The operating procedures to enrich or deplete select cell subsets from a leukapheresis product are standardized and provided by the manufacturer of the CliniMACS^® device (Miltenyi Biotec). Upon arrival in the GMP facility, the leucapherisate is diluted 1 + 3 with PBS–HSA–EDTA buffer and subsequently centrifuged at 200 g for 15 min at 22°C. After centrifugation, the supernatant is removed, the leucapherisate is resuspended in PBS–HSA–EDTA and centrifuged at 300 g for 15 min at 4°C. After this centrifugation step and subsequent removal of the supernatant, the leucapherisate is resuspended in 380 ml of cold PBS–HSA–EDTA and labeled with CliniMACS^® CD25 reagent. The CliniMACS^® CD25 bead reagent specifically labels up to 600 × 10⁶ CD25⁺ cells within a total population consisting of maximal 40 × 10⁹ white blood cells. These CliniMACS^® acceptance criteria are provided by Miltenyi Biotec. If one of these acceptance criteria is not met, a maximum of two portions, instead of one portion, of CliniMACS CD25 bead reagent are used to specifically label the leucapherisate. CD25-labeling is performed during 15 ± 2 min at 2–8°C at a cell shaker programmed at 25 rpm. After labeling is completed, the cell suspension is washed with PBS–HAS–EDTA, diluted in 100 ml PBS–HSA–EDTA and transferred into a cell collection bag. CD25⁺ enrichment is performed by using the automatic CliniMACS^® ENRICHMENT 3.2 program of the CliniMACS^® device according to the manufacturer’s instructions. The CD25⁺ enriched cell fraction is used for further manufacturing.

At day 4, fresh IL-2 (1,000 IU/ml) and rapamycin (100 ng/ml) were added to the cell cultures to substitute for cellular consumption. Cell cultures were gently mixed and incubated at 37 ± 1°C, 5 ± 1% CO₂, >70% r.h. for 3 days.

At day 7, cell culture plates were collected from the incubator, and the total cell culture volume transferred into a T75 flask. Cell count and viability of the CD25⁺ cells was determined on two samples of 40 µl by trypan blue staining according to Ph. Eur. 2.7.29. Depending on cell number, the cell density was adjusted to 0.5 × 10⁶ cells/ml by adding fresh complete autologous culture medium, and the cell suspension was seeded in new culture plates. To substitute for cellular consumption, fresh IL-2 (1,000 IU/ml) and rapamycin (100 ng/ml) were added. Cell cultures were gently mixed and incubated at 37 ± 1°C, 5 ± 1% CO₂, >70% r.h. for 4 days.

Analogous to day 7, cell culture plates were collected from the incubator, and the cell suspension, depending on the volume, transferred into a T75 or 1 l cell culture flask. Cell count and viability of the CD25⁺ cells was determined on two samples of 40 µl by trypan blue staining according to Ph. Eur. 2.7.29. Depending on cell number, the cell density was adjusted to 0.5 × 10⁶ cells/ml by adding fresh complete autologous culture medium. Depending on total volume, the cell suspension was seeded in T75 or T175 cell culture flasks. To substitute for cellular consumption, fresh IL-2 (1,000 IU/ml) and rapamycin (100 ng/ml) were added. Cell cultures were gently mixed and incubated at 37 ± 1°C, 5 ± 1% CO₂, >70% r.h. for 3 days.

