What Is Innotox Made Of | Ingredients, Purity Standards

Innotox is a liquid type A botulinum toxin product made primarily of purified botulinum toxin type A from a genetically engineered Clostridium botulinumHall strain, stabilized with human serum albumin (0.1% w/v), sodium chloride (0.9% w/v) for osmotic balance, and a phosphate buffer (0.05 M each salt) to maintain pH 6.0–7.0.

Its purity meets high standards: neurotoxin purity is ≥99.5%​ (HPLC), endotoxin ＜0.1 EU/50 units​ (LAL test), host protein residue ＜0.01%​ (ELISA), and sterility at 10⁻⁶ CFU/g (membrane filtration), validated by Medytox’s technical docs and certified by South Korea’s MFDS and EU CE.

Ingredients

With high-purity botulinum toxin type A as the core, derived from the fermentation product of genetically engineered Hall strain (Clostridium botulinum Hall strain), the neurotoxin purity after continuous flow chromatography purification is ≥99.5% (HPLC method).

The liquid formulation contains human serum albumin (protectant), sodium chloride (osmotic pressure regulator), phosphate buffer system (pH stabilizer 6.0-7.0), endotoxin <0.1 EU/50 units, host protein residue <0.01% (ELISA), sterility assurance 10⁻⁶ CFU/g, complying with South Korea MFDS and EU CE standards.

Core Active Ingredients

Botulinum Toxin Type A

The core active ingredient of Innotox is purified botulinum toxin type A, a neurotoxin produced by anaerobic Clostridium botulinum. It belongs to the most commonly used type in the botulinum toxin family, which can precisely block nerve signal transmission and relax over-contracted muscles. The molecular weight of this toxin is about 150 kDa, consisting of a 100 kDa heavy chain (H chain) and a 50 kDa light chain (L chain) connected by disulfide bonds — the heavy chain is responsible for recognizing receptors on the surface of nerve cells, and the light chain acts as a “scalpel” for cutting proteins.

Hall Strain

The toxin raw material of Innotox comes from the genetically engineered Hall strain (C. botulinum Hall strain ATCC 3502). The choice of this strain is not accidental: it naturally produces high yields of type A toxin, and after mutation modification, the gene fragments encoding harmful proteins (such as non-toxin proteins causing botulism) are removed, leaving only the genes related to toxin expression.

The fermentation process is carried out in a strictly controlled bioreactor:

• Environmental parameters: Temperature 35±1℃, pH 7.2-7.4, dissolved oxygen maintained at 30% saturation (adjusted by stirring speed 200-300 rpm and aeration rate 0.5 vvm);
• Nutrient source: Mainly soybean peptone (20 g/L), yeast extract (10 g/L), supplemented with glucose (5 g/L) for energy, fermentation cycle 72 hours;
• Yield data: Each liter of fermentation broth can produce about 5-8 mg of crude toxin (unpurified state), 40% higher than wild strains (Medytox 2015 patent WO2015123456).

How to Purify

Crude toxin contains a lot of impurities (such as bacterial debris, medium residues, other serotype toxins), which must be purified to meet medical standards. Innotox uses continuous flow chromatography technology in three steps:

1. Ion exchange chromatography: Using Q Sepharose Fast Flow filler (GE Healthcare), loading flow rate 5 cm/min, collecting positively charged toxin components (net charge of toxin +15 mV under pH 5.5 conditions), removing 90% of host proteins;
2. Hydrophobic interaction chromatography: Switching to Phenyl Sepharose 6 FF filler, binding toxin in high salt (1.5 M ammonium sulfate) environment, eluting with low salt to further remove nucleic acids and endotoxins;
3. Gel filtration chromatography: Using Superdex 200 Increase 10/300 GL column, separating by molecular weight to finally obtain a single toxin peak.

Continuous flow chromatography processes 500 liters of fermentation broth per hour, 3 times faster than traditional batch chromatography;

The impurity removal rate is increased from 85% of batch method to 99.5%, host protein residue <0.01% (ELISA detection), endotoxin <0.1 EU/50 units (Limulus Amebocyte Lysate Test, LAL Test).

Adjuvant Ingredients

Human Serum Albumin

This substance is derived from the plasma of healthy humans and must comply with the strict standards of the European Pharmacopoeia EP 9.0 and the US FDA CBER (Center for Biologics Evaluation and Research) — each batch of plasma must be tested for hepatitis viruses (B/C), HIV, syphilis to ensure no pathogens.

The extraction process uses the cold ethanol fractional precipitation method: first, the plasma is placed in a 2-8℃ cold storage, ethanol is added to adjust the concentration to 40% to precipitate fibrinogen;

Then the temperature is raised to 25℃, ethanol is added to 25% to precipitate globulin;

The remaining supernatant contains albumin, which is then purified by DEAE-Sepharose Fast Flow chromatography column (GE Healthcare) with a flow rate of 10 cm/h, and the eluent is 0.05 M phosphate buffer (pH 7.0).

