hIgG Titer (g/L) 0 1 2 3 4 ExpiFectamine CHO (with Enhancer and Feed) PEI (no Enhancer or Feed) 30fold TIter (mg/L) Roland Leathers, Chao Yan Liu, Virginia Spencer, Shyam Kumar, Jian Liu, Ping Liu, Henry Chiou * , Jonathan F. Zmuda Thermo Fisher Scientific, 7335ExecutiveWay, Frederick MD, * 5791 Van Allen Way, Carlsbad, CA. Figure 3. Characterization ofExpiCHOS Cells (A) ExpiCHOS cells. (B) Stability of protein exp ression ov er 18 pass ages. (C ) Growth c urve fo r ExpiCHOS cells grown in standard shake flask culture. ABSTRACT & INTRODUCTION C HO c ell s are the predominanthostfor biotherapeutic protein expressi on,with roughl y 70% of licensed bi ologi cs manufac tured i n CHO. Multi ple attri butes make CHO cell s desirable for bioproduction incl uding the ability to adapt to highdensit y s us pens ion culture in serumfree and chemicallydefined media and the incorporation of post translational modifications that are biol ogi call y ac ti ve in humans. For these reas ons, the ability to produce transi entCHOderi ved proteins earl y on during drug devel opment is highl y adv antageous to minimize, as muc h as pos s i ble, changes in protei n qualit y /func ti on observedwhen movi ng fromR&D to bioproduction. Unfortunatel y , C HO cell s express lower level s ofprotein than HEK293 cell s in exi sting transi ent sy s tems , in some instances 50100 times less than the best293based systems,and onl y modest titer improvements are obtained through the optimi zation of indi vidual component s of exi s ting trans ient C HO work flows . To address the s i gnifi cant unmet need for higher trans ient C HO protein titers , s ys tems bas ed approac hes w ere employ ed whereby the latest advances in cell cult ure media, feeds, transfection reagents and expression enhancers were optimized in conjunction with a new highexpressing CHOcell clone to generate a si mple and robust workflow for transient protein expression i n CHO cell s capabl e ofgenerating gram per liter protein titers in 1014 days. These advances will allow for unprecedent ed access to CHOderived proteins early on during candidate selection and may serve to revolutionize the use of CHO cells for transient protei n expressionduring thedrugdevelopmentprocess. CONCLUSIONS We descri be a s ys tems bas ed approac h for enhanc ing l evels of transi ent protein production i n CHO cells t hat all ows for the produc tion of rec ombi nant proteins at l evels exc eeding thos e of the Expi293 sys tem while mai ntaini ng acti vity,purityand gl yc osy lation patterns comparabl e to thos e obs erved i n s tably trans fec ted C HOS c ell s. This performance enhancement w as made pos s i ble through the incorporation ofmultiple novel reagents including: (1) a hi ghex pres s i ng CHO cell cl one,(2) a CD/AOF culture media that allows for hi gh dens ity CH O grow th and trans fec tion, (3) an optimi zed CHO cell trans fec ti on reagent, (4) a novel CHO feed optimi z ed for trans i ent trans fec tion c ult ure conditions, (5)a posttransfection enhancer solution and (6) a simple to perform workflow. ACKNOWLEDGEMENTS We woul d like to thank Mi chael Gillmeister for performing the glycan analysi s on the human IgG samples and Brian Paszkiet for assisti ng with the transfection effici enc y studies. ExpiCHO™: Surpassing the Performance of 293 in a Transient CHO Expression System Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad,CA 92008 • lifetechnologies.com Figure 5. Characteristics ofExpiFectamineCHO Transfection Reagent (Top panel) Ti me c ourse of GFP expr ession in ExpiCHOS c ells. Insets show flow cytom etry data fo r corresp onding cultu res. (A ) When used in conjunc tion with ExpiCHO Feed and E nhancer , ExpiFectamineCHO g ener ates gre ater th an 30f old highe r titers tha n PEI alone. (B) Despite the hig h density of cells at the time of t ransfecti on, plasmid DNA levels as low as 0.43 µg/ mL of final cultur e volume gene rate maxim al p rotein titers , co rresp onding to less than half of the indust ry sta ndar d o f 1. 