JOURNAL OF INTERFERON & CYTOKINE RESEARCH 22:1181–1184 (2002)© Mary Ann Liebert, Inc.

A Comparison of the Biologic Activity of Two RecombinantIFN-b Preparations Used in the Treatment of Relapsing-

Remitting Multiple Sclerosis

FRANCESCO ANTONETTI,1 ORNELLA FINOCCHIARO,1 MICHELE MASCIA,1

MARIA GRAZIA TERLIZZESE,1 and AMER JABER2

ABSTRACT

Recent clinical trials with interferon-b (IFN-b) in relapsing-remitting multiple sclerosis (RRMS) have clearlydemonstrated that the IFN-b dosing regimen affects the clinical efficacy, thereby highlighting the importanceof determining the relative biologic activities of the IFN-b products currently available. Although studies havebeen published that examine the biologic activities of the two structurally different forms of recombinant IFN-b,IFN-b1a (Rebif®, Serono, Geneva, Switzerland) and IFN-b1b (Betaseron®/Betaferon®, Berlex [Montville, NJ]/Schering [Berlin, Germany]), there have been few direct comparative studies. Therefore, to obtain a more ac-curate estimate of the relative biologic activities of Rebif and Betaseron, this study examined the antiviral ac-tivities of these two products within the same assay system and against the same natural human IFN-b stan-dard. Whereas the manufacturers’ information suggests that the bioactivity of Betaseron is only about 8.7-foldless than that of Rebif, the results of the present direct, comparative study show that Rebif has an antiviralactivity 14 times greater than that of Betaseron. This may have important clinical implications, because onthe basis of the results reported here, Rebif at 44 mg t.i.w. is approximately double the maximal licensedweekly dose for Betaseron.

1181

THREE RECOMBINANT INTERFERON-b (IFN-b) preparations arecurrently available for treatment of relapsing forms of mul-

tiple sclerosis (MS): three times weekly (t.i.w.) IFN-b1a(Rebif®, Serono, Geneva, Switzerland), once weekly (q.w.)IFN-b1a (Avonex®, Biogen, Cambridge, MA), and every otherday (q.o.d.) IFN-b1b (Betaseron®/Betaferon®, Berlex[Montville, NJ]/Schering [Berlin, Germany]), although thereare differences between the products (Table 1).(1,2) Recent ev-idence has shown that dosing regimen impacts clinical efficacy,with higher and more frequent (t.i.w.) doses being more effica-cious than lower, less frequent doses.(3–6) The two preparationsof IFN-b1a, Rebif and Avonex, have been shown previously tohave identical specific activities within the same assay.(7) Thepresent study was designed to compare the biologic activities ofIFN-b1a (Rebif) with IFN-b1b (Betaseron/Betaferon).

IFN-b1a (Rebif) is a glycosylated product that is expressedin mammalian (Chinese hamster ovary [CHO]) cells and is vir-tually identical to the native protein.(8) In contrast, IFN-b1b(Betaseron/Betaferon) is a nonglycosylated product expressedin Escherichia coli.(9) In addition to its lack of glycosylation,it differs from the native protein in that it has a serine rather

than a cysteine residue at position 17 and lacks N-terminal me-thionine. These structural differences may account for the lowerpotency of IFN-b1b compared with IFN-b1a(10) and the lowerproportion of patients who develop neutralizing antibodies withIFN-b1a compared with IFN-b1b.(4)

To date, only a handful of studies have compared the bio-logic activities of different IFN-b preparations.(10–13) The spe-cific activity for Betaseron provided by the manufacturer is 32MIU/mg, and that of Rebif is 270 MIU/mg. However, differ-ent IFN-b standards were used in determining these specific ac-tivities (Rebif is measured against a WHO natural IFN-b stan-dard NIH 531; for Betaseron, a WHO reference standard ofrecombinant IFN-b1b is used), and they may not be directlycomparable. If these standards are compared against each other,the natural standard measured vs. recombinant IFN-b1b givesdouble the labeled bioactivity. Conversely, the recombinantstandard gives less than half the labeled biologic activity whenmeasured against the natural standard. The aim of this study,therefore, was to compare the antiviral activity of these twoIFN-b products within the same assay system against the samehighly purified natural human fibroblast-derived IFN-b stan-

1Instituto di Ricerca Cesare Serono, Ardea, Rome, Italy.2Laboratoires Serono, 1267 Coinsins (VD), Switzerland.This study was conducted at the Instituto di Ricerca Cesare Serono, Rome, Italy.

