BIOTARGET™-1 Xeno-Free Medium for Immunology

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BIOTARGETTM is optimized for cell growth, viability and functionality

BIOTARGETTM delivers consistent results under serum-free conditions

BIOTARGETTM is xeno-free, reducing the risk of immunogenicity

BIOTARGETTM eases the transition to clinical research

BIOTARGETTM is the cost-effective alternative for growing human mononuclear cells.

BIOTARGETTM medium is available in many customized packaging configurations,

including Sartorius-Stedim Cultibags

BIOTARGET™-1: Advancing the field of Immunotherapy

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Standardizing immunology research and elucidating the complex cellular interactions

involved in immune response, requires effective and consistent tools.

BIOTARGET™-1 medium is one such tool, developed and optimized specifically for

culturing peripheral blood mononuclear cells (lymphocytes and monocytes).

Currently, mononuclear cells are grown in standard media and supplemented with

either human serum (A, AB) or foetal bovine serum. To eliminate the drawbacks

involved in serum supplementation, BI has developed BIOTARGET™-1, a serum-free

alternative.

The advantages of animal component-free media in culturing mononuclear cells are:

Elimination of non-specific growth factors and/or inhibitors, which interfere with ּ

cell activation

Elimination of potential introduction of pathogens

Allowing a reliable evaluation of the antigenic reaction (e.g. quantity of the

lymphokines)

Lot-to-lot consistency

During development, we screened hundreds of possible formulations variations,

and compared them (for viability, induction by mitogens, and other key parameters)

to serum supplemented media, as well as to a leading competition’s serum-free

medium.

Introduction

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• Xeno-free

• Chemically defined

• No added cytokines

• Comprehensive validation studies

• CE Mark

• Manufactured in cGMP compliant, ISO 13485:2003 and ISO 9001:2008 certified

facility

1. Mitogen mediated activation of mononuclear cells (PHA, CON.A, OKT-3)

2. Lymphoid cell mediated activation of mononuclear cells (RAJI, PEER, BA,

MOLT-4, JURKAT)

3. IL-2 and IL-3 production from mononuclear cells

4. Long-term post- activation culture of mononuclear cells

5. LAK / TIL cell generation by mononuclear cell interleukin-2 mediated activation

6. Natural killer cell (NK) generation by mononuclear cell activation

7. Cytotoxic T cell generation by mononuclear cell activation

8. Activation of macrophages

9. Investigating various cytokines’ influence on the production of mononuclear cell

sub-populations

10. HIV proliferation

11. Retrovirus proliferation in T cells for vaccine development

12. Cytokine production in human mixed leukocyte reaction (MLR), BIOTARGET™-1

was found as effective as serum containing medium in supporting leukocyte

proliferation and IL-2 secretion7

BIOTARGET™-1 advantages:

Applications

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Multiple evaluation protocols were used in the development of BIOTARGET™-1.

For evaluation, mononuclear cells were prepared by pooling peripheral blood

received from Israel’s Central Blood Bank. After stringent testing for Hepatitis B and

HIV viruses, cells were centrifuged in Ficoll-paque containing tubes. Mononuclear

cells were thus concentrated in a layer between the Ficoll and the plasma. Cells

were then washed 3x in PBS and divided into medium containing microwells at a

concentration of 2x105 cells per well.

1. Mitogenic Activation of Mononuclear Cells (Figures 1-9)

Activation was evaluated using different mitogens such as PHA, CON.A and

OKT-3. Proliferation determined through radioactive thymidine uptake. In order

to provide comprehensive measurements, several mitogen concentrations were

tested and thymidine uptake was determined over several days

Figures 1-2: Mitogenic activation of mononuclear cells by varying concentrations of

CON.A

Evaluation Protocol

Figure 2: Day 7Figure 1: Day 5

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Evaluation Protocol

Figures 3-4: Effects of CON.A concentration on mononuclear cell activation

Figure 9: Effects of OKT-3 concentration on mononuclear cell activation

Figure 4: Day 7

Figure 6: Day 5

Figure 8: Day 8

Figure 3: Day 5

Figure 5: Day 3

Figure 7: Day 7

Figures 5-8: Effects of PHA concentration on mononuclear cell activation

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Evaluation Protocol

2. Lymphoid Cell activation of Mononuclear Cells (Figures 10-11)

Mononuclear cell were activated using various lymphoid cells, such as:

JURKAT, RAJI, MOLT-4, and BA. A number of tumor:mononuclear

cell ratios were examined, and proliferation was measured through

radioactive thymidine uptake.

