Biological Production of Recombinant Proteins-

Transcription

1 Biological Production of Recombinant Proteins- Lower cost of goods, increase yield and quality with faster time, through the use of custom affinity resin Yong Wang, Wayne Sawlivich, Andrew Keefe, Douglas Gunzelmann, Chun Zhang, Christa Tauer Arron Tolley 1, and David Bunka 1 Pim Hermans 2, Frank Detmers 2, and Bruce Dawson 2 Biologics OPU Process Development, Process Development & Technical Services, Shire 1 Aptamer Group, Suite , Bio Centre, Innovation Way, Heslington, York, YO10 5NY 2 BAC BV, a Thermo Fisher Scientific Company, 1411 GP Naarden, The Netherlands Our purpose We enable people with life-altering conditions to lead better lives 1

2 Shire is Focused in Multiple Therapeutic Areas Rare Diseases Neuroscience Gastrointestinal Ophthalmology Hematology Oncology Symptomatic rare diseases, excluding oncology Platform technologies Drug delivery technologies Developmental / Behavioral Disorders Mood and Related Disorders Disorders of Cognitive Impairment Specialty Neurology Indications Rare Diseases Esophageal Disorders GI Health Liver Diseases Functional GI Disorders Inflammatory Bowel Disease Front of the Eye Innovative approaches to intervention Back of the Eye Innovative therapeutic approaches to retinal diseases Rare Diseases Hematological Malignancies Myelodysplasia Hemoglobinopathies Hemophilia Microangiopathies Anemias Small Molecules Biologics Next Generation Therapies Delivery Innovation 2

3 Non-Mab Biologics, A Purification Challenge No purification platform Heavy resource demand for Tox/Phase I purification process development Timeline impact for First Patient In (FPI) Multiple chromatographic steps required to separate the target protein from host cell impurities Each step not fully discriminative for the target protein of interest Peak cutting frequently needed Overall product yield dependent on number of chromatography steps Product quality risks/robustness risks 3

4 Non-Mab Biologics, Purification Challenges for a Legacy Product Harvests UF Capture Upstream P-22 / DF-103 Ultrafiltration & Diafiltration Thawing, pooling and depth filtration P-8 / C-102 Chrom 1 HA Chromatography P-22 / DF-103 P-8 / C-102 P-8 / C-102 P-8 / C-102 P-8 / C-102 Chrom 2 Chrom 3 UF/DF Chrom 4 Chrom 5 HA Chromatography HA Chromatography HA Chromatography HA Chromatography Ultrafiltration & Diafiltration Drug Substance UF/DF P-22 / DF-103 Ultrafiltration & Diafiltration Viral Filtration Chrom 6 P-8 / C-102 HA Chromatography P-22 / DF-103 UF/DF Ultrafiltration & Diafiltration Challenging purification process with multiple chromatography steps and UFDF operation to reach product quality target Impurity challenges/process robustness concerns Lower yield and higher COGs--- Lower profit margin 4

5 Opportunity for Affinity Chromatography, Simplified Downstream Process Conventional process Affinity based Clarified Harvest Turn recombinant protein purification into Mab-like platform Simplify MFG operations Reduce development time/fte/cogs while increasing yield/product quality Support QbD effort for S/F studies Capture Affinity HTS Chrom 1 Polish 1 Chrom 2 Polish 2 Chrom 3 UFDF UFDF Support QbD effort for cell culture process development 5

6 Presentation Agenda Outline: Technology Evaluation Case study: Synthetic affinity ligand (DNA Aptamers) Case study: Biological affinity ligand (camelid antibody, VHH) Goal: Evaluate/Implement custom affinity ligand technology for recombinant proteins to support process development, manufacturing and high throughput studies 6

7 Technology Evaluation, Biological vs Synthetic Synthetic Affinity Ligands (Chemicals, DNA, RNA, Peptide) No immune response required Library already established Shorter timeline to support early stage process development need Completely synthetic; Simpler structure and lower molecular weight (< 10 kda) Allow for selection of mild elution conditions Better stability of ligand Biological affinity ligands (Abs or Fragments) Biological origin Requires successful immune reaction to build the library Time consuming due to immunization need Ligand stability concern Allow for selection of mild elution conditions More mature technology 7

8 Technology Evaluation, Biological vs Synthetic Ligand Chemistry Evaluated with External Vendor Company Company 1 Company 2 Aptasol Company 3 Thermo (BAC) Technology Company size Company age Combinatorial Chemistry Peptide aptamer DNA/RNA aptamer DNA/RNA aptamer V H H antibody Large Small Small Small Large Format Resin Resin Diagnostic Therapeutic Some Resin Diagnostic Therapeutic Some resin Resin Wide range of development times and cost Collaboration with Aptasol and Thermo Fisher initiated DNA/RNA Aptamer vs Biological ligand 8

