The Simple Western Approach to Vaccine and Clinical Protein Research

Transcription

1 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

2 The Simple Western Approach to Vaccine and Clinical Protein Research Your Moderator John Sterling Editor-in-Chief Genetic Engineering & Biotechnology News

3 The Simple Western Approach to Vaccine and Clinical Protein Research Peter A. Fung, Ph.D. Product Manager Simple Western

4 Introducing the Simple Western Gel-free. Blot-free. Hands-free. October 4, 2012 Peter A. Fung, Ph.D.

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6 Western Blots are Tedious

7 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

8 Simple Western Delivers Automation Reproducibility Quantitation %CV = 5 n = 11 Gel-free, Blot-free, Hands-free

9 Providing New Capabilities In a single experiment: Use µl sample volumes Test multiple antibodies Generate 96 data points

10 Efficiently Generate Results Prepare samples Press start Results automatically

11 How the Simple Western Works

12 Multiple Views of Results Capillary Image View Electropherogram View Lane View

13 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

14 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

15 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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17 The Simple Western Approach to Vaccine and Clinical Protein Research Melissa Hamm Research Biochemist Vaccine Analytical Development Merck & Co.

18 A New Approach to Western Analysis in Vaccine Development Melissa Hamm Merck Research Labs

19 Outline Assay overview General sample preparation Potential Applications Advantages and Disadvantages of the technology Method development considerations Case studies Conclusions

20 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

21 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) 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

22 Potential Applications Concentration analysis Antibody screening Fermentation and process monitoring Identity Host cell protein analysis Any many more

23 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

24 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 µg/ml Fermentation samples can usually be diluted to between µg/ml or run undiluted.

25 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.

26 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

27 Project 1: Protein A and B Protein B Protein B-clipped Protein A (94kDa) (75kDa) (48kDa) p106 p86 p52

28 Peak area Project 1: Linearity of Protein A Standards Samples y = 58524x R 2 = Conc (ug/ml) Protein A is linear over a concentration range of 0.5-7µg/ml The protein is ~48kDa

29 Project 1: Protein B Dynamic Range and Linearity Dynamic Range ( ~ 2 orders) ( 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 ug/ml Protein B

30 Project 1: Protein B Capillary to Capillary Repeatability Repeatability (n=11) Peak Area %CV = 6.8%

31 Project 1: Protein B Fermentation Sample Analysis Protein B Standards D4 D5 D6 p106 p86 Protein B clipped Samples

32 Luminescences Luminescences Project 1: Fermentation Sample Electropherograms p86 p D D D4 Protein B clipped Protein B Standards Minutes 0

33 Project 1: RP-HPLC of Fermentation Samples OD (280 nm) F D4 F D5 F D6 Protein B Protein B clipped Time (min)

34 Project 1: Optimization of separation of Protein C Protein C Protein D Protein B Protein A Protein B Protein A Protein C Protein D

35 Project 1: Trivalent mixture Protein B Protein A Protein D mix Protein D Protein B Protein A

36 Project 1: Antibody screening for Protein D

37 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.

38 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.

39 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

40 Peak area Project 2: Protein H linearity 20µg/ml 1µg/ml y = 45851x R 2 = Conc (ug/ml) Linearity is demonstrated between 1-20µg/ml Molecular weight is ~39kDa

41 Project 2: Protein I linearity 10µg/ml 1µg/ml Linearity is demonstrated between 1-10µg/ml. The molecular weight is ~140kDa

42 Project 2: Protein H and I mixture mix S1 S2 S3 Protein I Protein H

43 Project 2: Protein H concentration Sample ID Simon (µg/ml) % RSD Mass Spec (µg/ml) S S Concentration range = µg/ml. All samples are tested neat. % RSD over 3 days < 2%

44 Project 2: Protein I concentration Standards S1 S2 S3 Sample ID Simon (µg/ml) % RSD S S S Protein I is linear between µg/ml. S1 is diluted 200-fold S2 is diluted 4-fold S3 is neat

45 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

46 Project 3: Simon vs Traditional Western S1 S2 S3 S4 RS blank Z Z

47 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.

48 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.

49 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

50 The Simple Western Approach to Vaccine and Clinical Protein Research Alice Fan, M.D. Instructor, Medicine and Oncology Stanford University School of Medicine

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91 The Simple Western Approach to Vaccine and Clinical Protein Research The Simple Western Approach to Vaccine and Clinical Protein Research Q&A

92 The Simple Western Approach to Vaccine and Clinical Protein Research Your Moderator John Sterling Editor-in-Chief Genetic Engineering & Biotechnology News

93 The Simple Western Approach to Vaccine and Clinical Protein Research Peter A. Fung, Ph.D. Product Manager Simple Western

94 The Simple Western Approach to Vaccine and Clinical Protein Research Melissa Hamm Research Biochemist Vaccine Analytical Development Merck & Co.

95 The Simple Western Approach to Vaccine and Clinical Protein Research Alice Fan, M.D. Instructor, Medicine and Oncology Stanford University School of Medicine

96 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