HemaCare Your partner in Cell Therapy and Healthcare Research

Cryopreserved Leukopaks Maintain

Cell Viability and Functionality: A

Solution for Cell Therapy Logistics

Enabling allogeneic cell therapy product

and supply chain development in Europe

Lou Juliano, SVP, Global Sales & Business Development

Barcelona - September 29, 2015

Focus Session 3

A presentation on the results from an experiment

investigating viability and functionality over time of

leukapheresis collections procured within an FDA-

registered cGMP donor facility.

To determine whether cryopreserved leukapheresis materials can be a viable

and functional option for use in autologous and allogeneic cell therapies.

The Objective:

It all begins with quality starting material

Apheresis expertise

Quality of leukapheresis collection

Donor center that meets FDA cGMP/cGTP requirements

AABB Accreditation Donor eligibility that meets EU Directives

Process control using stringent SOPs

Fresh vs. cryopreserved starting material?

We performed an experiment on three normal healthy

leukapheresis collections...

We investigated the viability and functionality of lymphocytes and monocytes,

both fresh and cryopreserved, from leukopaks (leukapheresis collections)

procured within our FDA-registered cGMP donor collection facility

We evaluated fresh leukopaks (LP) from healthy donors for cell viability via

flow cytometry

Our goal: determine whether cryopreserved leukapheresis materials can

be a viable and functional option for use in autologous and allogeneic

cell therapies

Experiment Materials and Methods

Fresh LP were stained for cell count and cell distribution, then split into two

equal halves

One half incubated at room temperature for 24 hr (Day 1 LP) to simulate

shipping, the other half was cryopreserved in CryoStor® CS10 (BioLife

Solutions, Bothell, WA) the same day as draw

Cell count and distribution were quantified on the Day 0 LPs, Day 1 LPs,

and the Post-thaw cryopreserved LPs

CD3 and CD14 were isolated from the Day 0 LPs, Day 1 LPs, and Post-

thaw LPs (7 days later) using magnetic beads (Miltenyi Biotec, Bergisch

Gladbach, Germany)

Observed and compared T-cell proliferation ability by monitoring CFSE

division between Day 0 LPs, Day 1 LPs, and Post-thaw cryopreserved LPs

Observed and compared the differentiation ability of CD14 cells into

dendritic cells (DC) from Day 0 LPs, Day 1 LPs, and Post-thaw LPs

Results: White Blood Cell Counts

Total lymphocyte (CD45) counts between Day 0, Day 1, and Post-thaw

cryopreserved LPs were not statistically significant using a paired student t-test

(2.64 billion, 2.26 billion and 2.19 billion average cells per time point, p= 0.73).

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

3.50E+09

White Blood Cell (WBC) Counts

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

3.50E+09

White Blood Cell (WBC) Counts DAY 0 DAY 1 POST-THAW

Results: CD3 Counts

The number of CD3 in Day 0, Day 1, and Post-thaw cryopreserved LPs were not

statistically significant using a paired student t-test (1.03 billion, 0.93 billion,

and 0.92 billion average cells per time point, p=0.81).

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

3.50E+09

White Blood Cell (WBC) Counts

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

3.50E+09

White Blood Cell (WBC) Counts

DAY 0 DAY 1 POST-THAW

Results: CD14 Counts

The number of CD14 cells in Day 0, Day 1, and Post-thaw cryopreserved

LPs were not statistically significant using a paired student t-test (0.40

billion, 0.38 billion, and 0.40 billion average cells per time point, p=0.61).

DAY 0 DAY 1 POST-THAW

Results: Viability

Viability of the

cells was greater

than 99% in all

cases when gated

on FSC/PI.

Results: Post Culturing Cell Counts

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

Post Culturing DC Cell Counts

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

Post Culturing CD3 Cell Counts

Day 1

Post 7 day culture Post Thaw

Post 7 day culture

Day 1

Post 7 day culture

Post Thaw

Post 7 day culture

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

Post Culturing DC Cell Counts

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

Post Culturing CD3 Cell Counts

Day 1

Post 7 day culture Post Thaw

Post 7 day culture

Day 1

Post 7 day culture

Post Thaw

Post 7 day culture

Dendritic cell

generation was

similar between

the CD14 cells

isolated from

both the Day 1

(24 hr fresh)

(32.7 million)

and Post-thaw

cryopreserved

LPs (37.8

million), p=0.75.

