Fu­el­ing More Break­throughs: Man­u­fac­tur­ing In­no­va­tion to Ex­pand Gene Ther­a­py’s Reach

Break­throughs are on the rise in the gene ther­a­py in­dus­try af­ter decades of man­u­fac­tur­ing chal­lenges. Ge­net­ic ther­a­pies of­fer the po­ten­tial to treat pa­tients with some of the most com­plex and de­bil­i­tat­ing dis­eases. The in­dus­try has re­spond­ed to these promis­ing ther­a­pies with in­creased ca­pac­i­ty for man­u­fac­tur­ing and de­vel­op­ment and an ever-grow­ing body of high­ly trained sub­ject mat­ter ex­perts. How­ev­er, tech­ni­cal hur­dles—par­tic­u­lar­ly in the com­mon­ly used HEK293 pro­duc­tion sys­tem such as lim­it­ed scal­a­bil­i­ty, in­con­sis­tent or low yields, and time-con­sum­ing process de­vel­op­ment path­ways—still pose chal­lenges for de­vel­op­ers try­ing to en­ter the clin­ic or even­tu­al­ly of­fer a com­mer­cial­ly avail­able prod­uct. As more gene ther­a­py pro­grams ad­vance past ear­ly clin­i­cal stages and new pro­grams be­gin tar­get­ing preva­lent in­di­ca­tions, the pres­sure to scale ef­fec­tive­ly and ef­fi­cient­ly to meet this de­mand has in­ten­si­fied.

De­spite ad­vance­ments in gene ther­a­py, ac­ces­si­bil­i­ty re­mains lim­it­ed by the need for high­er pro­duc­tion vol­umes to meet de­mand. Scal­ing up with larg­er biore­ac­tors can in­crease pro­duc­tion ca­pac­i­ty but can al­so po­ten­tial­ly raise costs of goods sold (COGS) and fails to ad­dress the crit­i­cal ef­fi­cien­cies re­quired to make these ther­a­pies broad­ly ac­ces­si­ble. Over­com­ing man­u­fac­tur­ing ef­fi­cien­cy bar­ri­ers is crit­i­cal to en­sur­ing gene ther­a­pies ful­fill their po­ten­tial to ad­vance more pro­grams and reach broad­er pa­tient pop­u­la­tions. In­no­v­a­tive man­u­fac­tur­ing ap­proach­es like Forge’s FU­EL™ plat­form aim to over­come these chal­lenges by en­hanc­ing pro­duc­tiv­i­ty and scal­a­bil­i­ty.  The FU­EL™ plat­form re­duces the to­tal pro­duc­tion vol­ume need­ed to meet pro­gram re­quire­ments, as il­lus­trat­ed in Fig­ure 1, mak­ing crit­i­cal progress to­ward more ac­ces­si­ble man­u­fac­tur­ing.

Pri­or­i­tiz­ing In­no­va­tion and Prod­uct Spe­cif­ic De­vel­op­ment to Un­lock Ef­fi­cien­cy in AAV Man­u­fac­tur­ing

Forge is not on­ly strate­gi­cal­ly scal­ing up and out to tack­le the need­ed ca­pac­i­ty but has al­so in­vest­ed ex­ten­sive time and re­sources in­to its foun­da­tion­al plat­form tech­nolo­gies with man­u­fac­tur­ing re­search and de­vel­op­ment (R&D) to ad­dress man­u­fac­tur­ing ef­fi­cien­cy bar­ri­ers such as COGS. The new FU­EL™ plat­form in­cor­po­rates mol­e­c­u­lar im­prove­ments in Ad helper and rep/cap plas­mids in­to Forge’s proven and adapt­able man­u­fac­tur­ing process. The FU­EL™ plat­form can al­so ac­com­mo­date prod­uct spe­cif­ic process op­ti­miza­tions or al­ter­na­tive en­hancers and ad­di­tives as they be­come avail­able in the field. Forge’s FU­EL™ plat­form pro­vides an ad­vanced start­ing point to ac­cel­er­ate gene ther­a­py de­vel­op­ment and lays the ground­work for pro­gram suc­cess.

