In­no­v­a­tive An­a­lyt­i­cal Ul­tra­cen­trifu­ga­tion Tech­niques for the Char­ac­ter­i­za­tion of AAV Vec­tors

The ther­a­peu­tic use of ade­no-as­so­ci­at­ed virus (AAV) ac­counts for the largest share of the glob­al gene ther­a­py in­dus­try due to its safe­ty pro­file and proven ef­fi­ca­cy in treat­ing ge­net­ic dis­eases. Over the past 30 years, sci­en­tists, clin­i­cians, and biotech in­dus­try pro­fes­sion­als have worked with con­tract de­vel­op­ment and man­u­fac­tur­ing or­ga­ni­za­tions (CD­MOs) to har­ness the nat­ur­al abil­i­ties of AAV to de­liv­er ge­net­ic in­for­ma­tion to spec­i­fied cell types. The col­lab­o­ra­tion be­tween AAV gene ther­a­py de­vel­op­ers and CD­MOs has re­sult­ed in scal­able man­u­fac­tur­ing so­lu­tions to tran­si­tion “a gene and a dream” in­to a bi­o­log­i­cal ther­a­peu­tic poised for suc­cess­ful pa­tient out­comes and a greater num­ber of com­mer­cial­ized treat­ments.

Gen­er­a­tion, Char­ac­ter­i­za­tion, and Re­moval of Emp­ty AAV Cap­sids

Man­u­fac­tur­ing process­es are con­tin­u­ous­ly op­ti­mized to en­sure ef­fi­cient, safe, and pure AAV gene ther­a­py prod­ucts are con­sis­tent­ly de­liv­ered to pa­tients. How­ev­er, with­in a pro­duc­tion cell line, AAV as­sem­bly and DNA pack­ag­ing process­es nat­u­ral­ly lead to a mix­ture of full, emp­ty, and some par­tial genome-con­tain­ing cap­sid pop­u­la­tions. While it has been demon­strat­ed that AAV con­tain­ing par­tial genomes can re­tain a lev­el of ther­a­peu­tic ben­e­fit, it is of­ten with re­duced po­ten­cy in com­par­i­son to full genome con­tain­ing cap­sids1. Ad­di­tion­al­ly, emp­ty AAV par­ti­cles con­tain no ther­a­peu­tic ben­e­fit while in­creas­ing the over­all vi­ral load and risk of im­mune re­sponse. Fur­ther­more, a het­ero­ge­neous pop­u­la­tion of genome frag­ments with­in a fi­nal AAV drug prod­uct can be ex­pect­ed to show in­con­sis­ten­cies from lot-to -lot and cause is­sues when at­tempt­ing to de­fine crit­i­cal qual­i­ty at­trib­ut­es of the prod­uct. For this rea­son, cap­sids lack­ing a full vec­tor genome are con­sid­ered process im­pu­ri­ties and are re­duced through var­i­ous meth­ods dur­ing down­stream pro­cess­ing.

Op­ti­miz­ing full cap­sid en­rich­ment tech­niques, which may lead to achiev­ing high­er ther­a­peu­tic ef­fi­ca­cy with less resid­ual im­pu­ri­ties, is an area of con­cen­trat­ed fo­cus for the down­stream process and an­a­lyt­i­cal de­vel­op­ment teams at Forge Bi­o­log­ics. Our teams strate­gi­cal­ly em­ploy ad­vanced tech­nolo­gies such as an­a­lyt­i­cal ul­tra­cen­trifu­ga­tion (AUC) tech­niques to un­der­stand the im­pact of process mod­i­fi­ca­tions and to char­ac­ter­ize the ge­net­ic con­tents pack­aged with­in an AAV cap­sid. An­a­lyt­i­cal ul­tra­cen­trifu­ga­tion analy­sis can iden­ti­fy full, emp­ty, and par­tial cap­sids from in-process and fi­nal AAV sam­ples, with high pre­ci­sion and ac­cu­ra­cy, al­low­ing this method to be con­sid­ered the gold stan­dard for quan­ti­fy­ing dis­tinct cap­sid pop­u­la­tions.

