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Engineering and Purification of Surrogate Markers of Biological Pharmaceuticals to Enable Visualization and Quantification of Pharmacological Assessments of Drug Delivery Jared , Cathryn Collin Timothy P. 1 2 Dillard University , Department of Microbiology, Immunology and Parasitology , LSU Health Sciences Center 2 Miller , EXPERIMENTAL PLAN Step 4: Large scale expression 1.5kb pBAD Nluc 7157 billion Avastin VEGF Mab, rDNA 6596 billion Rituxan CD20 Mab, rDNA 6596 billion Herceptin HER2 receptor Mab, rDNA 5770 billion Lantus Insulin glargine, rDNA 5190 billion Neulasta G-CSF, rDNA, PEG- 3950 billion Epogen/Procrit EPO, rDNA 3730 billion Lucentis VEGF Mab Fab, rDNA 3723 billion PHARMACOLOGICAL NEEDS, PROBLEM, & HYPOTHESIS •NEED: There is an intense need for developing carrier compounds of biological drugs that alter their immune recognition, drug availability, activity, delivery, retention, and pharmacological properties. •PROBLEM: Accurate assessment of pharmacological properties of specific carrier compounds for biological drugs is a complex process. Complications in modifying biological drugs for visualization and detection slows carrier drug development and assessment. Step 6: Use of surrogate markers to quantify biological pharmaceutical delivery compounds RESULTS: Induction of Surrogate Biological Pharmaceuticals Expression Green Fluorescent Protein Nano Luciferase Engineering Recombinant Surrogate Markers of Biological Drugs Nano Luciferase 3.5kDa Purified GFP Detection with Luminometer Nano Luciferase Activity is maintained for hours Large Scale Protein Expression PCR Amplification Nano Luciferase Gene PCR Cloning Green Fluorescent Protein Pure Protein Wash Through Flow Through GFP Soluble Supe. Uninduced Marker Green Fluorescent Protein Characterization of Nano Luciferase Enzymatic Activity Nano Luciferase Scaled up to 1 Liter cultures Analysis of Nano Luciferase Enzymatic Activity Every 1 Minute Over 3hr Time Period: Nano Luciferase Activity is STABLE! GFP has no Activity Utilization of Nano Luciferase to Assess Efficacy of Biological Carrier Compounds 160kDa 110kDa 80kDa 60kDa 50kDa 40kDa •HYPOTHESIS: Bioluminescent and fluorescent proteins can be utilized as surrogate markers of biological pharmaceuticals to assist in development of carrier compounds. Pure Protein 15kDa 10kDa Detection of Nano Luciferase TNF Mab, rDNA Marker Remicade pBAD 202 N. Luc. open #2 Arabinose induced 7870 billion pBAD 202 N. Luc. open #2 over night induced TNF Receptor-IgG Fc, rDNA pBAD 202 N. Luc. open #2 3hr induced Enbrel Step 5: Purification of surrogate biological markers pBAD 202 N. Luc. open #2 uninduced 7.932 billion Purified Nano Luciferase 2011 Revenues Marker TNF Mab, rDNA, human Wash Through 30kDa 20kDa 1kb 0.25kb Humira Flow Through 60kDa 50kDa 40kDa pBAD 0.5kb Description Marker 160kDa 110kDa 80kDa 0.75kb Trade Name GFP Solube Supe. Induced PROTEIN (GFP) Bound to Column Nano Luc. elution #3 Nano Luc. elution #2 Marker Isolate Individual Bacterial Colonies Clone #1 Clone #2 Clone #9 Clone #10 10. pBAD 8. DD 10kb 8kb 6 kb 5 kb 4 kb 3 kb 2.5kb 2kb 9. MW Marker 7. Hind III 6. Nco I 5. undigested 4. DD 3. Hind III 2011: Top 10 Biological pharmaceuticals $90.6 billion 2. Nco I Carriers for efficient therapeutic delivery of already developed biological pharmaceuticals 1. undigested Step 3: Induce expression of surrogate biological pharmaceuticals Clone #3 PCR Screen for Positive Clones Restriction Digestion Confirmation Clone #4 Therapeutic Need Diagnostics for Positive Clones Clone #5 Step 2: Generate Recombinant E. coli that contains marker genes Hydrophobic (GFP) or AFFINITY (NanoLuciferase) Interaction Chromatography Column Marker GFP Clone #6 NLuc Step 1: Engineer Surrogate Marker Genes of Biological Drugs Clone #7 2010: Top 30 Biological pharmaceuticals $84.6 billion Purification of Surrogate Biological Markers Transformed E. coli Florescence Bioluminescence 2 Foster Generation of Recombinant E.coli That Contains Marker Genes Clone #8 BACKGROUND 2 Garvey , Nano Luc. elution #1 1 Gambrell 30kDa 20kDa SDS- PAGE of Nano Luciferase Expression 15kDa 10kDa 3.5kDa Student subsistent allowance and related expenses for this summer research have been provided by a NIH/NIMHD grant P20MD004817: Dillard-LSUHSC Minority Health and Health Disparities Research Center. Analysis of Various Carrier Compound’s Ability to Retain Nluc on Vero Cells Analysis of NLuc Enzymatic Stability in Potential Carrier Compounds Some Carrier Compounds Tested Can Increase Enzymatic Activity of the Nano Luc Surrogate Biological Although all Carrier Compounds Enhanced Biological Drug Binding to Cells, the ALCON203 Formulation Was Significantly More Efficient in Cellular Retention of Biological Drugs
