The Use of Disposable and Alternative Purification Technologies for

Biopharmaceuticals

Tom RansohoffBioProcess Technology Consultants

July 22, 2005IIR 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 biopharmaceuticalmanufacturing:• 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

1000 kg/year

Capital29%

Direct Labor16%Materials

29%

Quality & Indirect20%

Utilities & Waste6%

100 kg/year

Capital40%

Direct Labor16%

Materials & Consumables

13%

Overhead/Indirect Labor22%

Utilities & Waste9%

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

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 1 2 3 4 5 6 7 8 9 10

Time (yr)

Probability of Success Cost of Capital *

Preclin

Phase I

Phase II

Phase III

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 inearly-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,” BioProcessingJournal, July/Aug 2004.

• Zhang, S et al, “Large Scale Production of Clinical Grade Adenovirus Vectors – Introgen’sExperience,” 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

LimitedWave Bioreactors

Fermentation

Media Prep/Storage

Purification

Formulation/Fill

Buffer Prep/Storage Stedim Celsius

Stedim Bags, Hynetics Mixers

* * -- Suppliers listed are examples, notSuppliers listed are examples, notan endorsement of any specific company or technologyan endorsement of any specific company or technology

Downstream processing unit operations

Cell Breakage/Homogenization• For recovery of products

expressed intracellularlyRefolding• For some E. coli products

Crystallization/PrecipitationChromatography and adsorptive separations• Typical downstream process

includes 3 – 4 chromatography and/or membrane adsorber steps− Ion exchange− Hydrophobic interaction− Affinity− Size exclusion− Reverse phase

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

Commercial product requirement trends -biopharmaceuticals

2004 Sales by Mfg Technology/Prod Type

Mammalian Mab, $13.1

Mammalian rProtein, $17.8

Microbial rProtein, $15.4

• 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

Mammalian Products

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

Amount Required (kg/yr) N Ave. Sales Price ($/mg)

rProteinsMAbs

Source: BPTC 2004 Annual Manufacturing Capacity Analysis

Clinical biopharmaceutical pipeline is weighted towards antibody-based products

Pipeline Number of Products

0

10

20

30

40

50

60

70

Market BLA Phase III Phase II Phase I

Stage

Num

ber o

f Pro

duct

s

MammalianMicrobial

Pipeline Fraction MAb-based

0%10%20%30%40%50%60%70%80%90%

100%

Market BLA Phase III Phase II Phase I

Stage

Perc

ent M

Ab-

base

d

MammalianMicrobial

• 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 onlySource: BPTC 2004 Annual Manufacturing Capacity Analysis

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 dependentFor 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 <10 kg/yr• Most antibody products require 10-500 kg/yr

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 tosuccessful MAbs:

0.0100.0200.0300.0400.0500.0600.0700.0800.0900.0

Ritu

xan

Rem

icade

Enb

rel

Her

cepti

n

Ava

stin

Erb

itux

All o

thers

(38)

Product

Bul

k R

eqm

ts 2

004

(kg/

yr)

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

18

Biotechnology DivisionMembrane AdsorbersRemember when...Things change - now

Sartobind® Membrane Adsorbers

•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

19

Biotechnology DivisionMembrane AdsorbersRemember when...Things change - now

DNA removal

AfterAffinityStep

After DNAremoval

step

0

100

200

DNA (pg/mgprotein)

Average of eight 2,000 liter batches using 70 ml Sartobind

Average of three 12,500 liter batches using 500 ml Sartobind

...clears the DNAbelow detection limit

.J. K. Walter, Boehringer-IngelheimPharma in: Bioseparation andBioprocessing, G. Subramanian (ed.) Wiley VCH, 1998, Vol. II p. 447-460

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,” BiotechnolBioeng, 88(1) (2004).Slepushkin V et al, “Large-scale Purification of a LentiviralVector by Size Exclusion Chromatography or Mustang Q Ion Exchange Capsule,” Bioprocessing Journal, Sep/Oct 2003.

Column SpecificationsPressure Rating 10-20 bar

w/ module 33 barDiameters 1.2, 8, and 20 cmBed Length 5 – 30 cmBed Volume 5 mL – 5+ L

Courtesy of BioSepTec, Inc.

