The Use of Disposable and Alternative Purification Technologies for ...

The Use of Disposable and Alternative
Purification Technologies for
Biopharmaceuticals
Tom Ransohoff
BioProcess Technology Consultants
July 22, 2005
IIR Biopharmaceutical Production Conference

Presentation outline
� Driving forces for use of disposable and alternative
purification technologies
� Examples of current technologies
� Obstacles to implementation
� Future possibilities

Driving Forces

Driving forces for disposable and alternative
purification technologies
� Business drivers for alternative approaches to biopharmaceutical
manufacturing:
• Need to reduce capital investment in high-risk projects
• Speed to clinical proof-of-concept and commercial launch
� Operational trends favoring disposables:
• Increasing focus on cleaning and cleaning validation
• Increasing availability of suitable disposable processing equipment
throughout the biopharmaceutical flowpath:
− Flexible bioreactors
− Media and buffer bags
− Disposable aseptic filling equipment
� Trend towards large volume monoclonal antibody processes:
• Increasing productivity and scale of upstream processes
• Purification increasingly becoming a bottleneck

The majority of manufacturing costs for most
biopharmaceuticals are fixed costs
Overhead/Indirect
Labor
22%
Utilities & Waste
9%
Materials &
Consumables
13%
100 kg/year
Direct Labor
16%
Capital
40%
Quality & Indirect
20%
Utilities & Waste
6%
Materials
29%
1000 kg/year
Capital
29%
Direct Labor
16%
Data from a process economic model of a typical
mammalian cell culture-derived monoclonal antibody

Project risk and capital cost decrease
significantly as development progresses
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Preclin
Phase I
Phase II
Phas e III
0 1 2 3 4 5 6 7 8 9 10
Time (yr)
Probability of Success Cost of Capital *
BLA Approval
* - Cost of Capital estimate based on discount factors
from a risk-adjusted NPV analysis

Technologies that
• reduce capital investment and/or
• increase speed to risk-reduction milestones
are inherently more valuable in high-risk projects
⇒There is a compelling
financial and risk-management argument for
disposable manufacturing technologies in
early-stage biopharmaceutical development

Examples of Process Use of Disposable
Bioreactors
� Use of disposable bioreactors for production at 100L+ scale:
• Pierce, LN and Shabram PW, “Scalability of a Disposable Bioreactor from 25 L – 500 L
Run in Perfusion Mode with a CHO-Based Cell Line: A Tech Review,” BioProcessing
Journal, July/Aug 2004.
• Zhang, S et al, “Large Scale Production of Clinical Grade Adenovirus Vectors – Introgen’s
Experience,” presentation at WilBio Viral Vectors and Vaccines Conference (2004).
� Use of disposable bioreactors for production at smaller scales:
• Jenderek, S et al, “Development of a Production and Purification Method for Type 5
Adenovirus,” BioProcessing Journal, May/June 2005.
� Use of disposable bioreactors to replace conventional
bioreactors in the inoculum train of large scale processes:
• Knevelman, C et al, “Characterisation and Operation of a Disposable Bioreactor as a
Replacement for Conventional Steam in Place Inoculum Bioreactors for Mammalian Cell
Culture Processes,” ACS poster presentation (2002).

Disposable products* exist across most of
the biopharmaceutical manufacturing flowpath
Wave Bioreactors
Fermentation
Media Prep/
Storage
Limited
Purification
Buffer Prep/
Storage
Stedim Bags, Hynetics Mixers
Formulation/
Fill
Stedim Celsius
* - Suppliers listed are examples, not
an endorsement of any specific company or technology

Downstream processing unit operations
� Normal Flow Filtration
• Depth filtration for
clarification
• Nanofiltration for virus
removal
• Sterile filtration
� Tangential Flow Filtration
• Ultrafiltration for
concentration and buffer
exchange
• Microfiltration for
clarification
� Centrifugation
• Clarification
• Inclusion body isolation
� Cell Breakage/Homogenization
• For recovery of products
expressed intracellularly
� Refolding
• For some E. coli products
� Crystallization/Precipitation
� Chromatography and adsorptive
separations
• Typical downstream process
includes 3 – 4 chromatography
and/or membrane adsorber steps
− Ion exchange
− Hydrophobic interaction
− Affinity
− Size exclusion
− Reverse phase

