A Manufacturer’s Perspective on Innovations in

Biomanufacturing

Abhinav A. Shukla, Ph.D. Vice President

Process Development & Manufacturing KBI Biopharma, Durham NC

IBC’s Biopharmaceutical Development & Production Week, Huntington Beach, CA, 2013  

Incremental vs. Disruptive Changes •  Incremental changes improve upon existing

technology • Disruptive changes offer a new way of doing things •  Both are important drivers of innovation

Gottschalk, Brorson & Shukla, Nature Biotechnology, 30(6), 489-491, 2012

Biopharmaceutical Innovation • ROI on biopharmaceutical products steadily

decreasing in the last 30 years •  Approaching 8-10% cost of capital levels •  Paradigm shifts occur most commonly when there is a

driver

What is driving change in the biopharmaceutical world?

•  Increased demand for biopharmaceuticals •  Both number of products and quantity produced •  Biosimilars

• Reducing ROI on pharmaceutical investment •  Increased competition

•  Multiple drugs for the same target/indication •  Expanding geographies for production

•  Lower scales for commercial production •  Personalized medicine •  Higher cell culture titers

• Novel proteins and other biologics •  PAT & QbD initiatives – maintaining high quality is an

ever present constraint

Increased demand for biopharmaceuticals Larger number of products •  > 900 biopharmaceuticals in development for > 100

diseases •  > $ 114 billion sales

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PhRMA Biotechnology Report, 2011

Increased demand for biopharmaceuticals Follow-on biologics

• All major markets have biosimilar legislation now • Comparability hurdles are being overcome • Large players are increasingly entering this segment

Decreasing ROI in pharmaceutical R&D investment •  Average Internal Rate of Return on Investment (IRR)

7.5% vs. 12% in the late 1990’s • Heading towards cost of capital

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Deloitte Thomson Reuters, Fundamental Productivity Challenge in Pharma, 2011

Increased competition Multiple drugs for the same target/indication

Rheumatoid arthritis biologic drugs • Enbrel • Remicade • Humira • Orencia • Simponi • Cimzia • Actemra • Rituxan

Increased competition Expanding geographies

89 results out of 710 cannot be plotted.

Lower scales for commercial production Higher cell culture titers

• Increase in cell culture titers will drive downstream process innovation • Continuous separations & Non-chromatographic separations are growing

Lower scales for commercial production Personalized medicine • Smaller product volumes • Omics, metabolic profiling and systems biology will all grow personalized therapeutics • Cost of sequencing 1 Mbp of DNA now <$1 instead of $10,000 in 2001

-Confidential-

Novel proteins based on protein engineering

Human antibody:

Superposition

QbD/PAT initiatives •  Increased emphasis on product safety •  Increased emphasis on process understanding •  Increased use of statistics in the bioprocess space •  Innovations in biosensors & “measure to control”

strategies

Innovations in the biopharmaceutical process & manufacturing space

•  > 100 fold titer improvement in cell culture since rDNA technology began to be used for biopharmaceutical production

•  Single-use manufacturing •  Increasing dynamic binding capacity (DBC) of chromatographic media •  Introduction of parvoviral grade filters & improvement of flux

properties of viral filters •  Membrane chromatography •  Continuous processing in bioprocessing •  Cell free protein synthesis •  Pseudo affinity separations on non-affinity stationary phases •  Non-chromatographic separations •  Scale-down process characterization & validation studies as a key

component of BLA/MAA filings

-Confidential-

Innovations in the biopharmaceutical process & manufacturing space

•  > 100 fold titer improvement in cell culture since rDNA technology began to be used for biopharmaceutical production

•  Single-use manufacturing •  Increasing dynamic binding capacity (DBC) of chromatographic media •  Introduction of parvoviral grade filters & improvement of flux

properties of viral filters •  Membrane chromatography •  Continuous processing in bioprocessing •  Cell free protein synthesis •  Pseudo affinity separations on non-affinity stationary phases •  Non-chromatographic separations •  Scale-down process characterization & validation studies as a key

component of BLA/MAA filings

• 1996: Introduction of the Wave bioreactor • 1998: Introduction of first membrane adsorbers • 2004: First 250 L disposable stirred-tank bioreactor • 2006: First 1,000 L disposable stirred-tank bioreactor • 2009: First 2,000 L disposable stirred-tank bioreactor • Advantages:

