Platforms for mAb commercialization

Abhinav A. Shukla, Ph.D.Senior Vice President

Development & ManufacturingKBI Biopharma, Durham NC

BPI West, San Francisco, February 27 – March 2, 2017

Durham, North Carolina- Cell Line Development- Cell Culture cGMP Manufacturing- Analytical QC, Formulation, Stability- Mass Spec Core Facility

RTP, North Carolina- Mammalian Process Development- Analytical development

Boulder, Colorado- Cell Line Development- Microbial Process Development- Microbial cGMP Manufacturing- Analytical, QC, Formulation, Stability- Particle Characterization Core Facility

The Woodlands, Texas- Cell Therapy

Manufacturing- Cell based assays

Contract Development & Manufacturing Organization

Programs in mammalian clinical manufacturing at KBI

• Primary emphasis 2011 – 2016 on clinical entry stage programs• ~ 10-14 IND filings per year supported via development &

manufacturing efforts• Also supported several stand-alone programs for process

characterization studies• Now emphasis shifting to include commercial launch process

development & manufacturing for mammalian cell culture programs• Boulder Colorado site already commercial ready for microbial

products

KBI Biopharma

Upstream Train I

Upstream Train II

ProA

VI

PolishVF

Bulk fill

KBI’s Cell Culture Manufacturing Facility

Purification Suite

2000L Prodn BRX200L Seed BRXWaveSF Harvest

2000L Prodn BRX200L Seed BRXWaveSF Harvest

3/9/20175 |

Mammalian cell culture expansion

• Leveraging significant capabilities in cell line development, analytical methods

• Development, formulation development & process development• Adding additional 2000L bioreactor in train II• Dedicated downstream purification for each train• Expanded buffer and media preparation integral with commercial suite• Increasing capacity to > 50 batches per year

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2013‐14 2015 2016

New Products Mfg Batches

Cell Culture Manufacturing in KBI Biopharma (Durham, NC)

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Biologics Commercialization

Pre-Clinical Phase I Phase II Phase III

Process DevelopmentProcess

CharacterizationProcess

ValidationProcess Monitoring

& Improvement

FIH Process• Deliver clinical process

quickly• Platform process• Clinical Supply

Submission & Approval

Lifecycle management

BLA Prep & PAI

Commercial Process• Deliver manufacturing process for

registrational trials and market• Design keeping large-scale manufacturing in

mind• Improve productivity, efficiency, robustness,

manufacturability, COGs• Analytical characterization and method

development

Process Characterization and Validation• Develop IPC strategy through understanding of process inputs and

outputs (design space)• Scale-down characterization and validation studies• Large-scale process validation to demonstrate process consistency• BLA preparation• Supporting documents for licensure inspections• Post-commercial process improvements (CI)• Post-commercial process monitoring

FIH process Commercial process

7Improvements in platform technology can enable one process development cycle

• Essential for biosimilars or highly accelerated programs

• Streamlined process characterization/validation effort

ProcessCharacterization

LifecycleManagementPlatform Application

IND BLA Commercial

Platform Technology Development

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Outline• Where are mAb platforms today and do they enable a

single cycle of development?• FIH process• Commercial process

• Can a platform approach be taken for commercialization• Process characterization studies leading to definition of an in-

process control strategy (IPC) – how fast can these be completed?

• Scale-down validation studies• Conformance lots

• Commercialization in single-use manufacturing facilities

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Investments in fundamental understanding of bioprocesses• Robust platforms can only be developed if there is a

strong understanding of the science of developing bioprocesses

• Multimodal chromatography• Platforms for non-mAbs (HIV vaccine proteins)• Improved Protein A resins

• Creates the ability to react quickly if an “unusual” observation is made

• All process decisions need to be made keeping large-scale production in mind

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050

100150200250300350400450500

0.0% 20.0% 40.0% 60.0% 80.0% 100.0%

HCP

 (ppm

)

Recovery

Capto MMC HCP Clearance25mM Tris pH 7.0 (baseline)

25mM Tris pH 7.0, 5% ethylene glycol

25mM Tris pH 7.0, 50mM arginine

25mM Tris pH 7.0, 50mM NaSCN

25mM Tris pH 7.0, 1M urea

25mM Tris pH 7.0, 1M ammonium sulfate

25mM Tris pH 7.0, 0.1M NaCl

25mM Tris pH 7.0, 0.5M ammonium sulfate

25mM Tris pH 7.0, 0.1M NaCl, 1M urea

Washes that disrupt protein-protein interactions

Conventional washes

log k’ = A – Blog(csalt) + C(csalt) k’ = (tr – tm )/tm

Wolfe, L., Barringer, C., Mostafa, S., Shukla, A. Multimodal chromatography: characterization of protein binding and selectivity enhancement through mobile phase modulators, Journal of Chromatography A, 1340, 151-156, 2014.