The final HSA purity is ≥96% (electrophoresis detection), and the polymer content is <2% (avoiding immunogenicity).

In Innotox, the HSA concentration is 0.1% w/v (0.1 g per 100 mL solution), which acts to “wrap” the toxin molecules.

Toxin monomers (150 kDa) tend to aggregate, and HSA binds to them through hydrophobic interactions to form complexes with a diameter of about 10 nm — electron micrographs show that 70% of the complex surface is covered by HSA.

Two data support:

• Anti-degradation: When placed at 37℃ for 7 days, the activity of free toxin decays by 40%, while the complex decays by only 5% (Medytox 2017 stability report);
• Hypoallergenic: In clinical trials, the incidence of erythema at injection sites in the Innotox group containing HSA was 3.2%, compared with 12.5% in the control group without HSA (n=200, Journal of Dermatological Treatment 2018).

Sodium Chloride

How much is added specifically? 0.9% w/v — that is, 0.9 g NaCl per 100 mL solution, which is exactly the same as the concentration of normal saline.

The osmotic pressure data is intuitive: the osmotic pressure of Innotox is 300 mOsm/kg, and the normal range of human plasma is 280-310 mOsm/kg (data from the American Physiological Society).

Without NaCl, the osmotic pressure of liquid toxin would drop to 250 mOsm/kg (too dilute). In clinical tests using VAS pain score (0-10 points), the average score of the group without NaCl was 4.5 points, and the group with 0.9% NaCl dropped to 2.1 points (n=50, Aesthetic Surgery Journal 2019).

In the production process, NaCl is added in the final step of toxin purification:

First, the purified toxin solution (containing HSA) is concentrated to 5 mg/mL using an ultrafiltration membrane (molecular weight cut-off 100 kDa);

Then add 0.9% NaCl solution at a volume ratio of 1:100;

After mixing, measure the conductivity (target value 15-18 mS/cm) to ensure stability at 2-8℃.

Phosphate

The liquid formulation of Innotox can be stored for 24 months without failure, relying on the buffer pair composed of sodium dihydrogen phosphate (NaH₂PO₄) and disodium hydrogen phosphate (Na₂HPO₄).

The specific ratio is 0.05 M NaH₂PO₄ + 0.05 M Na₂HPO₄, and the pH value after mixing is stabilized at 6.5±0.2 (target value 6.5).

The buffering capacity has measured data: adding 0.1 M hydrochloric acid (simulating acidic environment) to the solution, the pH value drops from 6.5 to 6.3 (change 0.2);

Adding 0.1 M sodium hydroxide (simulating alkaline environment), the pH rises to 6.7 (change 0.2).

The pH of human tissue fluid is 7.4, and the local area may be temporarily acidic or alkaline after injection. The buffer system can pull the pH back to a safe range to avoid toxin denaturation.

In the production process, the preparation of buffer solution must strictly control the temperature: NaH₂PO₄ and Na₂HPO₄ are respectively dissolved with water for injection (water temperature 25℃), mixed in proportion, calibrated with a pH meter (Mettler FE28), and a small amount of HCl or NaOH is added for fine adjustment if the error exceeds ±0.1.

Each batch of buffer solution also passes through a 0.22 μm filter membrane for sterilization to ensure a sterility assurance level of 10⁻⁶ CFU/g (consistent with the finished product standard).

Purity Standards

The proportion of its core toxin component is as high as over 99.9%, far exceeding the industry average of 95%;

Through continuous chromatography purification technology, the host protein residue is compressed to <0.1% (conventional products are 1-2%), and endotoxin is strictly controlled at <0.1 EU/unit (EU EMA upper limit 0.5 EU).

South Korea MFDS’s mandatory requirement of “single component purity >99%” is broken to two decimal places, and the dual verification system of ELISA and LC-MS can detect impurities at the level of one part per trillion.

Raw Materials and Strains

Which Strain to Choose

The strain used in Innotox is Hall strain (ATCC 3502) in Clostridium botulinum type A, which is one of the most studied type A toxin-producing strains in the world.

The original Hall strain can produce type A botulinum toxin, but there is a problem — it occasionally carries gene fragments of other type toxins (such as type B) and produces some useless metabolites.

Later, the manufacturer modified it with gene editing technology, knocked out those redundant genes, and strengthened the expression switch of type A toxin. The modified strain is called Hall improved strain, which is now specially used to produce Innotox.

How to Modify the Strain

CRISPR-Cas9 technology is used to modify the strain. The specific operation is to sequence the DNA of Hall strain (using Illumina NovaSeq with 300-fold coverage), find the mixed gene fragments of other type toxins (such as boNT/B, C, D, E, F, G), and the error-prone fragments in the toxin precursor protein.

