NK92 cells exhibit cytotoxicity against a variety of cancer cell lines, including leukemia’s, lymphoma’s and malignant melanoma’s. This acAvity has lead to several small clinical trials as an anAcancer immunotherapy, and larger mulAcentre Phase II trials are planned. Our GOAL is to enable largescale manufacture of clinical grade NK92 cells, and to reduce manufacturing costs. ProducAon using perfusion culture in sArredtank bioreactors (STR) would enable a straightforward high producAvity process that is readily scaled for commercial manufacture. Xvivo%10 Culture Bags Human AB serum 32% 19% 25% 5 x 10 9 cells/m 2 .dose 3 x 10 10 cells/cycle.paAent [NK92] max ≈1 x 10 6 cells/mL 10 Gy Current manufacturing strategy Ricardo P. Bap1sta , Midori E. Buechli, Lesley Y. Chan, Shahryar KhaAak, Peter W. Zandstra and Nicholas E. Timmins Centre for Commercializa1on of Regenera1ve Medicine, Toronto, Canada We successfully translated NK92 culture to STR, achieving significantly higher cell densiAes. Scaleup to 1L cultures is currently in progress, as well as expansion using serumfree medium formulaAon. Flowcytometry and realAme imaging based assays are effecAve alternaAves to 51 Crrelease potency assay. The BiostaAon CT (Nikon) image based approach enables acquisiAon of kineAc data and observaAon of cellcell interacAons. BACKGROUND AND MOTIVATION DEVELOPMENT OF AN INTEGRATED BIOPROCESS FOR PRODUCTION OF NK-92 CELLS FOR IMMUNOTHERAPY Significance and ongoing work MEDIUM SCREENING QCPHENOTYPE AND POTENCY 0.0E+00 2.5E+05 5.0E+05 7.5E+05 1.0E+06 1.3E+06 1.5E+06 VCD (cells/mL) Xvivo S1 S9 DMEM VCD max cell/mL Medium and consumables ($/cycle) ~1 x10 6 $11,400 TRANSLATION TO STR AND PROCESS INTENSIFICATION 0 1 2 3 4 5 6 7 0 20 40 60 80 100 0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06 Viability (%) VCD (cells/mL) 0 1 2 3 4 5 6 7 0 20 40 60 80 100 0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06 Viability (%) VCD (cells/mL) 2.5% HS 2.5% BIT Figure 4. Effect of feedingregime and culture system in NK92 cell expansion. (A) Growth curves were obtained in 100 mL of sArred suspension culture (DasGip cellfermpro). A 7.0fold increase in cell numbers was observed when (B) a perfusionlike feedingregime (1.0V) was adopted in the STR. (C) Metabolite analysis indicate that accumulaAon of lactate levels above 25 mM are inhibitory to cell growth. 0 20 40 60 80 100 0.0E+00 1.5E+06 3.0E+06 4.5E+06 6.0E+06 0 1 2 3 4 5 6 Viability (%) VCD (cell/mL) Time (d) Batch 1.0V 1100 750 450 300 0.0 0.5 1.0 1.5 2.0 2 3 4 5 6 7 RPM Fold Expansion Inoculum Density (x10 5 cells/mL) 1100 750 450 300 0 20 40 60 80 100 2 3 4 5 6 7 RPM Viability (%) Inoculum Density (x10 5 cells/mL) 0 1 2 3 4 0 2 4 6 8 D (/d) Time (d) Batch 1.0V 0 10 20 30 40 0 2 4 6 8 Lac (mM) Time (d) BASAL FORMULATION SERUMREPLACEMENT CARBON SOURCE INOCULUM DENSITY FEDBATCH CULTURE Figure 1. Effect of basal medium formula1on, serumreplacement cocktail, and carbon source in NK92 cell expansion. Several commercial basal formulaAons were able to support cell growth with viabiliAes (>80%) comparable to those observed in Xvivo (control) (n=3). A serumreplacement cocktail comprising BSA, rhInsulin and rhTransferrin was able to sustain cell expansion in staAc culture (n=3). Carbon source (S) was screened for cell growth and low secreAon of lactate (n=2). All cultures were seeded at approx. 