User Manual-ENZ-51023-KP002
Contents
1. covered by US and foreign patents and patents pending Contents VI VII VIII Introduction unsseisdaeteidandeutnddermeieee 1 Reagents Provided and Storage 1 Additional Materials Required 2 Safety Warnings and Precautions 2 Methods and ProceduUres 1 2 A REAGENT PREPARATION ii 2 Bi STAINING aa 3 C CREATING A STANDARD CURVE iii 4 Appendices serre 5 A MICROPLATE SETTING SELECTION 5 B EXPECTED RESULTS uses 5 C REAGENT COMPATABILITY WITH ASSAY i 8 References rrrnnsnsavnnnnnnnnnnnnnnnvnnnnnnnnnnnnnnnnennnnnnnnnnnnner 10 Troubleshooting Guide 11 Introduction Biochemical and biophysical assays for monitoring protein aggregation are often cumbersome relying upon ultracentrifugation size exclusion chromatography gel electrophoresis dynamic light scattering or turbidity measurements None of the above mentioned techniques works well for every protein nor are the assays ideal for tackling the wide range of aggregation problems that can arise during the manufacture of protein pharmaceuticals Enzo Life Sciences ProteoStat Protein Aggregation Assay provides a simple homogenous assay format for monitoring peptide and protein aggregation The assay can be em2. 484 239 E _ info be enzolifesciences com www enzolifesciences com incorporating NTERNA TONAL UK amp IRELAND ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK T 08456011488 UK customers T 44 0 1392 825900 from overseas F 44 0 1392 825910 E info uk enzolifesciences com www enzolifesciences com FRANCE ENZO LIFE SCIENCES c o Covalab s a s 13 Avenue Albert Einstein FR 69100 Villeurbanne France T 33472440655 F 33 437 484 239 E info fr enzolifesciences com www enzolifesciences com ALEXIS assay designs Stressgen
3. are intrinsically low throughput and usually require a long period of time to perform as well as many manual interventions during the evaluation period The development of new formulations is costly in terms of time and resources Moreover even for a known protein formulation batch to batch quality control analysis is often less than optimal using the current state of the art methods Therefore a versatile reliable rapid and resource efficient analytical method is useful for both developing novel protein formulations and identifying protein stability in quality control procedures Protein aggregates are characterized by a cross beta spine quater nary structure In this structure beta strands of stacked beta sheets emanating from different protein monomers are aligned perpendicu lar to the axis of the fibril The resulting quaternary structure consists of a double beta sheet with each sheet formed from parallel seg ments stacked in register Side chains protruding from the two sheets form a dry tightly self complementing steric zipper bonding the sheets together Within each sheet every segment is bound to its two neighboring segments through stacks of both backbone and side chain hydrogen bonds The structure highlights the overall stability of amyloid fibrils their self seeding characteristics and their tendency to form polymorphic structures The ProteoStat Protein Aggregation Assay kit contains a proprietary fluorescent probe
4. D ZE NAA RTRT gt K og og Coes om Cyc oon ra GEIT EIEE OOO Cyc EG OG 0 d QO oo Cocos Cec Go GOOG 7 Enzo Enabling Discovery in Life Science ProteoStat Protein Aggregation Assay for microplates Instruction Manual Cat No ENZ 51023 KP002 for 2 x 96 well plates For research use only Rev 1 5 0 August 2011 Notice to Purchaser This product is manufactured and sold by ENZO LIFE SCIENCES INC for research use only by the end user in the research market and is not intended for diagnostic or therapeutic use Purchase does not include any right or license to use develop or otherwise exploit this product commercially Any commercial use development or exploitation of this product or development using this product without the express prior written authorization of ENZO LIFE SCIENCES INC is strictly prohibited Limited Warranty These products are offered under a limited warranty The products are guaranteed to meet appropriate specifications described in the package insert at the time of shipment Enzo Life Sciences sole obligation is to replace the product to the extent of the purchase price All claims must be made to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo and ProteoStat are trademarks of Enzo Life Sciences Inc Grenier is a registered trademark of Grenier Bio One Several of Enzo s products and product applications are
