Comparison of botulinum neurotoxin type A formulations in Asia

Abstract

Results
All protein-based therapeutics, such as botulinum neurotoxin type A (BoNT/A), are potentially immunogenic and can lead to anaphylaxis, autoimmunity, or diminished or complete absence of therapeutic efficacy, especially if administered repeatedly. Therefore, the protein quantity in BoNT/A products is an important consideration when selecting products for treatment. However, essential formulation data are not always publicly accessible.

Materials and methods
The neurotoxin protein content of products newly introduced in Asia, such as (listed alphabetically) Botulax®, Meditoxin®, Nabota®, and Relatox®, was measured by sandwich enzyme-linked immunosorbent assay with antisera directed against BoNT/A compared to Xeomin®.

Results
Compared to Xeomin with no inactive neurotoxin, although Botulax and Nabota contained 844 and 754 pg of neurotoxin protein, respectively, the percentage of inactive neurotoxin was calculated to be 103 and 81, respectively, while the potency per pg of neurotoxin was 0.118 and 0.133 U, respectively. Meditoxin and Relatox had 575 and 578 pg of neurotoxins, respectively, marginally higher than that of Xeomin, while the percentage of inactive neurotoxins was 38 and 33, respectively, and the potency per pg of neurotoxin was 0.174 and 0.173 U, respectively. However, Xeomin, which has 416 pg/vial of purified neurotoxin and 0.240 U of efficacy per pg of neurotoxin, has the lowest neurotoxin protein content and consequently the highest specific potency compared to the four Asian BoNT/A preparations in this study.

Conclusion
Although Botulax and Nabota had more neurotoxin than Xeomin in an equivalent volume, they contained greater amounts of inactive neurotoxin. In addition, although Meditoxin and Relatox had slightly more neurotoxin than Xeomin, both contained greater amounts of inactive neurotoxin.