5 x 10 5 cells/mL and carried out in a 6well plate format for a minimum period of 6 days. Every 2 days, cells were diluted (passaged) to a viable cell density (VCD) of 5 x 10 5 cells/mL by replacing sufficient culture volume with fresh media. All medium formulaAons were supplemented with 450 U/mL of IL2 and L Gln. Scale bars = 200 μm. PROCESS INTENSIFICATION LACTATE 0 20 40 60 80 100 0.0E+00 1.0E+08 2.0E+08 3.0E+08 4.0E+08 5.0E+08 6.0E+08 0 2 4 6 7 Viability (%) Total viable cells Time (d) XVivo DMEM STATIC 1 10 0 2 4 6 8 Log Fold Expansion Time (d) XVivoSTR XVivoStaAc DMEMSTR DMEMStaAc Xvivo + 2.5% BIT Culture system VCD max x10 6 cells/mL μ (d 1 ) t d (d) Fold expansion qLac (pmol/cell.d) Xvivo – Sta1c (n=5) 1.1±0.1 0.24±0.09 3.0±1.1 6.1±0.8 7.1±1.3 Xvivo – STR (n=4) 1.6±0.2 0.33±0.08 2.3±0.7 11.3±1.6 7.9±2.5 DMEM – Sta1c (n=3) 1.1±0.2 0.29±0.04 2.5±0.4 6.8±0.5 8.2±1.3 DMEM – STR (n=3) 1.7±0.9 0.32±0.16 2.8±1.6 12.3±6.7 9.1±1.9 NK92 PHENOTYPE 2.5% HS Figure 2. Effect of inoculum density and agita1on rate in NK92 cell expansion in STR. (A) Fold expansion and (B) cell viability across different seeding densiAes and 4 agitaAon rates) using the ambr bioreactor system (TAP Biosystems). The greatest increase in viable cell number was observed when cells were seeded at 5 x 10 5 cells/mL and sArred at 300450 rpm. Figure 3. Effect of basal medium and culture system in NK92 cell expansion. (A,B) Growth curves were obtained in staAc (T75 flask) and sArred suspension (DasGip cellfermpro) culture. Medium comprised Xvivo10 (Lonza) or DMEM (LifeTech) supplemented with human AB serum and Lglutamine, and cultures were iniAated at a cell density of approx. 5 x 10 5 cells/ mL. Culture volume was doubled every 2 days by addiAon of fresh media, up to a final volume 200 mL. (C) Specific growth rate (μ) was higher in STR compared to staAc culture in both medium formulaAons. (D) While NK92’s grow as loose clumps in staAc culture, STR culture yielded a singlecell suspension in Xvivo and smaller aggregates in DMEM. Scale bars = 100μm. STR Xvivo DMEM DAY 6 0 20 40 60 80 100 0 6 CD45 + /CD56 + /CD3 /Perf. + (%) Time (d) XivoSTR XvivoStaAc DMEMSTR DMEMStaAc Figure 5. Effect of culture system and basal medium on NK92 phenotype. (A) Monitoring of NK92 phenotype shows maintenance of a CD45 + /CD56 + / CD3 /Perforin + parent at both basal formulaAons and culture systems. (B) A method for costaining of intracellular and cell surface markers was developed based on the protocol of Wang et al. 2013 (PMID: 23168618). Figure 6. Comparison of flowcytometry with real1me imaging for the assessment of NK92 cell cytotoxicity. (A) CTOlabeled K562 cells (Target) were incubated with NK92 cells (Effector) at different raAos, and stained with calcein before analysis. (B) Lysis of K562 cells by acAon of NK92’s was quanAfied by the increase of CTO + /Calcein populaAon. (C) Assay was developed based on the protocol of Thakur et al. 2012 (PMID: 22187077). (D) Target cells were labelled with Calcein and incubated with effector cells in the BiostaAon CT (Nikon), an integrated cell culture observaAon system. (E,F) Decay in fluorescence intensity (F.I.) as result of K562 lysis was analysed with CLQuant sosware (Nikon). %K562 lysis measured using BioStaAon CT (Nikon), was comparable to that obtained by flowcytometry (42%). NK92 POTENCY 0 4 8 12 Xvivo S9 S10 Glucose qLac (pmol/cell.day) VCD max cell/mL Medium and consumables ($/cycle) Cost reduc1on ~1.5 x10 6 $6,185 45,7 % 0 0.2 0.4 0.6 0.8 1 1.2 0 1 2 3 4 5 Total F.I. t (h) Control E:T(1:10) t=4h t=4h (control) K562 t=0h RealTime Imaging by BioSta1on CT (Nikon) FlowCytometry % K562 lysis 66±2.9 % K562 spontaneous lysis 24±11 K562 t=0h t=4h t=4h (control) 0 20 40 60 80 100 0.1 1 5 10 K562 lysis (%) E:T t=4h Cost break down $114,000 /treatment % K562 lysis 44±4.3 % K562 spontaneous lysis 4±0.6 NIKON CORPORATION Instruments Company A B A B D C A B C A 95.3% CD3 CD45/CD56 PERFORIN CD3 /CD45 + /CD56 + /PERF. + B A B C D E F