Figures 10 and 11 Activation of mononuclear cells with lymphoid cells

Figure 12: CON. A

Figure 10: (RAJI) Figure 11: (BA)

Figure 13: PHA

3. Production of Lymphokines by Activated Mononuclear Cells (Figures 12-15)

The levels of lymphokines IL-2 and IL-3 were measured in mononuclear cell

cultures after activation with various mitogens. Relative IL-2 concentrations

were estimated using CTLL-2 cells, mouse cytotoxic T-cells that grow only in the

presence of IL-2.

Figures 12 and 13: Production of IL-2 by CON.A / PHA activated mononuclear cells (Supernatant on CTLL cells)

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Evaluation Protocol

Figures 14 and 15: Production of IL-3 by CON.A / PHA activated mononuclear cells

(supernatant on 32 D-CL-23 cells)

Figure 15: PHAFigure 14: CON. A

Tables 1 & 2. RAJI cell mediated activation of mononuclear cells (5 days)

Table 1. Proliferation of the mononuclear cells.

Relative proliferation denoted in counts per minute of radioactive Thymidine.

Mononuclear cells only Mononuclear cellsRatio RAJI cells

1/5 1/10FBS 3193 1154 4190

COMPETITOR A 361 314 2519BIOTARGET™-1 2939 771 5680

Table 2. Measurement of Cytotoxicity using RAJI cells as target cells.

Results expressed as percentage of specific release of radioactive

Chromium (Total release minus spontaneous release).

Mononuclear cells Ratio RAJI cells

1/5 1/10FBS 3.0 21

COMPETITOR A 2.5 6.0BIOTARGET™-1 12 11

4. Cytotoxicity (see Tables 1 and 2) Mononuclear cells were seeded

at a concentration of 106 cells per well together with RAJI cells treated with mitomycin C. Varying ratios of the two cell types were examined. Following activation (5-7 days), lymphocytes were collected, centrifuged, suspended in medium and seeded in microwells in order to measure proliferation and cytotoxicity. RAJI cells were labeled with radioactive chromium (100 ci in a volume of 0.2 ml), washed three times, suspended at a concentration of 105 cells per ml, and divided into microwells containing the activated lymphocytes. Cytolytic activity was evaluated after 18 hours of incubation by measuring the radioactive chromium released from the target (RAJI) cells.

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Custom Manufacturing and Formulation Services

Biological Industries (BI) is positioned to become your product formulation

and manufacturing partner.

Our extensive experience in liquid manufacturing and packaging for the

biological and biopharmaceutical markets makes partnering with BI an

obvious choice as your products progress from the laboratory and pilot

scales to full scale production.

Sterile filtration and aseptic filling in a controlled environment

clean rooms (graded from ISO 8 up to ISO 5)

Flexible packaging in industrial single-use bags (0.5-2000 Liter)

- Consistent quality

- Rapid delivery

Upstream and downstream formulation (i.e. buffers)

Optimization of media and liquid formulations

Research and pilot scale freeze-drying

Extensive range of Quality Control services

Manufacturing under stringent quality system

- ISO 9001:2008 and ISO 13485:2003 certification

- cGMP compliant facility

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References

1. E. Elinav, N. Adam, T. Waks and Z. Eshhar. Amelioration of Colitis by genetically engineered murine regulatory T cells redirected by antigen-specific chimeric receptor. Gastroenterology, Vol. 136, Issue 5, pp. 1721-1731, 2009

