*4.16. F(ab')2-Based Antitoxin Production*

Concentrated anti-ricin F(ab')2 preparations were generated from pooled horse hyperimmune antisera by one hour of pepsin (1200 U/mL, pepsin A from porcine stomach mucosa, Sigma, Steinhaim, Germany) cleavage of the Fc fragments at 30 ◦C and pH 3.2. The cleavage step was finalized by increasing the solution pH to 7.4 and reducing the temperature to 18 ◦C. Purification of the F(ab')2 fragments was carried out in several stages: First, the contaminating proteins were precipitated with ammonium sulfate (25% saturation for 20 h at 18 ◦C). Then, the sediment that contains contaminating proteins was separated from the suspension that contains the F(ab')2 fragments using crossflow microfiltration cassettes 0.2 μm (Tangenx technology, Shrewsbury, MA, USA). The filtrate of the microfiltration was washed (from small contaminating proteins and peptides), concentrated, and dialyzed (against 50 mM phosphate buffer pH 8) with crossflow ultrafiltration 30KD cassettes (Sartocon Cassette polyethersulfon 30KD, Sartorius, Goettingen, Germany). The dialyzed solution was applied on a Q-sepharose anion-exchange column (Q sepharose fast flow, GE healthcare, Uppsala, Sweden). The F(ab')2 fragments fraction eluted with the flow through was collected, while the contaminating proteins remained bound to the column, which were then regenerated with 1 M NaCl. The flow through solution was adjusted to pH 6 and applied onto a SP-sepharose cation-exchange column (SP sepharose fast flow, GE healthcare, Uppsala, Sweden). The F(ab')2 fragments fraction was eluted with the flow through and were collected, while, the contaminating proteins remained bound to the column, which were then regenerated with 1 M NaCl. All the chromatographic processes were carried out using an AKTA Process Instrument (GE healthcare, Uppsala, Sweden). Finally, under grade A conditions, the antitoxin solution was concentrated and dialyzed (against 300 mM glycine buffer, pH 7.4), and filtered with nanofilter (Kleenpak-Nova, PALL life science, MI, USA).