5. anner by mixing them with detection reagent and using a fluorescence microplate reader to measure the intensity val ues As with any protein assay different protein aggregates will elicit greater or lesser fluorescence intensity response based upon their inherent amino acid composition and sequence It is recommended that a standard curve be prepared each time the assay is performed For best results the protein aggregation standard should always be prepared in the same buffer as the sample The best choice for a standard is a highly purified version of the aggregated protein being analyzed in the test samples If a highly purified version of the protein of interest is not available or if it is too expensive to use as the stan dard the alternative is to choose a protein that will produce a similar fluorescence response curve with the ProteoStat Protein Aggrega tion Assay For example when analyzing IgG aggregation an inexpensive goat anti mouse antibody may be substituted for generat ing the standard curve Once the standard protein is selected it is necessary to generate a fully aggregated form of it This is typically achieved by thermal denaturation as described below 1 Create a fully aggregated standard protein This is typically performed by heating the protein with shaking in a buffer that promotes aggregation of the protein For example an IgG anti body should be diluted to 0 9 mg mL in 100 mM HCI and incu bated at 37 C overnig
6. creening of compounds that promote or inhibit protein aggregation and potentially for the sensitive measurement of molecular chaperone activity Reagents Provided and Storage Reagent Quantity ProteoStat Detection Reagent 22 ul ProteoStat Positive Control Aggregate 300 pg lyophilized lysozyme ProteoStat Negative Control Monomer 300 ug lyophilized lysozyme 10X ProteoStat Assay Buffer 15 mL All reagents are shipped on dry ice Upon receipt the kit should be stored upright and protected from light at lt 20 C When stored properly these reagents are stable for at least twelve months Avoid repeated freezing and thawing The reagents provided in the kit are sufficient for 2 x 96 well microplates III Additional Materials Required e Fluorescence microplate reader with a filter set or monochromator setting of Excitation 550 nm Emission 600 nm e 96 well microplate black wall microplate preferably with a clear bottom e Calibrated adjustable precision pipetters preferably with disposable plastic tips e Deionized water IV Safety Warnings and Precautions e This product is for research use only and is not intended for diagnostic purposes e Some components of this kit may contain hazardous substances Reagents can be harmful if ingested or absorbed through the skin and may cause irritation to the eyes Reagents should be treated as possible mutagens and should be handled with care and disp
7. d protein If desired higher con centrations of protein may be diluted in 1X Assay Buffer or buffer of interest prior to assaying for protein aggregation The final con centration of the ProteoStat detection dye is about 3 uM in the assay Be certain to run negative and positive control samples as well as 1X Assay Buffer alone no protein as a blank sample 3 Incubate the microplate containing test samples in the dark for 15 minutes at room temperature 4 Read generated signal with a fluorescence microplate reader us ing an excitation setting of about 550 nm and an emission filter of about 600 nm NOTE DO NOT wash the sample after incubation with ProteoStat detection dye The fluorescence value of 1X Assay Buffer alone should be subtracted from the values for wells contain ing proteins C CREATING A STANDARD CURVE The ProteoStat Protein Aggregation Assay can be used to deter mine the percentage aggregated protein in a sample by comparing the assay response of a sample to that of a standard whose concen tration is known For convenience Enzo Life Sciences offers pre formulated aggregation standards ENZ 51039 The stabilized high quality reference samples allow easy generation of trace protein ag gregate levels in concentrated monomeric IgG However it is also possible to prepare standards directly in the laboratory Regardless protein samples and protein aggregation standards should be ana lyzed in the same m
8. ee of dust and dirt Protein signal is saturated The concentration from protein sample is too high Dilute the sample further with 1X Assay Buffer High fluorescence signal observed in negative control Sample contains interfering substances The assay is compatible with commonly used buffers PBS Tris HEPES and excipients trehalose and sucrose but not with high concentration of Tween 20 e g gt 0 2 There is no apparent protein in the controls Sometimes the dried pro tein is difficult to see Resuspend the protein as recommended 11 Life Sciences www enzolifesciences com Enabling Discovery in Life Science NORTH SOUTH AMERICA ENZO LIFE SCIENCES INTERNATIONAL INC 10 Executive Boulevard Farmingdale NY 11735 4710 USA T 1 800 942 0430 631 694 7070 F 631 694 7501 E info usa enzolifesciences com www enzolifesciences com SWITZERLAND amp REST OF EUROPE ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 41 0619268989 F 41 0619268979 E info ch enzolifesciences com www enzolifesciences com GERMANY ENZO LIFE SCIENCES GMBH Marie Curie Strasse 8 DE 79539 L rrach Germany T 49 0 7621 5500 526 Toll Free 0800 664 9518 F 49 07621 5500 527 E info de enzolifesciences com www enzolifesciences com BENELUX ENZO LIFE SCIENCES BVBA Frankrijklei 33 BE 2000 Antwerpen Belgium T 32 034660420 F 33 0 437