2. Yang-Ming Tseng, Sheng-Yi Chen, Chien-Hung Chen, Yi-Ru Jin, Shih-Meng Tsai, Ing-Jun Chen, Jang-Hwa Lee, Chzng-Cheng Chiu and Li-Yu Tsai. Effects of Alcohol-Induced Human Peripheral Blood Mononuclear Cell (PBMC) Pretreated Whey Protein Concentrate (WPC) on Oxidative Damage. Journal of Agricultural and Food Chemistry, 56 (17), pp 8141–8147, 2008

3. Q. Leng, Z. Bentwich and G. Borkow. Increased TGF-ß, Cbl-b and CTLA-4 levels and immunosuppression in association with chronic immune activation. International Immunology 18(5):637-644, 2006

4. D. Melamed, O. Messika, L. Glass-Marmor and A. Miller. Modulation of matrix metalloproteinase-9 (MMP-9) secretion in B lymphopoiesis. International Immunology 18(9):1355-1362, 2006

5. C. Rabinowitz and B. Rinkevich. Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges. Methods in Cell Science, Vol. 25, Numbers 3-4, 2004

6. F. Martí, E. Bertran, M. Llucià, E. Villén, M. Peiró, J. Garcia and F. Rueda. Platelet factor 4 induces human natural killer cells to synthesize and release interleukin-8. Journal of Leukocyte Biology 72:590-597, 2002

7. A. Bishara, R. Malka, C. Brautbar, V. Barak, I. Cohen and E. Kedar. Cytokine production in human mixed leukocyte reactions performed in serum-free media. Journal of Immunological Methods, Vol. 215, Issues 1-2, pp.187-190, 1998

8. G. Kampen , L. Poulsen, C. Reimert and P. Skov. A method for production and determination of histamine releasing activity from human

peripheral blood mononuclear cell. Journal of Immunological Methods, Vol. 210, Issue 2, pp. 185-193, 1997 9. S. Morecki, Y. Gelfand, S. Levi, A. Nagler, R. Condiotti, C. Nabet, A. Ackerstein, S. Slavin.

Activated long-term peripheral blood cultures as preparation for adoptive alloreactive cell therapy in cancer patients. Journal of Hematotherapy, 6 (2), pp. 115-124, 1997

10. Malka R.; Brautbar C.; Kedar E.; Cohen I.; Bishara A. Human mixed leukocyte reaction (MLR) performed in serum-free media and serum-containing medium. Human immunology, vol. 44, supp. 1, pp. 137, 1995

Product Name Catalogue

No.Unit Size

StorageTemp.

EZ Lympho-Sep™- Lymphocyte SeparationTubes

01-899-U 18 Or 30Tubes/Box

AMB

Human Serum Albumin (HSA Solution 10%),

05-720-1B 100ml -20ºC

05-720-1C 20ml -20ºC

05-720-1D 10ml -20ºC

Phytohemagglutinin-M (PHA-M), Lyophilized 12-006-1H 5ml 2-8ºC

Serum-Free Cell Freezing MediumProtein-free, Animal Component-Free

05-065-1A 500ml 2-8ºC

05-065-1C 20ml 2-8ºC

DCCM-1A high protein serum-free medium, designed for hybridoma cell growth and monoclonal antibody production.

05-010-1A 500ml 2-8ºC

05-010-1B 100ml 2-8ºC

DCCM-2A low protein serum-free medium, designed for hybridoma cell growth and monoclonal antibody production.

05-015-1A 500ml 2-8ºC

05-015-1B 100ml 2-8ºC

Low Protein Medium (LPM)Serum-free medium for the growth of a wide variety of hybridomas and other lymphocytes

05-040-1A 500ml 2-8ºC

05-040-1B 100ml 2-8ºC

BIOMPM-1, Multi-purpose SFMserum-free medium formulation for a wide variety of anchorage-dependent cells

05-060-1A 500ml 2-8ºC

05-060-1B 100ml 2-8ºC

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