•  Reduced risk of contamination •  Reduced need for SIP (Steam in Place) •  Reduced need for cleaning validation

• Now: •  Several manufacturing facilities and production trains with end-to-end disposable technologies •  Stainless steel facilities also make significant use of disposables for capacity extension and flexibility •  Multiple vendors for each single-use unit operation

Disposable Manufacturing

Why are single-use systems growing? •  Lower capital and utility costs (up to 40% reduction*) •  Increasing titers driving bioreactor scales smaller

•  Single-use bioreactors now up to 2000L volume

•  Increased universalization of biomanufacturing •  Co-location of manufacturing with markets •  Biosimilars (estimated $ 17 billion market by 2020) •  Smaller market sizes for novel drugs in niche/personalized

applications •  Market fragmentation making large single-product

manufacturing facilities redundant

•  Single-use systems finding application in stainless steel facilities for enhanced operational flexibility

Laukel et al, BioProcess International, May 2011 Supplement, pp. 14-21.

Ever-present need for accelerated early-stage process development & manufacturing

-Confidential-

Media and Feed preparation utilizing disposable mixing, filtration and storage systems

Disposable shake flasks or disposable spinner flasks

MCB or WCB vial

Disposable expansion reactor

Disposable seed bioreactor

Disposable production bioreactor

Disposable fluid path centrifuge

Disposable depth filtration system

0,2 µm  filter

Hold vessels (Bags)

Hold vessel (bag)

Disposable fluid path purification system

Disposable mixing tank

0,2 µm  filter

Retentate

Permeate

PD

Disposable fluid path purification system

Disposable mixing tank

0,2 µm  filter

BPC

Virus  filter

BPC

0,2 µm  filter

BPC

BPC

Sterile bulk fill and sampling bags

Buffer preparation utilizing disposable mixing, filtration and storage systems

0,2 µm  filter

Disposable fluid path UF/DF system

Aseptic connection

Hold vessel (bag)

Hold vessel (bag)

Hold vessel (bag)

Hold vessel (bag)

Hold vessel (bag)

Process Reproducibility

4 manufacturing runs in Single Use Bioreactors

Highly consistent process

-Confidential-

Scalability

• 4 different scales • 3L and 15L scales in non-disposable bioreactors

• Process performance with different working volumes is also reproducible

Single-use technologies in downstream processing • Centrifugation (kSep® Systems)

•  Closed, continuous centrifuge with class VI product contact surfaces

•  Counteraction of Centrifugal force and fluid flow force •  Very low shear •  Continuous operation •  Reversal of flow direction empties the chamber •  Up to 7.2 L/min

Single-use technologies in downstream processing • Depth filtration:

•  Harvest depth filters have traditionally been single-use except for their holders

•  Based on particle entrapment in a fibrous bed •  Can be used as the primary cell separation step for smaller cell

culture harvest volumes •  Millipore – POD® system •  Pall - Stax® system •  Sartorius – Sartoclear P ® •  Cuno – Zeta Plus ®

Pall – Stax System

Millipore - POD

Single-use technologies in downstream processing • Chromatography

•  Membrane adsorbers •  Mustang® (Pall), Sartobind® (Sartorius), Chromasorb® (Millipore),

Adsept® (Natrix), •  Q, S, HIC and salt-tolerant ion-exchange functionalities •  Most widely used for trace impurity removal in a flow-through mode

(DNA, endotoxin, viral clearance) •  Pre-packed chromatography columns

•  ReadyToProcess (GE Healthcare), Opus (Repligen), GoPure (Life Technologies)

•  Monoliths •  CIM monoliths (BIA Separations), Uno monoliths (Biorad)

Up to 20 cm D available

What is next for single-use systems? •  Further expansion of scale (up to 5000L?) •  Better systems for integrating unit operations

seamlessly • More vendors for downstream single-use technologies

(columns, UF/DF) •  Improved biosensors for single-use systems •  Improved standardization of systems

•  Extractables studies from vendors •  IQ/OQ documentation and system controls from vendors

-Confidential-

Shukla, A., Mostafa, S., Wilson, M., Lange, D. Vertical Integration of Disposables in Biopharmaceutical Drug Substance Manufacturing, Bioprocess International, 10(6), 34-47, 2012. Gottschalk, U., Shukla, A. Single-use disposable technologies for biopharmaceutical manufacturing, Trends in Biotechnology, 31(3), 147-154, 2013.

