Optimization of Protein Purification Using Small-Scale Separation Columns

Transcription

1 Optimization of Protein Purification Using Small-Scale Separation Columns Chris Suh, Ph.D Application Scientist, Inc. San Jose, Ca 2 nd Therapeutics & Drug Discovery Conference

2 Presentation Outline 1. Advantage of small-scale purification & PhyTip Technology 2. Technology Application Areas 3. Case Studies

3 Presentation Outline 1. Advantage of small-scale purification & PhyTip Technology 2. Technology Application Areas 3. Case Studies

4 Current Protein Purification & Downstream Assays Current Purification Volume/Mass needed Purification volume 20mL to 1 L Protein mass purified mgs to grams Assay Description Concentration A280 ~0.2 ug Coomassie Staining 0.5 ug Silver Stain > 0.2 ng Mass Spec > fg CE-LIF > pmol ELISA > 10 pg/ml Cell-base assays Variable SPR > 2.5 pmol HPLC > 15 ng

5 Comparison: Current vs Small-Scale Purification Current Purification Large culture/sample volume Large amt of reagents & materials Labor intensive Long purification time Low throughput Low parallel processing Expensive Small-scale Purification Small culture/sample volume Small amt of reagents & material Automation Short purification time High throughput High parallel processing Cost Savings in materials and labor

6 Top screen Resin maintained in discrete area Bottom screen * Supported Platforms: MEA, Caliper, Beckman, PE & Tecan

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8 Manual Handheld Electronic The MEA 96-channel robotics ME200 / ME1000

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10 Recommended Use: Automated Manual In Development 5 µl 10 µl 320 µl 160 µl 40 µl 1 ml 80 µl 5 ml PhyTip 20 µl 5 µl * 5 µl 1 µg 10 µg 100 µg 1 mg 10 mg 100 mg 1 g

11 Ion Exchange Resins Affinity purification resins Desalting Resins

12 PhyTip Column Flexibility & Performance Variable sample volumes 10 µl to 25 ml High yield of recovered protein Up to a few mgs of recombinant protein isolated using larger resin bed columns High concentration of functional protein -- Up to 10 mg/ml with 5 µl resin bed columns Scale Up

13 Presentation Outline 1. Advantage of small-scale purification & PhyTip Technology 2. Technology Application Areas 3. Case Studies

14 Target Discovery & Validation Drug Screening & Lead Optimization Development Clinical Trials Manufacturing Protein Interaction IP, Co-IP Pathway Activation ChIP Phosphoprotein enrichment Target Expression and Purification Screening Biomarker Discovery Biomarker Validation Selected Current Customers: Raven Biotechnologies, Cell Signaling Inc, Pfizer, Astra Zeneca, Domantis, Target Discovery, Sareum

15 Target Discovery & Validation Drug Screening & Lead Optimization Development Clinical Trials Manufacturing Library synthesis Library Screening and Selection Assay Development Lead Characterization Ab specificity, selectivity, affinity Lead Optimization Affinity maturation Structural Analysis Crystallization screening Selected Current Customers: GSK, Merck, Amgen, Dyax, EMD, Wyeth, Genentech, CAT, Pfizer, Astra Zeneca

16 Target Discovery & Validation Drug Screening & Lead Optimization Development Clinical Trials Manufacturing Process Development Protein Analytics Pre-Clinical Assays Selected Current Customers: Merck, Genentech, Inhibitex, FoldRX, Lonza, EMD, Talecris

17 Target Discovery & Validation Drug Screening & Lead Optimization Development Clinical Trials Manufacturing Immunogenicity Selected Current Customers: Talecris

18 Target Discovery & Validation Drug Screening & Lead Optimization Development Clinical Trials Manufacturing Selected Current Customers: Merck Wenger et al., An automated microscale chromatographic purification of virus-like particles as a strategy for process development, Biotechnol Appl. Biochem, 47, , 2007

19 Presentation Outline 1. Advantage of small-scale purification & PhyTip Technology 2. Technology Application Areas 3. Case Studies

20 Case1:Protein A PhyTip Columns Maximum yield as a function of column size Maximum PhyTip column capacity = manufacturer reported resin capacity (ProA = 29µg/µl) Miniaturization does not affect resin performance Performance due to processing method - Low dead volume - Back-and-forth flow

