GMP-compliant expansion of mesenchymal stromal cells (MSCs) using the CliniMACS Prodigy® and xeno-free MSC-Brew GMP Medium

Kathrin Godthardt¹, Claudia Schreiner¹, Christian Schmidt², Ronny M. Schulz², Andreas Bosio¹, and Sebastian Knoebel¹, ¹Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, ²Universität Leipzig, Medizinische Fakultät, Klinik und Poliklinik für Orthopädie, Unfallchirurgie und Plastische Chirurgie, Leipzig, Germany

A B C 10³ 10³ Viable leukocytes 0.03%-2 191 events Introduction 425 events/mL 10² 10² A A 10¹ 10¹ Human mesenchymal stem cells (MSCs) hold great tegrated cell processing platform CliniMACS Prodigy® PE- PE- 100 promise for clinical use and cell therapy applications. To for i) density gradient centrifugation (DGC) of human Before DGC 1 1 0 0 Lorem ipsum 100 80 -1 -1 ensure highest quality and safety of the resulting cellu- bone marrow, ii) cultivation of MSCs starting from bone -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ 80 60 lar products, MSCs have to be maintained using stan- marrow mononuclear cells (BM-MNCs), and iii) passag- CD45-FITCFITC-A Lorem ipsum Anti-MSCA-1-APCAPC-A Lorem ipsum 60 10³ 10³ 40 dardized cultivation conditions and procedures. To this ing of MSCs. Viable leukocytes 0.05%-2 40 121 events

469 events/mL Recovery [%] 20 end, we have developed our xeno-free MSC-Brew GMP MSCs from human bone marrow were isolated by 10² 10² 20 [%] Residual content 0 Medium following the recommendations of USP <1043> plastic adherence using MSC-Brew GMP Medium and A 0 10¹ 10¹ Lymphocytes Monocytes MSCs Erythrocytes Platelets PE- After DGC on ancillary materials, thus enabling isolation and ex- automated feeding and harvesting procedures in a CD271-PE 1 1 pansion of MSCs from various tissue sources (e.g. hu- closed, single-use tubing set. Subsequently, these cells 0 0

CD271-PE -1 -1 man bone marrow, adipose tissue, and umbilical cord). could be replated and expanded within the closed -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³

To increase the level of process standardization and system, illustrating the feasibility of an automated cell CD45-FITCCD45-FITC Lorem ipsum Anti-MSCA-1-APCMSCA-1-APC Lorem ipsum product safety we developed a procedure using the in- production. Figure 2 Methods 2 Expansion of MSCs using the CliniMACS Prodigy® Human MSCs were isolated by plastic adherence from