Full text

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Clinical, Cosmetic and Investigational Dermatology 2018:11 327–331
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ORIGINAL RESEARCH
open access to scientific and medical research
Open Access Full Text Article
http://dx.doi.org/10.2147/CCID.S160723
Comparison of botulinum neurotoxin type A
formulations in Asia
Jürgen Frevert1
Ki Young Ahn2
Mee Young Park3
Owen Sunga4
1Merz Pharmaceuticals GmbH,
Frankfurt, Germany; 2Dr. Ahn’s
Plastic and Aesthetic Surgical Clinic,
Daegu, South Korea; 3Department
of Neurology, Yeungnam University
School of Medicine, Daegu, South
Korea; 4Merz Asia Pacific, Singapore
Introduction: All protein-based therapeutics, such as botulinum neurotoxin type A (BoNT/A),
are potentially immunogenic and can lead to anaphylaxis, autoimmunity, or diminished or
complete absence of therapeutic efficacy, especially if administered repeatedly. Therefore, the
protein quantity in BoNT/A products is an important consideration when selecting products for
treatment. However, essential formulation data are not always publicly accessible.
Materials and methods: The neurotoxin protein content of products newly introduced in
Asia, such as (listed alphabetically) Botulax®, Meditoxin®, Nabota®, and Relatox®, was mea-
sured by sandwich enzyme-linked immunosorbent assay with antisera directed against BoNT/A
compared to Xeomin®.
Results: Compared to Xeomin with no inactive neurotoxin, although Botulax and Nabota con-
tained 844 and 754 pg of neurotoxin protein, respectively, the percentage of inactive neurotoxin
was calculated to be 103 and 81, respectively, while the potency per pg of neurotoxin was 0.118
and 0.133 U, respectively. Meditoxin and Relatox had 575 and 578 pg of neurotoxins, respectively,
marginally higher than that of Xeomin, while the percentage of inactive neurotoxins was 38
and 33, respectively, and the potency per pg of neurotoxin was 0.174 and 0.173 U, respectively.
However, Xeomin, which has 416 pg/vial of purified neurotoxin and 0.240 U of efficacy per pg
of neurotoxin, has the lowest neurotoxin protein content and consequently the highest specific
potency compared to the four Asian BoNT/A preparations in this study.
Conclusion: Although Botulax and Nabota had more neurotoxin than Xeomin in an equivalent
volume, they contained greater amounts of inactive neurotoxin. In addition, although Meditoxin
and Relatox had slightly more neurotoxin than Xeomin, both contained greater amounts of
inactive neurotoxin.
Keywords: botulinum neurotoxin type A, purity, potency, immunogenicity, Asia
Introduction
Botulinum neurotoxin type A (BoNT/A) is a leading tool in the treatment of
neuromuscular diseases and has also been used for cosmetic purposes for a long
time. During immunogenic responses, “neutralizing” antibodies develop against
the toxin, inhibiting the interaction between BoNT/A and its presynaptic mem-
brane binding site,1–3 causing an inadequate or no response to BoNT/A.4 BoNT/A
products, especially new toxin formulations, typically undergo rigorous evalua-
tions for use as prescription-only medicines. Clinicians should, therefore, select
less immunogenic, highly purified toxins to obtain successful results for long-term
repeated treatments.5
Correspondence: Jürgen Frevert
Merz Pharmaceuticals GmbH,
Hermannswerder 15, 14473 Potsdam,
Germany
Tel +49 331 230 0116
Fax +49 331 230 0199
Email Juergen.Frevert@merz.de
Journal name: Clinical, Cosmetic and Investigational Dermatology
Article Designation: ORIGINAL RESEARCH
Year: 2018
Volume: 11
Running head verso: Frevert et al
Running head recto: Comparison of botulinum neurotoxin type A formulations in Asia
DOI: http://dx.doi.org/10.2147/CCID.S160723
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New toxin formulations have recently emerged in Asia
but investigations to quantify these neurotoxins such as
their purity (defined as being complexing protein-free), the
impact of their dose on efficacy, or adverse events, have
been limited.6 Therefore, we analyzed the composition of the
