利用高通量圆二色性系统测量短路径样品池的再现性

二级和三级结构的评估在基于蛋白质和肽的生物制品的质量测试中是重要的。CD对生物分子不对称性的敏感性是药物稳定性和加工研究的理想选择，在这些研究中，分子或其环境的微小变化也会导致结构变化，改变其功能。虽然已知CD测量快速简单，但高通量系统显著增加了从两个96孔微孔板进行自动测量所获得的数据量。由于CD是一种基于Beer’s Law的吸收技术，因此样品浓度和路径长度对于获得良好的CD数据至关重要。强吸收样品和/或缓冲液可以通过降低浓度或样品池路径长度来测量。然而，对于某些样品，不能修改工作浓度。0.2mm的路径长度池允许用高通量CD系统在远紫外线中精确且可重复地测量强吸收样品。结果：显示了184个BSA样品的远紫外CD光谱。说明了HTCD系统在192.5、208和210nm下使用0.2mm路径长度电池的测量再现性。Application Note Measurement Reproducibility of a Short Pathlength Cellusing the High-Throughput Circular Dichroism System2/3Application Note Measurement Reproducibility of a Short Pathlength Cel l using the High-Throughput Circular Dichroism System Introd uc ti on T h e e val u atio n o f s e conda r y a n d tert i ary structur e i s im p o r t an t i n th e qual i ty testing of p rotein and pe p tide based b iologics . CD’s sensit i vity to a biomolec ul e's asymmetry i s i dea l for pharmaceutical stabil i ty and processing studies， where eve n slight c h anges t o the molecule or its e n viro n me n t can i n d u ce structur a l changes， a lter i n g it s functio n . Wh i le CD measur e me n ts ar e known to be quick and e asy， the high -thr oughput system HTCD drama ti cal l y i nc r easeAs t h e amount of data utom at ed Hig h -T h roughp u t CDobtained wi t h automated measuremen t s f r om two， 96-wel l mic r oplates. Si n ce CD is an absorptio n t echnique b ased on Beer 's Law， t h e sam p le co n ce ntr atio n and pat h length are c r itical t o obtaining good CD d a ta . St r ongly a b sorbi n g sam p l e s and/or b uff e rs can b e m e asur e d b y ei t he r d e cr e a s ing th e concen t ratio n or cell pathlength. Howeve r ， for some samp l es the working concentrat i on ca n not be modif i ed. The 0.2mm pathle n gt h cell allows for strongly absorbi n g samples to be accurate l y and reproduc i bly m easured in the f ar-UV with the high-throug h pu t CD system . T h is a ppl i c a tio n n ote demon s t ra tes t h e me a surement reproducibi l ity of the 0.2 mm pathlength ce l l usi n g the HTCD syst e m . Keywo r ds High -t h ro u g h put C ir c ul a r Di c hr o i sm (H TC D )， Pha rmaceuti ca l s ， B ioc h e mi s t ry E xperimen t al Data Pitch 0.1nm D.I.T. 4 sec Bandwidth 1nm Scanning speed 100 nm/min Accumulations 3 Concentration 0.5 mg/mL Syringe Pump Settings Wash Settings 1st Air Volume 200 pL Wash 1 200 uL x 2 times (0.5%detergent) Sample Volume 100 uL Wash 2 200 uL x 2 times (Water) Waiting Time 5 sec Wash 3 300 uL x 2 times (Ethanol) Syringe Speed for Load/ Medium Dry Time 120 sec Drain Syringe Speed for Flush Medium 184 a liquots of a 0.5 mg/mL BSA (b ovi n e se r um a lbumi n ) solution we r e dis tr ibuted i n the sample wells ma r ked by t h e gr e e n S i n Figur e 1. Th e orange B's d e signat e th e we l ls us e d t o o b tai n b aselin e measur e ments of wate r . Figure 1. Th e ra ck d isplay f i eld in d i ca te s th e p lac em en t of sa mp l es an d b asel in es on th e t w o m i cro p lat e s . Results Th e far -UV CD spec tr a of the 184 BSA sa m pl e s ar e shown i n Figure 2. Tabl e s 1-3 illustr a te t h e m e as u r e ment reproducib i l i ty of the HTCD syste m at 192.5， 208， and 210 nm， using a 0.2 mm pat h length cell . Figure 2. F a r -U V s pe c tr a of 184 b o v in e ser u m a l b u m i n s a mp les. Table 1. H TCD m eas u r e m e n t r e p ro d ucib il it y a t 192.5 n m. Samples 1-23 24-46 47-69 70-92 93-115 116-138 139-161 162-184 1-184 Average 34.60357 34.741 34.52855 34.11767 34.50529 34.1459 34.50052 34.5547 34.58715 Maximum 35.2961 35.7233 34.0444 35.0716 35.2649 35.8348 35.7714 35.1974 35.8348 Minimum 33.443 33.5198 33.9521 33.1163 33.7889 33.983 33.3085 32.4659 32.4659 S.D. 0.416472 0.44273 0.328491 0.418049 0.406204 0.418935 0.467622 0.535832 0.502431 RSD 0.012036 0.012744 0.009514 0.012253 0.011772 0.01192 0.013554 0.015507 0.014527 Table 2. H TCD m e as u r e m e n t re p ro d uc i b i li t y at 208 n m . Samples 1-23 24-46 47-69 70-92 93-115 116-138 139-161 162-184 1-184 Average -19.26734 -19.21747 -19.44108 -19.75571 -19.63827 -19.31892 -19.36812 -19.26523 -19.40902 Maximum -18.9835 -18.4109 -19.204 -19.1672 -19.1612 -18.8735 -18.6892 -18.1287 -18.1287 Minimum -19.4804 -19.7724 -20.1067 -20.4039 -20.3477 -19.8898 -19.7292 -196747 -20.4039 S.D. 0.136446 0.245101 0.20623 0.356229 0.225121 0.233698 0.238288 0.322388 0.307592 RSD -0.007082 -0.012754 -0.010608 -0.018032 -0.011463 -0.012097 -0.012303 -0.016734 -0.015848 Table 3. HTCD mea s ure m en t rep r oduci b il i t y a t 210 nm . Samples 1-23 24-46 47-69 70-92 93-115 116-138 139-161 162-184 1-184 Average -19.98659 -19.87717 -20.09943 -20.48393 -20.33072 -20.00834 -20.16703 -19.97124 20.11556 Maximum -19.7322 -19.0526 -19.8503 -19.9427 -19.8844 -19.497 -19.5355 -18.7035 -18.7035 Minimum -20.2018 -20.4108 -20.6848 -21.248 -21.0578 -21.05693 -20.6122 -20.3677 -21.248 S.D. 0.12906 0.262219 0.193207 0.342251 0.229245 0.24014 0.267663 0.348132 0.319071 RSD -0.006457 -0.01319 -0.009613 -0.016708 -0.011276 -0.01200 -0.013272 -0.017432 -0.015862