Multimodal chromatography

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High capacity Protein A chromatography resins

Resin Vendor Matrix Ligand Modified Protein A domain

Mean particlesize (µm)

MabSelect SuReTM GE HealthcareHighly cross‐linked 

agaroseAlkali‐stabilized 

rProtein AB domain 85

MabSelect SuReTM LX GE HealthcareHighly cross‐linked 

agaroseAlkali‐stabilized 

rProtein A B domain 85

ToyopearlTM AF‐rProteinA HC‐650F† Tosoh Polymethacrylate

Alkali‐stabilized rProtein A 

C domain 45

EshmunoTM A† EMD MilliporeCross‐linked

Polyvinyl EtherAlkali‐stabilized 

rProtein A C domain 50

AmsphereTM A3† JSR Life Sciences Polymethacrylate

Alkali‐stabilized rProtein A

C domain 50

1 2 3 4 5 60

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80mAb2

DB

C (g

/L)

Residence Time (min)

MabSelect SuRe MabSelect SuRe LX rProtein A HC-650F Eshmuno A Amsphere A3

mAb1 mAb2 mAb3 mAb40

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P Le

vel (

ppm

)

MabSelec SuRe MabSelect SuRe LX rProtein A HC-650F Eshmuno A Amsphere A3

CH505 Envelopes selected as vaccine immunogens

CH505 transmitted-founder (TF) and Env variants generated during viral evolution drove affinity maturation

of CH103 bnAb lineage

Antibody: UCAT/F gp120 Kd = ~200 nM

Env:

CH103

CH505 wk53

CH505 wk78

CH505 wk100

CH103 lineage intermediateantibodies

CH505 TF

CH505 wk136

12H.X. Liao et al. Nature 496: 469; 2013

• Parameters shaded in gray are defined across molecules. Parameters shaded in yellow require molecule specific optimization

• For all Env molecules the operating parameters, basal medium, feed type and some of the supplement additions have defined

• The need for additional supplements is molecule specific

• Reasons for supplement addition:

» Biocompatability in SU bioreactors

» Increase in productivity

ScaleTemperature Set point 37.0 ± 0.5°C Temperature Shift 33.0 ± 0.5°C on Day 6DO Set point 30%pH Set point 6.90 ± 0.1

Agitation (1-impeller) 50 rpm → 55 rpm

Air overlay 1.6 SLPMAir Sparge 0.5 SLPM Max. Oxygen Sparge 5 SLPMMax. CO2 Sparge 5 SLPM

Medium CD OptiCHO + 8 mM Glutamine

Target VCD 0.50 x 106 cells/mLBase 1M Sodium carbonate

Feed Type: LTI Feed A+B (1:1) 15% on Day 0, 10% current wv each on Days 3, 6, and 9

Supplement 1 addition: HT Supplement 1X current wv each on Days 0 and 4

Supplement 2 addition: CystineSupplement 3 addition: TyrosineSupplement 4 addition: Soy:Yeastolate Hydrolysate (2:3) 5g/L current wv each on Days 4 and 8

Supplement 5 addition: C1615Harvest Add 10g/L Hydrolysate on harvest

• Parameters shaded in gray are defined across molecules. Parameters shaded in yellow require molecule specific optimization

• Load and elution conditions for three of the unit operations require molecule specific definition given the heterogeneity of this class of molecules

• Env antigens structurally sensitive to hydrophobic surfaces, hence HIC not employed

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Log HCP

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Downstream Process

Platform HCP Clearance

TF Demo

TF ENG

TF GMP

w100 Demo

w100 ENG

w100 GMP

w78 Demo

w78 GMP

SEC‐HPLC % Main Peak

Sample ID TF Demo

TF ENG

TF GMP

w100 Demo

w100 ENG

w100GMP

w78Demo

w78 GMP

BDS 99.3% 98.9% 98.8% 98.9% 99.3% 99.2% 99.5% 99.6%

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MAB PLATFORMS

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Next generation mAb platforms• Driver

• High cell culture productivity is increasing interest in ultra-high loading polishing steps (> 100 mg/mL loading)

Genentech Biogen Millipore proposalProtein A

Viral Inactivation

Cation-exchange chromatography

Anion-exchange chromatography

Viral Filtration

UF/DF

Protein A

Viral Inactivation

AEX flowthrough

No salt Hydrophobic Interaction

Chromatography flowthrough

Viral Filtration

UF/DF

Protein A

Viral Inactivation

Anion-exchange flowthrough

Overloaded cation-exchange

chromatography

Viral Filtration

UF/DF

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Which platform should I use?

