Industry Perspective: Advantages of Open Access and Walkup LC/ MS Supporting Protein Drug Discovery and Development

Transcription

1 Industry Perspective: Advantages of Open Access and Walkup LC/ MS Supporting Protein Drug Discovery and Development Dawn Stickle, Agilent Technologies Originally presented by Eric Fang, Novartis

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3 Overview Recombinant proteins in drug discovery- role of the Protein Sciences group and the production workflow Intact protein LC-MS as a primary QC criterion and characterization tool in protein production Advantages of the Open Access approach over a conventional analyst-centered implementation Application examples in which Open Access contributes to usability and project success

4 The goal of the Protein Sciences group in drug discovery and development Produce well-characterized proteins for multiple purposes Active enzymes and substrates for biochemical assay and library screens Crystallography and NMR Biophysical assays (ITC, DSC, DSF, SPR...) Antigens for antibody discovery Therapeutic protein candidates for in vitro and in vivo assays, e.g. peptide hormones, cytokines, antibodies

5 The recombinant protein production workflow Design, Expression, Purification and Analysis Protein and construct design Molecular biology and expression Purification, processing and analysis Project goals Sequence analysis Literature review Experience Addition of fusion partners and purification tags Insertion into vector(s) Primary QC: DNA sequencing of expression cassette Protein purification from cell lysate or culture supernatant Tag removal and/or in vivo and in vitro modifications Primary QC: Verification of expected intact mass 5

6 The manual intact protein LC/MS workflow 11-5 min. per sample 11-5 min. per TIC peak or region of interest 11-5 min. per sample Total ion chromatogram with selected peak Extracted m/z spectrum from TIC peak Full range zero charge mass deconvolution 6

7 Intact mass provides supporting evidence of sequence identity The recombinant protein design process results in an expected specific primary sequence Most (not all) deviations from that expected sequence will result in a change in expected mass beyond experimental error Allowing for a limited number of common post-translational modifications, an observed mass consistent with calculated mass supports identity An observed deviation outside of experimental error requires explanation ü ü Sequence Name Target mass Mass Delta ppm Rule Z Count MinZ MaxZ Fit Score M yog lobin (horse) Intact protein Deconvoluted Spectrum (Zoomed) 7

8 Intact mass is especially helpful when multiple modifications are expected and need confirmation ü ü ü 8

9 It is also useful for in-process monitoring, such as in vitro enzymatic processing 9

10 Failure to provide confirmatory evidence at protein level can cause problems! Variants across a family of constructs/proteins can be mixed up without your knowledge, e.g.: Amino acid point mutations Truncation variants Different antibodies (how are you confirming the identity of your antibodies?) What is the state of post-translational modification of your protein? Is the expected activating or functional modification present, and in what relative amount? - Phosphorylations, biotinylation, In vitro chemical modifications SDS-PAGE gels, Western blots and activity assays are often (usually?) insufficient to answer these questions If the point of your experimental design is to compare protein variants, it s advisable to confirm that you are comparing the right proteins In real life, mix-ups and mutations can happen in between construct design (DNA sequence) and purified protein (protein sequence with PTMs) 10

11 What is Open Access? and why do we want it? Open Access is a software package that manages an analytical workflow to provide direct access to end users rather than through trained analysts Typically presents a defined menu of analyses to the end user, with the technical details managed by experts Data analysis may also be automated to varying degrees, depending on technique and analysis Housekeeping procedures, especially startup and shutdown, may also be automated We are running Agilent Easy Access in conjunction with MassHunter and MassHunter BioConfirm

12 Advantages of Open Access in the Intact LC-MS workflow Accessibility and Ease of Use- Two sides of the same coin Open Access increases the availability of the technique and makes it easy for users to support themselves Reduced demands on skilled analysts pays dividends Open Access makes it easy for skilled analysts to support more users, more samples and more applications

13 3 simple steps to result! Simple and convenient sample submission

14 Automating the intact mass workflow saves time 11-5 min. per sample 11-5 min. per TIC peak or region of interest 11-5 min. per sample Open Access software handles user sample submission, method selection and sequence selection LC-MS data acquisition Peak selection and extraction of m/z spectrum Zero charge mass deconvolution of peaks Automatic matching of found protein masses to provided sequence and variants Most vendors stop here Agilent and one other competitor Agilent MassHunter Bioconfirm + Easy Access The ideal Open Access workflow automate as much of intact mass workflow as possible Automatically performing zero-charge mass deconvolution saves operator time and tedium- only two vendors offer this capability Automated data annotation offers great potential for time savings 14

15 Diversity of protein constructs presents a challenge in keeping track of sequences Our group produces many different sequences at many different scales of production From 100 micrograms to 100s of milligrams to gram scale Diverse protein construct sources from locally designed constructs to transfers from internal and external collaborators Most researchers must keep track of dozens of sequences across multiple projects at one time Many proteins will be produced only once or a few times As a group, our custom sequence database numbers in the thousands Allowing individual users to keep track of their own sequences is helpful

