Forge Bi­o­log­ics’ cGMP Com­pli­ant and Com­mer­cial­ly Vi­able Be­spoke Affin­i­ty Chro­matog­ra­phy Plat­form

Forge Bi­o­log­ics has de­vel­oped a be­spoke affin­i­ty chro­matog­ra­phy plat­form ap­proach that fac­tors in unique vec­tor com­bi­na­tions to stream­line de­vel­op­ment time­lines and as­sist our clients in ef­fi­cient­ly en­ter­ing the clin­ic. By lever­ag­ing our ex­pe­ri­ence with nat­ur­al and nov­el serotypes and trans­gene con­for­ma­tions, we are able to ac­cel­er­ate affin­i­ty chro­matog­ra­phy de­vel­op­ment by near­ly 3-fold. Many down­stream pu­rifi­ca­tion mod­els are serotype-de­pen­dent, de­mand­ing unique and time-con­sum­ing de­vel­op­ment strate­gies for each AAV gene ther­a­py prod­uct¹. With the in­creas­ing de­mand to pro­pel AAV gene ther­a­pies to mar­ket, plat­form pu­rifi­ca­tion meth­ods that sup­port com­mer­cial-scale man­u­fac­tur­ing of high-qual­i­ty vec­tors with ex­cel­lent safe­ty and ef­fi­ca­cy pro­files are es­sen­tial.

Forge has es­tab­lished a be­spoke pu­rifi­ca­tion process ap­plic­a­ble across a va­ri­ety of AAV vec­tors. When affin­i­ty chro­matog­ra­phy process de­vel­op­ment or tar­get­ed op­ti­miza­tion is re­quired for a spe­cif­ic vec­tor, the Forge team ap­proach­es this in a cal­cu­lat­ed man­ner mak­ing da­ta-dri­ven de­ci­sions based on our plat­form de­vel­op­ment stud­ies. Rather than start­ing from scratch and eval­u­at­ing com­bi­na­tions of resin, bind­ing ca­pac­i­ty, and buffer com­po­si­tions, on­ly mi­nor ad­just­ments are need­ed, for ex­am­ple, a change in the pH of the elu­tion buffer.

With three AAV gene ther­a­pies on the mar­ket and sev­er­al more an­tic­i­pat­ed reg­u­la­to­ry de­ci­sions this year, scal­able, com­mer­cial-ready down­stream process­es are need­ed to meet cur­rent and fu­ture man­u­fac­tur­ing de­mands. Fur­ther­more, with the in­dus­try rapid­ly ad­vanc­ing, rare and high preva­lence dis­ease in­di­ca­tions, ca­pac­i­ty, as well as scal­a­bil­i­ty of process­es are re­quired in tan­dem. Forge has built a state-of-the-art man­u­fac­tur­ing fa­cil­i­ty com­mit­ted to 200,000L+ per year that us­es sin­gle-use affin­i­ty chro­matog­ra­phy flow paths to en­hance re­pro­ducibil­i­ty be­tween pro­duc­tions and mit­i­gate risks as­so­ci­at­ed with cross-prod­uct con­t­a­m­i­na­tion.

Affin­i­ty Chro­matog­ra­phy: A Com­mer­cial­ly Vi­able Plat­form Ap­proach to a Pow­er­ful Down­stream Pu­rifi­ca­tion Tech­nique

Affin­i­ty chro­matog­ra­phy pu­rifi­ca­tion meth­ods pro­vide high se­lec­tiv­i­ty, res­o­lu­tion, and ca­pac­i­ty by ex­ploit­ing a pro­tein’s bi­o­log­i­cal struc­ture to pu­ri­fy it from so­lu­tion. Affin­i­ty chro­matog­ra­phy is the first step in Forge’s plat­form down­stream AAV pu­rifi­ca­tion process and is com­prised of an affin­i­ty resin sta­tion­ary phase and mo­bile phas­es pro­mot­ing cap­ture or elu­tion of the AAV prod­uct. Com­mer­cial­ly avail­able affin­i­ty resins, such as Cy­ti­va AVB, and POROS™ Cap­ture­S­e­lect™ AAV9, AAV8, and AAVX, re­main re­li­able as­sets to the AAV man­u­fac­tur­ing field and are rou­tine­ly screened and used at Forge dur­ing all stages of de­vel­op­ment.

Af­ter ini­tial cap­ture of AAV, the dis­so­ci­a­tion and re­cov­ery of vi­ral genomes from chro­matog­ra­phy resins in­volves a dis­rup­tion in bind­ing in­ter­ac­tions of the AAV from the im­mo­bi­lized lig­and by al­ter­ing the buffer com­po­si­tion. Forge’s uni­ver­sal buffer ma­tri­ces for equi­li­bra­tion, wash­ing, and elut­ing AAV par­ti­cles from affin­i­ty resins were con­scious­ly de­signed for broad ap­plic­a­bil­i­ty to a va­ri­ety of cap­sid serotypes. Fig­ure 2 be­low high­lights the pro­fi­cien­cy of Forge’s affin­i­ty plat­form across a va­ri­ety of serotypes.