0 µg/mL plasmid DNA. Figure 6. Identification of OptimalPostTransfection Enhancers (A) Combinatio ns of differe nt expr ession enh ancers we re tested by multifact orial DOE for effects o n protein titers. Data f rom a sub set o f enh ancer combi nations are s hown, in dicating t he im pact of vario us enhance r for mulations o n pro tein titers . (B ) P rotein tit ers ar e reduc ed by 50% or more wit hout th e addition of the E xpiFectamineCHO E nhancer reagent. II. ExpiCHO Expression Medium and ExpiCHO Feed III. ExpiFectamineCHO Transfection Reagent VI. Kinetics of Protein Production Figure 7. Transient Transfection Protocol – 7 or 8Stepsto Protein Production Figure 1. SystemsBased Approach toIncreasedTransientProtein Expression I. Generation of HighExpressing ExpiCHOS Cells Transfection of CHO cells with Protein X Selection of high expressing clones by Clone Pix Clone expansion into 6 well dishes Screen clones for Protein Y expression Clone expansion into 30 mL shake flasks Screen clones for Protein Z expression Master Cell Bank Generation Figure 11. Glycosylation Patterns in CHO (Transient and Stable)and HEK293 Human IgG sup erna tant sam ples were collect ed and p urified usin g POROS® MabCaptu re® A resin . Following PNGase digestio n and A P TS labeling, glyca n profiles we re analyz ed on an Applie d B iosystems® 3500 S eries Genetic A nalyzer by capillary electrophoresis. Figure 4. Optimization of ExpiCHO Feed Addition (A) Fo r the Max Titer Protocol, additio n of s econd feed can be a dded on Day 4, 5, or 6 pos ttr ansfectio n with similar perfo rma nce. (B) Two equal volume fee ds on Days 1 and 5 p osttr ansfectio n do uble protei n titers. Figure 2. Workflow for Identifying HighExpressing CHOClones (A ) CHO cells were transiently tr ansfecte d and evalu ated fo r prot ein expressi on using th e ClonePix system ( Molecula r Devices ). (B) Selec ted cl ones w ere furt her evaluat ed via tr ansfectio n with plasmids for multiple protei ns. Control h uman IgG titers using an existi ng CHO clone a re shown as well as Control titers for the preclonal pool. B Figure 8. Kinetics of hIgG Expression (Green line ) Standar d Protocol consisting of on e feed and n o tempe ratu re shift. (Blue line) High Tite r Protocol co nsisting of one fee d a nd temp eratu re shift to 32°C. (R ed lin e) hIgG kinetics using the M ax Titer P rotocol consisting of two feeds and temperature shift to 32°C. A Media Cells Optimized Transient Expression V. Workflow IV. ExpiFectamineCHO Enhancer VII. Expression Levels of Various Transient Systems VIII. Glycan Analysis Days in Culture VCD (x 10 6 cells/mL) (solid line) % Viability (dotted line) 0 1 2 3 4 5 6 7 8 0 5 10 15 20 25 0 50 100 ExpiCHO Expression Media Attributes • One media for growth and transfection • Formulated specifically for transient transfection • Chemicallydefine d (CD) • A nimal originfree (A OF • S erumfree • P roteinfree • Manufactured under cGMP • S upports highdensity cell growth • Matched to a specific feed • Free from regulatory/im po rt/e xp ort limitations ExpiCHOSCellLine Attributes • Derived from GMP CHOS cells • A dapted for highdensity culture • Nonclumpy, singlecell phenotype • S hort doubling time (~17 hours) • S table growth and expression over 20+ passages Figure 10. Titers in FreeStyleCHO,Expi293 and ExpiCHO E xpression levels of hu man I gG, Rabbit IgG a nd E ryth ropoi etin in FreeS tyleCHO, E xpi2 93 an d E xpiCHO transient expr ession syste ms ar e shown . E xpiCHO titers ran ge fr om 2 5x – 1 60x th ose of Fr eeS tyleCHO and 2x – 4x those obtained using the Expi293 system. hIgG Titer (mg/L) One Feed Two Feeds Day 3 Day 5 Day 7 B A C A B A B hIgG (mg/L) ExpiFectamine Enhancer 0 1000 2000 3000 4000 + Figure 9. Scalability of the ExpiCHO System 160x 3x 95x 4x 25x 2x 0.14 0.28 0.43 0.57 0.71 0.86 0 1 2 3 Titer (g/L) DNA Concentration (μg/mL) Expi293 ExpiCHO Stable CHOS 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% G0 A1 G2F Man5A1F G1F_2 G1F_1 G0F D4 D5 D6 D7 0 1 2 Titer (g/L) Timing of Second Feed 125mL 250mL 500mL 1L 2L 3L 0 1000 2000 3000 4000 Titer (hIgG) mg/L 125rpm 70rpm 25m L 50m L 100m L 200m L 400m L 750m L Flask Size Transfection Volume Shake Speed 32°C E nhancer 2 feeds 32°C E nhancer 1 feed 37°C E nhancer 1 feed Days of Culture Titer (g/L) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 Max Titer Protocol High Titer Protocol Standard Protocol Expi293 ExpiCHO