Prac­ti­cal im­pli­ca­tions of re­cent FDA guid­ance on im­muno­genic­i­ty risk as­sess­ments on your IND sub­mis­sions

Com­pre­hen­sive bio­an­a­lyt­i­cal strat­e­gy has be­come an im­por­tant part of safe­ty as­sess­ment of bi­o­log­ic ther­a­pies, and un­less spon­sors ex­e­cute ad­e­quate test­ing to as­cer­tain im­muno­genic­i­ty of can­di­date bi­o­log­ic drugs, reg­u­la­to­ry agen­cies may place a clin­i­cal hold on a pro­gram, cost­ing a com­pa­ny valu­able time.

In Jan­u­ary 2019, the FDA up­dat­ed its guid­ance for the de­vel­op­ment and val­i­da­tion of im­muno­genic­i­ty test­ing as­says, rec­om­mend­ing a risk-based ap­proach to eval­u­at­ing and man­ag­ing im­mune re­spons­es elicit­ed by ther­a­peu­tic pro­teins. There are many up­dates in the 2019 guid­ance com­pared to the ear­li­er guid­ance, but up­dates in three ar­eas – de­ter­mi­na­tion of the cut point, de­vel­op­ment of as­says for neu­tral­iz­ing an­ti­bod­ies (NAbs) and changes in doc­u­men­ta­tion – car­ry sig­nif­i­cant con­se­quences for build­ing the IND pack­age.

Neu­tral­iz­ing An­ti­bod­ies

Dur­ing the course of im­muno­genic­i­ty test­ing, if an­tidrug an­ti­bod­ies are iden­ti­fied, it’s very im­por­tant to as­sess if any of these an­tidrug an­ti­bod­ies are NAbs that would cause ad­verse ef­fects on the ther­a­peu­tic pro­tein. NAbs have the po­ten­tial to dra­mat­i­cal­ly in­ter­fere with clin­i­cal ac­tiv­i­ty of a ther­a­peu­tic pro­tein in­clud­ing, chang­ing PK, PD, safe­ty and ef­fi­ca­cy.

The FDA rec­om­mends that com­pa­nies car­ry out as­says to de­ter­mine whether NAbs are present if ear­ly im­muno­genic­i­ty test­ing re­sults in sta­tis­ti­cal­ly sig­nif­i­cant (above cut point) ADA re­spons­es. As with all im­muno­genic­i­ty test­ing, in­ad­e­quate test­ing ahead of first in-hu­man tri­als risks clin­i­cal hold, which can af­fect pro­gram bud­gets and time­lines.

De­vel­op­ing re­li­able neu­tral­iza­tion as­says can be chal­leng­ing es­pe­cial­ly when it’s crit­i­cal that any NAb as­say has the ca­pa­bil­i­ty to re­li­ably de­tect NAbs with ad­e­quate sen­si­tiv­i­ty, speci­fici­ty, se­lec­tiv­i­ty and pre­ci­sion(2). The dif­fer­ing as­say for­mats – whether cell-based or non-cell-based – can car­ry dis­tinct chal­lenges. For ex­am­ple, pre­vi­ous guid­ance rec­om­mend­ed us­ing cell-based as­says as much as pos­si­ble for NAb de­tec­tion, yet cer­tain mech­a­nisms of ac­tion – en­zy­mat­ic re­place­ment that doesn’t re­quire cel­lu­lar up­date, for ex­am­ple – are not amenable to cell-based as­says. Giv­en this, the FDA’s up­dates al­low greater flex­i­bil­i­ty for de­sign­ing the most ap­pro­pri­ate as­say to de­tect NAbs.

Sta­tis­ti­cal Ap­proach to De­ter­min­ing Cut Point

The cut-point of the as­say de­ter­mines whether the sam­ple re­sponse is pos­i­tive or neg­a­tive, and es­tab­lish­ing the ap­pro­pri­ate cut-point is crit­i­cal to re­duc­ing false-neg­a­tive re­sults, and as­says should be de­signed such that they gen­er­ate a 5% false pos­i­tive rate, which is im­por­tant for en­sur­ing the as­say iden­ti­fies all sub­jects who may de­vel­op an­ti­bod­ies to a ther­a­peu­tic pro­tein.

The im­por­tance of the cut point is fur­ther em­pha­sized by up­dates in the guid­ance that re­quire spon­sors to de­vel­op a sta­tis­ti­cal­ly-sound method to de­ter­mine the cut point, where­as old­er guid­ance rec­om­mend­ed es­ti­mat­ing the cut point us­ing a small num­ber of sam­ples.

In de­vel­op­ing the cut point for a screen­ing as­say, the guid­ance sug­gests a sta­tis­ti­cal ap­proach that ap­plies a 90% one-sided low­er con­fi­dence in­ter­val for the 95th per­centile of the neg­a­tive con­trol pop­u­la­tion. This as­sures at least a 5% false-pos­i­tive rate with a 90% con­fi­dence lev­el.