Commercial product requirement trends -
biopharmaceuticals
Microbial
rProtein, $15.4
2004 Sales by Mfg Technology/Prod Type
Mammalian
rProtein, $17.8
Mammalian Mab,
$13.1
Mammalian Products
• Fastest growing commercial biopharmaceutical segment is
monoclonal antibodies at 40%+/year
• Trend for commercial products is from agonists to antagonists ->
larger volume, less expensive ($/g) products
3000.0
2500.0
2000.0
1500.0
1000.0
500.0
0.0
Amount Required (kg/yr) N Ave. Sales Price ($/mg)
Source: BPTC 2004 Annual Manufacturing Capacity Analysis
rProteins
MAbs

Number of Products
Clinical biopharmaceutical pipeline is weighted
towards antibody-based products
70
60
50
40
30
20
10
0
Pipeline Number of Products
Market BLA Phase III Phase II Phase I
Stage
Mammalian
Microbial
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Pipeline Fraction MAb-based
Market BLA Phase III Phase II Phase I
• Biopharmaceutical pipeline is 70% mammalian cell culture
• Significant trend towards antibody-based products is ~ 85%
of mammalian cell culture pipeline
• This analysis covers recombinant protein and monoclonal
antibody product candidates only
Percent MAb-based
Stage
Source: BPTC 2004 Annual Manufacturing Capacity Analysis
Mammalian
Microbial

Typical material requirements – clinical
development
� Pre-clinical development 0.1 - 2 kg
� Phase I 0.1 – 1 kg
� Phase II 0.5 – 5 kg
� Phase III 1 – 20 kg

Material requirements – commercial
biopharmaceuticals
� Highly product dependent
� For microbial fermentation-derived products,
commercial requirements range from:
• ~10 g/yr (e.g., Infergen) to…
• ~5,000 kg/yr (Novolin)
• Most products require 0.5-50 kg/yr
� For mammalian cell culture-derived products,
commercial requirements range from:
• ~10 g/yr (e.g., Bexxar/Zevalin) to…
• ~750 kg/yr (Rituxan)
• Most non-antibody products require

2004 bulk product requirements (kg/yr) –
mammalian commercial products
� Total estimated bulk product requirements for mammalian cell
culture commercial products (2004):
• All mammalian products – 2,875 kg/yr
• Monoclonal antibodies – 2,810 kg/yr
• rProteins – 65 kg/yr
� Significant growth in bulk requirements over recent years due to
successful MAbs:
Bulk Reqmts 2004 (kg/yr)
900.0
800.0
700.0
600.0
500.0
400.0
300.0
200.0
100.0
0.0
Rituxan
Remicade
Enbrel
Herceptin
Product
Avastin
Erbitux
All others (38)
Source: BPTC 2004 Annual Manufacturing Capacity Analysis

Current Approaches

Examples of current “disposable format”
purification products and technologies
� Membrane adsorbers
� Pre-packed chromatography columns or cartridges
� Disposable flow-path system concepts

Membrane Adsorbers
18
Remember when...
Things change - now
Sartobind ® Membrane Adsorbers
Biotechnology Division
•Matrix: stabilized and cross-linked cellulose >3µm
pore size
•Q,S,C,D and affinity ligands (ProtA, IDA, pABA, etc)
•Very low unspecific adsorption
•High chemical resistance against solvents,acids and
caustic solutions and autoclaving

Membrane Adsorbers
DNA removal
...clears the DNA
below detection limit
.
J. K. Walter,
Boehringer-Ingelheim
Pharma in:
Bioseparation and
Bioprocessing, G.
Subramanian (ed.)
Wiley VCH, 1998, Vol.
II p. 447-460
19
Remember when...
Things change - now
DNA (pg/mg
protein)
200
100
0
After
Affinity
Step
After DNA
removal
step
Biotechnology Division
Average of eight 2,000 liter batches using 70 ml Sartobind
Average of three 12,500 liter batches using 500 ml Sartobind

Other examples of process use of membrane
adsorbers
� Martin J, “Case Study – Orthogonal Membrane Technologies
for Viral and DNA Clearance,” presented at SCI Membrane
Chromatography Conference (2004).
� Haber C et al, “Membrane chromatography of DNA:
conformation-induced capacity and selectivity,” Biotechnol
Bioeng, 88(1) (2004).
� Slepushkin V et al, “Large-scale Purification of a Lentiviral
Vector by Size Exclusion Chromatography or Mustang Q Ion
Exchange Capsule,” Bioprocessing Journal, Sep/Oct 2003.

