Transcript BIOMAN 2011 CHO-tPA Production System Upstream Processing

BIOMAN 2011
CHO-tPA Production System
Downstream Processing
Mike Fino
MiraCosta College
Unit Operations
Many decisions to be made at each
step in the process
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Downstream Example
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Harvest Separation (Clarification)
• There are two technologies for removing the cell
mass from the solution containing the target protein
prior to loading onto columns:
– Centrifugation (e.g. disk stack)
– Filtration
• Dead-ended filtration (aka normal flow: membrane + depth)
• Crossflow membrane filtration (aka tangential flow)
• Crossflow membranes are preferred for large scale
operations and have many advantages
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Media and Cells In, Clarified Media Out
CLARIFIED
BROTH
SLUDGE
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NORMAL FLOW FILTRATION (NFF):
Traps contaminants larger than the pore size on the top surface of the membrane.
Contaminants smaller than the specified pore size pass through the membrane.
Used for critical applications such as sterilizing and final filtration.
MEMBRANE
DEPTH
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Sterilizing Filters:
Industry/Regulatory standard
• Capable of achieving an LRV >7 for a B. diminuta
challenge using ASTM methodology (per FDA
Guidelines)
– > 7 LRV means <1 microbe / 107 microbe challenge
– Doesn’t specify pore size or filter type
– B. diminuta model organism
• Sterilizing filters must be able to retain all
challenge microorganisms at a maximum
bioburden
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Tangential Flow Filtration
Clarification/Purification
Concentration
Buffer Exchange
Uses Crossflow to reduce build up
of retained components on the
membrane surface
Allows filtration of high fouling
streams or high resolution
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Different Size Pores in TFF
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What is Membrane Integrity?
Integral Membrane
Contaminants
larger than
pores upstream
No downstream
contamination
Non-Integral Membrane
Contaminants
larger than expected
pores upstream
Downstream
contamination
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Principles of Integrity Testing
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A benefit of membrane filters is the ability to
perform a non-destructive integrity test.
Testing ensures filtration SYSTEM integrity before,
during, or after filtration.
Membrane prefilters and depth filters cannot be
integrity tested with precision or accuracy because
of wide pore distribution.
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Reasons to Integrity Test
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Confirms manufacturers specifications
Assures integrity after steaming or autoclaving
Assures integrity before sterilization
Detects system leaks due to o-rings, gaskets,
faulty seals
Assures the correct pore size filter
Part of corporate standard operating procedure
GMP requirement
Audit requirement
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Two Basic Types of Integrity Test

Destructive
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Provided as a manufacturers
assurance of microbial retention.
 Bacterial Challenge
Non-Destructive

Provided to allow in-situ testing
 Pressure hold
 Bubble Point
 Diffusion
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Basic Elements of a
Bacterial Retention Test
Saline lactose
media w/
B. Diminuta
Test Filter
0.22 or 0.1 m disc or
filter cartridge
Assay Filter
(47mm MEC
disc)
MEC = mixed esters of cellulose
47mm disc
on TSA
TSA = tryptic soy agar
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Non-Destructive Integrity Test
Bubble Point
Open
pore
space
View of the membrane cross-section


Fully wetted membrane filters
hold liquid in their pores by
surface tension and capillary
forces.
Bubble point pressure is
inversely related to largest
pore diameter
Water
held
with
surface
tension
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What is Pressure Hold/Bubble Point?
Water Wet
Integral Membrane
Air pressure
upstream
greater than
specification
Water in pores is a
barrier to gas flow:
No gas flow observed
downstream until
upstream pressure
exceeds critical value
Water Wet
Non-Integral Membrane
psi
Air pressure
upstream
less than
specification
psi
Gas will flow through
large opening and is
easily observed downstream
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Inverse Relationship:
Pore size v. Bubble Point
• A sterilizing
filter has a
log
reduction
value of
greater than
7
Decreasing
pore size
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TFF System
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Retentate
Flow
Outlet Pressure
Permeate
Flow
Hollow Fiber
Feed Flow
Inlet Pressure
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PERISTALTIC PUMP:
Creates a gentle squeezing
action to move fluid through
flexible tubing.
