium sulfate for hydrophobic binding and a pH of 4 to
induce attraction between negatively charged ligand and positively
charged product. Elution is achieved by reducing the ammonium
sulfate concentration and increasing the pH. This sets a dual gradient (Fig. 5) and resulted in 97% purity and a yield of 49% based on
densitometry (Table 7). The separation between the impurities
Fig. 4 The structure of multimodal resins Nuvia cPrime and CMM Hypercel
Table 7
Purification of rotavirus antigen A using 0.6 mL Nuvia™ cPrime™ and CMM Hypercel™ under MMC
buffered conditions performed on the Tecan EVO
® 200
Resin
Scale (mL) Conditions used to purify Purity (%) Yield (%) OD 260/280
CMM Hypercel™
0.6
MM
97
49
0.59
Nuvia™ cPrime™
0.6
MM
97
56
0.59
HTPD Purification of Viral Antigens
129