9. ht 2 Monitor the aggregation process using the ProteoStat dye assay method described in section B at different time points In the 4 example given in Figure 1 different stages are generally observed during the formation of IgG protein aggregates First a lag time is observed during which no aggregates are formed After a certain lag time nucleation occurs and aggregates start to form growth phase Eventually the growth rate decreases and becomes zero indicating a limitation in monomer supply The protein is considered fully aggregated when the fluorescence intensity no longer increases Continued incubation may lead to large insolu ble aggregates For this reason it is best to employ the fully ag gregated protein as soon as it reaches the plateau phase for fur ther use 3 The standard curve is generated from different concentrations of aggregated protein in monomeric protein The protein concentra tion used is typically about 30 uM but this may vary significantly depending upon the needs of the investigator A typical curve can be created using 100 80 40 20 10 5 2 5 1 25 0 6 0 3 and 0 aggregated protein The different solutions of aggregated protein are prepared by mixing different ratios of aggregated protein and monomeric protein keeping the total protein concentration the same For example a 20 aggregated protein can be prepared by adding 10 uL of 100 aggregated protein and 40 uL of monomeric prote
10. in to each well A 10 aggregated protein can be prepared by mixing 5 uL of 100 aggregated protein and 45 uL of monomeric protein in a well See Figure 3 and Table 1 Typically standard curves are constructed using at least three replicates for each point on the curve VI APPENDICES A MICROPLATE SETTING SELECTION The selection of optimal settings for a fluorescence microplate reader application requires matching the monochromator or optical filter specifications to the spectral characteristics of the dyes employed in the analysis Please consult your instrument or filter set manufacturer for assistance in selecting optimal filter sets Pre designed filter sets for Texas Red should work well for this application For monochro mator based detection we recommend a slit width of approximately 9nm Excitation around 550 nm gives slightly higher signal for pro tein aggregates relative to properly folded monomeric protein B EXPECTED RESULTS The formulation of protein drugs is a difficult and time consuming process mainly due to the structural complexity of proteins and the very specific physical and chemical properties they possess Most protein formulations contain excipients added to stabilize protein structure such as a particular buffer system isotonic substances metal ions preservatives and one or more surfactants with various 5 concentration ranges to be tested The conventional instrumentation intensive analytical methods
11. ix interference and compatible with high throughput screening workflows 9000 r 8000 ProteoStat Detection Reagent y 291 2x 0 988 7000 Thioflavin T y 4 56x 6000 R 0 517 5000 RFU 4000 3000 2000 1000 0 5 10 15 20 of Aggregate Figure 3 Effective linear dynamic range for antibody aggregate detection using Proteo Stat Detection Reagent compared with Thioflavin T Relative fluorescence unit values RFUs may differ depending upon the microplate reader employed for the analysis C REAGENT COMPATIBILITY WITH ASSAY The following table summarizes a sampling of reagents and their con centrations that have been demonstrated to be compatible with the ProteoStat Protein Aggregation Assay The values represent the highest concentration of reagent tested with the protein sample Reagents were tested individually in the protein samples and were ProteoStat 3uM ThioflavinT 3uM sea Antibody Aggregated Conc 0 00 pg ml 24 0 19 pgiml 0 3 EE EEE 0 38 ug ml 0 6 67 0 0 0 84 0 75 pg ml 1 3 1 50 jig ml 2 5 3 00 pg ml 5 0 6 00 pg ml 10 0 34 0 2 0 91 12 00 pg ml 20 0 AS EAN SR 40 598 e Table 1 Detection limit comparison between ProteoStat Detection Reagent and Thioflavin T 42 0 0 0 82 considered compatible if the monomer signal didn t increase by more than 2 fold This information is only provided as a guideline and actual sa
