BIOREACTOR SYSTEM DESIGN

CELL CULTURE SYSTEM DESIGN & SCALE-UP.INDUSTRY TRENDS FOR IMPROVED RELIABILITY AND

PERFORMANCE EQUIVALENCE.

By: Ted DeLoggio

BIOPROCESS CONSULTANTS

The Institute for International Research

BIOREACTOR SYSTEMS

Philadelphia, April 20, 1998

CURRENT DESIGN TRENDS

MAJOR TOPICS

• PROCESS DESIGN & SCALE-UP• BIOREACTOR• PIPING• BIOREACTOR SYSTEM

INTEGRATION• BIOPROCESS SYSTEMS

INTEGRATION

PROCESS DESIGN & SCALE-UP

COMMON CELL CULTURE SCALE-UP PROBLEMS“OBSTACLE OR OPPORTUNITY”

• [CO2] TOXICITY

• HYDRODYNAMIC SHEAR STRESS

• INTERFACIAL SHEAR

• BLEND TIME

• OTR REQUIREMENTS

• METABOLIC EQUIVELENCE

[CO2] TOXICITY

DESCRIPTION OF PROBLEM[CO2] sensitivity begins at 75-100 mmHg

Gas exchange occursas bubbles rise. Sizeand composition ofbubbles change.

BALANCE

•OTR•CTR

Gas exchange at surface.

Cells consume O2and produce CO2.CO2 reacts with water to form HCO3

-

CO2O2

CO2O2

HCO3-

+H+

CO2O2

CO2O2

[CO2]74 ppm

OUROTR

CO2O2

CTRCER

CO2O2

air overlay ~0.05 VVM

[CO2] TOXICITYSCALE-UP IMPACT OF “OUR” ON [CO2]

025

5075

100

125

150

0 2000 4000 6000 8000 10000 12000

Bioreactor Volume, liters

[CO

2], m

mH

g

0.5 OUR 1.0 OUR 2.0 OUR (mmol/L/hr)

[CO2] TOXICITY

Design and Operating Actions to Reduce the Riskof [CO2] Toxic Effects

• PREVENTIVE ACTIONS– USE DRILLED HOLE SPARGERS (>3000l)– MAXIMIZE P/V, REDUCE SUPPLIMENTAL O2– IMPELLER TYPE, CONSIDER (K/Np) RATIO ↓– CONSIDER STRIPPING SPARGER IF SINTERED

ELEMENTS ARE USED

• REMEDIAL ACTIONS– EVALUATE BUFFER SALT COMPOSITION– CONTROLLED CARBON SOURCE ADDITION– CONSIDER USE OF STRIPPING GAS– OPTIMIZE pH CONTROL LOOP TUNING

MIXING SCALE-UP PROCEDURES IN BIOTECHNOLOGY

All Procedures Require Geometric Similarity in Scale Up

CRITERIA PROCEDURE PROCESS EXAMPLE

Equal Mass Transfer Coefficients

(P/V)2 ≅ 1 (P/V)1

Microbial FermentationCell Culture

Equal Physical Shear to particles

(σ)2 ≅ 1 (σ)1

Cell culture

Equal Blend time or Vessel Turnover

(N)2 ≅ 1 (N)1

Rapid Kinetic Reactions

DESIGN SENSITIVITY PRESERVING VESSEL GEOMETRY IN SCALE -UP

Intrinsic Dependencies of Highlighted Parameters

- Tank geometry- Sparge Rate & Bubble Size- impeller geometry- baffles & internals

OTR = kLaSPG/H (C* - CL) + kLaSUR/H (Cov - CL)

kL - mass transfer coefficient

a - air-liquid interfacial area

kLaSPG = C(P/V)α(vs)β

P0 = Np ρ N3 di5

shear rate = K Ndi

Mass Transfer and Shear Functionality depend on Vessel Geometry

MAX

MIXINGESTABLISH THE REQUIRED AGITATION FOR YOUR SYSTEM

TipSpeed(ft/min)

