Protein crystallization seeding techniques

Carsten Jacobsen (Novo Nordisk) presented results on protein crystallization in preclarified, concentrated fermentation broths. In particular, the impact of filtration rate on the formation of favorable large diamond versus rod shapes was examined. By adding seed crystals just above the solubility curve, where no nucleation occurred, the authors were able to produce 30% larger crystals as compared to an unseeded crystallization. Although there was minimal recovery and characterization data, this technique may prove very beneficial for dealing with difficult feed streams. While the work presented in this talk was done at the laboratory scale, scale-up experiments will be required to confirm the suitability of this approach for industrial process applications. 

Thaller, C., Weaver, L. H., Eichele, G., Wilson, E., Karlsson, R., and Jansonius, J. N. 1981. Repeated seeding technique for growing large single crystals of proteins. J. Mol. Biol. 147 465-469. 

Apart from the seed zone method, a conventional seeding technique can also be used for preparing microcrystals for inhalation. This method involves the creation of seeds by suspending protein particles in a suitable buffer solution and centrifuging this suspension at about 10,000rpm for about lOmin. The supernatant can then be stored at 4°C, to be used as a seed solution. Protein particles can then be dissolved in another buffer solution but at a much lower pH to facilitate the dissolution of the protein. The amount of protein is then slowly increased to achieve supersaturation. The solution is then filtered and the initially prepared seed solution is added to the filtrate. The mixture is sealed and incubated at 37°C. The only issue with this conventional seeding method is that crystals above 5 pm could be formed, which are not suitable for pulmonary delivery (see Note 2). 

In cases where protein microcrystals are available, seeding techniques can be utilized to increase the size of the crystal to a range acceptable for X-ray diffraction studies. An excellent review of seeding techniques typically employed for protein crystals is presented by Stura and Wilson (1992). 

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