For es­ti­mat­ing the cut point for con­fir­ma­to­ry as­says, FDA ad­vis­es the use of an 80 to 90% one-sided low­er con­fi­dence in­ter­val for the 99th per­centile. The goal of the con­fir­ma­to­ry as­say is to elim­i­nate false-pos­i­tive sam­ples aris­ing as a re­sult of non-spe­cif­ic bind­ing. There­fore, FDA rec­om­mends de­sign­ing the cut point to a more strin­gent 1% false pos­i­tive rate.

These new sta­tis­ti­cal cal­cu­la­tions rec­om­mend­ed by the FDA of­fer more clar­i­ty than the 2016 guid­ance. To date, there is no gen­er­al­ly-ac­cept­ed stan­dard method for these de­ter­mi­na­tions, which can be chal­leng­ing for com­plex ther­a­peu­tic pro­grams.

One method de­vel­oped by WuXi AppTec and its part­ner In­te­grat­ed Med­ical De­vel­op­ment CRO, is a sta­tis­ti­cal­ly-sound es­ti­ma­tion pack­age in­clud­ing out­lier ex­clu­sion and cut-point es­ti­ma­tion, sat­is­fy­ing the new FDA re­quire­ments. The sta­tis­ti­cal method­ol­o­gy is based on or­der sta­tis­tics – Bayesian and Monte Car­lo meth­ods – and can be ap­plied to any as­say da­ta with at least 50 sam­ples. These are im­ple­ment­ed us­ing stan­dard sta­tis­ti­cal pro­gram­ming lan­guages SAS and R, is de­signed to sat­is­fy or ex­ceed the FDA con­fi­dence re­quire­ments with­out be­ing too con­ser­v­a­tive.


A ma­jor up­date in the 2019 guid­ance for de­vel­op­ment of im­muno­genic­i­ty as­says con­cerns how im­muno­genic­i­ty da­ta is pre­sent­ed in doc­u­men­ta­tion to the FDA. Pre­vi­ous­ly, im­muno­genic­i­ty da­ta was dis­persed through­out the elec­tron­ic com­mon tech­ni­cal doc­u­ment (eCTD), the stan­dard for­mat spon­sors use for sub­mit­ting da­ta to the FDA. This pre­sent­ed chal­lenges for re­view­ers try­ing to un­der­stand a ther­a­peu­tic drug can­di­date’s im­muno­genic­i­ty pro­file.

New guid­ance di­rects spon­sors to add an in­te­grat­ed im­muno­genic­i­ty sum­ma­ry re­port that clear­ly sum­ma­rizes the da­ta gen­er­at­ed in sup­port of a po­ten­tial ther­a­peu­tic pro­tein’s reg­u­la­to­ry fil­ings, al­low­ing FDA re­view­ers to un­der­stand im­muno­genic­i­ty da­ta up front.

The sum­ma­ry re­port should be di­vid­ed in­to dis­tinct sec­tions:

  1. Im­muno­genic­i­ty Risk As­sess­ment
  2. Tiered Bio­an­a­lyt­i­cal Strat­e­gy and As­say Val­i­da­tion Sum­maries
  3. Clin­i­cal Study De­sign and De­tailed Im­muno­genic­i­ty Sam­pling Plans
  4. Clin­i­cal Im­muno­genic­i­ty Da­ta Analy­sis
  5. Con­clu­sions and Risk Eval­u­a­tion and Mit­i­ga­tion Strate­gies (REMS)

The Right Out­sourc­ing Part­ner Can Help Avoid Pit­falls with the FDA

A mul­ti­dis­ci­pli­nary out­sourc­ing part­ner fa­mil­iar with the lat­est reg­u­la­to­ry changes and up­dates can help nav­i­gate pit­falls that can stymie a ther­a­py’s first in-hu­man tri­als. WuXi AppTec has the knowl­edge and ex­pe­ri­ence to de­vel­op, ex­e­cute and an­a­lyze the im­muno­genic­i­ty as­says that are es­sen­tial to max­i­mize sub­mis­sion suc­cess of your de­vel­op­ment pro­grams.


  1. FDA, Guid­ance for In­dus­try: Im­muno­genic­i­ty Test­ing of Ther­a­peu­tic Pro­tein Prod­ucts – De­vel­op­ing and Val­i­dat­ing As­says for An­ti-Drug An­ti­body De­tec­tion (Sil­ver Spring, MD, Jan­u­ary 2019).
  2. Wu, B, et. al., “Strate­gies to De­ter­mine As­say For­mat for the As­sess­ment of Neu­tral­iz­ing An­ti­body Re­spons­es to Bio­ther­a­peu­tics,” AAPS J, 18(6): 1335-1350, 2016.


John Pirro

Head of Bioanalysis