T-cell Enrichment using MagCloudz platform

• Peripheral blood mononuclear cells (PBMC) were isolated from whole blood

• T-cell populations were enriched using bio-tinyated anti-CD3

• Data sets include Human Peripheral Blood and Umbilical Cord Blood

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MagCloudzStreptavidin

Target Cells

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T-Cell Enrichment from PBMC Fractions

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APPLICATION NOTE

100 Cummings Center, Suite 438N | Beverly, MA 01915 info@quadtechnologies.com | www.quadtechnologies.com

NOVEL BEAD-RELEASE TECHNOLOGY FOR MAGNETIC ENRICHMENT AND PURIFICATION

OF CD3+ T-CELLS FROM PERIPHERAL BLOOD

The isolation of T-cells or other immune-derived cells is an emerging interest for the development of immunotherapies and

translational clinical research where isolated cells must be magnetic label-free for the intended downstream application.

Quad Technologies’ MagCloudz™ Streptavidin with QuickGel™ technology is a unique platform, which enables the

isolation of label-free target cell populations in a biologically-friendly manner while preserving cell viability and native

morphology. Here, we present representative data highlighting the enrichment, purity and viability of T-cell populations

isolated from peripheral blood using our MagCloudz™ Streptavidin cell separation kit with biotinylated-anti-CD3 antibody

(clone UCHT1, eBioscience). Detailed protocols are available at www.quadtechnologies.com.

Figure 1: Representative flow cytometry dot plots for T-Cell enrichment and purification from peripheral blood.

Cell populations were stained with PerCP-conjugated anti-CD3, PE-conjugated anti-CD45, annexin-V-FITC (apoptosis

marker) and propidium iodide (viability marker) following the MagCloudz Streptavidin cell separation protocol. A) The

forward and side-scatter profile of the initial PBMC population; B) 56% CD3+/CD45+ T-Cells (target cell population) were

present in the initial sample; C) 92% T-Cell uptake as indicated by the low percentage of T-Cells remaining in the un-bound

cell fraction following MagCloudz Streptavidin binding; D) forward and side-scatter profile of the recovered CD3+ T-Cell

population; E) High purity T-Cells were recovered from the MagCloudz Streptavidin, enriched from 56% in the initial

population to 98% in the final isolated/released population and F) high viability (99%) of the recovered T-Cells, indicated

by the absence of annexin-V and propidium iodide staining in the bottom left quadrant of the plot. A total of 80% of the

target T-Cell population was recovered as magnetic label-free T-Cells from the MagCloudz Streptavidin in this assay.

80% recovered T-cells

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• Human umbilical cord blood (HUCB) was enriched for T-Cell populations:

T-Cell Enrichment from HUCB Fractions

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Confidential Information

CD

3

CD45

An

nV

7AADCD45

CD3

Attributes of Quickbeads

BF

FITC IgG-PE

Plug and play (easy to use) chemistry

Bio-friendly release

Mono-dispersed

Very-low settling velocity

100x great magnetic moment than conventional beads

Recovery Benchmark

Rec

ov

ery

[%

]

Strategic demonstrates protein purification: Quad’s MagCloudz yield much cleaner results

Positive Control

ConventionalApproach

Quad’sMagCloudz

Cell Sorting Features for CAR-T Immunotherapy

CellViability

≥ 70% ✔ ✔ X

Purity ≥ 80% ✔ ✔ ✔

Residual Magnetic Particles

100 magnetic particles ≤ 3,000,000 cells

✔ X ✔

High Throughput

> 5,000,000 cells/s

✔ ✔ X

Sterile Yes ✔ ✔ ✔

Ability to Multi-sort

CD4+CD8+ ✔ X X

DesiredFeatures*

Confidential Information *Carl June et. Al New England Journal of Medicine (2014)

Company M Company L

What is in the Box?

Summary

• First viable magnetic separation product with cell release capabilities

• Simple, short workflows with time and cost savings over competitors

• Our MagCloudz system achieves high purity, efficiency & excellent viability of isolated cells

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Confidential Information

Sean Kevlahan, Ph.D.skevlahan@quadtechnologies.com