neurotoxin component of each product relative to Xeomin®
using a sandwich enzyme-linked immunosorbent assay
(ELISA) with antisera directed against the purified BoNT/A,
to better understand these newer formulations. We seek to
provide clinicians with valuable information to enable safe
and effective treatment with BoNT/A.
Materials and methods
Materials
The different protein and neurotoxin contents of Botulax®
(Batch HUA 15133; Hugel Inc., Seoul, Korea), Innotox®
(Batch LAE 1401; Medytox Inc., Seoul, Korea), Meditoxin®
(Batch FAA 1587; Medytox Inc., Seoul, Korea), Nabota®
(Batch 084962; Daewoong Pharmaceutical Co. Ltd., Seoul,
Korea), Relatox® (Batch 0615; Microgen, Russia), as well as
Xeomin (Batch 31149; Merz Pharmaceuticals GmbH, Rein-
heim, Germany), were measured using a sandwich ELISA
with antisera (Table 1). Because the manufacturing process
for a biologic therapeutic should be consistent and every
batch should be representative, the batches were, therefore,
selected arbitrarily. The batches were purchased from a South
Korean pharmacy (ShinOn Pharmacy Co. Ltd., Seoul, South
Korea). Care was taken to transport and store the samples
at 2°C–8°C, except samples of Xeomin that can be stored
at room temperature. The composition of the neurotoxin
elements of each product compared to those of Xeomin was
then analyzed in duplicate to determine the mean amount of
neurotoxin protein.
Methods
All analyses were carried out with an ELISA approved by
the FDA (Food and Drug Administration, Silver Spring, MD,
USA) and several other health authorities. The facility in which
samples were analyzed was inspected by the FDA regularly and
fulfilled current good manufacturing practice requirements.
For incubation, PBS + 0.1% bovine serum albumin
(solution 1) and PBS + 6% gelafusal (Serumwerke Bern-
burg, Bernburg, Germany; solution 2) (Merck, Darmstadt,
Germany, or Riedel-de-Haen, Seelze, Germany) were used.
Additional reagents included O-phenylenediamine dihydro-
chloride (Sigma-Aldrich Corp, St. Louis, MO, USA) and
horse anti-serum reacting with the neurotoxin complex of
BoNT/A (UK National Institute for Biological Standards
and Control, NIBSC). Following a modified protocol,7 the
150 kDa neurotoxin purified from the “Hall Strain”, C. botu-
linum type A, strain ATCC 3502, was confirmed by western
blot as complexing protein-free and detoxified by 0.4%
formaldehyde treatment to produce the nontoxic antigen for
antibody preparation. Complexing proteins (excluding the
botulinum neurotoxin protein) were prepared as previously
published.8 The purified toxin was dialyzed against 50 mM
TRIS (tris[hydroxymethyl] aminomethane)/HCl pH = 7.9,
Q-sepharose column chromatography-purified (GE Health-
care, Munich, Germany) and column-bound complexing
proteins were eluted. Antibodies against BoNT/A were
immobilized on a CNBr sepharose matrix (GE Healthcare).
BoNT/A was removed through affinity chromatography,
eluted, and its composition checked for integrity.
ELISA
The amount of BoNT/A in pharmaceutical formulations of
Botulax, Meditoxin, Nabota, or Relatox was measured in
Table 1 Properties of botulinum neurotoxin type A products analyzed in this study
Product name Innotox®Botulax®Meditoxin®/
neuronox®
Nabota®Relatox®Xeomin®
Manufacturer Medytox Hugel Inc Medytox Inc Daewong Microgen Merz
Dosage (U) 25 100 100 100 100 100
Composition Complex Complex Complex Complex
(900 kDa)
Complex
(900 kDa)
Puried toxin (150 kDa)
Appearance Liquid Lyophilizate Lyophilizate Lyophilizate Lyophilizate Lyophilizate
Formulation Polysorbate
(No HSA)
0.5 mg HSA;
0.9 mg NaCl
0.5 mg HSA;
0.9 mg NaCl
0.5 mg HSA;
0.9 mg NaCl
6 mg gelatine;
12 mg maltose
4.7 mg sucrose; 1 mg HSA
Storage 2°C–8°C 2°C–8°C 2°C–8°C 2°C–8°C 2°C–8°C Room temperature (20°C–25°C)
Clostridial protein per 100 U (pg) N/A 5,00012 N/A N/A N/A 416
Notes: In other countries, Meditoxin is sold as Neuronox. As polysorbate prevents accurate ELISA readings, Innotox was not reported further in our work.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; HSA, human serum albumin; N/A, information not publicly available.

