Frankaer Christian G, Moroz Olga V, Turkenburg Johan P, Aspmo Stein I, Thymark Majbritt, Friis Esben P, Stahl Kenny, Nielsen Jens E, Wilson Keith S, Harris Pernille
Department of Chemistry, Technical University of Denmark, Kemitorvet 207, DK-2800 Kgs. Lyngby, Denmark.
Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5DD, England.
Acta Crystallogr D Biol Crystallogr. 2014 Apr;70(Pt 4):1115-23. doi: 10.1107/S1399004714001497. Epub 2014 Mar 21.
A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Å and a = 52.72, b = 57.13, c = 65.86 Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Å with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.
对工业大规模生产过程中产生的迟缓芽孢杆菌枯草杆菌蛋白酶（诺维信435）微晶悬浮液进行了X射线粉末衍射（XRPD）和X射线单晶衍射（MX）分析。XRPD确定，代表整个生产悬浮液的大量微晶样品属于空间群P212121，晶胞参数a = 47.65、b = 62.43、c = 75.74 Å，与已知的正交晶型（PDB条目1ndq）相同。随后，使用钻石光源的同步加速器光束线（光束尺寸为20×20 µm）的MX来研究悬浮液中存在的最大晶体，从两颗单晶（约20×20×60 µm）收集衍射数据，分辨率分别为1.40和1.57 Å。这两种结构也都属于空间群P2(1)2(1)2(1)，但与XRPD鉴定的主要晶型有很大不同，晶胞参数分别为a = 53.04、b = 57.55、c = 71.37 Å和a = 52.72、b = 57.13、c = 65.86 Å，精修后的R值分别为10.8%和Rfree值为(15.5%)，以及R值为14.1%和Rfree值为18.0%。它们也与PDB中先前报道的任何晶型都不同。用高度纯化的诺维信435样品进行的可控结晶实验，得到了XRPD鉴定的晶型的单晶；其结构被解析并精修至分辨率为1.17 Å，R值为9.2%，Rfree值为11.8%。因此，生产悬浮液中至少存在三种多晶型物，尽管以类1ndq微晶为主。展示了这两种技术如何能为这样的生产悬浮液提供宝贵且互补的信息，并提出XRPD为这类悬浮液提供了出色的质量控制工具。