1000M

500

CC

0

Mixing Requirements

1. Hydrodynamic Shear

2. OTR

3. Cell Suspension

MIN

VFD

HYDRODYNAMIC SHEAR

The shear v. mass transfer scale-up dilemma

Shear Rate response to Equal P/V scale-upP/V=50 W/m^3

20KL5000L

1500L

600L100L

30L

3L

0

5

10

15

20

25

30

0 20 40 60 80 100 120

Bioreactor Diameter (in)

Shea

r Rat

e (1

/s)

Impeller Zone Shear Rate

Bulk Fluid Shear Rate

Scale-up issues in Cell Culture-Mixing

Once the Design Basis for Mixing is defined (e.g. equal P/V), the controlling shear damage equation must be determined

• Average Shear Rate ~ KN

• Maximum Shear Rate ~ K’ND

• Cumulative Shear Stress

• ISF

• Kolmogoroff Eddy Scale

• Energy Dissipation

INTERFACIAL SHEARDESCRIPTION OF PROBLEM

du/dxBulk Flow

INTERFACIAL SHEAR PROTECTION

Agent Cells Killed Cells Killedper bubble # per batch %

None 1600 >100PEG 1000 >100PVA 450 >100Methocel 30 45F-68 5 8

SECONDARY EFFECTS OF F68• Reduces KL (minor)• Increases a (major)• Cell membrane interaction• Influences foam formation

EVALUATION OF O2 TRANSFER REQUIREMENT

OTRMECH > OURMET

OUR = ( u /YX /O) X= (4.5 x 10-10 mmolO2/cell/hr)(6 x 109 cells/L)= 2.7 mmol/L/HR THE GOAL

OTR = KLa /H*(C * - CL)H = 101330 Pa/atm / 86000 Pa-L/mmolO2(C * - CL) = (Ci –Co )/LN[(Ci –CL )/(Co - CL )](C * - CL) = 0.250 atm @ 21% O2(C * - CL) = 0.800 atm @ 60% O2 (50/50:air/O2)

KLa = 2.7/1.17*0.250 = 9.2 1/h air spargeKLa = 2.7/1.17*0.800 = 3.0 1/h 50/50 air/O2 sparge

CURRENT DESIGN TRENDS

MAJOR TOPICS

• PROCESS DESIGN & SCALE-UP• BIOREACTOR• PIPING• BIOREACTOR SYSTEM

INTEGRATION• BIOPROCESS SYSTEMS

INTEGRATION

BIOREACTOR DESIGN METHODOLOGY

GOAL:

Reproduce the metabolic and physical environment achieved at the current scale of operation to produce product at the new scale with equivalent quality and quantity. To assure safety, consistency, robustness, and validatability of the process per the customers

established product & process requirements.

PROCESS OBJECTIVES

• Define mixing, mass transfer, heat transfer needs

• Define execution “building blocks”

• Define component and line sizing

• Define reliability, safety, cost & compliance needs

• Detail the equipment arrangement

BIOREACTOR DESIGN METHODOLOGYDESIGN MILESTONES

BIOREACTOR DESIGN METHODOLOGYDESIGN MILESTONES

INTEGRATED PROCESS OBJECTIVES:

• FDA regulations - product quality and consistency

• Containment - product and worker safety

• Validation execution and documentation

• Maintenance Access

• SIP/CIP protocols

• Controls integration

• Downstream process integration

• Flow of materials and personnel

BIOREACTOR VESSEL DESIGN

• DESIGN FOR PROCESS, CIP & SIP

• PRESERVE GEOMETRIC SIMILARITY– HL/Dt = 1.5 (cell culture)– HL/Dt = 2.5 (Microbial)

• NOZZLES– ORIENT TOP ASSEMBLY FOR SPRAYBALL COVERAGE.– USE “SHOP” WELDED VALVE ASSEMBLIES.– SLOPE TO DRAIN, AVOID DEADLEGS.– USE FLUSH MOUNTED SAMPLE AND HARVEST VALVES.