How can existing downstream process steps be made more efficient? • Most current chromatographic steps are designed to

remove impurities based on differential binding to the stationary phase surface • Conventional wisdom: wash conditions are between

binding and elution conditions • Orthogonal approach à disrupt impurity-product

interactions

Washes that disrupt protein-protein interactions

Conventional washes

Enhancing HCP clearance across Protein A • HCPs form a diverse set of impurities • HCP clearance is a key concern in biopharmaceutical

separation processes

• Conventional wisdom: use washes with a pH intermediate between load and elution solutions to wash the Protein A column post-loading

-Confidential-

Enhancing HCP clearance across Protein A

Washes can be developed to disengage HCPs from the product rather than disrupt product-Protein A ligand interactions

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MAbSelecteluate (load =

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Prosep A eluate(load = null

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Prosep A eluate(load = null

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Normalized Yield vs. normalized CHOP for a variety of washes on MAbSelect Protein A

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Enhancing HCP clearance across Protein A •  Use washes at high pH (pH > 7) to preserve Protein A –

mAb interactions •  Include chaotropes in washes to disrupt HCP-mAb

interactions E v a lu a tio n o f in te rm e d ia te w a s h e s a t p H > 7 .0

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Shukla, A. Hinckley, P. Host cell protein clearance during Protein A chromatography - development of an improved column wash step, Biotechnology Progress, 24, 1115-1121, 2008.

Mixed Mode Chromatography

•  Takes advantage of more than one type of interaction •  Can reduce process steps •  Provides enhanced selectivity, “pseudo-affinity” •  Several mixed mode resins have recently been developed with:

»  Increased loading capacities »  Higher ionic strength tolerance

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Mode

GE Healthcare, Capto MMC ligand

Ionic interactions

Hydrophobic interactions

Hydrophobic interactions

Ionic interactions

GE Healthcare, Capto Adhere ligand

Log k’ vs Log [NaCl]

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Log  k'  

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Lysozyme  

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1M  urea  

5%  ethylene  glycol  

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pH  7.0  

1M  urea  

5%  ethylene  glycol  

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Monoclonal  an6body  

pH  7.0  

1M  urea  

5%  ethylene  glycol  

50mM  arginine  

Wash development on mixed mode

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HCP  (ppm

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Recovery  

Capto  MMC  HCP  Clearance  25mM  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  

25mM  Tris  pH  7.0,  0.1M  NaCl,  1M  urea,  5%  ethylene  glycol  

25mM  Tris  pH  7.0,  0.1M  NaCl,  1M  urea,  5%  glycerol  

•  Selective wash strategies can eliminate one chromatographic step in non-mAb processes •  Pseudo-affinity separations by combining mixed mode interactions with highly selective mobile phase modulators

Process Analytical Technology •  PAT: “a system for designing, analyzing and

controlling manufacturing through timely measurements (i.e. during processing) of critical quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring final product quality” FDA Guidance

Hou, Y., Jiang, C., Shukla, A., Cramer, S. Improved Process Analytical Technology (PAT) for Protein A chromatography using predictive PCA tools, Biotechnology and Bioengineering, 108(1), 59-68, 2011.

-Confidential-

Innovations in Biopharmaceutical Process

Development & Manufacturing

“E pluribus unum”

Biochemical/Chemical Engineering/Biochemistry/Molecular Biology

Pharmaceutical Manufacturing

(continuous processing)

Statistics (QbD)

Materials Science (resins, membranes)

What does the plant of the future look like?

or