SHORT COMMUNICATION

dard (the in-house standard BILS-11 calibrated against the in-ternational standard NIH 531) (Table 2).

The antiviral activities of preformulated Rebif (RBIF, Serono,32005 process, 0.354 mg/ml protein content, specific activity2.8 3 108 IU/mg) and Betaseron (Berlex, batch BAP 3053, la-beled potency 9.6 3 106 IU in 0.3 mg) were assessed using thehuman WISH cell/vesicular stomatitis virus (VSV) cytopathic as-say (CPE) system.(14) The potencies of the preparations wereevaluated against natural human fibroblast-derived IFN-b (in-house standard BILS-11) using a 3 3 3 balanced parallel line as-say, based on the statistical parameters (regression, linearity, parallelism, and curvature) according to the United States Phar-macopoeia and European Pharmacopoeia.(15,16) The wholedose-response curves were run by performing eight serial (1.5-fold) dilutions of each IFN preparation. The starting concen-tration for Rebif was 1:500,000 (0.71 ng/ml) in each case. Be-

taseron was tested with starting concentrations of 12.5 ng/ml,10 ng/ml, and 6.25 ng/ml. The whole dose-response curve ofthe in-house standard BILS-11 (1:75 starting dilution, i.e., 200IU/ml) was run in the same plate as a control. Dose-responsecurves (calculated by averaging the results of three independentexperiments) were then prepared and used to calculate the massratio of the Rebif and Betaseron preparations.

The results obtained from these assays clearly showed thatthe antiviral dose-response curve for Betaseron, measured inthe WISH/VSV assay, lay to the right of that for Rebif (Fig. 1),indicating that Rebif is more potent than Betaseron. Further-more, when the preparations were assayed at starting concen-trations of 0.71 ng/ml for Rebif and 10 ng/ml for Betaseron,the dose-response curves could be superimposed (Fig. 2). Theabscissa of the overlapping curves shows that the highest con-centrations per well for Rebif and Betaseron were 0.236 ng/ml

ANTONETTI ET AL.1182

TABLE 1. IFN-b PRODUCTS CURRENTLY AVAILABLE

Betaseron®/Betaferon®

Rebif ® (Serono) Avonex® (Biogen) (Berlex/Schering)

Weekly dosing regimena 44 mg s.c. t.i.w. 30 mg i.m. q.w. 8 MIU s.c. q.o.d.International non- IFN-b1a IFN-b1a IFN-b1b

proprietary nameCell origin, molecular Mammalian, as Mammalian, as E. coli bacteria, not

structure natural IFN-b natural IFN-b natural IFN-bSpecific activity provided 270 3 106 IU/mg 200 3 106 IU/mg 32 3 106 IU/mg

by manufacturerb

Weekly dose 35.6 MIU (132 mg) 6 MIU (30 mg) 28 MIU (875 mg)Specific activity using 270 3 106 IU/mg 270 3 106 IU/mg 20 3 106 IU/mg

same bioassayc

Weekly dose 35.6 MIU (132 mg) 8.1 MIU (30 mg) 17.5 MIU (875 mg)

as.c., subcutaneous; i.m., intramuscular; t.i.w., three times weekly; q.w., once weekly; q.o.d., every other day; MIU, millioninternational units.

bManufacturers’ prescribing information.cIU/mg in same bioassay.