21 Case1:Protein A PhyTip Columns ProA Capacity (0.01mg/ml starting concentration) IgG recovery using various bed volumes Post-purification sample concentrations Recovered protein concentration and bed size migg2a (ug) ul ul ul ul ul ul ul Concentration (X 10-3 mg/ml) ul ul ul ul ul ul ul Resin bed volume (ul) Resin ben volume (ul) - Analyzed 500uL sample at 0.01mg/mL IgG concentration - Trade-off between mass yield and concentration

22 Case1:Screening Purification Conditions 24 conditions (capture, wash, elution) tested for each of 4 clones Yield measured by ELISA, activity by SPR

23 Case2:Ni-IMAC PhyTip Columns Recovery of His-ubiquitin 500uL of 0.1mg/mL starting conc. Elution conditions screening of His-ubiquitin

24 Case2:Ni-NTA IMAC Resin Screening with PhyTip Columns Resin: * Target protein

25 Case3:Gel Filtration Columns Protein recovery and salt removal Routinely obtain 80% protein recovery & remove >95% salt Separation of myoglobin (brown MW 16.7kD) from DNP-glutamate (yellow, MW 313D) Elution fractions collected in separate wells Tips from left: µl column µl sample load x100 µl chaser µl chaser Tubes from left: 1. Starting sample µl fraction µL fractions µL fraction

26 Conclusion: PhyTip column technology benefits: Scalability Screening Hits/Library High Performance (high yield, high concentration, reproducibility, retain protein functionality) High throughput / Automation Small scale Maturation Many applications with one system Affinity Desalting / Gel Filtration Ion exchange Normal and Reverse phase

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28 Vertex Pharmaceuticals Ni-IMAC purification platform comparisons: Magnetic Beads, Spin columns, PhyTip Columns

29 Vertex Pharmaceuticals Ni-IMAC purification platform comparisons: Magnetic Beads, Spin columns, PhyTip Columns

30 Merck Research Laboratories Comparison of PhyTip columns to filter plates and large scale lab columns Micro-batch adsorption (Filter plates) Micro-pipette column (PhyTips) Laboratory column Format Microplate with 96 wells Pipette tip, eight-channel arm Column 10 ml Sample input Cell lysate <1 g Cell lysate <1 g Cell lysate >10 g Sample presentation Batch vessel (can be mixed); Defined pressure-driven flow; Defined pressure-driven flow requires longer processing bi-directional Elution Step elution Step elution Step or linear gradient elution Flexibility High; adsorbent type and Moderate; requires custom High; adsorbent type and volume easily varied preparation with fixed bed volume easily varied Automation Complex; extensive plate Simple; little or no plate Semi-automated; serial movement in and out of manipulation; pre-packed operation; requires column filtration device; resin plaque pipette columns; no aspiration packing; larger operating device needed for accurate or mixing of resin required volumes resin aliquots a ; requires longer incubations for efficient mixing Relative throughput b Method robustness Moderate High High Marc D. Wenger, Peter DePhillips, Colleen E. Price and Daniel G. Bracewell, An automated microscale chromatographic purification of virus-like particles as a strategy for process development, Biotechnol. Appl. Biochem. (2007) 47,

31 Ion exchange purification conditions screening A matrix of binding buffers of varying ph and elution buffers of increasing salt concentration allow for rapid process optimization

32 Ion exchange purification conditions screening Recovery of lysozyme from CM Recovery of BSA from DEAE

33 Desalting Gel Filtration Column Automated method Requirements Consistent protein recovery (red), volume recovery (black) and protein concentration (blue): ul/% recov mg/ml Vol Rec ( ul) Conc [ mg/ ml] % IgG r ec PhyTip columns

34 320 µl Recommended Use: Automated 40 µl 160 µl 5 ml Manual In Development 5 µl 10 µl 80 µl 1 ml PhyTip 20 µl 5 µl * 5 µl 1 µg 10 µg 100 µg 1 mg 10 mg 100 mg 1 g Kingfisher 96/1mL* HisPur TM 0.2 ml HisPur 1 ml HisPur 3 ml Dynabead Talon* Ni-NTA Agarose Kit His MultiTrap TM 1mL HisTrap TM His SpinTrap TM 5mL HisTrap HisPrep TM HIS-Select TM HC Plate** Minimum final concentration: 1 mg/ml except: * 0.1 mg/ml ** 0.01 mg/ml HIS-Select Spin HIS-Select ilap HIS-Select Filter Plate HIS-Select HF 6.4mL HIS-Select HF 1.25mL HIS-Select Cartridge