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

    licensing terms.

  • 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%.

  • SUB Quality

    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

    Tox Manufacturing

  • Cell Line Development Technologies

    Vector Construction

    Transfection & Selection


    EnrichmentClone Isolation

    & ScreeningStability


    Strong vector w/ enhance element

    Transient Transfection


    Stable Pool


    High throughputcloning

    (FACS or ClonePix)

    Gene codonoptimization Shaker

    24-wp clone



    Process Development

    Process Development

    Cell Bank

    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




    by d


    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.

    1mL tips

    1mL or

    4mL tips

    Two culture stations; each holding 12 bioreactors.All 24 bioreactors independently controlled for pH and DO.

    Used Tips Discard


    ambrTM Technology

    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

    Bulk Fill

    UF/DF: formulation buffer exchange, concentration adjustment

    Virus filtration

    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

    structural stability

    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

    Wave 20/50

    Vial Thaw

    Xcellerex 2000L

    Xcellerex 200L

    Shake Flask


    Seed or Production Bioreactor

    Production Bioreactor

    Each passage 2 4 daysProduction 2 3 weeks

  • Production Options for Preclinical Tox Drug Substance Production

    Conservative Approach

    Engineering run


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