BIOFLASH 12™ and BIOFLASH 80™ Prepacked Columns

High performance packing in a disposable format

Ret. Time 4.77Efficiency 823Assym. 1.03

CM Toyopearl 650BioFlash 80(10 cm H)

Efficiency measured using 5% acetone in 0.1M NaClas mobile phase.

2.0 4.0 6.0 8.0

minutesCourtesy of BioSepTec, Inc.

Scale-up of recombinant protein separation on BioFlash pre-packed column

70 80

90 100

200

MM

NaC

l

1M NaCl

300

100

A280

Peak of Interest

200

MM

NaC

l

1M NaCl

300

100

A280

Peak of Interest

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.

Ransohoff, T et al, “Evaluation of BIOFLASH Prepacked ChromatographyColumns for the Large-Scale Purification of EPI-HNE-4 Protein over MacroPrep High S Media,” Purdue Chrom. Workshop (1999)

Introducing…

The Millipore Pod Filter PlatformImproves 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 HardwareSelf-contained, disposable PodsDisposable feed ports and fittingsNo product contact with endplates or process skid

Improved HandlingNo messy spent filtersLightweight, easy to set up and useNo 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+ m3 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 batchIncreasing 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 cultureprocesses,” 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 MaxProcess/Analytical Development Costs $1,500 $3,000Total Manufacturing Costs $475 $2,525 Cost per lot $400 $600 # of bulk lots $1 $4 Cost of DP mfg $75 $125TOTAL COSTS $1,975 $5,525

Scale 500 L Titer 1 g/L Yield 50% Output 0.5 g/L Clinical Requirements (g) 100 1000# of batches 1 4

Process Assumptions

Assumptions Cost of media $10,000 per L # of cycles per lot 10 Binding capacity 20 g/LVolume of media 2.5 LCost of use as a disposable $25,000 $100,000As a % of Total Costs 1.3% 1.8%As a % of Manufacturing Costs 5.3% 4.0%

Media cost of using disposable ion exchange chromatographyAssumptions Cost of media $1,000 per L

# of cycles per lot 10 Binding capacity 30 g/LVolume of media 1.25 LCost of use as a disposable $1,250 $5,000As a % of Total Costs 0.1% 0.1%As a % of Manufacturing Costs 0.3% 0.2%

Media cost of using disposable capture affinity chromatography

• 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

Mam Cell (Mab) Process Cost v. Scale

$0.00

$50.00

$100.00

$150.00

$200.00

$250.00

$300.00

$350.00

$400.00

$450.00

0 200 400 600 800 1000 1200

Scale (kg/yr)

Cost

of G

oods

($/g

)

Cost impact for disposable chromatography in large-scale manufacturing

Commercial Manufacturing Costs Min MaxTotal Manufacturing Costs ($M/yr) $40 $150 Cost per gram $400 $150 Annual requirements (kg/yr) 100 1,000Total Materials Costs ($M/yr) $5 $44 Materials Costs as % of Total Costs 13% 29%

Scale 15000 L Titer 1.5 g/L Yield 60% Output 0.9 g/L# of batches/yr 8 75

Process Assumptions

Assumptions Cost of media $10,000 per L # of cycles per lot 10 Binding capacity 20 g/LVolume of media per batch 112.5 LCost of use as a disposable ($M/yr) $9.0 $84.4Cost per g product produced ($/g) $90 $84As 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 chromatographyAssumptions Cost of media $1,000 per L # of cycles per lot 10

Binding capacity 30 g/LVolume of media per batch 56.25 LCost of use as a disposable ($M/yr) $0.5 $4.2Cost per g product produced ($/g) $5 $4As a % of Total Mfg Costs 1.1% 2.8%As a % of Total Matls Costs 8.7% 9.7%

Media cost of using disposable capture affinity chromatography

• 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 ComSource: Weidner J “Validation Considerations for Disposable Components and Systems,” presented at the ponents and Systems,” presented at the NC Biotech Symposium, RTP, NC Nov 18, 2004NC 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 challengesThere 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 303Acton, MA 01720

978 266-9154www.bioprocessconsultants.com

Carousel-type SMB evaluation of Protein A separation

Columns are mounted on a slowly rotating carouselThe columns on the carousel are connected to the pumps and vessels via a rotary valveSwitching 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)