Table 5.

Extraction, clarification and purification of VisA-anti-CD64 RIT and surrogate fusion protein from pPCPs and plants.

	RIT produced in pPCP		RIT produced in plants		Surrogate produced in plantsc
Process step [-]	Step recovery [%]	Purity [%]	Step recovery [%]	Purity [%]	Step recovery [%]	Purity [%]
Extraction	100.0 ± 0.0	0.4 ± 0.1	100.0 ± 0.0	0.2 ± 0.1	100.0 ± 0	0.5 ± 0.1
Clarificationa	100.0 ± 0.0	0.4 ± 0.1	100.0 ± 0.0	0.2 ± 0.1	46.1 ± 13.6	0.6 ± 0.1
Sterile filtration	90.5 ± 3.6	0.4 ± 0.1	85.5 ± 8.2	0.2 ± 0.1	93.1 ± 5.1	0.6 ± 0.1
Protein L flow-through	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
Protein L elutionb	84.5 ± 5.1	78.1 ± 2.7	92.4 ± 4.6	71.2 ± 6.6	97.2 ± 2.8	85.5 ± 26.2

^aFor RITs, this consisted of a single centrifugation step (4200 × g, 10 min, 10°C), whereas for the surrogate it consisted of a combined bag and depth filtration step. ^bFor pPCPs, elution consisted of a two-step low and high pH elution (pH 2.1/pH 12.3), whereas only the low pH elution step was applied for whole plants. ^cWe used mCherry-anti-CD64 as a surrogate for RITs to reduce the rating from BSL-2 to BSL-1 during process development. Data are presented as means (n ≥ 2 biological replicates) with ± indicating the standard deviation.