Process Chal­lenges and So­lu­tions for Scal­ing Affin­i­ty Chro­mato­graph­ic Pu­rifi­ca­tion of AAV Vec­tors

Pu­rifi­ca­tion of AAV at scale re­mains chal­leng­ing and cost­ly due to the han­dling of large work­ing vol­umes, the dif­fi­cul­ty in re­mov­ing im­pu­ri­ties, and the com­plex­i­ties in re­cov­er­ing vec­tor genomes. Top con­sid­er­a­tions dur­ing the scale up of affin­i­ty process­es in­clude, but are not lim­it­ed to, 1) AAV sta­bil­i­ty, 2) resin bind­ing ca­pac­i­ty, and 3) res­i­dence time.

1. AAV Sta­bil­i­ty: Pro­cess­ing time is a key con­sid­er­a­tion when mov­ing di­rect­ly in­to affin­i­ty chro­matog­ra­phy from clar­i­fied har­vest, as ex­tend­ed pro­cess­ing times can im­pact prod­uct sta­bil­i­ty. While there are meth­ods to com­bat the ex­tend­ed pro­cess­ing times of large-scale affin­i­ty chro­matog­ra­phy (such as an ul­tra­fil­tra­tion / di­afil­tra­tion (UF / DF) step post-clar­i­fi­ca­tion), ad­di­tion­al pro­cess­ing steps in­tro­duce risk for vec­tor loss and in­crease the cost of goods per batch.
2. Resin Bind­ing Ca­pac­i­ty: The bind­ing ca­pac­i­ty of an affin­i­ty chro­matog­ra­phy resin dic­tates the con­cen­tra­tion range of vi­ral genomes that can be ef­fec­tive­ly loaded on­to the resin. Uni­ver­sal and high-speci­fici­ty resins ad­ver­tise a bind­ing ca­pac­i­ty of 10¹² – 10¹⁴ vg/mL resin. While the up­per lim­it of the bind­ing ca­pac­i­ty is cost-ef­fec­tive by re­quir­ing less resin, the low­er lim­it of the bind­ing ca­pac­i­ty can pro­vide pro­cess­ing time ef­fi­cien­cy.
3. Res­i­dence Time: The ex­po­sure time of AAV to the resin in the affin­i­ty col­umn (i.e., res­i­dence time) al­so im­pacts the to­tal pro­cess­ing time of the affin­i­ty unit op­er­a­tion.

Forge’s ap­proach to a scal­able affin­i­ty chro­matog­ra­phy step main­tains a man­age­able and con­sis­tent to­tal pro­cess­ing time. As our da­ta sup­ports, the in­crease in res­i­dence time with in­creas­ing scale does not neg­a­tive­ly im­pact the affin­i­ty chro­matog­ra­phy process, as seen in Fig­ure 3, and is a pa­ra­me­ter that is op­ti­mized when lin­ear ve­loc­i­ty de­creas­es. Es­tab­lish­ing large-scale chro­matog­ra­phy pro­cess­ing sys­tems and pro­to­cols for cGMP man­u­fac­tur­ing that are com­pa­ra­ble to bench-scale mod­els is a fa­mil­iar ob­sta­cle that Forge con­sid­ers ear­ly dur­ing process de­vel­op­ment. Forge’s affin­i­ty chro­matog­ra­phy plat­form lever­ages the Repli­gen ARTeSYN and Cy­ti­va ÄK­TA Ready™ 450 sin­gle-use chro­matog­ra­phy sys­tems and has demon­strat­ed process ef­fi­cien­cy and in­creased re­cov­er­ies at scale.

Forge Bi­o­log­ics in­creas­es safe­ty and ef­fi­ca­cy by pro­vid­ing a com­mer­cial-ready process through:

• The use of sin­gle-use flow paths, recipe-dri­ven, and au­to­mat­ed affin­i­ty chro­matog­ra­phy meth­ods to mit­i­gate risks as­so­ci­at­ed with cross-prod­uct con­t­a­m­i­na­tion, as well as to en­hance re­pro­ducibil­i­ty be­tween pro­duc­tions
• Process con­ti­nu­ity and con­sis­tent pro­cess­ing time across scales
• In­creased re­cov­ery and im­pu­ri­ty re­moval us­ing the 21 CFR part 11 com­pli­ant Repli­gen ARTeSYN and Cy­ti­va ÄK­TA Ready™

In­creased Prod­uct Qual­i­ty and Down­stream Suc­cess

A pro­duc­tive and con­sis­tent affin­i­ty chro­matog­ra­phy method is a crit­i­cal pro­cess­ing step for the suc­cess of fur­ther down­stream pu­rifi­ca­tion. Host cell im­pu­ri­ties, resid­u­als, and “emp­ty” cap­sids, which lack the gene of in­ter­est and there­fore do not pro­vide ther­a­peu­tic ben­e­fit², are byprod­ucts of AAV man­u­fac­tur­ing that must be re­moved or re­duced dur­ing down­stream pu­rifi­ca­tion. While affin­i­ty chro­matog­ra­phy is un­able to dis­tin­guish and re­move emp­ty cap­sids¹, its abil­i­ty to re­move and re­duce process im­pu­ri­ties aids in the ef­fi­cien­cy of the sub­se­quent pol­ish­ing step (i.e., CsCl ul­tra­cen­trifu­ga­tion gra­di­ent or an­ion ex­change chro­matog­ra­phy).

