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Pulse refolding is a strategy to overcome concentration dependent aggregation, assuming that aggregation is significantly suppressed under diluted conditions. When a typical 2nd or higher order aggregation kinetics is assumed, kinetics over predicted yields at low refolding concentrations. Using GFP as our model protein, we found a transition in aggregation kinetics from 2nd to 1st order when intermediates deplete from 100 to 60 µg/ml. Taking this transition into account, the model can better predict refolding yields in batch and pulse refolding strategies. This model is suited for the design of refolding processes since this deviation from 2nd or higher order aggregation was also previously observed in other proteins.

The separation of C3-carbohydrates (DL-glyceraldehyde and dihydroxyacetone) present after the aldol-condensation of formaldehyde and glycolaldehyde was examined using the membrane processes nanofiltration (NF) and reverse osmosis (RO), which exemplifies the crucial problem area of the retention of small organic compounds. Two RO- and one NF-membrane were tested in order to obtain two fractions, namely a C3-enriched and a fraction containing mainly formaldehyde, methanol, and formic acid for a subsequently repeated aldol-condensation. In the case of a RO-process, operated until a volumetric concentration factor of 4 and two sequenced diafiltration steps, approximately 80% of formaldehyde, methanol, and formic acid were removed. Meanwhile, only about 40% of C3-carbohydrates were found in the permeate.