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Comparison of botulinum neurotoxin type A formulations in Asia
parallel with Xeomin using a sensitive sandwich ELISA with
anti-150 kDa neurotoxin antisera raised in rabbit and guinea
pigs. Coated microtiter plates were incubated with antisera
diluted 1:1,000 in 100 mmol/L sodium carbonate, pH = 9.5.
Except for the liquid Innotox formulation, two vials from
the same batch of Botulax, Meditoxin, Nabota, Relatox, and
Xeomin (from 100 U vials each) were reconstituted with 1
mL of solution 1. One hundred microliters of each preparation
was analyzed in antisera-coated plates. Innotox was supple-
mented with human serum albumin to establish the reaction
conditions validated for the ELISA. Each vial of reconstituted
Botulax, Meditoxin, Nabota, Relatox, or liquid Innotox was
analyzed in duplicate. Following incubation, the wells were
washed with solution 1, and incubated with the guinea pig
antiserum (1:2,000 dilution in solution 2). Unbound 150 kDa
neurotoxin antibodies were washed off. Anti-guinea pig
immunoglobulin G peroxidase conjugate (1:5,000; Sigma,
A7289) was used to detect bound antibodies by colorimetric
quantification with o-phenylenediamine (16 mmol/L) in
10 mmol/L citrate buffer (pH = 5.0). Optical density was
measured at 490 nm using a microtiter plate reader and
SoftMax Pro GxP (Spectra-Max Plus, Molecular Devices,
San Jose, CA, USA). A standard curve of between 0.2 and
1.6 ng/mL of neurotoxin was produced in 0.2 ng/mL inter-
vals (see Frevert 20108 for representative standard curves).
Internal controls were performed for each plate to ensure
assay validity. Standard curve linearity and ELISA specific-
ity, accuracy, and robustness were performed according to
International Conference on Harmonisation guidelines.9 The
ELISA was shown to be specific for the BoNT/A neurotoxin
and did not detect complexing proteins.
Results
Table 1 describes the properties of Innotox, Botulax, Medi-
toxin, Nabota, and Relatox.10
Highly sensitive sandwich ELISA was used to quantify
the amount of BoNT/A protein in Botulax, Meditoxin,
Nabota, and Relatox (Table 2). Xeomin was independently
analyzed in parallel as a control and found to have a mean
toxin content of 416 pg/vial, comparable to reports from
another batch.8 It should be noted that this variation from
published values is due to these batches of toxin being no
longer available for the present analysis and the use of a dif-
ferent batch of Xeomin, as well as a 5% intervial variability
during the manufacturing process (unpublished data 2018,
Merz Pharmaceuticals GmbH).
Botulax and Nabota contained 844 and 754 pg of neuro-
toxins, respectively, which are nearly twice the neurotoxin
content of Xeomin (416 pg) in an equivalent 100 U vial.
However, the percentage of inactive neurotoxins was also
calculated to be much higher at 103 and 81, respectively,
than that of Xeomin with no inactive neurotoxin. The potency
per pg of neurotoxin in Botulax and Nabota was found to be
0.118 and 0.133 U, respectively. This was less potent than
Xeomin’s 0.240 U per pg of neurotoxin. Meditoxin and
Relatox contained 575 and 578 pg of neurotoxins, respec-
tively, which were slightly higher than that of the Xeomin,
as was the calculated percentage of inactive neurotoxins at
38 and 33, respectively. The efficacy per pg of neurotoxin in
Meditoxin and Relatox was found to be 0.174 and 0.173 U,
respectively, which were also lower than Xeomin’s 0.240 U
of efficacy per pg of neurotoxin.
Discussion
All BoNT/A formulations contain the 150 kDa neurotoxin,
which is the active molecule. Xeomin, however, consists
solely of the 150 kDa neurotoxin. All products are based
on the botulinum toxin complex with about sixfold more
additional bacterial proteins, assuming a molecular weight of
≈900 kDa for the complex. Some of the complexing proteins
Table 2 Determination of content of botulinum neurotoxin type A protein in products by ELISA
Product name Batch name Dosage Amount of neurotoxin
protein per 100 units
(pg)
Specic potency
(U/pg neurotoxin)
Calculated
proportion (%) of
inactive neurotoxin*
Botulax®HUA 15133 100 U/vial (Lyo) 844 ± 43‡0.118 103
Meditoxin®/Neuronox®FAA 1587 100 U/vial (Lyo) 575 ± 6 0.174 38
Nabota®084962 100 U/vial (Lyo) 754 ± 11‡0.133 81
Relatox®0615 100 U/vial (Lyo) 578 ± 48 0.173 33
Xeomin®31149 100 U/vial (Lyo) 416 ± 6 0.240 Not found
Notes: Innotox® (not reported in this table) contains the surfactant polysorbate,25 which can interfere with antibody–antigen binding during ELISA and lead to inaccurate
and low concentrations. Innotox’s toxin content, therefore, could not be accurately measured using standard ELISA, which is validated for experimental conditions without
polysorbate. *Calculation based on claim that Xeomin contains only the active neurotoxin (=100%); ‡Value above standard curve.
Abbreviations: ELISA, enzyme-linked immunosorbent assay; Lyo: lyophilized.

