The Simple Western Approach to Vaccine and Clinical Protein Research The Simple Western Approach to Vaccine and Clinical Protein Research Broadcast Date: Thursday, October 4, 2012 Time: 1 pm ET, 10 am PT Sponsored by
The Simple Western Approach to Vaccine and Clinical Protein Research Your Moderator John Sterling Editor-in-Chief Genetic Engineering & Biotechnology News
The Simple Western Approach to Vaccine and Clinical Protein Research Peter A. Fung, Ph.D. Product Manager Simple Western
Introducing the Simple Western Gel-free. Blot-free. Hands-free. October 4, 2012 Peter A. Fung, Ph.D.
Western Blots are Tedious
Simple Western Product Family Sally Size Separation 96 Assays NanoPro 1000 Charge Separation 96 Assays Simon Size Separation 12 Assays Peggy Size and Charge Separation 96 Assays
Simple Western Delivers Automation Reproducibility Quantitation %CV = 5 n = 11 Gel-free, Blot-free, Hands-free
Providing New Capabilities In a single experiment: Use µl sample volumes Test multiple antibodies Generate 96 data points
Efficiently Generate Results Prepare samples Press start Results automatically
How the Simple Western Works
Multiple Views of Results Capillary Image View Electropherogram View Lane View
NFkB Pathway Profiling With Sally Rep 1 Rep 2 WC NE WC NE TNFa - + - + - + - + NFkB-p65 IkB-a C-Rel NFkB2 (p100/p52) NFkB1 (p105/p50) IKKa Caspase 9 atubulin (Loading Control) Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 5 µl samples at 1 µg/µl
The Simple Western is the Future Gel-free, Blot-free, Hands-free Sally Size Separation 96 Assays NanoPro 1000 Charge Separation 96 Assays Simon Size Separation 12 Assays Peggy Size and Charge Separation 96 Assays
Simple Western Delivers More Feature Simple Western Western Blot Capillary Electrophoresis HPLC ELISA Size-based Separation Charge-based Separation Target ID/Specificity Quantitation Reproducibility FTE Efficiency Throughput
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The Simple Western Approach to Vaccine and Clinical Protein Research Melissa Hamm Research Biochemist Vaccine Analytical Development Merck & Co.
A New Approach to Western Analysis in Vaccine Development Melissa Hamm Merck Research Labs
Outline Assay overview General sample preparation Potential Applications Advantages and Disadvantages of the technology Method development considerations Case studies Conclusions
Assay Overview Step 1: Load Load stacking and separation matrices and samples Step 2: Separate Separate protein components based on size under high voltage Step 3: Immobilize Immobilize the proteins to the capillary walls using UV light Step 4: Immunoprobe Detect the proteins of interest with specific antibodies followed by an HRPconjugated secondary Step 5: Quantitate Proteins that are bound by the antibody complex are visualized as an electropherogram
General Sample Preparation Samples are denatured and reduced in the presence of SDS and DTT at either 70 C or 95 C for 10 minutes. Samples and reagents are loaded onto a 384-well plate. The plate and other reagents are loaded onto the system and the run starts. Vol (ml) 1 2 3 4 5 6 7 8 9 10 11 12 15 15 A B A B 5 C X S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 10 D D 10 E D Primary 10 F Y Secondary 10 G luminol / peroxide mixture A = stacking matrix X = biotinylated ladder B = separation matrix Y = streptavidin-hrp D = blocking buffer S = standards or samples
Potential Applications Concentration analysis Antibody screening Fermentation and process monitoring Identity Host cell protein analysis Any many more
Advantages / Disadvantages Advantages Quantitative Fast analysis times (about 3-5 hrs per run) Simple method development Easy sample preparation (plate can be prepared in about 1 hr) Low volume of samples and reagents required Disadvantages Not designed for larger proteins ( 250kDa) Limited availability of secondaries (only rabbit and mouse) Need program specific primary antibodies
Method Development Considerations Primary Antibody dilution Westerns typically use dilutions of 500-fold or more Simon uses dilutions of 20- to 1000-fold for most mabs Sample preparation Incubate the samples under the same conditions that are normally used for SDS- PAGE Purified proteins can be diluted between 0.5-20µg/ml Fermentation samples can usually be diluted to between 50-500µg/ml or run undiluted.
Method Development Considerations For improved sensitivity, the sample injection and stacking matrix loading can be changed. Sample injection can be increased to 12 sec Stacking matrix loading can be increased to 16 sec This is useful for low abundance proteins to demonstrate clearance.