Im­pu­ri­ty re­moval dur­ing affin­i­ty chro­matog­ra­phy is re­liant on de­vel­op­ing ef­fec­tive wash step con­di­tions. The goal of the wash step is to re­move un­bound par­ti­cles and oth­er pro­teins that bind non­specif­i­cal­ly to the affin­i­ty col­umn. A few con­sid­er­a­tions for de­vel­op­ing a wash step for affin­i­ty chro­matog­ra­phy in­clude op­ti­mal buffer con­di­tions, flow ori­en­ta­tion, and wash col­umn vol­umes. Salt com­po­si­tion and con­cen­tra­tion must be care­ful­ly bal­anced in the wash buffer to avoid pre­ma­ture vec­tor elu­tion while pro­mot­ing ef­fi­cient im­pu­ri­ty re­moval. Flow ori­en­ta­tion can al­so play an im­pact­ful role in re­mov­ing non­spe­cif­ic pro­teins which be­come more con­cen­trat­ed at the col­umn en­trance and less con­cen­trat­ed at the col­umn ex­it. Uti­liz­ing up- and down-flow func­tions of your chro­matog­ra­phy sys­tem dur­ing the wash step pre­vents non­spe­cif­ic pro­teins from re­bind­ing through­out the col­umn and in­stead re­leas­es them from the resin in­to the wash frac­tion. The amount of wash buffer that is passed over the resin in the two flow ori­en­ta­tions (up-flow & down-flow) can dras­ti­cal­ly af­fect the pu­ri­ty pro­file of the fi­nal AAV prod­uct.

Forge’s be­spoke plat­form demon­strates note­wor­thy re­moval of host cell DNA, host cell pro­tein, and to­tal pro­tein im­pu­ri­ties at the affin­i­ty chro­matog­ra­phy step as shown in Fig­ure 4 be­low, pro­vid­ing op­ti­mal ma­te­r­i­al for fur­ther suc­cess­ful down­stream pro­cess­ing.

Con­clu­sion

The AAV gene ther­a­py in­dus­try is suc­cess­ful­ly tack­ling mul­ti­ple ob­sta­cles for es­tab­lish­ing scal­able, com­mer­cial-ready down­stream pu­rifi­ca­tion process­es. Time­lines for affin­i­ty chro­matog­ra­phy process im­prove­ments can be re­duced with­out neg­a­tive­ly im­pact­ing prod­uct qual­i­ty by ap­proach­ing de­vel­op­ment from a da­ta-backed start­ing point sup­port­ed with process de­vel­op­ment ground­work com­plet­ed by Forge on be­half of clients. Forge has over­come affin­i­ty chro­matog­ra­phy pro­cess­ing chal­lenges while main­tain­ing or in­creas­ing re­cov­er­ies when mov­ing from bench­top to GMP scale sys­tems. This demon­stra­tion of scal­a­bil­i­ty uti­liz­ing sin­gle-use tech­nolo­gies for the pu­rifi­ca­tion of clin­i­cal ma­te­r­i­al and Forge’s re­cent fa­cil­i­ty ex­pan­sion are es­sen­tial mile­stones in es­tab­lish­ing and pro­vid­ing clients with a com­mer­cial­ly vi­able process at 1,000L and greater.

Ref­er­ences

1. Uni­ver­sal Method for the Pu­rifi­ca­tion of Re­com­bi­nant AAV Vec­tors of Dif­fer­ing Serotypes Shel­ley A. Nass,¹ Maryellen A. Mat­ting­ly,¹ Denise A. Wood­cock,¹ Bren­da L. Burn­ham,¹ Jef­frey A. Ardinger,¹Shay­la E. Os­mond,¹ Amy M. Fred­er­ick,¹ Abra­ham Scaria,¹ Seng H. Cheng,¹ and  Cather­ine R. O’Ri­or­dan^1,∗https://www.ncbi.nlm.nih.gov/pmc/ar­ti­cles/PMC5767896/#bib10

2. AAV Emp­ty Cap­sids: For Bet­ter or for Worse? J Fras­er Wright¹ https://www.ncbi.nlm.nih.gov/pmc/ar­ti­cles/PMC3978789/

Co-Au­thor: Bri­an­na Bar­rett, Ph.D., As­so­ciate Di­rec­tor, Tech­ni­cal Sales & Sci­en­tif­ic Ad­vi­so­ry, Forge Bi­o­log­ics

Con­trib­u­tors: Bryant Yung, Ph.D., Se­nior Sci­en­tist, Process De­vel­op­ment, Forge Bi­o­log­ics and Mikhail Gavrilin, Ph.D., As­so­ciate Di­rec­tor, An­a­lyt­i­cal De­vel­op­ment, Forge Bi­o­log­ics