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are hemagglutinins, which are glycoprotein binding proteins
(so-called lectins). In contrast to the 150 kDa neurotoxin,
these complexing proteins have the potential to bind to
dendritic cells,11,14 the sentinel cells of the immune system.
These cells must be activated as the first step of the initia-
tion of an immune response.12,14 In this regard, complexing
protein-containing products have a higher potential to cause
an immune response. Indeed, the formation of antibodies in
patients treated with complexing protein-containing products
in esthetic medicine has been reported.7 In contrast, antibody
formation was not observed in patients treated with Xeomin
free of complexing proteins. A further important factor
determining the potential for immunogenicity of BoNT/A
formulations and subsequent treatment failure is the amount
of neurotoxin protein present.5,15 This is associated with
increased antigen levels and, consequently, a greater risk
of antibody production.16 It was demonstrated in cervical
dystonia patients treated with BoNT products that the spe-
cific potency (U per pg neurotoxin) is correlated with the
antibody-induced therapy failure.17 It is, therefore, helpful
for the clinician to receive information about the specific
potency of different botulinum toxin products.
Currently, BoNT/A preparations approved for several
indications in adults in Asia, Europe, and the USA include
onabotulinumtoxinA (Allergan Inc., Irvine, CA, USA; also
known as Botox® or Vistabel®), abobotulinumtoxinA (Ipsen
Ltd, Slough, UK/Galderma, Paris, France; also known as
Dysport® or Azzalure®), and incobotulinumtoxinA (Merz
Pharmaceuticals GmbH, Reinheim, Germany; also known
as Xeomin or Bocouture®), each of which is uniquely formu-
lated. These variable manufacturing, formulation, and testing
processes have produced preparations with different potency,
dosage, constituents, and immunogenicity.18,19
As Xeomin’s manufacturing process isolates only the
active 150 kDa neurotoxin, Xeomin is entirely free of com-
plexing protein.20 Besides the active 150 kDa neurotoxin,
Botox and Dysport contain complexing proteins that form a
high-molecular-weight complex with the 150 kDa neurotoxin.
That is, Botox comprises one 150 kDa neurotoxin molecule
within a 900 kDa protein complex.21 Although Dysport’s
precise biochemical composition remains undefined, it is also
likely to contain the 500–600 kDa L-complex protein within
the 900 kDa complex protein as well.22 Xeomin/Bocouture
remains the only BoNT/A product marketed as containing
“purified neurotoxin” that has been registered with regulatory
authorities in the USA and Europe.
As reported, Botox contains 5,000 pg of toxin per 100
U vial23 (including complexing proteins), Dysport contains
4,350 pg of toxin (including complexing proteins) per 500 U
vial,24 and Xeomin contains 440 pg of neurotoxin per 100 U
vial.8 Here, the mean concentration of BoNT/A neurotoxin
was 730 pg in a 100 U vial of Botox (batches C2344C3,
C2384C3, C2419, and C2385), 650 pg in a 100 U vial of
Dysport (batches 678F and 689X), and 440 pg in 100 U
vials of Xeomin (batches 61,111, 70,604, and 81,208).8 The
specific potency, defined as the potency in units associated
with a specified amount of the 150 kDa toxin in each prod-
uct, was 0.137 U/pg for Botox, 0.154 U/pg for Dysport, and
0.227 U/pg for Xeomin,8 which suggested that Xeomin was
the most potent because it has the highest amount of toxin
protein among those tested.
Comparing the different products, Botulax and Nabota
showed a similar specific potency with 0.118 and 0.133 U/
pg, respectively. Meditoxin and Relatox have less neurotoxin
protein than Botulax and Nabota but higher specific poten-
cies (0.174 and 0.173 U/pg). However, their specific poten-
cies are lower than that of Xeomin (0.240 U/pg). One can
conclude that the lower specific potency of Botulax, Nabota,
Meditoxin, and Relatox may actually indicate the presence of
significant amounts of inactive, rather than active, neurotoxin.
Thus, high neurotoxin protein levels detected in this study
were not due to biologically efficacious neurotoxin, but due
to inactive toxin provided that all products were equipotent
in containing 100 U per vial. This inactive neurotoxin cannot
be taken up by neurons but might represent an immunogenic
impurity.17 These inactive components, which have no clinical
efficacy, per se, may stimulate antibody production.11 They
can reduce the efficacy of the neurotoxin by inducing immu-
noreactions in patients who then need to receive a higher
dosage at later sessions, ultimately increasing their risk of
becoming nonresponders.
Conclusion
Four BoNT/A formulations being used in Asia have shown
lower neurotoxin purity and specific potency but higher
neurotoxin protein concentrations than Xeomin in this study.
Although Botulax and Nabota had more neurotoxin than Xeo-
min in an equivalent volume, they contained greater amounts
of inactive neurotoxin. In addition, although Meditoxin and
Relatox had slightly more neurotoxin than Xeomin, both
contained greater amounts of inactive neurotoxin. In the
future, it will be necessary to conduct a comparative study
on the efficacy, effective duration, and safety profile of all
neurotoxin products, particularly on the incidence of second-
ary treatment failures due to antibody formation in patients
undergoing long-term treatment with BoNT/A.

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Comparison of botulinum neurotoxin type A formulations in Asia
Authors contribution
All authors contributed toward data analysis, drafting and
revising the paper and agree to be accountable for all aspects
of the work.
Disclosure
JF and OS are employee of Merz Pharmaceuticals. The
authors report no other conflicts of interest in this work.
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