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Introduction: TFF Layout & Operation
• Operating Steps:
–
–
–
–
–
–
–
–
Flush
Clean Water Flux
Pump curve
Integrity Test
Buffer Flush
Microfilter
Or Concentrate
Or Diafilter
initial
feed
diafiltrate
retentate
reservoir
feed
product
recovery
permeate
filter
feed
pump
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Key Parameters
• Feed Flow rate
– Flow rate leaving the pump
– Set by pump speed
• Transmembrane pressure (TMP)
– Average of inlet/outlet pressures
– Set by backpressure (retentate)
• Permeate control
– Flow rate through the fibers
– Set by backpressure (permeate)
– We don’t use this control in this cllass
• Membrane area
– Scales linearly
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Transmembrane Pressure (TMP)
Inlet Feed Pressure
Retentate Pressure
TMP = (Pin + Pout)/2 - Pperm
Pin = 30psi
Filter membrane
Pout = 20psi
Permeate
Pressure
We leave this
line
Pperm = 0psi unrestricted
TMP = (30 + 20)/2 - 0 = 25 PSI
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System Operation
Initial Feed
Diafiltration Buffer
Flush
Steps
•
•
•
•
Clean water flux
Pump Curve
Integrity Test
Filtration
Retentate
Tank
Pump
Membrane
Feed
Permeate
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Operation: Microfiltration
Trash
Collect and
Keep
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Operation: Microfiltration
Trash
Collect and
Keep
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Operation: Microfiltration
Trash
Collect and
Keep
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Operation: Microfiltration
Trash
Collect and
Keep
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Operation: Microfiltration
Trash
Collect and
Keep
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Operation: Microfiltration
Trash
Collect and
Keep
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Operation: Concentration
• Dewater the
retained solutes
• Procedures
Initial Feed
– Fill tank with
process fluid
– Start pump and
adjust system to
recommended
flows/pressures
– Remove permeate
Diafiltration Buffer
Flush
Retentate
Tank
Pump
Membrane
Feed
Permeate
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Operation: Concentration
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Operation: Concentration
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Operation: Concentration
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Operation: Concentration
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Operation: Concentration
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Operation: Concentration
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Operation: Concentration
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Operation: Concentration
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Operation: Diafiltration
• “Wash out”
permeable solutesproduct or
contaminants
• Procedure:
– Add diafiltration
buffer to the feed
tank at the same
rate that permeate
is being removed
from the system
Initial Feed
Diafiltration Buffer
Flush
Retentate
Tank
Pump
Membrane
Feed
Permeate
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Operation: Diafiltration
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Operation: Diafiltration
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Operation: Diafiltration
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Operation: Diafiltration
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Operation: Diafiltration
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Operation: Diafiltration
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Operation: Diafiltration
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Operation: Diafiltration
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Background: Virus Safety
Effective Clearance Steps
• Virus Filtration
– Large (enveloped) & small (non-enveloped) viruses
– Smallest parvovirus is about 50% bigger than an antibody
• Inactivation
– Low pH or Solvent detergent (enveloped)
• Chromatography
– Protein A Affinity for MAbs (enveloped & non-enveloped)
– Anion Exchange Flow through for MAbs (enveloped & nonenveloped)
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Types of Chromatography
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Column Chromatography
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Commonly employed downstream
processing methods
Processing
Method
Attributes
Benefits
Limitations
Clarification:
Sedimentation based
clarification
Continuous centrifugation
Normal flow Filtration
Microporous
Capable of handling very large
harvest volumes
Open process- contamination and
safety issues
Volume and throughput limited
Charged filter media
Cellulose pads
Tangential flow filtration
Contained systems
Capable of handling large harvest
volumes
Protein A Affinity
High throughput, high purity
High initial cost
Other affinity ligands
High throughput
Purity, regulatory acceptance
Cation exchange
Low cost media
Low throughput, feedstock
preconditioning
Chromatography
Ion exchange, HIC, IMAC,
hydroxyapatite
Variety of selectivities, high
capacity, robust
Often flow rate limited
Adsorptive membrane
Charged membranes
High throughput, contained, suited