Down­stream Pu­rifi­ca­tion Strate­gies for Full Cap­sid En­rich­ment

CsCl Den­si­ty Gra­di­ent Pu­rifi­ca­tion
Ce­sium chlo­ride (CsCl) den­si­ty gra­di­ent ul­tra­cen­trifu­ga­tion tech­niques ex­ploit dif­fer­ences in buoy­an­cies of emp­ty and full cap­sids to achieve sep­a­ra­tion. CsCl and AAV move to­geth­er as cen­trifu­gal force moves heavy den­si­ties of CsCl and full cap­sids to­ward the bot­tom of the gra­di­ent, while lighter den­si­ties of CsCl and emp­ty cap­sids re­main clos­er to the top, as seen in fig­ure 2A. Ad­di­tion­al species oc­ca­sion­al­ly en­coun­tered are in­ter­me­di­ate bands of par­tial genomes be­tween the bands of full or emp­ty cap­sids, as well as high-den­si­ty bands be­low the full cap­sid band. Af­ter ex­tend­ed ul­tra­cen­trifu­ga­tion, the bands con­tain­ing full cap­sids are col­lect­ed from the tube and titered by ddPCR. The sam­ples con­tain­ing full AAV are then pooled and fur­ther pu­ri­fied.

Full cap­sid en­rich­ment via CsCl ul­tra­cen­trifu­ga­tion con­sis­tent­ly re­sults in a fi­nal prod­uct with greater than 80-90% full cap­sids. In ad­di­tion, this method is cost-ef­fec­tive and can be per­formed with­out in­ten­sive de­vel­op­ment steps, which speeds up time­lines to achieve pre-clin­i­cal and ear­ly clin­i­cal mile­stones. How­ev­er, CsCl ul­tra­cen­trifu­ga­tion re­quires open pro­cess­ing steps and be­comes a la­bo­ri­ous process on large pro­duc­tion scales.

Ion Ex­change Pu­rifi­ca­tion
An al­ter­na­tive method for sep­a­rat­ing emp­ty and full par­ti­cles is ion ex­change chro­matog­ra­phy (IEX) with a mono­lith or resin-packed col­umn. This method dif­fer­en­ti­ates AAV par­ti­cles based on minute dif­fer­ences in the iso­elec­tric point amongst emp­ty, full, or par­tial­ly full cap­sids. This re­sults in elu­tion from the col­umn with the ap­pli­ca­tion of a con­duc­tiv­i­ty gra­di­ent, as seen in fig­ure 2B. This method al­lows for a ful­ly closed sys­tem and scal­able process for sep­a­rat­ing emp­ty, full, and par­tial AAV par­ti­cles. How­ev­er, ma­te­ri­als are cost­ly, and the de­vel­op­ment time re­quired to op­ti­mize the sep­a­ra­tion of vec­tor species with a nar­row range in iso­elec­tric points can cost months.

Fig­ure 2. Emp­ty/Full Sep­a­ra­tion via CsCl den­si­ty ul­tra­cen­trifu­ga­tion or AEX Chro­matog­ra­phy. AAV was pro­duced us­ing Ig­ni­tion cells (HEK293 Sus­pen­sion Cell Line) in a shake flask at 1 L work­ing vol­ume (A) or a 50 L biore­ac­tor (B). Cells were triple trans­fect­ed with Rep/Cap, a sin­gle strand­ed gene of in­ter­est un­der 4.7kb, and pEM­BR (Ad Helper Plas­mid) to pro­duce AAV, lysed, har­vest­ed, and clar­i­fied. (A) Clar­i­fied AAV9.GFP was processed through affin­i­ty chro­matog­ra­phy, elut­ed, loaded on­to a CsCl gra­di­ent, and spun overnight. Vis­i­ble full (bot­tom band) and emp­ty band­ing (top band) was present af­ter CsCl den­si­ty ul­tra­cen­trifu­ga­tion. (B) A styl­ized rep­re­sen­ta­tion of a chro­matogram pro­duced from an op­ti­mized an­ion ex­change chro­matog­ra­phy process. Typ­i­cal prepa­ra­tion for an AEX run in­cludes cap­ture of clar­i­fied AAV vec­tor with affin­i­ty chro­matog­ra­phy, elu­tion, and di­lu­tion in a buffer com­pat­i­ble with AEX chro­matog­ra­phy. Vec­tor is then loaded on­to a CIMQA mono­lith­ic an­ion-ex­change col­umn (or al­ter­na­tive resin) at a con­cen­tra­tion of ~1E+13 vg/mL and elut­ed over a step gra­di­ent with in­creas­ing con­cen­tra­tions of sodi­um sul­fate. Peak 1 rep­re­sents an en­riched pop­u­la­tion of emp­ty cap­sids, as in­di­cat­ed by UV ab­sorbance at 280nm ex­ceed­ing UV ab­sorbance at 260nm. Peak 2 rep­re­sents an en­riched pop­u­la­tion of full genome con­tain­ing cap­sids, as in­di­cat­ed by UV ab­sorbance at 260nm ex­ceed­ing UV ab­sorbance 280nm.