The Tech­nol­o­gy Ad­vance­ments of FU­EL™

Op­ti­mized pEM­BR 2.0™ Ad Helper Plas­mid

The ade­n­ovi­ral (Ad) el­e­ments that are re­quired for the suc­cess­ful pro­duc­tion of re­com­bi­nant ade­no-as­so­ci­at­ed virus (AAV) have been well es­tab­lished for 27 years¹. These crit­i­cal Ad com­po­nents fa­cil­i­tate ef­fi­cient repli­ca­tion and pack­ag­ing of AAV vec­tors in mam­malian cells. How­ev­er, Forge has gone be­yond con­ven­tion­al un­der­stand­ing, be­ing the first in iden­ti­fy­ing an­oth­er crit­i­cal re­gion en­cod­ing the Ad L4 22/33K pro­teins, which ex­pands the in­dus­try’s un­der­stand­ing of AAV man­u­fac­tur­ing re­quire­ments. Ex­per­i­ments con­duct­ed by Forge in 2023 con­firmed that Ad helper plas­mid vari­ants lack­ing L4 22K pro­tein en­cod­ing se­quence were un­able to sup­port AAV pro­duc­tion. Yet, by sup­ply­ing the L4 22K gene, AAV yields were ful­ly re­stored. Ad­di­tion­al­ly, Forge’s nov­el stud­ies showed that the L4 33K pro­tein, when ex­pressed syn­er­gis­ti­cal­ly, could fur­ther boost AAV pro­duc­tion². These find­ings have since been ver­i­fied by Doshi, Jiten et al. and Jo­hari, et al.^3,4.

Build­ing on these find­ings, Forge pro­ceed­ed to re­fine and op­ti­mize its orig­i­nal pEM­BR™ Ad helper plas­mid. By re­mov­ing ex­tra­ne­ous Ad genes, Forge cre­at­ed a stream­lined se­ries of re­duced size pEM­BR 2.0™ helper plas­mids (from 7 to 9 kb) that could en­hance the AAV man­u­fac­tur­ing process. The fi­nal pEM­BR 2.0™ can­di­date was test­ed in small, mid-sized, and large-scale pro­duc­tions across var­i­ous AAV serotypes and vec­tor trans­genes, show­ing ex­cep­tion­al per­for­mance in both sin­gle-strand­ed and self-com­ple­men­tary AAV pro­duc­tion. The strate­gic mod­i­fi­ca­tion of key ge­net­ic el­e­ments in the small­er pEM­BR 2.0™ Ad helper plas­mid re­sult­ed in sig­nif­i­cant in­creas­es in AAV yield, while po­ten­tial­ly im­prov­ing the over­all safe­ty of the de­sign. pEM­BR 2.0™ demon­strates a sub­stan­tial im­prove­ment in the ef­fi­cien­cy, man­u­fac­tura­bil­i­ty, and safe­ty of AAV pro­duc­tion.

Mod­i­fied Rep/Cap Plas­mids

Forge has al­so made mol­e­c­u­lar im­prove­ments in tra­di­tion­al rep/cap plas­mid de­sign, em­ploy­ing tech­niques such as gene shuf­fling, codon se­quence al­ter­ations, and site-di­rect­ed mod­i­fi­ca­tions. These im­prove­ments have led to sub­stan­tial in­creas­es in AAV yields across mul­ti­ple serotypes, in­clud­ing the com­mon­ly uti­lized AAV2, AAV6, AAV9, AAVrh10, and AAVrh74. In ad­di­tion to the yield in­crease seen with mod­i­fied rep/caps alone, when com­bined with pEM­BR 2.0™, an ad­di­tion­al 3 to 15-fold in­crease in AAV yield has been ob­served, de­pend­ing on the serotype, as seen in Fig­ure 2. This ad­vance­ment with­in the mol­e­c­u­lar foun­da­tion of AAV pro­duc­tion marks an es­sen­tial boost to AAV man­u­fac­tur­ing ef­fi­cien­cy, mak­ing high yields at­tain­able in man­age­able vol­umes. While both rare and preva­lent in­di­ca­tions stand to ben­e­fit from a sub­stan­tial re­duc­tion in man­u­fac­tur­ing COGS, preva­lent in­di­ca­tions will al­so ben­e­fit from a move­ment to­wards clin­i­cal and com­mer­cial man­u­fac­tur­ing with­in a man­age­able foot­print.

Ig­ni­tion Cells™   

Forge’s pro­pri­etary sus­pen­sion HEK293 Ig­ni­tion Cells™ are a foun­da­tion­al tech­nol­o­gy avail­able for all clients to use as part of the new FU­EL™ plat­form. The cell line has a well-doc­u­ment­ed his­to­ry and has been op­ti­mized for ro­bust tran­sient trans­fec­tion, en­abling stream­lined pro­duc­tion of AAV vec­tors at scale. Forge’s GMP Mas­ter Cell Bank (MCB) is ful­ly qual­i­fied and de­liv­ers con­sis­tent, high-qual­i­ty AAV pro­duc­tion per­for­mance.