Case Study 1 Project 1 is a multivalent vaccine consisting of 4 different proteins ranging in size from about 50kDa 300kDa. Goals for method development: Concentration analysis monovalent and tetravalent Identity Host cell protein clearance Samples are incubated at 95 C for 10 minutes
Project 1: Protein A and B Protein B Protein B-clipped Protein A (94kDa) (75kDa) (48kDa) p106 p86 p52
Peak area Project 1: Linearity of Protein A Standards Samples 450000 400000 350000 300000 250000 y = 58524x - 363.5 R 2 = 0.9917 200000 150000 100000 50000 0 0 1 2 3 4 5 6 7 8 Conc (ug/ml) Protein A is linear over a concentration range of 0.5-7µg/ml The protein is ~48kDa
Project 1: Protein B Dynamic Range and Linearity Dynamic Range ( ~ 2 orders) (0.2-17 mcg/ml) 1.4e+6 1.2e+6 Peak Area 1.0e+6 8.0e+5 6.0e+5 4.0e+5 2.0e+5-7.0e+0 0 10 20 30 40 50 ug/ml Protein B
Project 1: Protein B Capillary to Capillary Repeatability Repeatability (n=11) Peak Area %CV = 6.8%
Project 1: Protein B Fermentation Sample Analysis 1 2 3 4 5 6 7 8 9 10 11 12 Protein B Standards D4 D5 D6 p106 p86 Protein B clipped Samples
Luminescences Luminescences Project 1: Fermentation Sample Electropherograms 150000 12 p86 p106 150000 11 10 D6 100000 9 100000 D5 50000 8 7 6 5 4 3 2 1 50000 D4 Protein B clipped Protein B Standards 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Minutes 0
Project 1: RP-HPLC of Fermentation Samples 0.5 0.4 OD (280 nm) 0.3 0.2 0.1 0.0 F11 602 D4 F11 602 D5 F11 602 D6 Protein B Protein B clipped 0 5 10 15 20 25 Time (min)
Project 1: Optimization of separation of Protein C Protein C Protein D Protein B Protein A Protein B Protein A Protein C Protein D
Project 1: Trivalent mixture Protein B Protein A Protein D mix Protein D Protein B Protein A
Project 1: Antibody screening for Protein D
Project 1: Host cell protein clearance Host cell protein clearance needs to be demonstrated in the process to ensure that very little of the proteins from the host cells are present in the final vaccine for safety reasons. A commercially available monoclonal exists for HCP analysis and also a new mab was made against a more relevant capsid protein. To enhance the sensitivity of the method, the sample injection was increased to 12 sec from 6 sec and also the stacking matrix loading was increased to 16 sec.
Project 1: HCP clearance Commercially available mab Capsid protein mab Column feed product HCP is detected in the sample, but about a 3 log clearance is seen up to this step using either antibody.
Case Study 2 Project 2 is an OMV based vaccine that contains at least 5 antigens of interest. Our study will focus on 2 of the antigens that are 40kDa and 140kDa in size. Goals for method development: Concentration analysis Samples are incubated at 70 C for 10 minutes
Peak area Project 2: Protein H linearity 20µg/ml 1µg/ml 1000000 900000 800000 700000 600000 500000 400000 300000 200000 100000 0 y = 45851x + 31296 R 2 = 0.992 0 5 10 15 20 25 Conc (ug/ml) Linearity is demonstrated between 1-20µg/ml Molecular weight is ~39kDa
Project 2: Protein I linearity 10µg/ml 1µg/ml Linearity is demonstrated between 1-10µg/ml. The molecular weight is ~140kDa
Project 2: Protein H and I mixture mix S1 S2 S3 Protein I Protein H
Project 2: Protein H concentration Sample ID Simon (µg/ml) % RSD Mass Spec (µg/ml) S1 2.0 1.4 1.4 S2 2.7 1.2 2.1 Concentration range = 0.25-5µg/ml. All samples are tested neat. % RSD over 3 days < 2%
Project 2: Protein I concentration Standards S1 S2 S3 Sample ID Simon (µg/ml) % RSD S1 256.7 0.7 S2 8.5 3.9 S3 1.9 0.7 Protein I is linear between 0.25-5µg/ml. S1 is diluted 200-fold S2 is diluted 4-fold S3 is neat
Case Study 3 Project 3 is a live virus vaccine consisting of 3 major capsid proteins that are between 60kDa 130kDa. Goals: Detect and quantitate major capsid proteins in fermentation samples Samples are incubated at 95 C for 10 minutes
Project 3: Simon vs Traditional Western S1 S2 S3 S4 RS blank Z Z
Project 3 Multiplexing Y X Z X Z X Y Z It was expected that all 3 proteins would be resolved since Y and Z are expected to be ~12kDa apart, but they are not resolved. Only X and Y or X and Z can be multiplexed.
Conclusions Multiplexing can be useful, but is not applicable in all cases. Linearity has been demonstrated for all proteins tested to date and the quantitative results obtained are in line with other techniques. Clearance of unwanted proteins can be monitored and quantitated. Fermentation samples can be easily assessed with minimal sample preparation.
Acknowledgements Merck: Sha Ha Richard Rustandi Richard Peluso Bioprocess Group for all material Basic Research for project specific antibodies Protein Simple: Chris Heger Annegret Boge Peter Fung
The Simple Western Approach to Vaccine and Clinical Protein Research Alice Fan, M.D. Instructor, Medicine and Oncology Stanford University School of Medicine
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research
The Simple Western Approach to Vaccine and Clinical Protein Research The Simple Western Approach to Vaccine and Clinical Protein Research Q&A
The Simple Western Approach to Vaccine and Clinical Protein Research Your Moderator John Sterling Editor-in-Chief Genetic Engineering & Biotechnology News
The Simple Western Approach to Vaccine and Clinical Protein Research Peter A. Fung, Ph.D. Product Manager Simple Western
The Simple Western Approach to Vaccine and Clinical Protein Research Melissa Hamm Research Biochemist Vaccine Analytical Development Merck & Co.
The Simple Western Approach to Vaccine and Clinical Protein Research Alice Fan, M.D. Instructor, Medicine and Oncology Stanford University School of Medicine
The Simple Western Approach to Vaccine and Clinical Protein Research Thank You For Attending The Simple Western Approach to Vaccine and Clinical Protein Research Broadcast Date: Thursday, October 4, 2012 Time: 1 pm ET, 10 am PT Sponsored by