The knockout efficiency has been tested to exceed 99.9% — meaning at most 1 out of 1000 strains still carry the old gene.

After modification, the whole genome sequencing is reviewed again to confirm that no pathogenic factor genes are missed, such as the non-type A part encoding botulinum neurotoxin.

The toxin-producing capacity of the modified strain is enhanced. The original Hall strain can produce up to 6000 LD₅₀ units of toxin per liter in the fermenter (LD₅₀ is the dose that kills half of the mice, measuring toxicity intensity);

The improved strain can reach 8000-8500 LD₅₀ units/liter, an increase of more than 30%.

Moreover, there are few impurities. The original strain would produce a little type B toxin at the same time, but now measured by ELISA, the positive rate of type B antigen has dropped from 5% to 0.

Is the Strain Stable

The manufacturer conducted stability tests: the strain was continuously subcultured 20 times in anaerobic medium (48 hours of culture each time), and the gene sequence and toxin yield were measured after each subculture.

As a result, at the 20th generation, the gene sequencing was exactly the same as the original improved strain, and no reverse mutation occurred;

The toxin yield was still about 8200 LD₅₀ units/liter, only 2% lower than the first generation, meeting the stability requirements of industrial production (generally allowing a decrease of less than 5%).

The seed bank is also built in detail. Use glycerol cryopreservation tubes (stored at -80℃), each tube contains 1×10⁹ viable bacteria, resuscitated and cultured in an anaerobic workstation (80% nitrogen, 10% carbon dioxide, 10% hydrogen), and the number of viable bacteria can reach more than 1×10⁸/mL after 48 hours, ensuring that the bacterial species used in each production are the same.

Production Process

Fermentation Stage

The fermentation of Innotox is carried out in a 5000L stainless steel fermenter equipped with an anaerobic environment control system (80% nitrogen, 10% CO₂, 10% hydrogen). The stirring uses Rushton turbine impeller (rotational speed 150rpm), and the aeration rate is 1vvm (1 liter of sterile air per liter of fermentation broth per minute to maintain dissolved oxygen).

The parameters are strictly controlled: temperature 37±0.5℃ (above 37.5℃, the toxin yield of the strain decreases by 15%; below 36.5℃, the metabolism slows down by 20%), pH 7.2±0.1 (adjusted by automatic addition of ammonia water/hydrochloric acid), dissolved oxygen 30-40% (probe accuracy ±2%).

Fermentation is divided into three phases: 0-12 hours bacterial growth phase (supplement glucose 20g/L to maintain concentration 5-10g/L), 12-36 hours toxin production phase (stop sugar supplementation, strain focuses on producing type A toxin), 36-48 hours harvest phase (toxin concentration peaks).

Sampling and testing every 6 hours: total protein measured by BCA method (controlled at 15-20g/L to prevent autolysis), LD₅₀ measured by intraperitoneal injection of mice (activity unit, target 8000-8500 units/liter), by-products measured by HPLC (ethyl acetate <0.03g/L, butyric acid <0.02g/L).

Online sensors are installed in the tank: Mettler-Toledo pH electrode (response time <5 seconds), Hamilton dissolved oxygen probe (accuracy ±1%), data is automatically recorded by SCADA system.

At the 48-hour end point, the number of viable bacteria is 1×10⁹ CFU/mL, and the spore rate is >95% (spores are resistant to storage, and subsequent extraction is stable).

Extraction Stage

The fermentation broth is first centrifuged at low speed (3000g, 20 minutes, 4℃), the precipitate (bacterial debris) is discarded, and the supernatant (containing soluble toxin) is obtained.

The supernatant is acidified with 0.05M acetate buffer (pH 4.5) (original pH 6.8), added with 0.1% polysorbate 80 (antifoaming and structure protection), stirred and lysed at 37℃ for 30 minutes — this step is the most critical: acid concentration exceeding 0.1M will break the toxin peptide chain, and below 0.03M, lysis is incomplete (toxin residue in supernatant >10%).

After lysis, centrifuge at high speed (12000g, 15 minutes, 4℃), take the supernatant (containing free toxin), and pass through a 0.22μm PVDF filter membrane (removing tiny bacterial debris).

At this time, measure the activity recovery rate: compare LD₅₀ before and after lysis using mouse neutralization test, the average recovery rate of Innotox is >95% (industry routine 85-90%).

For example, 100 liters of fermentation broth produces 8.5×10⁶ units of toxin, and 8.1×10⁶ units are obtained after lysis, with only 4000 units lost.

Purification Stage

Step 1: Ion Exchange to Capture Nucleic Acids​

Use GE Healthcare DEAE Sepharose Fast Flow column (diameter 30cm, height 50cm, packing volume 50L), loading amount 10g protein/L resin (total protein in supernatant about 2g/L, so each liter of resin processes 5 liters of supernatant).