16 Open Access, along with appropriate instrumentation, allows flexibility of application LC System: Agilent 1290 Infinity UHPLC with dual temperature zone oven and 6-column selector valve Agilent 6530 QToF 1 Protein and 1 peptide reverse phase columns at elevated temperature for standard LC/MS and MS-MS 2 different columns at high temperature for antibody applications 2 additional positions available Common buffer system (formic acid/acetonitrile/water) but potential for second buffer system on the binary pump 16 Open Access LC/MS in recombinant protein production Eric Fang 26 September 2013

17 Protein Open Access is a success! Ease of use and accessibility increase usage The Open Access environment easily saves 5-10 analyst minutes per intact mass sample Sample preparation and submission are off-loaded onto the end user Peak extraction and zero-charge mass deconvolution are fully automated If the user provides a sequence at submission, basic mass matching and results annotation are provided automatically System introduced February 1, 2012 with almost 2400 samples processed through the end of year, and on track to double usage in 2013 A busy month in the old environment might have been ~80 samples 17 Open Access LC/MS in recombinant protein production Eric Fang 26 September 2013

18 Open Access Protein MS changes the relationship between the user and the technique Reduces the burden of sample submission Reduces the turnaround time to usable results Increases the overall availability of the resource Skilled analysts become even more of an enabler, and less of a gatekeeper Skilled analysts are more free to make contributions in other ways In the research environment, few have dedicated technical roles Open Access changes MS from a service to a tool 18 Open Access LC/MS in recombinant protein production Eric Fang 26 September 2013

19 Monitoring of purification fractions Open Access allows end user to develop optimized purification Protein was expressed as a heterogeneously phosphorylated population in insect cells In vitro dephosphorylation was tried but deemed insufficient or ineffective in producing homogeneous pool The ability to rapidly and independently assess phosphorylation state of different fractions on her own assisted in the development of a purification to molecular homogeneity Courtesy Kelly Yan 19

20 Covalent inhibitor studies Studying the formation kinetics and stability of a covalent adduct End users had minimal to no experience with intact protein MS Training was limited to approximately 5 minutes of login instruction, method selection and autosampler and vial loading This data was acquired by someone trained by another minimally trained user Useful data/low barrier to entry Parent Adducted 20 Courtesy Brian Feng and Lisa Cunden Open Access LC/MS in recombinant protein production Eric Fang 26 September 2013

21 Reaction Monitoring: Target phosphorylation by protein kinase monitored at the intact protein level Protein kinase phosphorylates its substrate twice. Compound binds to the substrate, not the activating kinase Open Access MS makes this type of experiment easier and more appealing to conduct Reduced data analysis demands directly enable the analysis of the effects of multiple compounds at multiple time points, as well as multiple experimental replicates when necessary phosphorylations phosphorylations minutes minutes Control Compound Conclusion: compound binding to substrate greatly slows the full (double) phosphorylation of the substrate 21

22 Reaction Monitoring: Target phosphorylation by kinase monitored at the peptide level Open Access peptide LC-MS/MS was used to identify accurate mass-retention time pairs for the two possible singly phosphorylated activation peptides Open Access peptide LC-MS was then used to monitor the site 1 and site 2 phosphorylations of the activating peptide by accurate mass-retention time Easy Access + MassHunter automated the acquisition and analysis so that it was only necessary to record and plot the intensities vs. time of the 4 monitored species Conclusion: Compound binding does not change the ordered phosphorylation (site 1 then site 2), but greatly slows the process 22 Open Access LC/MS in recombinant protein production Eric Fang 26 September 2013

23 Peptide LC-MS/MS for Protein ID in Open Access Automatically run data dependent MS/MS on a protein digest and output Mascot-ready.mgf Could fully automate searching using Mascot Daemon Mascot search against SwissProt can identify target protein as well as host cell contaminants Mascot search against our custom internal database can differentiate between our recombinant constructs 23 Open Access LC/MS in recombinant protein production Eric Fang 26 September 2013

24 Summary Protein intact mass is an essential tool in the production and characterization of recombinant proteins Increasing access and usability through Open Access improves the ability of users to enhance and understand their processes and make contributions to research projects Open Access reduces the burden on trained analysts and allows increased support of users, applications and projects 24

25 Acknowledgments Novartis Eric Fang, Original Presenter Dazhi Tang, Analytical Sciences Co-owner of Protein Open Access support Gavin Dollinger, Director Analytical Sciences Isabel Zaror, Director Protein Sciences Harry Sterling, Analytical Sciences Protein Sciences, NIBR Emeryville Kelly Yan John Fuller Steven Martin, Global Head Analytical Sciences Agilent Taegan Cleary, Market Director- Pharma Xiaoling Wu Robert Yang Maithilee Samant Ning Tang Edmund Neo Wilfred Tang Andy Gieschen Jose Meza Kristin Swanson Donghui Yi 25