to trace contaminant removal
Low capacities
Capture:
Chromatography
Purification:
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Typical contaminant clearance values
from each chromatography stage
Contaminant
Affinity
load
Host cell protein (ng/ml) 105
Intermediate
purification
load
Polishing load
103
10
Endotoxin (EU/ml)
106
10
<1
DNA (pg/ml)
106
103
102
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Common process constituents and
methods of removal or purification
Component
Culture harvest
level
Final product
level
Conventional
method
Therapeutic Antibody
0.1-1.5 g/l
1-10 g/l
UF/Cromatography
Isoforms
Various
Monomer
Chromatography
Serum and host proteins
0.1-3.0 g/l
< 0.1-10 mg/l
Chromatography
Cell debris and colloids
106/ml
None
MF
Bacterial pathogens
Various
<10-6/dose
MF
Virus pathogens
Various
virus filtration
DNA
1 mg/l
<10-6/dose (12
LRV)
10 ng/dose
Endotoxins
Various
<0.25 EU/ml
Chromatography
Lipids, surfactants
0-1 g/l
<0.1-10 mg/l
Chromatography
Buffer
Growth media
Stability media
UF
Extractables/leachables
Various
<0.1-10 mg/l
Purification reagents
Various
<0.1-10mg/l
UF/
Chromatography
UF
Chromatography
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Downstream Design
Overall Yield (%)
100
80
60
95% yield/step
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90% yield/step
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85% yield/step
0
1
2
3
4 5 6 7 8
Number of Steps
9 10
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Ion Exchange Chromatography
• If the charge on the bead is positive, it will
bind negatively charged molecules.
– This technique is called anion exchange.
• If the beads are negatively charged, they bind
positively charged molecules
– This technique is called cation exchange.
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IEC (cont’d)
• Thus, a scientist picks the resin to used based on the
properties of the protein of interest.
• During the chromatography, the protein binds to the
oppositely charged beads.
• Once the contaminant protein is separated from the
protein of interest, a high salt buffer is used to get the
desired protein to elute from the column.
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Ion Exchangers
• Ion exchange chromatography is based on
adsorption and reversible binding of charged
sample molecules to oppositely charged groups
attached to an insoluble resin
• The pH value at which a biomolecule carries no
net charge is called the isoelectric point (pI)
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IEX (cont’d)
• When exposed to a pH below its pI, the biomolecule
will carry a positive charge and will bind to a cation
exchanger.
• At a pH above its pI, the protein will carry a negative
charge and will to bind to an anion exchanger
• Depending on what pH the biomolecule is more stable
at will decide whether an anion or cation exchanger is
used
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Background for IEC of tPA
• SP Sepharose is a cation resin, which means
that positively charged molecules will bind to
the negatively charged resin.
• The extent of binding is dependent on the
cationic strength of the protein of interest and
can be manipulated by changing the pH and/or
conductivity of the buffers used in the
chromatography process.
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• The main proteins in the media used to grow
tPA are tPA, Bovine serum albumin (BSA),
insulin, and transferrin. Each protein has a
specific isoelectric point called the pI.
– BSA has a pI of 4.9
– tPA is 7.5 - 8.5
– transferrin is 5.9
– insulin is 5.3
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• We are able to selectively bind the tPA to the resin by
controlling the pH and ionic strength of the equilibration buffer
(aka Buffer A).
• At a pH of 6.0, tPA is more cationic (positively charged) than
either BSA or Transferrin.
• Therefore, the more positive charged tPA will bind to the resin
and the others will flow through the column and out to waste.
• tPA is then removed from the column using a high concentration
of salt, which competitively "bumps" the protein off the resin as
the sodium ions bind.
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Steps in Chromatography
•
•
Prime and de-bubble the system
Condition the column resin with a solution that promotes the binding of your protein
– Called Equilibration
•
Pump your sample solution over the column resin, which should bind as much of your
protein as possible
– Called Applying Sample
•
•
•
Everything that doesn’t bind goes to the drain.
At this point, your protein will stay bound to the resin indefinitely.
Now pump a solution over the resin that competes for binding on the resin with the
proteins from your solution.
– Called Elution
•
•
At some point, the competing solution will beat out the various proteins for position
on the resin and they will let go of the resin.
You will collect fractions along the way that can be frozen and analyzed later.
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ÄktaPrime Liquid Chromotography System
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ÄktaPrime Flow Path
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