magnetic- and centrifugal-based cell sorting for clinical trials · •julio cotte •aleksandra...
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Magnetic- and Centrifugal-Based Cell
Sorting for Clinical Trials
Bruce Levine, Ph.D.
Division of Transfusion Medicine
Dept. of Pathology and Laboratory Medicine
University of Pennsylvania
• Following chemotherapy or stem cell transplants, T
cell counts and immune function is depressed for
months to years
• “Tumor and Viral Darwinism”: Tumors and many
viruses have evolved sophisticated means to avoid
immune detection.
• Hypothesis: Select, re-educate and expand
T cells ex vivo outside of tumor milieu and
re-infuse to prevent relapse and/or infections
Rationale for T Cell Therapy
Some Methods for Immune Cell
Separation/Enrichment
• Sedimentation (centrifugation)
• Gradient centrifugation (ficoll, percol)
• Centrifugation/filtration
• Counterflow centrifugal elutriation
• Adherence
• Magnetic based adherence
• Flow cytometry
COBE 2991 During Start/Spin
Hydraulic fluid (in green) pushes against membrane to force out supernatant. Closed
system disposable set. Limitation is not in cell number, but in volume since chamber
holds max of ~550mls with ~50mls dead volume in supernatant out mode
COBE 2991
Strengths Weaknesses
•Washing only, but can separate
cells with ficoll
•Very simple design and operation
•Flexible for variety of washing
protocols
•Closed system disposable set
•Kits relatively inexpensive
•Dead volume in chamber means
that washing residuals not nearly
as efficient as other systems
•If large volume of fluid, washing is
relatively slow because of load and
superout cycle, rather than flow
through design
Baxter CytoMate
Fully automated device designed for washing and concentrating white blood cell
products and fluid transfer. ~100-150mls/minute continuous flow
Baxter CytoMate
Strengths Weaknesses
•Efficient reduction of residuals
•Can deplete platelets and some red
cells through spinning membrane filter
•Closed system sterile disposable kit
•Relatively expensive instrument, kits
are less expensive
•Fairly slow, not useful for large
volumes above ~5 liters
•With large numbers of cells,
spinning membrane filter becomes
less efficient and concentration and
washing slower
•Discontinued
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CaridianBCT Elutra
Counter Flow Centrifuge
• Separation of cells based on size and
density into lymphocyte and monocyte
fractions
• Entire apheresis product can be
separated in 1-2 hrs
• Depending on settings, purity in
fractions can range ~70-99%
CaridianBCT Elutra
Strength Weakness
•Untouched cells, no antibodies or
reagents required
•Elutriation with Beckman instrument
has ~25 year history in research and
clinical labs
•Closed system disposable kit
reduces risk and cross-contamination
•Excellent technical support and
training from company
•Can only separate lymphocytes
from monocytes, and deplete
RBC’s, platelets
•Large space footprint
•Upfront $ investment, though less
than flow cytometry sorters,
CliniMACS, some other instruments
CaridianBCT Elutra
• Company provided:
– multi-day technical training
– Feedback on data collected during validation runs
– Letter of cross-reference to DMF
• Procedure modified for optimal lymphocyte
collection based on company experience with
settings for optimal monocyte collection
Equipment Validation Plan to Optimize
Lymphocyte Yield and Purity
Process cells through the Elutra using different
configuration profiles (alter flow rate, fraction
volume, # of fractions)
Count and size white blood cells, monocytes and
lymphocytes in apheresis product and each
fraction collected
Perform flow cytometry assays on apheresis
product and each fraction
Consider time, volume collected and ease of use
Dynal/Invitrogen Magnetic Beads
• Old instrument- Baxter MaxSep,
replacement- ClinExVivo is magnet
only, no pump, is designed to work
with any bag and closed system
tubing and bags, can be gravity flow
or attached to a pump
• Ab can be conjugated to beads
• Uncoated beads can deplete
monocytes.
Dynal/Invitrogen Magnetic Beads
Strengths Weaknesses
•Large magnetic moment, no
need to pass cells over column
•Safety: device is only a magnet,
no software
•Research reagents of similar
composition for scale-up or
validation studies
•Binding GMP Abs must be
obtained separately
•Cost for high cell number
separations
Miltenyi CliniMACS and Magnetic Beads
Clinical Grade Reagents
CD3 CD4 CD8
CD133 CD34 CD1c
CD19 CD25 CD304
CD271 CD56 CD45RA
Biotin IFN-g
•Magnet with clamps and pumps driven by software for each individual reagent, positive or negative separation
•Single-use closed system disposable kit.
•Magnetic beads are nanoparticles, smaller magnetic moment requires that bound cells be run through column filled with iron wool
•Selection time varies depending on % of target selection- for some ~2hrs, others may take ~6hrs
Miltenyi Magnetic Beads
Strengths Weaknesses
•Flexible system, most clinical
reagents
•Nanoparticles able to be infused on
positively selected products, can
process up to 120e9 cells
•Research reagents based on
similar platform allow for scale-up
•Closed system, have Device Master
File with FDA
•Support for clinical trials of their
new/selected reagents
•Cost, if depleting based on multiple
markers or sequential negative and
then positive selection.
•Depending on frequency of
population can be long process
•Ability to repetitively infuse
positively selected cells not
established, Abs are mouse, if
residual on infused cells, could
result in human anti-mouse
antibodies
Biosafe Sepax centrifugal separation in a closed system, capable of density gradient
centrifugation
variable volume (process products from 8 ml
and above)
closed system, sterile and single use
plasma
Buffy coat RBC
Biosafe Sepax
Strength Weakness
•closed system kit, flexible system
•Used for ficoll and small volume
separations of UCB, BM
•Relatively low volume throughput,
but this OK for the applications for
which it is marketed
•Instrument cost
Flow Cytometry Sorting
20,000 to 50,000 cells per second with advanced systems.
Disposable/autoclaveable tubing available on some sorters
can install in laminar flow for sample collection
Flow Cytometry Sorting
Strength Weakness
•Extremely pure cells from any subset of
cells for which antibodies are available
•Ability to deplete or enrich to defined
degree
•Widley accepted in research market
•Upfront very expensive investment,
$500K or more, and large footprint
•Reagents very expensive for large scale
separation
•Slow for large scale separations
compared with magentic or density
methods
•Positive selection can activate or
inactivate cells
•Removal of Ab may be an issue
•Lack of GMP reagents
Intraoperative Blood
Transfusion Equipment
•Dideco –Compact
–Electa Essential
load
reservoir
waste
product wash
Cell Separation Equipment Needs
and Items for Discussion
• Replacement for Cytomate?
• Efficient residual reduction
• Flow through >120 mls/min desirable
• Large Volume Harvester to Replace Fenwal
• Is Haemonetics sufficient?
• 10-25 L capacity?
• Multiparameter separation
• Magnetic or Flow cytometric?