Process and AAV Prod­uct Char­ac­ter­i­za­tion by An­a­lyt­i­cal Ul­tra­cen­trifu­ga­tion 

It is cru­cial to uti­lize an­a­lyt­i­cal tech­niques that pro­vide ac­cu­rate and re­pro­ducible mea­sure­ments of emp­ty, full and/or par­tial cap­sids. Sev­er­al meth­ods that have been ex­plored in­clude high-per­for­mance liq­uid chro­matog­ra­phy (HPLC), trans­mis­sion elec­tron mi­croscopy (TEM), or cal­cu­la­tion of vec­tor genome titer by qPCR ver­sus to­tal cap­sid quan­tifi­ca­tion by serotype-spe­cif­ic ELISA. While each method has pros and cons, lim­i­ta­tions in­clude long de­vel­op­ment time­lines, in­ac­cu­ra­cy, or re­quired up­front in­vest­ment in­to spe­cial­ized equip­ment & ex­per­tise. One no­table draw­back of the com­mon­ly uti­lized qPCR/ELISA cal­cu­la­tion for E/F quan­tifi­ca­tion is the lack of abil­i­ty to dis­tin­guish par­tial from full genomes lead­ing to high­er over­all er­ror. Den­si­ty-based sep­a­ra­tion by AUC has proven to be a suc­cess­ful tool in re­solv­ing emp­ty, full, and par­tial cap­sids and quan­ti­fy­ing each sub­set with un­par­al­leled pre­ci­sion and re­pro­ducibil­i­ty2.

The ba­sic prin­ci­ples of AUC re­ly on full cap­sids hav­ing greater buoy­ant den­si­ty than emp­ties, al­low­ing them to sed­i­ment more quick­ly through so­lu­tion dur­ing cen­trifu­ga­tion. Based on the ra­tio be­tween ve­loc­i­ty and cen­trifu­gal field, the sed­i­men­ta­tion co­ef­fi­cient (c(S)) is cal­cu­lat­ed. Full AAV cap­sids have a c(S) of ~100 S, emp­ty AAV cap­sids have a c(S) of ~55-65 S, and par­tial AAV cap­sids have a c(S) be­tween ~100 S and ~55 S.

To qual­i­fy the AUC method­ol­o­gy for each unique prod­uct, sam­ples of AAV with vary­ing ra­tios of full and emp­ty cap­sids are an­a­lyzed. In a sam­ple con­tain­ing 15% emp­ty and 85% full cap­sids (Fig­ure 3A & 3B) a sig­moidal curve is seen in the sed­i­men­ta­tion ve­loc­i­ty (SV) scan, but the dis­tri­b­u­tion plot shows dis­tinct peaks at 55 S and 100 S, in­di­cat­ing the pres­ence of both emp­ty and full cap­sids, re­spec­tive­ly. In a sam­ple con­tain­ing 30% emp­ty and 70% full cap­sids, a top curve, bot­tom curve, and cen­tral in­flec­tion point are seen in the SV scan (Fig­ure 3C), as well as a dis­tri­b­u­tion plot with dis­tinct full, emp­ty, and par­tial species (Fig­ure 3D). A prepa­ra­tion of AAV, en­riched for full cap­sids us­ing a CsCl ul­tra­cen­trifu­ga­tion method shows an SV scan with a uni­form sig­moidal curve (Fig­ure 3E), as well as a dis­tri­b­u­tion curve with an el­e­vat­ed full peak and near­ly un­de­tectable par­tial or emp­ty peaks (Fig­ure 3F).

