Micronization of Proteins by Jet Milling
Micronization of Proteins by Jet Milling
复制标题
通过喷射研磨对蛋白质进行微粉化
DOI：
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
A. Göpferich
中科院分区：
文献类型：
--
作者：
A. Ehmer;Stefan Kolb;J. Teßmar;T. Blunk;A. Göpferich
本文的目的是提供更多关于蛋白质射流铣削的信息，确定重要参数及其对所研究蛋白质的粒径、化学稳定性和生物活性的影响。
The aim of this thesis was to provide more information about the jet milling of proteins, to identify important parameters and their effect on resulting particle size, chemical stability and bioactivity of the investigated proteins.
To perform the planned micronization experiments the MC One® of Jetpharma had to be modified. A cryogenic cooling system had been established facilitating milling gas temperatures of about -60°C in reproducible quality. To exclude possible effects of air humidity the whole setup was integrated into a glovebox filled with dry nitrogen. Therefore, it was possible to process amounts starting from 200 mg with yields of more than 80 % under constant environmental conditions. This way it was possible to attribute all the observed effects to controlled parameters (chapter 3).
The micronization of three different model proteins proved the size reduction potential of jet milling. It was possible to achieve narrow particle size distributions with d90 values below 10 µm for each of the three proteins. Chosen pressure and number of milling cycles had a significant impact on the milling results. For the first cycle the size distribution of the bulk material played an important role. Especially for the coarse BSA powder some larger particles were still present after the first milling cycle. A grinding limit existed for all model proteins at about 3-4 µm (d90), implicating that additional milling cycles would have no further effect on the particle size. The effect of the third investigated parameter, the milling gas temperature, was different for the proteins. No effect was detected for insulin; for lysozyme the particle size increased slightly under cryogenic conditions, while a clear decrease was detected for BSA. By applying statistical design it was possible to fit mathematical models to all the three micronization processes, describing the qualitative and quantitative impact of the parameters on the resulting particle size. So the prediction of milling results of not tested parameter combinations within the investigated range was enabled (chapter 4).
It was the second focus of the thesis was to investigate the effect of the jet milling process on the chemical stability and the bioactivity of proteins. For insulin no chemical changes were observed. The analysis of secondary and tertiary structures revealed slight changes. Nevertheless, they seemed to have no effect on the bioactivity investigated, using a chondrocyte proliferation assay (chapter 5).
After micronization chemically no changes were detected for lysozyme compared to the unprocessed bulk material. The analysis of the secondary protein structure by CD-spectroscopy showed no difference either. Regarding the tertiary structure fluorescence analysis revealed significant changes. Especially the relative surface hydrophobicity was affected by a 12-fold increase after three milling cycles at 14 bar. Due to these changes the bioactivity decreased depending on applied pressure and milling cycles. For three cycles at 14 bar 87 % of the former activity were measured. In particular repeated milling seemed to have a negative effect. The milling gas temperature had no significant effect. An influence of increased iron content due to attrition during the milling process was excluded by ICP measurements. The attempt of reducing the decrease in bioactivity by using crystalline lysozyme brought no advantage for the process (chapter 6).
For BSA the most prominent effect of the micronization process was the formation of an insoluble fraction of the protein. Experiments revealed that all the three investigated milling parameters affected this result. With higher pressure and more milling cycles the insoluble fraction increased to 14 %. By applying cryogenic conditions the amount had been decreased to 6-9 %. This indicates that heat creation on the particle surface may play a role in this process, inducing changes within the particles. Different possible reasons for this phenomenon were investigated. Covalent protein aggregates due to thiol-disulfide interchange could be excluded as a reason as well as oxidation processes of this free thiol group. Increased surface hydrophobicity by rearrangement of fatty acids present at the molecule surface could be neglected by micronizing fatty acid free BSA. However, changes in relative surface hydrophobicity were detected by fluorescence analysis. The micronization process resulted in a significant decrease of this value while the intrinsic fluorescence was not affected. A correlation to the insoluble BSA fraction was not possible. Attempts to analyse the insoluble fraction itself brought no results. Nevertheless, beside the cryomilling approach of reducing this fraction, the co-lyophilization of BSA with PEG showed good results. With increasing amounts of PEG in the mixture the insoluble fraction decreased. At a BSA PEG ratio of 2:1 no insoluble BSA could be detected anymore. This effect was referred to the separation of the BSA molecules by PEG, preventing intermolecular reactions, and of the hydrophilic properties of PEG (chapter 7).
Processing of glycerol tripalmitate revealed that it is possible to micronize even such a soft material by jet milling. It resulted in d90 values below 20 µm. During storage an increase in particle size had been observed due to changes in surface structure of the micronized particles. DSC analysis revealed that the micronization process induces an activation at the surface. During storage these activated areas recrystallized. It was possible to correlate this effect to the number of milling cycles and the milling gas temperature. With more milling cycles the effect was more prominent, which was significantly reduced by applying cryogenic conditions (chapter 8).
In conclusion the micronization process of proteins by jet milling was clarified in more detail. Particle size reduction was possible for all the tested substances, but different effects on chemical stability and bioactivity were revealed, strongly depending on the investigated substance. Especially repeated stressing of the materials seemed to have a negative effect and should be avoided for sensitive substances. Some effects like the formation of insoluble fractions for BSA or structural changes in glycerol tripalmitate were induced by heat formation at the breakage surface. In these cases cryomilling had a protective effect and it may be worth the additional costs. Therefore, for each new substance the milling conditions should be tested carefully. Application of design of experiments for these investigations proved to be a useful and cost saving tool.
影响因子：
2.9
作者：
KIM, YJ;SAH, RLY;GRODZINSKY, AJ
通讯作者：
GRODZINSKY, AJ