Results: Purity of Dendritic Cells

Dendritic cell purity

was greater than

99% in all cases

when gated on

CD1c/CD45.

CD14 Cells Isolated from Day 1 Fresh Leukopak

CD14 Cells Isolated from Cryopreserved Leukopak

Results: Proliferation of CFSE Labeled CD3 T-cells

CD3 cells isolated from both the Day 1 and post-thaw cryopreserved LPs showed

similar high proliferation function. Starting counts were 30 million and the

proliferation amounted to 42 million and 39 million respectively, p=0.61.

CD3 T-cells Isolated from 24 hr Fresh Leukopak

CD3 T-cells Isolated from Cryopreserved Leukopak

Experiment Summary

Leukopaks from healthy donors, cryopreserved per the HemaCare collection and processing model, maintain viability and functionality of target cells. This presents a scalable option for emerging autologous and allogeneic cell therapies that require apheresis shipments from collection centers to cell therapy processing facilities around the world.

Confidence, delivered.

In conclusion...

are Who

we? HemaCare is a premier global supplier of blood products

and services customized to the unique needs of the

healthcare and scientific community. For 37 years, we have

been committed to providing unparalleled access to high

quality biological materials that inform medical research and

help save lives. With service available globally, we are a

trusted partner to some of the world’s leading

pharmaceutical companies and research organizations.

Founded as a

blood bank

Began

therapeutic

apheresis

operations

Participated in

leukapheresis

clinical trial

collections and

stem cell

collection

programs

Partnered with

Dendreon to

provide

apheresis

materials for

test runs and

began cellular

therapy clinical

trial collections

Began Provenge®

patient collections

Created BioResearch

Products & Services

Launched Cell Therapy

Solutions portfolio

Ability to provide

apheresis starting

material for cell therapy

process development

1970s 1980s 1990s 2000s 2010s

Our History

The HemaCare Advantage

FDA-registered, cGMP/cGTP compliant donor collection center AABB and CLIA accredited

37 years experience

Confidence, delivered.

Renowned scientific expertise

Leading-edge technologies

Global reach

Our donors make the difference

Rigorously validated and tested per AABB guidelines and IRB-approved informed consent

Highly profiled “pedigreed” donor database

HemaCare recruits and develops its own healthy donor pool for any research or cell therapy project

Our products and services

HUMAN-DERIVED PRIMARY CELLS

BIOPRESERVATION MEDIA

CELLULAR THERAPY SOLUTIONS

• Peripheral Blood • Mobilized

Peripheral Blood • Bone Marrow • Cord Blood

• Cryostor CS10® • Hypothermosol®

Leukapheresis collections for: • Process development • Immuno-cellular therapy clinical trials • Commercial immuno-cellular therapy (i.e. PROVENGE®)

Cellular Therapy Solutions

Apheresis Collection

Network

Cold Chain and Logistics

Cryopreservation and

Biopreservation Services

Patient Collections

within a

Standardized

Apheresis Network

Process Development Clinical Trials Commercialization

A highly flexible, modularized portfolio of Cellular Therapy Solutions

Customized Apheresis

for Process

Development

• CAR-T comparability runs

• Fresh and frozen

• Customized to meet

varying donor specs (e.g.

high neutrophils)

In conclusion...

At Hemacare, we're not just about the science. We are committed to

charting a path of growth by expanding our comprehensive portfolio

of research and cell therapy products that will touch the lives of

patients everywhere.

We demonstrate this through a compelling value proposition that

cannot be easily replicated:

Vertically integrated supplier to the industry

FDA-registered, cGMP and cGTP clinical collection capabilities

Donor management and apheresis collection expertise

spanning 37 years of performing over 250,000 collections