At day 14, cell culture flasks were collected from the incubator, and the cell suspension, depending on the volume, transferred into a T75 or 1 l cell culture flasks. Cell count and viability of the CD25⁺ cells was determined on two samples of 40 µl by trypan blue staining according to Ph. Eur. 2.7.29. To deplete potentially contaminating CD8⁺ and CD19⁺ cells, the CD25⁺ cell product was magnetically labeled with CliniMACS^® CD8 and CliniMACS^® CD19 bead reagent according to the manufactures’ instructions. Specifically, up to 4 × 10⁹ CD8⁺ cells and up to 5 × 10⁹ CD19⁺ cells within a total population consisting of maximal 40 × 10⁹ cells may specifically be labeled by 7.5 ml of the CliniMACS^® CD8 bead reagent and CliniMACS^® CD19 bead reagent, respectively. These CliniMACS^® acceptance criteria were provided by Miltenyi Biotec. In general, CD25⁺ cell expansion on day 14 does not result in total cell numbers above 1 × 10⁹ total cells, and the relative contamination with CD8⁺ and/or CD19⁺ cells is assumed to be below 25%. Therefore, a predefined aliquot of 1.875 ml of CliniMACS^® CD8 bead reagent and CliniMACS^® CD19 bead reagent is used to label up to 1.0 × 10⁹ CD8⁺ cells and up to 1.25 × 10⁹ CD19⁺ cells within a total population consisting of maximal 10 × 10⁹ cells. For depletion of CD8⁺ and CD19⁺ cells, sterile CliniMACS^® PBS/EDTA buffer supplemented with human serum albumin is used. Depletion of CD8⁺ and CD19⁺ cells is performed by using the automatic CliniMACS^® DEPLETION 2.1 program of the CliniMACS^® device. This program facilitates automated magnetic depletion of CD8⁺ and CD19⁺ cells in a closed, sterile system. Two cell fractions are collected into bags according to the instrumental settings of the CliniMACS^® DEPLETION 2.1 program. The CD8⁺- and CD19⁺-depleted cell fraction 2 is used for further processing.

Cell number in fraction 2 is determined on two samples of 40 µl by trypan blue staining according to Ph. Eur. 2.7.29. CD25⁺/CD8⁻/CD19⁻ cells of fraction 2 obtained from the CliniMACS^® device are continued to be cultivated in vitro. Depending on cell number, the cell density was adjusted to 0.5 × 10⁶ cells/ml by adding fresh complete autologous culture medium. Depending on total volume, the cell suspension was seeded in T75 or T175 cell culture flasks. To compensate for any anti-CD3/anti-CD28 bead removal during the CD8⁺ and CD19⁺ cell depletion process, anti-CD3/anti-CD28 beads (analogously to the amount of beads used at day 0) are added to the cell cultures. Finally, fresh IL-2 (1,000 IU/ml) and rapamycin (100 ng/ml) were added. Cell cultures were gently mixed and incubated at 37 ± 1°C, 5 ± 1% CO₂, >70% r.h. for 4 days.

Analog to days 7 and 11, cell culture plates or flasks were collected from the incubator, and the cell suspension, depending on the volume, transferred into a T75 or 1 l cell culture flasks. Cell count and viability of the CD25⁺ cells was determined on two samples of 40 µl by trypan blue staining according to Ph. Eur. 2.7.29. Depending on cell number, the cell density was adjusted to 0.5 × 10⁶ cells/ml by adding fresh complete autologous culture medium. Depending on total volume, the cells are subsequently seeded in T75 or T175 cell culture flasks. To substitute for cellular consumption, fresh IL-2 (1,000 IU/ml) and rapamycin (100 ng/ml) are added. Cell cultures are gently mixed and incubated (37 ± 1°C, 5 ± 1% CO₂, >70% r.h.) for an additional 3 days.

At day 21, cell culture plates or flasks are collected from the incubator, and the total cell culture volume is transferred into a set of 50 ml centrifuge tubes. Culture flasks are washed once with approximately 10–20 ml of PBS/EDTA buffer supplemented with human serum albumin. Used washing buffer is also transferred into the 50 ml centrifuge tubes. Tubes are centrifuged, supernatants are discarded, and pellets are collected into a set of 50 ml centrifuge tubes by resuspending pellets with approximately 5 ml of PBS/EDTA buffer supplemented with human serum albumin. Cell count and cell viability of CD25⁺/CD8⁻CD19⁻ cells are determined on two samples of 40 µl by trypan blue staining according to Ph. Eur. 2.7.29.

A maximum of 2.04 × 10¹⁰ beads can be depleted from a maximum of 4 × 10¹⁰ CD25⁺ cells in a final concentration of 20 × 10⁶–400 × 10⁶/ml during the bead removal process. These CliniMACS^® acceptance criteria are provided by Miltenyi Biotec. In general, CD25⁺ cell expansion on day 21 does not result in total cell numbers above 4 × 10¹⁰ total cells. Therefore, the assumed maximum amount of added beads is 0.48 × 10¹⁰ [four times the maximum amount of isolated CD25⁺ cells at day 0 (1,200 × 10⁶)]. For bead removal, sterile CliniMACS PBS/EDTA buffer supplemented with human serum albumin is used. Bead removal is performed by using the automatic CliniMACS^® DEPLETION 2.1 program of the CliniMACS^® device. The CliniMACS^® device facilitates automated magnetic depletion of anti-CD3/anti-CD28 expander beads and CD25-, CD8-, and CD19-labeling beads in a closed, sterile system. Two cell fractions are collected into bags according to the instrumental settings of the CliniMACS^® DEPLETION 2.1 program. The bead-depleted cell fraction 2 is used for filling and storage.

