Mike Fino
MiraCosta College

Unit Operations
Many decisions to be made at each step in the process
2

Downstream Example
3

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
4

CLARIFIED
BROTH
SLUDGE
5

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

• Capable of achieving an LRV >7 for a B. diminuta challenge using ASTM methodology (per FDA
Guidelines)
– > 7 LRV means <1 microbe / 10 7 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
7

8

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
9

10

Different Size Pores in TFF
11

Integral Membrane
Contaminants larger than pores upstream
Non-Integral Membrane
Contaminants larger than expected pores upstream
No downstream contamination
Downstream contamination
12


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.
13









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
14


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

Saline lactose media w/
B. Diminuta
Test Filter
0.22 or 0.1
 m disc or filter cartridge
47mm disc on TSA
Assay Filter
(47mm MEC disc)
MEC = mixed esters of cellulose
TSA = tryptic soy agar
16

Open pore space
View of the membrane cross-section
Water held with surface tension

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
17

Water Wet
Integral Membrane
Air pressure upstream greater than specification psi
Water Wet
Non-Integral Membrane
Air pressure upstream less than specification psi
Water in pores is a barrier to gas flow:
No gas flow observed downstream until upstream pressure exceeds critical value
Gas will flow through large opening and is easily observed downstream
18

• A sterilizing filter has a log reduction value of greater than
7
Decreasing pore size
19

TFF System
20

Retentate
Flow
Outlet Pressure
Permeate
Flow
Hollow Fiber
Feed Flow
Inlet Pressure
21

22

PERISTALTIC PUMP:
Creates a gentle squeezing action to move fluid through flexible tubing.
23

• Operating Steps:
– Flush
– Clean Water Flux
– Pump curve
– Integrity Test
– Buffer Flush
– Microfilter
– Or Concentrate
– Or Diafilter diafiltrate product recovery initial feed reservoir feed pump feed filter retentate permeate
24

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
25

Inlet Feed Pressure
Pin = 30psi
TMP = (Pin + Pout)/2 - Pperm
Retentate Pressure
Pout = 20psi
Filter membrane
Permeate
Pressure
Pperm = 0psi
We leave this line unrestricted
TMP = (30 + 20)/2 - 0 = 25 PSI
26

System Operation
• Clean water flux
• Pump Curve
• Integrity Test
• Filtration
Initial Feed
Tank
Diafiltration Buffer
Flush
Retentate
Pump
Membrane
Feed
Permeate
27

Trash
Operation: Microfiltration
Collect and
Keep
28

Trash
Operation: Microfiltration
Collect and
Keep
29

Trash
Operation: Microfiltration
Collect and
Keep
30

Trash
Operation: Microfiltration
Collect and
Keep
31

Trash
Operation: Microfiltration
Collect and
Keep
32

Trash
Operation: Microfiltration
Collect and
Keep
33

Operation: Concentration
• Dewater the retained solutes
• Procedures
– Fill tank with process fluid
Initial Feed
– Start pump and adjust system to recommended flows/pressures
– Remove permeate
Tank
Diafiltration Buffer
Pump
Flush
Feed
Retentate
Membrane
Permeate
34

Operation: Concentration
35

Operation: Concentration
36

Operation: Concentration
37

Operation: Concentration
38

Operation: Concentration
39

Operation: Concentration
40

Operation: Concentration
41

Operation: Concentration
42

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
Tank
Diafiltration Buffer
Pump
Flush
Feed
Retentate
Membrane
Permeate
43

Operation: Diafiltration
44

Operation: Diafiltration
45

Operation: Diafiltration
46

Operation: Diafiltration
47

Operation: Diafiltration
48

Operation: Diafiltration
49

Operation: Diafiltration
50

Operation: Diafiltration
51

• 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)
52

53

54

Column Chromatography
55

Processing
Method
Clarification :
Sedimentation based clarification
Normal flow Filtration
Attributes
Continuous centrifugation
Benefits
Capable of handling very large harvest volumes
Limitations
Open process- contamination and safety issues
Volume and throughput limited Microporous
Charged filter media
Cellulose pads
Contained systems Tangential flow filtration
Capture:
Chromatography
Capable of handling large harvest volumes
Protein A Affinity
Other affinity ligands
Cation exchange
High throughput, high purity
High throughput
Low cost media
High initial cost
Purity, regulatory acceptance
Low throughput, feedstock preconditioning
Purification:
Chromatography
Adsorptive membrane
Ion exchange, HIC, IMAC, hydroxyapatite
Charged membranes
Variety of selectivities, high capacity, robust
Often flow rate limited
High throughput, contained, suited to trace contaminant removal
Low capacities
56

Contaminant
Affinity load
Intermediate purification load
Polishing load
Host cell protein (ng/ml) 10 5
Endotoxin (EU/ml) 10 6
DNA (pg/ml) 10 6
10 3
10
10 3
10
<1
10 2
57

Component
Therapeutic Antibody
Isoforms
Culture harvest level
0.1-1.5 g/l
Various
Serum and host proteins 0.1-3.0 g/l
Cell debris and colloids 10 6 /ml
Bacterial pathogens Various
Virus pathogens Various
DNA
Endotoxins
Lipids, surfactants
1 mg/l
Various
0-1 g/l
Buffer Growth media
Extractables/leachables Various
Final product level
1-10 g/l
Monomer
< 0.1-10 mg/l
None
<10 -6 /dose
<10 -6 /dose (12
LRV)
10 ng/dose
<0.25 EU/ml
<0.1-10 mg/l
Stability media
<0.1-10 mg/l
Conventional method
UF/Cromatography
Chromatography
Chromatography
MF
MF virus filtration
Chromatography
Chromatography
Chromatography
UF
UF/
Chromatography
UF Purification reagents Various <0.1-10mg/l
58

Downstream Design
100
80
60
95% yield/step
40
20
0
1 2 3 4 5 6 7 8 9 10
Number of Steps
90% yield/step
85% yield/step
59

• 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.
60

• 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 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

• 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

• 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.
64

• 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
65

• 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.
66

• 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.
67

68

69

ÄktaPrime Liquid Chromotography System
70

ÄktaPrime Flow Path
71