Proven Man­u­fac­tur­ing Process­es Honed by a Team of AAV Ex­perts

In the fast-paced field of gene ther­a­py, the race to­wards pa­tient ac­cess to treat­ment has led many de­vel­op­ers to re­ly on tra­di­tion­al plat­form process­es and their abil­i­ty to man­u­fac­ture ther­a­pies re­li­ably, at scale, and with con­sis­tent qual­i­ty. As the field reach­es be­yond ‘proof of con­cept’ and equips for long-term com­mer­cial vi­a­bil­i­ty, tech­ni­cal ad­vance­ments in man­u­fac­tur­ing are key. The FU­EL™ plat­form is in­ten­tion­al­ly de­signed to ad­dress some long­stand­ing chal­lenges in the HEK293 pro­duc­tion sys­tem for AAV man­u­fac­tur­ing, of­fer­ing the scal­a­bil­i­ty, flex­i­bil­i­ty, and ef­fi­cien­cy need­ed to bring these trans­for­ma­tive ther­a­pies to their next phase of growth.

By it­er­at­ing up­on its plat­form process­es and suc­cess­ful­ly com­plet­ing near­ly 500 runs across a wide range of serotypes—in­clud­ing nov­el cap­sids—Forge has ap­plied sub­stan­tial in­sights to the de­vel­op­ment of the FU­EL™ plat­form. Stan­dard up­stream pro­duc­tion and down­stream pu­rifi­ca­tion pa­ra­me­ters re­main large­ly un­changed, but for ad­just­ments to scale due to the ben­e­fits of the im­proved FU­EL™ plat­form.

Plat­forms Are Not a One-Size-Fits-All Mod­el: Op­ti­miza­tion Work Pack­ages to Meet In­di­vid­ual Pro­gram Needs

The FU­EL™ plat­form of­fers a ro­bust base­line man­u­fac­tur­ing process, along with a suite of op­tion­al op­ti­miza­tion pack­ages de­signed to re­fine and cus­tomize the process to meet spe­cif­ic pro­gram needs. These pack­ages tar­get im­prove­ments in yield, re­cov­ery, and prod­uct pu­ri­ty.

Trans­fec­tion Op­ti­miza­tion Pack­age:

Gene ther­a­py man­u­fac­tur­ers have in­creas­ing­ly fo­cused on max­i­miz­ing up­stream pro­duc­tiv­i­ty as a key strat­e­gy to en­hance to­tal pu­ri­fied pro­duc­tion yield. The Trans­fec­tion Op­ti­miza­tion Pack­age us­es a De­sign of Ex­per­i­ment (DOE) ap­proach to en­hance the base­line per­for­mance of the FU­EL™ plat­form by cre­at­ing a pre­dic­tive mod­el and screen­ing po­ten­tial trans­fec­tion con­di­tions aimed at boost­ing AAV pro­duc­tion yields such as DNA per cell, or plas­mid ra­tios, as well as screen­ing of reagents that may act as en­hancers of the AAV pro­duc­tion process. This op­ti­miza­tion pack­age in com­bi­na­tion with the FU­EL™ plat­form process has demon­strat­ed pro­duc­tiv­i­ty in­creas­es from 1.59E+14 vg/L to 2.00E+15 vg/L for AAV9, as seen in Fig­ure 3.

Down­stream Op­ti­miza­tion Pack­ages:

There are al­so prod­uct-spe­cif­ic down­stream pro­cess­ing op­ti­miza­tion work pack­ages that of­fer tai­lored so­lu­tions to en­hance both pu­ri­ty and re­cov­ery rates, with a fo­cus on de­vel­op­ing com­mer­cial­ly vi­able process­es.

Op­tions for op­ti­miza­tion of down­stream process­es in­clude:

• Affin­i­ty chro­matog­ra­phy op­ti­miza­tion: Forge’s be­spoke affin­i­ty chro­matog­ra­phy plat­form serves as both a ver­sa­tile frame­work and a tool for im­ple­ment­ing fur­ther prod­uct-spe­cif­ic process im­prove­ments. This work pack­age en­ables stud­ies to as­sess bind­ing ca­pac­i­ty and op­ti­mize elu­tion con­di­tions, sup­port­ing scal­able and cost-ef­fec­tive process de­vel­op­ment.
• Ce­sium chlo­ride gra­di­ent op­ti­miza­tion: CsCl cen­trifu­ga­tion stud­ies can be per­formed to as­sess load ca­pac­i­ty for scal­a­bil­i­ty, while an­ion ex­change chro­matog­ra­phy (AEX) stud­ies are con­duct­ed to op­ti­mize prod­uct-spe­cif­ic pro­to­cols for large-scale pu­rifi­ca­tion.
• Tan­gen­tial flow fil­tra­tion (TFF) op­ti­miza­tion: TFF stud­ies may be per­formed to in­crease the pu­ri­ty of the fi­nal prod­uct, en­sur­ing prod­ucts meet dos­ing re­quire­ments and are pre­pared for clin­i­cal ap­pli­ca­tions.