Fig­ure 3 AUC Analy­sis of Mixed Full/Emp­ty AAV or CsCl pu­ri­fied AAV. AAV sam­ples in fi­nal for­mu­la­tion buffer were pre­pared for AUC analy­sis by di­lut­ing the sam­ple to 0.5 O.D. in 1 mL of ref­er­ence buffer. (A&B) Pu­ri­fied sam­ples of emp­ty and full AAV were mixed at 15% emp­ty and 85% full and an­a­lyzed by AUC. (B) A styl­ized rep­re­sen­ta­tion of a chro­matogram pro­duced from an op­ti­mized an­ion ex­change (AEX) chro­matog­ra­phy process. (C&D) Pu­ri­fied sam­ples of emp­ty and full AAV were mixed at 30% emp­ty and 70% full and an­a­lyzed by AUC. In the SV scan, full cap­sids are rep­re­sent­ed at the bot­tom por­tion of the curve, par­tial cap­sids are rep­re­sent­ed in the mid­dle in­flec­tion point of each curve, and emp­ty cap­sids are seen in the top por­tion of the curve (C). A cor­re­spond­ing dis­tri­b­u­tion curve shows emp­ty cap­sids near 55 S and full cap­sids near 100 S (D). (E&F) An AAV sam­ple pu­ri­fied by CsCl ul­tra­cen­trifu­ga­tion for the pol­ish­ing step, shows a uni­form sig­moidal curve on the SV scan (E). A cor­re­spond­ing dis­tri­b­u­tion curve (F) shows a full cap­sid peak near 100 S with near­ly un­de­tectable lev­els of emp­ty or par­tial cap­sids. (G) Sam­ple de­scrip­tions with cor­re­spond­ing emp­ty and full cap­sid per­cent­ages as mea­sured by AUC and an­a­lyzed with SED­fit soft­ware.

Forge Bi­o­log­ics In-house AUC Ca­pa­bil­i­ties 

With Beck­man Coul­ter’s Op­ti­ma AUCs in-house, Forge Bi­o­log­ics quan­ti­fies full, par­tial, and emp­ty AAV species with high pre­ci­sion and ac­cu­ra­cy. By de­sign­ing a sys­tem­at­ic ap­proach to de­vel­op­ing down­stream pu­rifi­ca­tion process­es for each unique prod­uct, Forge achieves in­dus­try-lead­ing sep­a­ra­tion of emp­ty from full AAV par­ti­cles. In­te­grat­ed in the process de­vel­op­ment strat­e­gy, is the strate­gic use of AUC analy­sis to char­ac­ter­ize progress and im­prove­ments be­gin­ning ear­ly in the de­vel­op­ment process. The tac­ti­cal use of in­no­v­a­tive man­u­fac­tur­ing and an­a­lyt­i­cal equip­ment al­lows Forge to ef­fi­cient­ly pro­duce AAV drug prod­ucts of op­ti­mal pu­ri­ty. In ad­di­tion, Forge’s at­ten­tion to scal­able pu­rifi­ca­tion meth­ods has cre­at­ed read­i­ly avail­able, plat­form pro­to­cols to rapid­ly de­liv­er qual­i­ty vec­tor pro­duc­tions from less than 1 liter to 5,000 liters.

Con­tribut­ing Au­thor: Ju­lianne Bartz, Sci­en­tist II, Forge Bi­o­log­ics


1 Dong, B., Nakai, H., & Xi­ao, W. (2010). Char­ac­ter­i­za­tion of genome in­tegri­ty for over­sized re­com­bi­nant AAV vec­tor. Mol­e­c­u­lar ther­a­py18(1), 87-92.
2 Wer­le AK, Pow­ers TW, Zo­bel JF, et al. Com­par­i­son of an­a­lyt­i­cal tech­niques to quan­ti­tate the cap­sid con­tent of ade­no-as­so­ci­at­ed vi­ral vec­tors. Mol Ther Meth­ods and Clin Dev. 2021;1(23):254-262.


Brianna Barrett

Ph.D., Associate Director, Technical Sales, Forge Biologics