BIOFLASH 12 and BIOFLASH 80
Prepacked Columns
Column Specifications
Pressure Rating 10-20 bar
w/ module 33 bar
Diameters 1.2, 8, and 20 cm
Bed Length 5 – 30 cm
Bed Volume 5 mL – 5+ L
Courtesy of BioSepTec, Inc.

High performance packing in a disposable
format
CM Toyopearl 650
BioFlash 80
(10 cm H)
Efficiency measured using
5% acetone in 0.1M NaCl
as mobile phase.
2.0 4.0 6.0 8.0
minutes
Courtesy of BioSepTec, Inc.
Ret. Time 4.77
Efficiency 823
Assym. 1.03

Scale-up of recombinant protein separation on
BioFlash pre-packed column
A280
A280
70 80 90 100
Peak of Interest
Peak of Interest
Ransohoff, T et al, “Evaluation of BIOFLASH Prepacked Chromatography
Columns for the Large-Scale Purification of EPI-HNE-4 Protein over
MacroPrep High S Media,” Purdue Chrom. Workshop (1999)
1M NaCl
300
200
MM NaCl
100
1M NaCl
300
200
MM NaCl
100
1 cm (D) x 10 cm (H)
column
BioFlash 80 (8 cm D) x
10 cm (H)
Purification of EPI-HNE-4 on
Macroprep 25S at 100 cm/hr.

Introducing…
The Millipore Pod Filter Platform
� Improves process flexibility
� Decreases processing time
� More robust and scalable
� Easier to setup and use
� Minimizes product loss
� Enhances operator safety
� Reduces cleaning
requirements
� Improves process economics

Improved Handling and Ease of Use
� Improved CIP of Hardware
� Self-contained, disposable
Pods
� Disposable feed ports and
fittings
� No product contact with
endplates or process skid
� Improved Handling
� No messy spent filters
� Lightweight, easy to set up
and use
� No hoist or high ceiling
required

Impact of increasing cell culture titer and scale
on downstream processes
� Cell culture titers of 4 g/L are now achievable for monoclonal
antibodies:
• Wurm F, “Production of recombinant protein therapeutics in cultivated
mammalian cells,” Nature Biotechnology, 22(11) (2004).
• Smith M, “Strategies for the purification of high titre, high volume mammalian
cell culture batches,” presented at BioProcess International European
Conference and Exhibition (2005).
� New facilities are increasing bioreactor scale to 20+ m 3 to
meet requirements of large volume products:
• Lonza Portsmouth: 3 x 20,000 L (on-line) + 1 x 20,000 L (2006)
• Genentech Vacaville CCP-2: 8 x 25,000 L (2009)
� At 4 g/L, a 25,000 L bioreactor will yield 100 kg per batch
� Increasing production scale and titers present challenges and
opportunities for downstream process operation

Current approaches to managing high volume
processes
� At 4 g/L, a 25,000 L bioreactor yields 100 kg per batch
• At a loading capacity of 20 g/L during capture chromatography,
− 8 cycles on a 2 m (ID) x 20 cm (H) column (CV ~ 630 L) are required
− At 15 CV buffer per cycle, 75,000 L of buffer are needed per batch per
chromatography step
� Current approaches based on maximizing value of existing
conventional technology approaches (cf Smith*):
• On-line dilution to reduce buffer storage requirements
• Move to more rigid capture media to achieve higher velocities on
chromatography steps
• Multiple chromatography cycles (5-20) per batch
• Load chromatography columns at/near break-through
• Optimize UF steps to improve utilization of membranes
� These approaches work, but may have a limited lifespan ->
parallel track investment in novel technologies
* - Smith, M, “Strategies for the purification of high titre, high volume mammalian cell culture
processes,” presented at BioProcess International European Conference and Exhibition (2005)