9 Case study 1: Synthetic Affinity Ligand, DNA Aptamers 9

10 Synthetic Affinity Ligand, DNA Aptamers What is an Aptamer? Aptamer = Nucleic acid sequence selected for its ability to bind specifically to a target Sometimes referred to as nucleic acid antibodies 10

11 How are Aptamers Raised- The Starting Library Primer 1 T7 promoter Random Region Primer 2 Random region = 30 bases = 4 30 possible sequences 11

12 Library Screening- In Vitro Selection All steps of this process are carried out under controlled laboratory conditions Precisely control the nature of the aptamer-target interaction Aptamers can be isolated under a wider variety of conditions Aptamers binding to an unwanted target can be removed Specific release conditions can be included- Mild Elution 12

13 Aptamer Selection Scheme using Immobilized Target Schematic representation of a typical selection round. Negative selection involves incubation of the aptamer library with blocked beads (lacking target). Positive selection involves incubation of the aptamer library with blocked beads and target loaded beads. Unbound material is removed and discarded (along with washed off material). In early rounds, the bound aptamers are recovered by thermal denaturation. In later rounds this is replaced with an elution. Material recovered from target and non-target beads are then quantified and compared by qpcr. Material from the target beads is then amplified (by PCR) and used in the subsequent round of selection 13

14 qpcr Quantitation Aptamer Recovery for a Model Protein qpcr data from a single elution round of selection. Buffer eluted material (green) and heat eluted material (yellow) from the target loaded beads is compared with recovery from non-target beads (red) and a standard gradient (blue). Aptamer enrichment observed for buffer eluted material that binds to the protein target 14

15 Post Selection Evaluation of Final Aptamers Pool % Recovery demonstrated that final enriched Aptamer population binds to the target immobilized beads and recovered upon elution, but not to the negative beads Recovery of specific aptamers was observed with selection buffer alone (blue) or selection buffer +50% of mock clarified harvest Impurities in clarified harvest did not affect Aptamer binding to the target 15

16 Individual Aptamer Isolation and PCR Amplification Bacterial cloning The Aptamer population is inserted into a commercially available vector and then used to transform a lab strain of E. coli A single Aptamer per bacterial cell. Single colony is then picked and the aptamer insert is amplified by PCR. 16

17 Individual Aptamer Immobilization and Binding Kinetics to the Target Protein BLI (Biolayer Interferometry ) traces demonstrate that immobilised individual aptamer sequences have a range of affinities for target protein Selected Aptamers with appropriate binding and release properties were sequenced Selected ssdna aptamers were synthesized 17

18 Affinity Resin Development using Aptamer as Ligand Top performing Aptamers sequenced and generated with chemical modification on one end; Aptamers immobilized on activated beads; Unbound aptamers washed from resin followed by chemical blocking Affinity resin washed with buffer followed by a feed stock (additional blocking) Target protein then incubated with the affinity resin followed by wash and elution Analyzed by SDS-PAGE Aptamer clone 1 Unselected aptamer population or bland resin (similar result) Additional optimization for resin development/column development ongoing 18

19 Case study 2: Biological affinity ligand (camelid antibody, VHH), A CaptureSelect Technology from Thermo Fisher 19

20 CaptureSelect affinity ligands Technology based on single-domain [V H H] antibody fragments Combining antibody based selectivity and process robustness Unique screening technology for target specificity, mild elution & stability Animal origin free (AOF) production process in yeast Safe and well tolerated by humans Products in commercial purification processes (US and EU) Thermo Fisher S c i e n t i f i c 20

21 Library Screening for a Model Protein Llama Immunization Boost-1 Library Boost-2 Library ~200 clones Enrichment Pre-absorb with mock harvest Reducing concentration of target protein Selectivity Binding Profiles Binding KD (kon, koff) Elution screening Direct ELISA Capture ELISA IBIS MX96 (SPR) 12 candidates 4 top candidates DNA sequencing Clonality pi Sequence liability Family Thermo Fisher S c i e n t i f i c 3 prototype Affinity resins 21

22 Three Prototype Affinity Resins for Column Testing Testing with harvest material Testing with purified model protein A280 A280 Affinity resin 1 Affinity resin 2 Affinity resin 3 Affinity resin 2 Affinity resin 1 Affinity resin 3 Thermo Fisher S c i e n t i f i c Using either purified model protein or the harvest material containing the model protein, a clear elution peak was observed in all three prototype affinity resins SDS-PAGE revealed one step purification of the model protein from unpurified harvest material Lower binding affinity was observed (protein breakthrough) suggesting additional optimization of base beads and ligand density is needed Ligand stability upon base treatment 22

23 Summary and Path Forward Binding Mild Elution Immobilized Ligand Impurities Purification target Complex Impurities Purified sample Customer ligand development- Finding protein A alike for recombinant proteins Lower COGs, increased yield and quality, fast time to market CaptureSelect technology well established for affinity ligand generation and affinity resin development, even for difficult molecules DNA aptamer shows promise as a synthetic ligand technology for future cheaper, faster affinity resin generation Thermo Fisher S c i e n t i f i c 23