TABLE 2. EQUIVALENCE OF REBIF® AND BETASERON® ACTIVITY

Potency vs. BILS-11 (highly Mass ratio vs.Labeled Mass ratio purified natural human Rebif ® (based onpotency as per vs. Rebif® fibroblast-derived; potency assessedvial (based on in-house standard) vs. naturalMIU/vial/mla labeled MIU/ml human fibroblastMIU/mg potency) MIU/mg standard)

Rebif ® lot 23 — Rebif ® lot 23 —100 MIU/ml 97 MIU/ml2.8 MIU/mg 2.7 MIU/mg

Betaseron® 8.7 Betaseron® 149.6 MIU/ml 6.0 MIU/ml32 MIU/mg 20 MIU/mg

NIH 535 7.8 NH 535 standard 15standard (rIFN-b1b)(rIFN-b1b) 2958 MIU/ml6000 MIU/ml 1.8 MIU/mg3.6 MIU/mg

aMIU, million international units.

and 3.333 ng/ml, respectively, giving a Betaseron/Rebif massratio of 14.

In this study, the antiviral potency of Betaseron wasmarkedly lower than that of Rebif. A similar conclusion wasreached in a study comparing formulated Rebif with formulatedBetaseron. This evidence would imply that in clinical practice,the amount of protein required to achieve a biologic responsewould be considerably higher with Betaseron than with Rebif.The fact that IFN-b1b is not identical in structure to the nativeprotein, together with the need to inject a larger amount of pro-tein than with IFN-b1a, may contribute to a higher incidenceof neutralizing antibody formation with IFN-b1b than with IFN-b1a.(11)

The specific activity of Betaseron of 32 MIU/mg providedby the manufacturer was determined using the WHO recombi-

nant IFN-b1b standard, whereas this study shows it to be only20 MIU/mg when compared with natural fibroblast IFN-b (in-house standard BILS-11 calibrated against the WHO NIH 531standard). Betaseron, therefore, has a biologic potency (as as-sessed by antiviral activity) 14 times lower than that of Rebif,which has a specific activity of 270 MIU/mg. This differenceis in close agreement with the work of Runkel et al.,(10) whofound IFN-b1a (Avonex) to have a 10–15-fold greater potencythan Betaseron. The dose of Rebif (44 mg t.i.w.) is, therefore,equivalent to approximately double the total weekly dose ofIFN-b received by a patient on Betaseron (8 MIU q.o.d.). In-deed, pharmacodynamic studies have shown that single dosesof IFN-b1b, 8 MIU, and of IFN-b1a, 22 mg, resulted in equiv-alent pharmacodynamic effects on serum markers of immuneactivation.(11,12)

BIOLOGIC ACTIVITY OF TWO IFNs USED IN RRMS 1183

FIG. 1. Comparison of Rebif® and Betaseron® antiviral dose-response curves. The Rebif and Betaseron concentrations are plot-ted using the same scale. The results are means 6 SEM of three independent assays. OD, optical density.

FIG. 2. The antiviral dose-response curve for Betaseron® matches that of Rebif® when the starting concentrations of theseagents are 10 ng/ml and 0.71 ng/ml, respectively. This indicates that Betaseron is 14 times less potent than Rebif on a mass ba-sis. OD, optical density.

The results of this study show that administering Rebif, 44mg t.i.w., provides approximately double the antiviral activityof IFN-b compared with Betaseron, 8 MIU q.o.d., and a dose-dependent effect of IFN-b has been shown.(5,6,17) This analy-sis will provide additional information for clinicians who wishto use the maximal approved dose of IFN for patients with MS.

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Address reprint requests or correspondence to:Dr. Amer Jaber

Laboratoires SeronoCentre Industriel

1267 Coinsins (VD)Switzerland

Tel: 141 22 354 54 52Fax: 141 22 354 50 17

E-mail: amer.jaber@serono.com

Received 17 July 2002/Accepted 26 September 2002

ANTONETTI ET AL.1184

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