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

CaridianBCT COBE 2991

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

Separation of Lymphocyte from Monocytes:

Counterflow Centrifugal Elutriation

CaridianBCT Elutra

Counterflow Centrifugal Separation of

Lymphocytes and Monocytes

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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

Cell Distribution by Elutra Fraction

Powell DJ et al. Cytotherapy. 2009;11(7):923-35

Cell Number by Elutra Fraction

Powell DJ et al. Cytotherapy. 2009;11(7):923-35

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

AXP AutoXpress – UCB Washing

BioE

•process cord blood processing closed system disposable kit for

volume reduction

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

Haemonetics Cell Saver 5

Adjustable pump speed and centrifuge speed

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?

•Andrea Brennan

•Anne Chew

•Anisha Chirmule

•Julio Cotte

•Aleksandra Kowalczyk

•Dawn Maier

•Eric Murray

•Christopher Nowaczyk

•Dan Powell

•Tamara Tripic

•Ashley Vogel

•Zoe Zheng

Clinical Cell and Vaccine Production Facility http://www.med.upenn.edu/bmcrc/CCVPF/?CCVPF