Concept for clinical-scale MSC manufacturing using 5.0×10⁸ BM-MNCs using the CliniMACS Prodigy® and com- the CliniMACS Prodigy® pared to the manual laboratory standard (T175 flask). 1 4.5×10⁸ A clinically relevant number of MSCs was harvested The CliniMACS Prodigy® provides a range of ports for Bone marrow aspirate is transferred into the application 4.0×10⁸ after 14 days in the CliniMACS Prodigy using MSC- connecting bags containing buffer, media, and re- bag (Bag 1) of the tubing set in a sterile manner. The 3.5×10⁸ Brew GMP Medium (fig. 3). In this experiment we used agents. Various tubing sets allow for a multitude of ap- following steps are performed automatically by the 3.0×10⁸ 30 mL of bone marrow aspirate. We performed an au- plications. For the cultivation process, we chose the CliniMACS Prodigy using the Adherent Cell Culture Pro- 2.5×10⁸ tomated DGC as described in result 1 and continued tubing set CliniMACS Prodigy TS 730, which provides cess_Beta_0.4. These steps comprise the density gradi- the Adherent Cell Culture Process_Beta_0.4 with the 2.0×10⁸ up to eight connections for buffer and media and the ent centrifugation (DGC) of human bone marrow aspi- cultivation of BM-MNCs for isolation of MSCs. Subse- Number of MSCs 1.5×10⁸ possibility to pre-warm solutions during transfer from rate with the possibility of taking samples to determine Start: quently, all steps were performed within the closed an external 4 °C storage compartment to the cultiva- the cell number. Furthermore, an initial expansion step 1.0×10⁸ 1.0×10⁸ BM-MNCs system of the CliniMACS Prodigy. These steps com- (1.6×10⁵ BM-MNCs/cm²) tion and centrifugation unit (CCU) as well as external was performed using external tissue culture (TC) ves- 5.0×10⁷ prised: i) an initial expansion step starting with a den- tissue culture (TC) vessels which are connected to the sels, which are connected in a sterile manner to the tub- sity of 1.6×10⁵ BM-MNCs/cm² using a 1-layer stack, ii) 1.2×10⁴ tubing set (fig. 1A). Bags can be connected in a sterile ing set and are placed in an incubator next to the 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 media exchange and washing of cells, iii) semi-auto- manner prior to the installation procedure or later via CliniMACS Prodigy. All liquid handling steps, including Days of cultivation mated harvesting of P1 MSCs from the 1-layer stack sterile welding. The MSC-Brew GMP Medium is provid- washing the CCU, inoculation, medium exchange, and D0 D10 D14 and transfer to three 5-layer stacks connected to the ed in a bag with a PVC tube for reliable, sterile welding cell harvest can be performed automatically, except po- tubing set in a sterile manner, and iv) semi-automat- to the tubing set. tential handling of the external culture vessel (fig. 1B). 1-layer TC three 5-layer TC ed harvesting of P2 MSCs from three 5-layer stacks. In 636 cm² 9540 cm² comparison, all steps were performed manually using A B CliniMACS Prodigy Manual processing standard T175 cell culture vessels.

Bag 1 (Bone marrow aspirate) Bag 2 Day 0: Density gradient centrifugation and Figure 3 Bag 3 (Target cell bag: BM-MNCs) culture start Bag 4 (Target cell bag: MSCs P1) • Install tubing set • Connect all media and solutions • Density gradient centrifugation Characterization of MSCs Dissociation reagent Dissociation Trypsin inhibitorTrypsin Ficoll® Paque GMP MediumMSC-Brew • Seeding of BM-MNCs in connected 1-layer stack 3

CliniMACS® Buffer + 0.5% HSA + 0.5% Buffer CliniMACS® Days 2 / 5 / 8: Culture maintenance • Closed-system cell wash and/or media exchange Bag 5 (Target cell bag: MSCs P2) Day 9: Split • Semi-automated harvest

CliniMACS® Buffer CliniMACS® • Automated cell wash 132 45678910 • Storage of cells in Target cell bag Side scatter Side scatter Side scatter Side scatter Side scatter (possible QC and cell count sampling) 99.92% 99.89% 99.77% 0.75% 0.23%

23 24 11 Relative cell number Relative cell number Relative cell number Relative cell number number cell Relative 12 • Seeding of P1 MSCs in max. 3 connected 5-layer stacks -1 01 10¹10² 10³ -1 01 10¹10² 10³ -1 01 10¹10² 10³ -1 01 10¹10² 10³ -1 01 10¹10² 10³ 13 XXXCD73-APCYYYxxxyy XXXCD90-FITCYYYxxxyy XXXCD105-PEYYYxxxyy Non-MSC-PerCPXXXYYYxxxyy Anti-HLA-DR-FITCXXXYYYxxxyy yYxYy yYxYy Day 12: Culture maintenance yYxYy yYxYy yYxYy 16 14 15 Figure 4 17 • Closed-system media exchange 18 19 20 21 22 Day 15: Split To confirm the quality of the cells processed with the CD105 while CD14, CD20, CD34, CD45 (Non-MSCs) as • Semi-automated harvest CliniMACS Prodigy®, MSC marker expression was ana- well as HLA-DR expression was low (fig. 4). Morphology • Automated cell wash • Harvest P2 MSCs in Target cell bag lyzed using flow cytometry. MSCs met ISCT criteria during MSC expansion was as expected (not shown). (possible QC and cell count sampling) showing high expression levels of CD73, CD90, and CCU Waste bag External culture device Figure 1 Conclusion and outlook