The bead-depleted cells are transferred into a set of 50 ml centrifuge tubes. Tubes are centrifuged, supernatants are discarded, and pellets are collected into 100 ml of PBS/EDTA buffer supplemented with human serum albumin. The final cell number in cell fraction 2 after bead removal is determined on two samples of 40 µl by trypan blue staining according to Ph. Eur. 2.7.29. Before filling and storage, a freezing medium is freshly prepared in a sterile bottle. The total volume is calculated based on the cell counting results obtained after the first centrifugation step after the anti-CD3/anti-CD28 bead removal step. The freezing medium consists of human serum albumin, DMSO, and 40% glucose solution (hereafter called freezing medium). After definition of the final CD25⁺ cell number, cells are centrifuged and depending on cell number resuspended in an appropriate volume of human serum albumin at a final concentration of 20 × 10⁶ viable cells/ml. Five hundred microliters of cell suspension are filled into each cryovial (1.0 ml total volume). After addition of 500 µl freezing medium (55.5 Vol.-% human serum albumin, 25.0 Vol.-% DMSO, and 20.0 Vol.-% glucose) to every vial, the closed vials are mixed gently and transferred immediately into a freezing container. The container is than stored immediately at −75 ± 10°C for 4–18 h. The vials are transferred to the gas phase of liquid nitrogen (≤−150°C) for up to 2 years.

Throughout the manuscript, Treg are defined as CD4⁺CD25⁺CD127⁻ cells (=Treg drug product identity) based on previously published data (3, 4). Treg drug product identity was determined before and after 21 days of expansion by staining with directly conjugated mouse antihuman antibodies (mAbs) against CD4 (FITC, clone RPA-T4), CD25 (FITC, clone M-A251), and CD127 (PE, clone RDR5). Treg drug product cellular composition was determined before and after 21 days of expansion by staining with directly conjugated mouse antihuman mAbs against CD8 (FITC, clone SK1), CD19 (FITC, clone SJ25C1), CD16 (PE, clone 3G8), and CD56 (PE, clone B159). Corresponding IgG_1,κ mouse isotype controls (FITC, clone MOPC-21 and PE, clone MOPC-21) were included to assess unspecific binding. Potential dead cells were excluded by labeling with propidium iodide (PI) (BD Biosciences) according to the manufacturer’s instructions. Cells were acquired using a FACS Calibur (BD Biosciences), and data were analyzed using CellQuest™ Pro (BD Biosciences) software. For information-only purposes, cells were intracellularly stained with FoxP3 (clone PCH101) using a FoxP3/Transcription Staining Buffer Set (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions.

In general, flow cytometry is performed as described in Ph. Eur. 2.7.24. The specific method for flow cytometry was validated, since it is not in detail described in the Ph. Eur. In brief, thawed dendritic cells were used to validate the method for flow cytometry. Thawed dendritic cells, thawed lymphocytes, thawed Treg, and thawed CD25⁻ cells were used to determine specificity. Lymphocytes were single stained with CD4, CD127, or CD8 antibody, respectively, fixed with 1% formaldehyde, washed twice and subsequently mixed with unstained lymphocytes of the same preparation. To determine specificity of the CD25 antibody, Treg were single stained with the CD25 antibody, fixed with 1% formaldehyde, washed twice and subsequently mixed with unstained Treg of the same preparation The FACS result for stained lymphocytes or Treg was set to 1 (=100% positive). For specificity purposes, optimal positive and negative cells for each antibody characterizing the Treg drug product were measured. Nominal negative cells shall show less than 0.04 (=4%) positive cells, and nominal positive cells shall show more than 0.04 (=4%) positive ones. Summarized results are shown in Table 1. In addition, the stringent cutoff of less than 0.1% contaminating CD8⁺ cells in the final Treg product was validated separately. To avoid false negative CD8⁺ values in the final Treg product, the number of to acquire cells was increased to 300,000. A total of six acquisitions were performed with mature monocyte-derived dendritic cells, which represented the CD8⁻ cell subset. The SD (s) was calculated from all acquired “CD8⁺” cells within the CD8⁻ mature dendritic cell subset, and the quantification limit (QL) was calculated based on the following formula: QL = 10s/a with a set to 0.973 (=linear regression value derived from the flow cytometry validation report) (data not shown).