12. lson R Sawaya MR Balbirnie M Madsen A Riekel C Grothe R Eisenberg D 2005 Structure of the cross beta spine of amyloid like fibrils Nature 2005 435 7043 773 778 10 VIII Troubleshooting Guide Problem Potential Cause Suggestion Poor fluorescent signal observed in the positive control Detection reagent has been exposed to strong light Protect samples from exposure to strong light and analyze them immediately after staining Kit reagent has degraded Band pass settings are too narrow or not optimal for the fluorescent probe Verify that the reagents are not past their expiration dates before using them Use correct monochromator setting or filter set for the fluoro phore Check Methods and Procedures section of this manual and Appendix A for recommendations Insufficient ProteoStat dye concentration Follow the procedures provided in this manual The aggregated protein is not in solution Extended centrifugation will cause the aggregate to precipitate Do not centrifuge the aggregated protein sample or control High fluorescence background in the well without protein sample Inappropriate dye dilution Follow the procedures provided in this manual It is important to make certain that there are no particles in the dye Centrifuge well before use Dust or solid material in well Make certain all solutions micro plates and tubes are clean and fr
13. mended to per form the assay with positive 20 uM aggregated Lysozyme and negative control 20 uM native Lysozyme Unused stock control samples may be stored in aliquots at 4 C for several weeks Do not centrifuge the positive control as the aggregates are in suspension not in solution 3 ProteoStat Detection Reagent Loading Solution NOTE The ProteoStat Detection Reagent is light sensitive Avoid direct exposure of the reagent to intense light Aliquot and store unused re agent at 20 protected from light Avoid repeated freeze thaw cycles For each 96 well plate prepare 200 uL of ProteoStat Detection Reagent Loading Solution as follows Add 10 uL of ProteoStat Detection Reagent and 20 uL of 10X Assay Buffer into 170 ul deionized water Mix well B STAINING 1 Dispense 2 uL of the prepared ProteoStat Detection Reagent Loading Solution see section A 3 page 3 into the bottom of each well of a 96 well microplate NOTE The ProteoStat detection dye is light sensitive Be sure to pro tect samples from light 2 Add 98 uL of the protein of interest to each well The recommended protein concentration range is 1 ug ml to 10 mg ml For higher concentrations of protein mg ml range fluores cence intensity readings in the presence of high amounts of ag gregate may become saturated and beyond the linear range of the assay This should not be a problem when detecting lower aggre gation levels lt 20 aggregate
14. mples which normally contain different buffers and other components may behave differently Tested Reagents and Validated Concentrations Sodium Chloride up to 1 M Ascorbic Acid up to 1 mM Calcium Chloride up to 200 mM Dithiothreitol up to 1 mM Ammonium Sulfate up to 300 mM Triton X 100 up to 0 01 Sorbitol up to 600 mM Tween 20 up to 0 01 Mannitol up to 600 mM Arginine up to 500 mM Trehalose up to 600 mM Glycine up to 2 Lactose up to 300 mM Ethanol up to 20 Higher concentrations of certain detergents such as 0 2 Tween 20 may produce an increase in background signal with the monomeric protein However the protein aggregate signal typically remains substantially larger Table 2 Substances tested for compatibility with the ProteoStat Protein Aggregation Assay VII References 1 Arakawa T Philo JS Ejima D Tsumoto K Arisaka F 2006 Aggregation analysis of therapeutic proteins part 1 Bioprocess International 4 10 32 42 Krishnamurthy R Sukumar M Das TK Lacher NA 2008 Emerging analytical technologies for biothererapeutics development Bioprocess International 6 5 32 42 Arakawa T Philo JS Ejima D Tsumoto K Arisaka F 2007 Aggregation analysis of therapeutic proteins part 2 Bioprocess International 5 4 36 47 Arakawa T Philo JS Ejima D Sato H Tsumoto K 2007 Aggregation analysis of therapeutic proteins part 3 Bioprocess International 5 10 52 70 Ne
15. osed of properly e Observe good laboratory practices Gloves lab coat and protective eyewear should always be worn Never pipet by mouth Do not eat drink or smoke in the laboratory areas All blood components and biological materials should be treated as potentially hazardous and handled as such They should be disposed of in accordance with established safety procedures e To avoid photobleaching perform all manipulations in low light environments in amber microcentrifuge tubes or protected from light by other means V Methods and Procedures A REAGENT PREPARATION NOTE Allow all reagents to thaw at room temperature before beginning the procedures Upon thawing gently hand mix or vortex the reagents prior to use to ensure a homogenous solution Briefly centrifuge the vials at the time of first use to gather the contents at the bottom of the tube 2 1 10X Assay Buffer Allow the 10X ProteoStat Assay Buffer to warm to room tempera ture Make sure that the reagent is free of any crystallization 2 Controls Prepare the ProteoStat Positive Control Aggregate and the ProteoStat Negative Control Monomer for monitoring and detection of protein aggregation Both controls are supplied as lyophilized powder 300 ug each and should be reconstituted in 500 uL deionized water to generate a 40 uM stock solution Gently mix the controls to re suspend them do not vortex or cause unnecessary bubbles It is strongly recom