Obstacles and Future Possibilities

Potential obstacles to implementation of
disposable or alternative technologies
� Cost
� Extractables and Leachates
� Inertia

Cost impact of disposable technologies
� The use of disposable-format purification
technology will generally:
• Reduce capital costs
• Reduce labor requirements for setup/cleaning/cleaning
validation
• Increase direct materials costs
� The magnitude of the direct material cost impact
for disposable chromatography is assessed for two
situations:
• Clinical manufacturing for an outsourced early-stage product
• Commercial large-scale manufacturing

Typical project costs – development and
clinical manufacturing (CMO)
Clinical Process and Analytical Development
Activity Time Approximate Cost
Cell Line Development (includes Vector
Construction, Transfection, Selection,
Identification of Final Clone)*
4 – 6 months $150,000 – 500,000
Master and Working Cell Bank Generation* 4 – 6 months $75,000 – 200,000
Process Development 8 months $500,000 – 1,500,000
Process Scale-up 3 months $300,000
Analytical Development and Qualification 9 months $150,000
Viral Clearance Validation* – (1-3 model
virus)
6 – 12 months $150,000 - $250,000
TOTAL - ~ $1.5-3 million
cGMP Manufacturing
• Clinical engineering and cGMP mfg lots ~$400k – 600k per batch (bulk)
• cGMP aseptic filling* of clinical lots ~$75k – 125k per lot
* - Activities that are frequently sub-contracted to other service providers

Cost impact for disposable chromatography
in clinical manufacturing
Total Project Costs - Clinical Manufacturing Min Max
Process/Analytical Development Costs $1,500 $3,000
Total Manufacturing Costs $475 $2,525
Cost per lot $400 $600
# of bulk lots $1 $4
Cost of DP mfg $75 $125
TOTAL COSTS $1,975 $5,525
Process Assumptions
Scale 500 L
Titer 1 g/L
Yield 50%
Output 0.5 g/L
Clinical Requirements (g) 100 1000
# of batches 1 4
Media cost of using disposable capture affinity chromatography
Assumptions
Cost of media $10,000 per L
# of cycles per lot 10
Binding capacity 20 g/L
Volume of media 2.5 L
Cost of use as a disposable $25,000 $100,000
As a % of Total Costs 1.3% 1.8%
As a % of Manufacturing Costs 5.3% 4.0%
Media cost of using disposable ion exchange chromatography
Assumptions
Cost of media $1,000 per L
# of cycles per lot 10
Binding capacity 30 g/L
Volume of media 1.25 L
Cost of use as a disposable $1,250 $5,000
As a % of Total Costs 0.1% 0.1%
As a % of Manufacturing Costs 0.3% 0.2%
• With non-disposable approach, initial investment in media will be at least
equivalent to single lot cost.
• Benefits of using disposable chromatography approach may be significant
enough in this application to justify modest additional operating costs:
•Improved manufacturing efficiency
•Improved process portability
•Reduced risk of cross-contamination

Summary of COG estimates for a “typical”
commercial-scale monoclonal antibody process
Cost of Goods ($/g)
$450.00
$400.00
$350.00
$300.00
$250.00
$200.00
$150.00
$100.00
$50.00
$0.00
Mam Cell (Mab) Process Cost v. Scale
0 200 400 600 800 1000 1200
Scale (kg/yr)

