Crystallization process
protein biosynthesis
Precipitation and initial purification
Example of laboratory experiment
60 mg/ml human serum albumin from Cohn fraction V. The samples are precipitated or crystallized with similar reagents: ammonium sulphate in phosphate buffer pH 5.8
A Instant amorphous precipitation
C Gradual well controlled crystallization
A C
Samples centrifuged 30 min 2000 rpm The crystal sediment C is much more compact than amorphous precipitate A
Process scale-up
Macrocrystal is specialized in development of complete protein crystallization processes starting directly from crude upstream product.
Initial purification
The crude starting material from the upstream biosynthetic step is partially purified and concentrated. Often as low as 30% protein purity is sufficient for batch process crystallization.
Adjusting protein concentration, reagents, pH, temperature
Conditions for crystallization are adjusted on the basis of phase diagrams. Carefully made phase diagram (crystal solubility) study is prerequisite for process planning.
Crystallization in stirred tank reactor
Protein is crystallized in thermostat tank under gentle propeller stirring. The process may proceed optimally when the reagent is added slowly and temperature is lowered gradually.
Crystal harvesting
Crystals are separated from the mother liquor by centrifugation or filtration. At the end of harvesting procedure the crystals are washed with pure crystallizing reagent solution in order to remove effectively the mother liquor which contains impurities. Single crystallization is usually sufficient for industrial use.
Recrystallization
Recrystallization is often performed in order to achieve superior purity. Crystals are dissolved in buffer which does not contain the specific crystallizing reagent. Thereafter the optimal crystallization conditions are adjusted again and the process is repeated. Recrystallized protein is requested as analytical reagent or pharmaceutical product.
Special notes about crystallization process
Crystals have much better separation properties than amorphous precipitate. In stirred tank process the batch may contain both amorphous precipitate of impurities and pure crystals of the product. In centrifugation the crystals sediment faster than amorphous protein precipitate and will make the most compact pellet. Crystals have good filtration properties and the cake can be washed on the filter plate.
Protein crystallization process for crude starting material
The industrial enzymes are concentrates directly from the filtered fermentation medium. These concentrates are dark brown viscous liquids which contain numerous macromolecular impurities in addition to the specific enzyme product. Macrocrystal has crystallized many enzymes directly from these crude industrial products.
Protein purity is not the primary factor for its successful crystallization. Crystallization is an extremely specific process when looked from the physical point of view. Protein will crystallize when the right physical and chemical conditions are achieved.
Advantages of crystallization
- Volumes of processes are small
- Consumption of chemicals is small
- Equipment is simple
- Scale up is unlimited
- Crystalline state stabilizes proteins in the storage and use