We have developed a method for DGC preparation of relevant numbers of MSCs were expanded, starting Results bone marrow and adherent, closed-system cultivation from a human bone marrow sample. MSC-Brew GMP of MSCs using the CliniMACS Prodigy® Adherent Cell Medium is xeno-free and meets the recommendations Culture Process and the CliniMACS Prodigy. Clinically of USP <1043> on ancillary materials. 1 Density gradient centrifugation using the CliniMACS Prodigy® Density gradient centrifugation (DGC) was performed BM-MNCs revealed a viability of 98.2% (±1.9%) (results Funding: iCARE has received funding from the German Federal Ministry of Education and Research (Fkz01EK1601D). Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. MACS® GMP Products are for research use and ex vivo cell culture processing only, with four human bone marrow samples (30–100 mL) not shown). MSCs were quantified using a flow-based and are not intended for human in vivo applications. For regulatory status in the USA, please contact your local representative. MACS GMP Products are manufactured and tested under a quality system certified to ISO 13485 and are in compliance with relevant GMP guidelines. They are designed following the recommendations of USP <1043> on ancillary materials. The CliniMACS® System components, including Reagents, Tubing Sets, Instruments, and using the CliniMACS Prodigy® Adherent Cell Culture quantification kit (MSC Enumeration Kit, Miltenyi Biotec) PBS/EDTA Buffer, are designed, manufactured and tested under a quality system certified to ISO 13485. + + – In the EU, the CliniMACS System components are available as CE-marked medical devices for their respective intended use, unless otherwise stated. The CliniMACS Reagents and Biotin Conjugates are intended for in vitro use only Process. The whole process including installation and based on a CD271 MSCA-1 CD45 gating strategy and are not designated for therapeutic use or direct infusion into patients. The CliniMACS Reagents in combination with the CliniMACS System are intended to separate human cells. Miltenyi Biotec as the manufacturer of the CliniMACS System does not give any recommendations regarding the use of separated cells for therapeutic purposes and does not make any claims regarding a clinical benefit. For the manufacturing and use of target cells in priming of the tubing set TS 730 took ~2 hours. Lym- (figure 2A). A median of 84.5% (±23%) MSCs were humans the national legislation and regulations – e.g. for the EU the Directive 2004/23/EC (“human tissues and cells”), or the Directive 2002/98/EC (“human blood and blood components”) – must be followed. Thus, any clinical application of the target cells is exclusively within the responsibility of the user of a CliniMACS System. phocytes and monocytes were quantified via flow cy- recovered after DGC (figure 2B). Just 0.3% (±0.15%) of all In the US, the CliniMACS CD34 Reagent System, including the CliniMACS Plus Instrument, CliniMACS CD34 Reagent, CliniMACS Tubing Sets TS and LS, and the CliniMACS PBS/EDTA Buffer, is FDA approved; all other products of the CliniMACS Product Line are available for use only under an approved Investigational New Drug (IND) application or Investigational Device Exemption (IDE). CliniMACS MicroBeads are for research use only and not for human ther- tometry using CD45, CD14, and side scatter as well as erythrocytes and 2% (±1.4%) of all platelets reside apeutic or diagnostic use. CliniMACS, CliniMACS Prodigy, MACS, and the MACS logo are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. All other trademarks men- tioned in this document are the property of their respective owners and are used for identification purposes only. propidium iodide detection for analysis of cell viability. within the BM-MNC fraction (figure 2C). Copyright © 2018 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.