低温对VHH抗体二级结构影响的分析

FTIR光谱已被广泛用于评估蛋白质如VHH抗体的结构。α-螺旋和β-薄片的二次结构估计（SSE）可以快速而容易地进行。在本申请说明中，我们讨论了FTIR测量与新型冠状病毒（SARS-CoV-2）结合的VHH抗体冷却过程和反复冷冻和解冻引起的二级结构变化的结果。样品三种抗严重急性呼吸系统综合征冠状病毒2型VHH  1号（6.27 mg/mL）  2号（7.22 mg/mL）  3号（5.15 mg/mL）  缓冲液：20mM PBS实验：1：由冷却过程引起的结构变化      使用装有Peltier控制的ATR PRO ONE（水冷式）的FT/IR-6600光谱仪进行测量，温度范围从室温到-10ºC（如本应用中图1）。对于1号样品，在从室温到略高于冰点的温度范围内测量IR光谱，并估计二级结构。          2：反复冻融过程中的结构变化结论：研究发现，本实验中使用的VHH抗体在冷却过程中以及冷冻和解冻过程中都不会显著改变其二级结构，显示出抗冷却的高结构稳定性。我们相信，类似的系统不仅可以用于抗体药物对抗环境变化的稳定性，还可以用于其他评估，例如与其他成分的反应性。Application Note200-AT-0267 Evaluation of HOS identity and VHH antibody binding tocoronavirus using circular dichroism spectroscopy2/4Application Note Analysis of the effects of low temperature on the secondary structure of VHH antibodies In t rod u ction VHH (var i able domain h eavy/heavy cha in antibodies ， lack in g i n l i g h t chains ) sing l e domain ant i bodies have h igh aff in ity，stability， product i vity， and processability for antigens . These ant i bod i es will highly l i kely offer a next-ge n eration drug d i scovery modality. The storage environment may correspond to a loss of act i v i ty i f the structure of a therapeu t ic antibody changes with temperature or freeze/thaw cyc l es. Therefo r e， it is essential to evaluate the structural stabil i ty in order to maintain quality control . Antibody based drugs are typical l y stored in a refrigerated or frozen state， but t h is may lead to denaturat i o n f r om changes i n tempe r ature or r epeated freezing a n d thawing F T IR spectroscopy has been wide l y used to evaluate the structu r e of prote in s such as VHH an t i bodies . Seconda r y structure estimation (SSE) of a-Hel i ces and B-Sheets can be performed qu i c k ly and easily. I n th i s application note we d i scuss the r esu l ts of FTIR measurements of seco n dary st r ucture cha n ges caused by t h e cooling process and repeated freezing and thawing of a VHH antibody that bi n ds to the novel co r onavirus (SARS-CoV-2). Samples T h r e e Speci e s of Ant i SARS-CoV-2 VHH*1 No.1 (6.27 mg/mL) No.2 (7.22 mg/mL) No.3 (5.15 mg/mL) Buffe e r r：：20mM PBS Keywords New corona virus， SARS-CoV-2， COVID-19， Biopharmaceu t icals， Antibody drug， BioSimilar， VHH antibody， HOS， Secondary Structure， Tertiary Structu r e E x perimen t al Structural change caused by the cooling process An FT/IR-6600 spectrometer f itted with a Peltier controlled ATR PRO ONE (water-cooled) wit h a tempe r ature range from r oom tempe r ature to -10 °℃ was used for the measurement (Figure 1). For sample No.1， IR spectra were measured at a r ange of tempe r atures from room temperature to just above freezing ， and the secondary structure was estimated. Measurement Conditions Main Unit FT/IR-6600 Accumulation 1 minute Attachment ATR PRO ONE withcooling option Filter Band path filter(<2000 cm-) Detector Mid-band MCT Purging Gas N2 Resolution 4cm1 Fi gure 2 s h ows th e percentage of a-Hel i x and B-Sheet structures at each temperature. Th e VHH a n tibody (sample No.1)i s considered to have high sta bili ty during cooling ， because the secondary structure does n ot cha n ge signi f icantly d u r i ng the cooling process. Figure 2. Chan g es i n sec ond ary str u c t ure o f VHH an t ibody No. 1 d ur i n g t he co o l in g p r o cess Structural changes during repeated freez i ng and thawing Sampl e