By fine-tun­ing both up­stream and down­stream process­es, these work pack­ages stream­line man­u­fac­tur­ing work­flows, re­duce costs, and help with faster time-to-mar­ket for crit­i­cal ther­a­pies.

Prepar­ing Your Ge­net­ic Ther­a­py for Com­mer­cial Longevi­ty

The mod­i­fied rep/cap and pEM­BR 2.0™ tech­nolo­gies with­in Forge’s FU­EL™ plat­form have demon­strat­ed the pow­er­ful abil­i­ty to ex­ceed in­dus­try stan­dards for AAV pro­duc­tion yields, while main­tain­ing safe­ty and qual­i­ty. When com­bined with prod­uct-spe­cif­ic op­ti­miza­tion pack­ages, the plat­form demon­strates en­hanced pro­duc­tiv­i­ty, fur­ther sup­port­ed by its scal­a­bil­i­ty from a 1L shake flask to a 200L sin­gle-use biore­ac­tor, en­sur­ing seam­less tran­si­tion across de­vel­op­ment stages as seen in Fig­ure 4. The re­sult for ther­a­peu­tic de­vel­op­ers is a man­u­fac­tur­ing plat­form that of­fers in­creased ef­fi­cien­cies while main­tain­ing high prod­uct qual­i­ty and safe­ty. These pro­duc­tiv­i­ty in­creas­es will be even more crit­i­cal as de­vel­op­ers move from clin­i­cal pro­duc­tion in­to com­mer­cial man­u­fac­tur­ing where the ef­fi­cien­cies gained at scale can help ad­dress the high COGS bar­ri­ers that have the po­ten­tial to lim­it a pro­gram’s com­mer­cial vi­a­bil­i­ty. With these in­creased ef­fi­cien­cies, Forge is unique­ly po­si­tioned to help de­liv­er high qual­i­ty ther­a­pies to gene ther­a­py de­vel­op­ers’ pa­tients world­wide, mak­ing our plat­form not on­ly a foun­da­tion for un­leash­ing ex­cel­lence but al­so a cat­a­lyst for mak­ing gene ther­a­pies more ac­ces­si­ble long term.

Learn more about how Forge can help fu­el your gene ther­a­py here.

Ref­er­ences

1. Mat­sushi­ta T, El­liger S, El­liger C, Pod­sakoff G, Vil­lar­real L, Kurtz­man GJ, Iwa­ki Y, Colosi P. (1998). Ade­no-as­so­ci­at­ed virus vec­tors can be ef­fi­cient­ly pro­duced with­out helper virus. Gene Ther. 1998 Jul;5(7):938-45. doi: 10.1038/sj.gt.3300680. PMID: 9813665
2. Ad­sero, A., Chest­nut, B., Shah­ne­jat-Bushehri, S., Sas­noor, L., Mc­Mur­phy, T., Swenor, M., Pasquino, R., Prad­han, A., Her­nan­dez, V., Padegi­mas, L., & Dis­muke, D. (2023). A nov­el role for the ade­n­ovirus L4 re­gion 22K and 33K pro­teins in Ade­no-As­so­ci­at­ed virus pro­duc­tion. Hu­man Gene Ther­a­py, 35(1–2), 59–69. https://doi.org/10.1089/hum.2023.146
3. Doshi, Jiten et al. (2024). E2A, VA RNA I, and L4-22k ade­n­ovi­ral helper genes are suf­fi­cient for AAV pro­duc­tion in HEK293 cells. Mol­e­c­u­lar Ther­a­py Meth­ods & Clin­i­cal De­vel­op­ment, Vol­ume 32, Is­sue 4, 12 De­cem­ber 2024, 101376
4. Jo­hari, Y. B., Pohle, T. H., White­head, J., Scar­rott, J. M., Liu, P., May­er, A., & James, D. C. (2024). Mol­e­c­u­lar de­sign of con­trol­lable re­com­bi­nant ade­no-as­so­ci­at­ed virus (AAV) ex­pres­sion sys­tems for en­hanced vec­tor pro­duc­tion. Biotech­nol­o­gy Jour­nal, 19, e2300685. https://doi.org/10.1002/biot.202300685 Mol­e­c­u­lar Ther­a­py Meth­ods & Clin­i­cal De­vel­op­ment, Vol­ume 32, Is­sue 4, 12 De­cem­ber 2024, 101376