• USE DIMPLED OR HALF-PIPE JACKET DESIGN– CONSIDER TWO SIDE WALL ZONES.– CONSIDER ONE BOTTOM ZONE

Large Scale Bioreactor DesignExample

• Operating Range• 18,000L –22,000L (Vw)• 28,000L (Vt)

• Aspect Ratio• HT/DT = 1.75• L/ DT = 1.43

• HL/DT• At 18,000L: 1.1• At 22,000L: 1.4

• Di/DT• Hydrofoil - 0.45• Hydrofoil - 0.45

• 4 Baffles • Width – 0.1DT• Clearance – 0.01DT

• Drilled Hole Sparge Element• 4 mm / 2 mm

193 (HT) 157 (L)

110 DT

HT= Total vessel inside height (in)

L = tangent to tangent height (in)

HL = Liquid height (in)

AGITATION CONSIDERATIONS

• USE BOTTOM MOUNTED DESIGN IF POSSIBLE

• DOUBLE MECHANICAL SEAL vs MAGNETIC– MAGNETIC OPTIONS IMPOSE SCALABILITY ISSUES.

SUITABLE FOR PROCESS VESSEL NOT BIOREACTOR– USE PRESSURIZED BARRIER FLUID– SELECT “BEST” SEAL NOT BIOREACTOR VENDOR

• PROVIDE “ALARM & RECOVERY METHOD FOR SEAL FAILURE

• USE DIRECT SPEED PICK-UP FOR ACCURACY

AGITATION CONSIDERATIONS

• IMPELLER TYPE, DIAMETER, AND NUMBER

• VESSEL, AGITATION, AND SPARGE DECISIONS ARE INTERDEPENDANT.

• CONSIDER ACCESS FOR MAINTENANCE

• SHAFT DESIGN & DEFLECTION CALCULATIONS.– Design for the worst case scenario and future impeller needs

• GEAR BOX FOR LOW SPEED OPERATIONS

BIOREACTOR SYSTEM INTEGRATION

• VESSEL ADDITIONS

• GAS MANAGEMENT AND CONTROL

• TEMPERATURE CONTROL

• EXHAUST

• AGITATION

• PROCESS CONTROL AND SENSORS

“BUILDING BLOCKS”

CURRENT DESIGN TRENDS

MAJOR TOPICS

• PROCESS DESIGN & SCALE-UP• BIOREACTOR• PIPING• BIOREACTOR SYSTEM

INTEGRATION• BIOPROCESS SYSTEMS

INTEGRATION

PROCESS PIPING

• SKID v. D&B v. GRAY SPACE OPTIONS• DESIGN AND SIZE FOR PROCESS, CIP & SIP• LOCATE CRITICAL COMPONENTS FOR

ACCESSIBILITY• REDUCE FIELD HAND WELDS• SLOPE AND DRAIN LOW POINTS• CONSIDER 3-D MODELING FOR INTERFERENCE• INTEGRATE DRAWINGS WITH “BOM” AND “PM”• CONSIDER 3rd PARTY FOR INSPECTION

– USE PHYSICAL WELD SAMPLES– AGREE ON SPECIFICATIONS AND INSPECTION

PROCESS PIPINGCIP DESIGN CONSIDERATIONS

• DEVELOP CIP SCENARIOS ALONG WITH PROCESS

• USE PARALLEL FLOW PATHS

• CONSIDER CIP SUPPLY AND RETURN FLOW REQUIREMENTS FOR LINE SIZING

• DEFINE STRATEGY TO VERIFY SECONDARY CLEANING PATH

• MODULARIZE AUTOMATION TO OPTIMIZE CLEANING CYCLES

PROCESS PIPINGSIP DESIGN CONSIDERATIONS

• USE AUTOMATIC TEMP. MEASUREMENT AND STER. VERIFICATION

• ALLOW 8-12” DISTANCE BETWEEN RTD AND TRAP

• GROUP RTDs FOR EASE OF CALIBRATION

• USE VACUUM STEP TO ELIMINATE AIR

• CONSIDER STEAM ADDITION AND VENT LOCATIONS

• ENSURE ADEQUATE SUPPLY AND VENTING