Cost impact for disposable chromatography
in large-scale manufacturing
Commercial Manufacturing Costs Min Max
Total Manufacturing Costs ($M/yr) $40 $150
Cost per gram $400 $150
Annual requirements (kg/yr) 100 1,000
Total Materials Costs ($M/yr) $5 $44
Materials Costs as % of Total Costs 13% 29%
Process Assumptions
Scale 15000 L
Titer 1.5 g/L
Yield 60%
Output 0.9 g/L
# of batches/yr 8 75
Media cost of using disposable capture affinity chromatography
Assumptions
Cost of media $10,000 per L
# of cycles per lot 10
Binding capacity 20 g/L
Volume of media per batch 112.5 L
Cost of use as a disposable ($M/yr) $9.0 $84.4
Cost per g product produced ($/g) $90 $84
As a % of Total Mfg Costs 22.5% 56.3%
As a % of Total Matl Costs 173.1% 194.0%
Media cost of using disposable ion exchange chromatography
Assumptions
Cost of media $1,000 per L
# of cycles per lot 10
Binding capacity 30 g/L
Volume of media per batch 56.25 L
Cost of use as a disposable ($M/yr) $0.5 $4.2
Cost per g product produced ($/g) $5 $4
As a % of Total Mfg Costs 1.1% 2.8%
As a % of Total Matls Costs 8.7% 9.7%
• Operating cost impact of disposable chromatography approach far greater
in this application.
• Alternative approaches required to significantly impact costs in large-scale
commercial manufacturing

Extractables and leachates
� Extractables and leachates are a potential concern with any
disposable technology.
� Level of concern increases as product purity increases.
� Weidner presented work at Biogen Idec to address regulatory
requests related to extractables:
• Risk-based assessment of extractables based on
− Process step
− Contact time
− Temperature
− Solvent
− Stage of development
− Vendor provided information on extractables and toxicological testing
• Conduct extractable tests where warranted based on potential risk
− Mass transfer principles guide test design
− Analytical methods for quantification and identification appropriate to situation
− Results assessed against acceptance criteria or by evaluation of toxicological risk
Source: Weidner J “Validation Considerations for Disposable Components Components
and Systems,” presented at the
NC Biotech Symposium, RTP, NC Nov 18, 2004

Inertia: driving forces against use of novel
technology and approaches
� There are significant costs and risks associated with process
innovation in any highly regulated industry
• Conservative approach to implementation of new technologies
• Security of supply is a concern that must be addressed
� The complexity of biopharmaceutical processes provide
additional challenges
� There is no good time to innovate: significant obstacles to
implementation of new technologies exist at every stage of
development
Result: New technologies often take longer than anticipated to
implement, even when a compelling need exisits

Future trends
� Increased use of disposable purification technologies,
particularly in smaller volume processes, driven by:
• Continued advances in and increased use of disposable systems across the
biopharmaceutical manufacturing flowpath
• Implementation of new materials that significantly reduce per liter costs and allow
increased throughput, potentially including:
− Cost-effective affinity media
− Novel membrane materials and adsorptive separation supports
� Increasing interest in alternative technologies and operating
approaches for large-volume processes as optimization of
conventional technologies matures, including:
• Operationg approaches that move away from “big batch” and towards continuous
purification processes (e.g., SMB-like processes)
• Integration of unit operations (i.e., semi-continuous capture and clarification)
• Implementation of novel (to biotech) technologies and unit operations

Summary
� Current existing technology meets some of the requirements
for cost-effective disposable purification solutions
• Membrane adsorbers
• Pre-packed columns
• Disposable-format systems
� Trends in biopharmaceutical manufacturing are increasing the
need for disposable or alternative purification technologies:
• Increasing use of disposable technologies to reduce capital investment
and product development times
• Increasing titer and scale of monoclonal antibody cell culture production
� The use of disposable and alternative purification technologies
will increase as:
• The trends towards disposable equipment and large volume processes
continue
• New technology and viable solutions are introduced that provide
solutions more broadly for purification unit operations

Thank you!
BioProcess Technology Consultants, Inc.
289 Great Road, Suite 303
Acton, MA 01720
978 266-9154
www.bioprocessconsultants.com

Carousel-type SMB evaluation of Protein A
separation
� Columns are mounted on a slowly rotating carousel
� The columns on the carousel are connected to the pumps and vessels via a
rotary valve
� Switching mode simulates continuous adsorbent flow
Courtesy of J. Thommes (Biogen Idec), from “Protein A Affinity Simulated Bed Chromatography for Continuous Monoclonal Antibody Purification,”
Presented at Recovery of Biological Products XI Conference, Banff Canada (2003)