16. ployed to streamline protein processing and formulation optimization procedures Relative to conventional protein aggregation detection dyes such as Thioflavin T Enzo s ProteoStat De tection Reagent can detect aggregates from a broader range of proteins yields a much brighter signal provides at least 2 orders of magnitude linear dynamic range and offers superior performance across a broad range of pH values 4 10 and buffer compositions Sensitivity of this assay is in the submicromolar range and less than 1 protein aggregate is detectable in a protein solution The assay provides a convenient mix and read format and delivers Z factor scores greater than 0 5 Therefore the assay is ca pable of providing quantitative analysis of protein aggregation in a robust and high throughput fashion Lyophilized native and aggregated lysozyme is provided as negative and positive controls for monitoring changes in protein aggregation status To facilitate quantitative analysis of protein aggregation The ProteoStat Protein Aggregation standards ENZ 51039 can be used to determine low levels of aggregated protein in a sample by comparing the assay response of a test sample to that of the standard curve comprised of standards with known concentrations of aggregated IgG The ProteoStat Protein Aggregation Assay enables monitoring of protein aggregate formation in solution This is useful for defining optimal storage formulations for proteins for s
17. t reagent or 3 uM Thioflavin T Protein o o o 9 9 o o a A DO N o N Figure 2 Absorption and fluorescence emission spectra for ProteoStat Detection Reagent All spectra were determined in 1X Assay Buffer 2 5 O r t r 400 450 500 550 600 650 700 was incubated with the dye for 15 minutes prior to determining the fluorescence using a BioTek Synergy Mx microplate reader with ex citation setting at 500 nm and emission setting at 603 nm both with a 9 nm slit width for ProteoStat reagent while Thioflavin T was detected with excitation setting at 435 nm and emission setting at 495 nm both with a 9 nm slit width Readings were taken in at least tripli cate in a Greiner uClear black clear bottom 96 well microplate As demonstrated in Figure 3 the signal generated from the ProteoStat Detection Reagent is 100 fold brighter than Thioflavin T and the concentration response curve is more linear In Table 1 the limit of antibody aggregate detection using ProteoStat Detection Reagent compared with Thioflavin T is shown ProteoStat Detection Reagent is able to reliably detect as little as 0 3 aggre gated IgG in concentrated IgG sample i e 0 19 ug mL of the 60 ug mL total IgG concentration Thus the kit provides an ideal analytical method enabling sensitive accurate and linear detection of protein aggregates over a broad concentration range Additionally the assay is resistant to sample 7 matr
18. that is minimally 90 80 Plateau Phase Region is considered 100 aggregated Growth Phase Lag Phase 60 40 Aggregate Signal ee ee ee eee eee o a 12hrs Time Figure 1 Different stages are generally observed during the formation of IgG protein aggregates First a lag time is observed during which no fibrils are formed Then nucleation occurs and fibrils start to form growth phase Eventu ally the growth rate decreases and becomes zero indicating a limitation in native protein supply The aggregation process can be accelerated by subjecting the proteins to elevated temperature Extensive aggregation will cause large insoluble aggregates fluorescent in the presence of the native form of a protein but dis plays a 20 90 fold fluorescence intensity enhancement upon binding to the cross beta spine quaternary structure of aggregated proteins Figure 3 demonstrates the linear dynamic range of detection for goat anti mouse IgG aggregate using ProteoStat Detection Reagent as compared with Thioflavin T Purified rabbit anti goat IgG 4 26 mg mL was incubated in 100 mM HCI pH 2 7 at 80 C for 90 minutes to form aggregates The signal from the aggregate was determined after mixing aggregated with monomeric proteins at different ratios such that the total IgG concentration remained 60 ug mL protein The readings were taken in 50 mM potassium phosphate pH 7 contain ing either 3 uM ProteoSta
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