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

Downstream Example

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
+ depth)

(aka normal flow: membrane

Crossflow membrane filtration (aka tangential

flow)

Crossflow membranes are preferred for

large scale operations and have many
advantages
4

Media and Cells In, Clarified

Media Out
CLARIFIED
BROTH

SLUDGE

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
6

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
Doesnt 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
Non-Integral Membrane
Contaminants
larger than
pores upstream

No downstream
contamination

Contaminants
larger than expected
pores upstream

Downstream
contamination

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Principles of Integrity
Testing

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

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

Provided as a manufacturers
assurance of microbial retention.

Bacterial Challenge

Non-Destructive

Provided to allow in-situ testing

Pressure hold

Bubble Point

Diffusion

15

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
surfac
e
tensio
n

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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

19

TFF System

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Retentate
Flow
Outlet
Pressure

Permeat
e Flow

Hollow Fiber

Feed Flow
Inlet Pressure
21

22

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 dont use this control in this cllass

Membrane area
Scales linearly

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Transmembrane Pressure
(TMP)
Inlet Feed Pressure

Pin = 30psi

Filter membrane

Retentate Pressure

TMP = (Pin + Pout)/2 Pperm

Permeate
Pressure

Pout = 20psi

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 fluxTank

Pump Curve
Integrity Test
Filtration

Retentate

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
Initial Feed

Dewater the
retained solutes
Procedures

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

36

Operation: Concentration

37

Operation: Concentration

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Operation: Concentration

39

Operation: Concentration

40

Operation: Concentration

41

Operation: Concentration

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Operation: Diafiltration
Wash out
permeable
solutes- product
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

44

Operation: Diafiltration

45

Operation: Diafiltration

46

Operation: Diafiltration

47

Operation: Diafiltration

48

Operation: Diafiltration

49

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 & nonenveloped)
Anion Exchange Flow through for MAbs
(enveloped & non-enveloped)

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Types of Chromatography

53

54

Column Chromatography

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Commonly employed downstream processing methods

Processing
Method

Attributes

Benefits

Limitations

Clarification:
Sedimentation based
clarification
Normal flow Filtration

Continuous centrifugation

Capable of handling very large

harvest volumes

Microporous

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
Other affinity ligands

High throughput, high purity

High throughput

High initial cost

Purity, regulatory acceptance

Cation exchange

Low cost media

Low throughput, feedstock

preconditioning

Ion exchange, HIC, IMAC,

hydroxyapatite
Charged membranes

Variety of selectivities, high

capacity, robust
High throughput, contained, suited
to trace contaminant removal

Often flow rate limited

Capture:
Chromatography

Purification:
Chromatography
Adsorptive membrane

Low capacities

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Typical contaminant clearance values from each

chromatography stage

Intermediate
purification
load

Polishing load

Host cell protein (ng/ml) 105

103

10

Endotoxin (EU/ml)

106

10

<1

DNA (pg/ml)

106

103

102

Contaminant

Affinity
load

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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

95% yield/step

90% yield/step

85% yield/step

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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
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exchange.

IEC (contd)
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.

61

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)
62

IEX (contd)
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

63

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 doesnt 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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ktaPrime Liquid Chromotography System

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ktaPrime Flow Path

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