s Nos. 1， 2 and 3 we r e f ro z en to -25°C， t hawed ， and m e a su r e d at room t e mpera tu re. Fr e ez e /thawing was r e p eat e d 3 t i mes. The f reezing t ime wa s set to 2 h ours for the f irst cycle and 90 hours f or t h e seco n d and third cycles.In order to measure a small amount of sample wi th high se n sit i vi t y， a pentagonal prism mu l t i-ref l ectio n ATR (ATR PRO PENTA)*2 was us e d (F i g u re 3.). Measurement Conditions Spectrometer FT/IR-6600 Accumulation 1 minute Accessory ATR PRO PENTA Sample Volume Several uL Detector MCT (PV type) Purging Gas Not used Resolution 4cm² JASCO INC. Figure 4 shows the difference spectrum between the sample and the buffer before freezing. The Amide l and Amide l l bands were con f irmed clearly even i n a small amount (a few pL) of di l uted aqueous protein solution. Figure 3. ATR P R O PE N TA Figure 4. Differ e nc e s pec t r um b e t w ee n t he VH H ant i bod y an d b u f f er Figur e 5 shows the SSE r e su l t u sing t h e ATR cor re ct e d dif fe r e nce spectrum. Numerical val u es in t h e f ig u r e ar e mean val u es for n =3 an d e rro r b a r s are standa r d d e v i at i o n s. Th e s e co n dary structur e s of t he th ree VHH anti b ody sam p l e s us e d h e r e were sim i lar ， and it was con fi r m ed t h at the secondary structure of all th r ee VHH ant i bod i es did not change s i g n if i cantly after th r ee f r e eze/t h aw cyc l es. Figure 5. Ch a nge s i n secon d ary s t r u ct u r e o f VHH anti b ody b y r epeated f r e ezing a nd t ha w in g Conclusion It was found that the VHH ant i body used in th i s exper i ment does not sign i fica n tly change i ts seconda r y str u cture during the cooling p rocess nor freezi n g and thawing， demons t rating h i g h str u ctural stability against cooling. We believe t h at similar systems can be u sed not only for stability of ant i bod y dr u gs agai n st environmental changes bu t a l so for other evaluations， suc h as react i vi t y wi th othe r compo n ents. *1： VHH antibody was kind l y provided by RePHAGEN https：//rephagen.com/ *2： Application note 140-AT-0263 ‘Prote i n secondary structure analysis i n low concentration aqueous solut i ons Appl i c a t i on Note System Configuration [Structural c h ang e duri n g t h e cooling process] Model Description Part Number Main Unit FT/IR-6600 FT/IR Spectrometer 7085-J002A*) Detector MCT-6000M Mid-band MCT detector withswitching mechanism 6888-J714A*2) Attachment ATR PRO ONE with coolingoption *) Software IR SSE-4000 Secondary structure estimationprogram 4880-7234A *1) T h e FT/IR-4600/4700 a n d The FT/IR-6700/6800 can a lso be used. *2) Choose the MCT -4000M with t h e F T/IR-4600/4700. *3) A cu s tom software package. [Str u ctural c h ange during r epeated freezing and t hawing] Model Description Part Number Main Unit FT/IR-6600 FT/IR Spectrometer 7085-J002A*) Detector MCT-6000PV MCT(PV) detector 6584-J345A*5) Attachment ATR PRO PENTA Multi reflection ATR 6584-J343A Software IR SSE-4000 Secondary structure estimationprogram 4880-7234A *4) The FT/IR-4600/4700 and The FT/IR-6700/6800 can also be used. *5) Choose the MCT-4000M with the FT/IR-4600/4700. JASCO INC. 28600 Mary's C o ur t ， E asto n， MD 21601US A T el ： (800) 333-5272 F ax： (410) 822-7526 A ppl ic a tio n Lib r ary： ja s c o i n c.com /appli c a t i o ns