Regulatory T cell drug product purity was determined after anti-CD3/anti-CD28 expander bead removal by staining with directly conjugated mouse antihuman IgG_3,κ Labeling Check PE (clone AC146, Miltenyi Biotec) and APC (clone AC146, Miltenyi Biotec). Samples were acquired using a FACS Calibur (BD Biosciences), and data were analyzed using CellQuest™ Pro (BD Biosciences) software.

The method to measure anti-CD3/anti-CD28 expander bead and CD25-, CD8- and CD19-labeling bead contamination was validated since it is not described in a Ph. Eur. Specifically, thawed lymphocytes were mixed with predefined amounts of anti-CD3/anti-CD28 expander beads and incubated for 1 h at 37°C at 5% CO₂. After 1 h of incubation, cells were lysed using saponin 0.2% (Sigma-Aldrich, St. Louis, MI, USA) to also capture potential intracellular beads. Subsequently, beads were stained by flow cytometry with Labeling Check Reagent PE and Labeling Check Reagent APC as described above. To determine bead contamination, all samples were acquired with the use of Trucount^® tubes (BD Biosciences) to assure standardized acquisition. Nominal samples negative for beads should show less than 400 beads, nominal samples positive for beads should show more than 400 beads. At least 100 × 10⁶ Treg drug product cells should be lysed, labeled with Labeling Check Reagent PE and Labeling Check Reagent APC and subsequently measured to define bead contamination in the final Treg drug product. The results of the validation are summarized in Table 2.

The method to assess Treg drug product function was validated, since it is not described in the Ph. Eur. Specifically, cryopreserved autologous CD25⁻ cells were thawed, washed, and labeled with 5 µM carboxyfluorescein succinimidyl ester (CFSE) (ThermoFisher Scientific, Carlsbad, CA, USA). Next, CFSE-labeled CD25⁻ cells (containing CD4⁺ and CD8⁺ cells; hereafter called responder cells) were cocultured with thawed day 21 Treg drug product cells (hereafter called Treg) at a Treg to responder cell ratio of 1 + 1, 1 + 5, and 1 + 10. Cocultures were stimulated with anti-CD3 and anti-CD28 coated beads (MACS GMP ExpAct Treg Kit, Miltenyi Biotec) at a bead-to-cell ratio of 1 + 1. Negative controls included responder cells alone and Treg + responders cells at a ratio of 1 + 1 without the addition of anti-CD3 and anti-CD28 coated beads. The positive control included responder cells alone with the addition of anti-CD3 and anti-CD28 coated beads at a bead + responder cell ratio of 1 + 4. The absolute cell concentration and cell density at the beginning of the coculture was 1 × 10⁶/ml and 1 × 10⁶/cm² per well, respectively.

Cocultures were routinely performed in triplicates in 48-well plates and harvested after 60–72 h of incubation at 37 ± 1°C, 5 ± 1% CO₂, >70% r.h. After 60–72 h of incubation, conditions were harvested, stained with PI and CD8 as described under flow cytometry and acquired using a FACS Calibur (BD Biosciences). Data were analyzed using CellQuest™ Pro (BD Biosciences) software. Treg-mediated suppression was calculated based on the percentage of divided cells in the first cell generation with the positive control set to 100%. One cell generation was defined to contain at least ≥10% divided cells. Based on all cell generations, negative control cells should show less than 5% proliferated cells, and positive control cells should show more than 30% proliferated cells. Lot-release is based on the amount of Treg-mediated suppression in cocultures with a Treg to responder cell ratio of 1 + 1, 1 + 5, and 1 + 10, respectively. For validation, more Treg to responder cell ratios were included. The results of the validation are summarized in Table 3.

Cell number, cell concentration, and cell viability are determined by trypan blue staining and microscopic examination using a hemocytometer according to the method described in Ph. Eur. 2.7.29. Sterility testing is routinely performed according to Ph. Eur. 2.6.1 by Bioservice Scientific Laboratories (BSL) GmbH, Planegg, Germany. Testing on bacterial endotoxins is routinely performed according to Ph. Eur. 2.6.14 by BSL. Testing on mycoplasma DNA is achieved by PCR by an in-house validated method by BSL.