next generation recombinant protein manufacturing
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Next Generation Processes: What Model Works Best to Manufacture Recombinant
Proteins in Asia?
Thomas Jung, M.S.Vice President, Business Development
KBI Biopharma Inc.
BioPharma Asia 2017Suntec Convention
March 22, 2017
Internal Manufacturing vs CMO
Internal vs CMO Considerations
Target Market(s)Manufacturing Yield
Facilities CostAccess to Expertise
Location of CMO
Type of Molecule Innovator vs biosimilar Therapeutic antibody, recombinant protein,
antigen, peptide Stage of development: preclinical, early
clinical, late stage, commercial Indication Route of administration Dose: known or estimated Dosage will influence manufacturing
Target Market(s) Selected country or countries, region, or
global? Regulatory requirements may influence
manufacturing strategy. Clinical / commercial drug substance / drug
product requirements. Market scope will influence DS / DP
requirements and manufacturing demand.
Manufacturing Yield Production clone developed? RCB Available? Yield of current cell line adequate? Consider development of higher yielding cell
line. Higher yielding cell line reduces
manufacturing scale. Higher productivity cell lines / more costly
Internal Manufacturing Considerations
Costs Facility cost Equipment cost Development and manufacturing personnel cost Quality systems cost
Development Strategy Stage of development Exit strategy: Sell asset? At what phase? Partner with large pharma, biotech?
Risk Postpone internal manufacturing costs? Build internal manufacturing later, if needed?
CMO Option Advantages of Using CMO
Contract vs build (rent vs buy) Access to existing facilities Access to CMO expertise
Selection Criteria Strong and relevant experience Scientific expertise Implementation of latest innovations Quality systems / regulatory history Program management
Geographic location of CMO Close proximity not essential Good communications are critical
Single Use Bioreactor vs Stainless Steel
SUB vs SS Considerations
Scale of Manufacturing
SUB vs SS Comparison
Scale of Manufacturing
*Yields are based on upstream expression levels.with downstream recoveries assumed to be 70%.
Single Use Bioreactors:
No CIP/SIP reduces changeover time.
Significantly decreases possibility of product carryover or cross-contamination from one campaign to the next.
Increased facility throughput. Reduced timelines for sequential
batch manufacturing. Flexible manufacturing scale
matches process scale to DS requirements, reducing materials costs.
SUB Flexibility Flexible manufacturing scale of GE xcellerex SUB with
a 5:1 turndown ratio 2000L SUB working volume scalable from 400L to 2000L.
SUB maximum working volume of 2000L could be limitation.
Multiple DS batches at 2000L may satisfy DS requirements e.g. 2 X 2000L and possible pooling for purification as single batch.
SUB shows good comparability to SS such that transfer to larger scale SS bioreactor can be made for late stage / commercial, if needed.
SUB vs Stainless SteelSingle Use Facility Relative to Stainless Steel
Water Usage 87% reductionCleaning Chemicals Usage 95% reductionEnergy (Electricity) Demand 30% reduction
Facility Footprint 38% lessSteelwork 62% lessHeadcount 21% lower
Plastic Waste 880 kg increaseCO2 Emissions 26% reduction
Comparison based on 3 X 2000L MAb Production Scale*
* The Environmental Impact of Disposable Technologies By Andrew Sinclair, Lindsay Leveen,Miriam Monge,Janice Lim,Stacey Cox
Accelerated Manufacturing Timelines
Time is of the Essence
Bring innovative therapies to patients sooner.
Innovative biologic drugs improve patient survival, quality of care, and quality of life.
Intense competition for share of biosimilar market requires early approval and entry into market to be
successful.High cost of drug development requires shortening time
to return on investment for venture capitalists and investors.
Need to exploit any opportunities to shorten clinical trial / regulatory approval process without sacrificing quality.
Opportunities to AccelerateCell Line Development
Cell Culture Process Development
Purification Process Development
Analytical Method Development
Drug Product Formulation Development
Cell Line Development Technologies
Transfection & Selection
Strong vector w/ enhance element
(FACS or ClonePix)
Gene codonoptimization Shaker
High Throughput clone selection (Clonepix, FACS) screens largernumber of clones and selects clones based on productivitycompared with traditional limited dilution cloning.
Shaker 24-Well Plate Clone Screening
0 5 10 15 20 25 30 35 40Cl
pClone Ranking in SF 7 day batch culture
New cloning screening process reduces timeline by 10 days. Shaker culture is introduced as early as possible, so that the clones screened out fit into the downstream scale up model.
Good correlation between two steps
High Throughput Cell Culture Process Development
Basal medium, feed medium, and process parameter studiescan be performed at 9-16 ml scale rather than 2L or 10Lworking volume. Conventional process development timelinesof 3-4 months can be reduced to 1-1.5 months using ambr.
Two culture stations; each holding 12 bioreactors.All 24 bioreactors independently controlled for pH and DO.
Used Tips Discard
Microbioreactor(9-16mL working volume)
Scale Up Studies
Cell Growth Titers Product Quality Attributes
Comparison across scales for the production of a recombinant glycoprotein in a recombinant CHO cell line.
The process decisions and results from ambrTM were reproducible to other scales.
Platform mAb Purification Process
UF/DF: formulation buffer exchange, concentration adjustment
Chromatography (can be flow through)
Purification IEX chromatography
Low pH viral inactivation
Protein A capture chromatography
Cell separation by depth filtration
High Throughput Analytics
LabChip GXII has capability to run specific assays for Protein Glycan, Protein Charge Variant (CZE), and Protein Molecular Weight
Octet instrument is used for quick turnaround ProA-based titer analysis of diluted, high concentration, in process cell culture samples.
High Throughput Analytics allow for rapid titer determinations and assessment of critical quality attributes, leading to faster decisions and reduced timelines in cell line development and process development.
Analytical Utilize Platform Methods as Appropriate
Protein Primary Structure Peptide Sequencing via LC/MS/MS Amino Acid Analysis Peptide Mapping
Biophysical Characterization CD, FTIR, DSC, DLS, fluorescence
Capillary and Slab Gel Electrophoresis CZE SDS-CGE cIEF and icIEF SDS-PAGE and IEF Western blot Microchip electrophoresis 2D gels and blots
Glycan Analysis Oligosaccharide mapping Monosaccharide composition Sialic Acid Quantitation
Process Residuals ELISA (HCP, protein A etc.) HPLC (antibiotics, IPTG, detergents, etc) qPCR (DNA)
HPLC Size Exclusion (with MALLS) Ion Exchange Reverse Phase Hydrophobic Interaction Affinity
Potency Assays Binding Assays via ELISA, Biacore and
ForteBio Cell Based Assays (e.g., proliferation,
cytokine release, etc.)
Mass Spectrometry Intact mass Peptide mapping with LC/MS or
LC/MS/MS Disulfide Mapping Post translational modifications (e.g.,
oxidation, deamidation) PEGylation site identification Glycan Identification & site identification
Preformulation: Design of Experiments (DOE)
30-40 Candidate Formulations:
Various buffer, pH, ionic strength, and excipient conditions
Biophysical Screening: DSC, DLS, CD, FTIR, Fluorescence
Select Candidate formulation(s)
with appropriate thermal and
High Throughput Preformulation screening approach selects optimal formulations to carry into forced degradation and final dosage development based on biophysical characteristics and guided by statistical analysis.
Platform Expansion Process
Seed or Production Bioreactor
Each passage 2 4 daysProduction 2 3 weeks
Production Options for Preclinical Tox Drug Substance Production