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Next generation mAb platforms• Platform processes for mAbs have hugely facilitated

the growth of mAbs as therapeutic agents• Rapid clinical entry with lower cost & resource burden

& significant time savings (gene to IND in ~ 12-14 months)

DS ProcessPlatform

DS ProcessPlatform

Cell line diversityCell line diversity

Media/feed type

diversity

Media/feed type

diversity

HCP level variabilityHCP level variability

Cell density variability

Cell density variability

HMW level variability

HMW level variability

Protein A

Viral Inactivation

AEX Based Polishing (Flow Through mode)

CEX Based Polishing

Viral Filtration

UF/DF

20Multimodal chromatography in next generation mAb platforms

• Mixed-mode has the simultaneous ability to clear HMW and HCP leading to mAb platforms with wider coverage

• Added advantage of ability to operate over wider conductivity range for loading

93.0

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Capto S ImpAct pH 5.0

Eshmuno CPX pH 5.0

Fractogel SO3 pH 5.0

Capto MMC pH 7.5

Selectivity Curves for HMW Clearance

Mai

n Pe

ak (%

)

Increase selectivity

Accumulated Yield (%)

Hydrophobicity scale: Capto S < Fractogel SO3 < POROS HS50 < Nuvia cPrime < Capto MMC

21Success of mAb platforms that include multimodal chromatography

• Can successfully accommodate wide range of cell lines and cell culture feed streams into a single downstream platform

• Cell lines from KBI, Bioceros, Selexis, Cellca, Excellgene, Antitope, Life Technologies

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ProA AEX CEX BDS

%H

MW

mAb Platform HMW Clearance

mAb A

mAb B

mAb C

mAb D

mAb E

mAb F

mAb G

mAb H

mAb I0

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ProA AEX CEX BDS

rHC

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mAb Platform rHCPClearance

mAb A

mAb B

mAb C

mAb D

mAb E

mAb F

mAb G

mAb H

mAb I

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MAPPING PROCESS DESIGN SPACE (PROCESS CHARACTERIZATION AND SCALE-DOWN VALIDATION)

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Quality by Design (QbD)• “Quality by design means designing and developing

manufacturing processes during the product development stage to consistently ensure a predefined quality at the end of the manufacturing process.” ICH Q10, FDA 2006

Process Design(Process Development)

Process ControlStrategyDefinition

ProcessValidation

Continued ProcessVerification

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Process Design Space

Higher level of assurance of product quality

Manufacturing Efficiency and Flexibility Continuous process

improvement while maintaining product quality

Characterization Space

Design space

Control space

Design Space (ICH Q8, 2006): The multidimensionalcombination and interaction of input variables (e.g., material attributes) and process parameters that have been demonstrated to provide assurance of quality.

Need a high throughputscale-down model forthe process

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Accelerating the Entire Product Development Lifecycle

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Accelerating process characterization & scale-down validation studies• Small-scale bioreactors (1-10L working volume) have

been the traditional scale-down model in industry till date

• Accelerating PC/PV studies requires a high-throughput scale-down model

• Ambr250 as a scale-down model for cell culture processes

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Mimicking “Cellular Environment” in SDMs.

Given the large sets of variables in a cell culture process, establishing a costand time-efficient SDM, mimicking a cellular environment similar to large scaleproduction bioreactor, is critical for conducting successful PC studies.

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Matching key process indicators in SDMs

Comparison of time courses for viable cell growth and lactate profiles for two recombinant CHO cell lines inambr™ SDMs for a mAb and a Biosimilar. Matching cell growth and lactate profiles for CHO cell linesproducing a mAb and Biosimilar respectively were key process indicators and in turn dictated the processyield and product quality.

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Comparison of SDMs across scales

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Accelerated Upstream PC Timelines with high-throughput SDMsMonth 1.5

SDMQ USP

Month 5.5

N-1/N-2 Screening(40 x 3L Seed)

Harvest PC Work12 -15 Harvest conditions

Month 0

Raw Materials and Worst Case (20 x 3L and 1 round of ambr250 runs: 24 vessels)

Main Stage PC (3 rounds of ambr250 runs: 72 vessels)

Inoculum Studies(100 Shake Flasks and 4 Wave Runs)

Worst-case Linkage USP/DSP

Month 7.0

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Scale-Down Process Validation Studies

• Scale-down validation studies in addition to large-scale process validation (conformance lots)

• Probe extremes in the process and demonstrate them to be acceptable

• Examples• Reprocessing validation – combine hold times with process

conditions that create the greatest stress on the protein• Intermediate hold times – combine hold times and

demonstrate releasable drug substance• Viral clearance studies• Impurity clearance studies

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Conclusions• Robust scientific understanding is a pre-requisite for

developing robust platforms that can make single cycle development possible

• Highly generic and manufacturing friendly mAb platforms have been designed (gene to IND in 12-14 months)

• Process characterization & scale-down validation studies can be accelerated (6-9 months) by using high throughput cell culture platforms as the scale-down model

• Single-use manufacturing is now a viable commercial launch platform (long term may combine single use manufacturing & higher productivity continuous manufacturing)

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mAb platforms

SU manufacturing

High through process development tools

Rapid process characterization & validation tools & approaches

Henry David Thoreau1817-1862, writer

Walden Pond Mahatma Gandhi1869 - 1948

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