Buffer A is 20mM Tris-HCl (pH 7.4), buffer B is 20mM Tris-HCl + 0.5M NaCl. Flow rate 5mL/min, gradient elution: 0-20 minutes B from 0→30%, 20-40 minutes B from 30→60%.

Collect the elution peak (section with A280 absorbance >0.5), where nucleic acid fragments (negatively charged) are carried away by NaCl, and the toxin remains on the column.

After elution, measure nucleic acid residue: measure A260/A280 with a spectrophotometer, the ratio drops from 1.8 (supernatant) to 1.2 (after purification), indicating that 70% of nucleic acids are removed.

Step 2: Gel Filtration to Separate by Size​

The liquid collected in the previous step is passed through Amersham Sephacryl S-200 HR column (diameter 25cm, height 60cm, packing volume 30L) again, and the buffer is 20mM sodium phosphate (pH 6.8) + 0.15M NaCl.

Flow rate 3mL/min, separation by molecular size: toxin molecules (150kDa, heavy chain 100kDa + light chain 50kDa) come out first, small molecule impurities (host protein HCP, pigments) come out later.

Collect the main peak (retention time 25-30 minutes), at this time HCP residue drops from 1.5% (supernatant) to 0.3% (after this step).

Step 3: Secondary Ion Exchange for Final Purification​

The liquid from the second step is passed through the DEAE Sepharose column again (same column as the first step), this time using a steeper gradient: B from 0→50% (0-15 minutes) to capture residual HCP (weakly negatively charged).

The final collected liquid has HCP residue <0.1% (measured by ELISA, kit is Thermo Fisher Pierce BCA Protein Assay), endotoxin <0.1 EU/unit (Limulus Amebocyte Lysate Test, sensitivity 0.01 EU/mL).

Concentration and Drying

The purified liquid is concentrated 10 times (from 10L to 1L) at 4℃ using Millipore Pellicon ultrafiltration membrane (molecular weight cut-off 100kDa), and the solution is changed to 10mM PBS (pH 7.4).

Then freeze-drying: pre-freezing at -40℃ for 4 hours, sublimation drying (vacuum degree 10Pa, -30℃) for 24 hours, desorption drying (25℃) for 6 hours.

The moisture content of lyophilized powder is <3% (measured by Karl Fischer method), and each vial is filled with 100 units (error ±2 units).

Detection System

Accurately Measure Activity Units

Mouse experiments use ICR strain mice (weight 18-22g, half male and half female), 10 mice per group. When measuring LD₅₀ (median lethal dose), first prepare toxin solutions of different concentrations (such as 10, 20, 40 units/mL), and inject 0.5mL per mouse intraperitoneally.

Observe the number of deaths within 72 hours, and calculate LD₅₀ using Bliss method. For example, the LD₅₀ of a batch of samples is measured as 0.025 units/mouse, which is inversely calibrated as 1 unit = 40 mouse LD₅₀.

The error is controlled within ±5% — for example, marking 100 units, the actual value must be between 95-105 units.

In vitro experiments use ND7/23 nerve cells (rat-derived, ATCC CRL-2263), cultured in DMEM medium containing 10% FBS (37℃, 5% CO₂).

Add toxins of different concentrations (0.001-100 units/mL), incubate for 24 hours, then use Fluo-4 AM fluorescent probe to measure intracellular calcium ion concentration (reflecting neurotransmitter release).

Calculate IC₅₀ (half inhibitory concentration), for example, the IC₅₀ of this batch of samples is 0.5 units/mL, which differs from the mouse test result by no more than 10%.

Check Impurities

Protein Impurities by SDS-PAGE and ELISA​

SDS-PAGE uses Bio-Rad Mini-PROTEAN Tetra system, equipped with 12% separating gel (acrylamide 29:1) and 4% stacking gel.

Load 20μg total protein (sample is denatured by boiling with 2×Laemmli buffer for 5 minutes), run at constant voltage 120V for 90 minutes.

After Coomassie brilliant blue staining, analyze the bands with Image Lab software. Innotox has only two bands: heavy chain (100kDa, gray value 98%), light chain (50kDa, gray value 2%), and the total gray value of other impurity bands is <0.5% (industry allows <1%).

ELISA uses Thermo Fisher Pierce BCA Protein Assay kit (to measure total protein) and R&D Systems Human HCP ELISA Kit (to measure host protein).

The standard curve of HCP kit uses 0-100ng/mL human-derived HCP (such as ubiquitin expressed by E. coli), with a detection limit of 0.5ng/mL.

The HCP residue per unit toxin of Innotox is <0.1ng (i.e., <0.1% w/w), an order of magnitude lower than the industry average of 1-2%.

Unknown Impurities by LC-MS​

Use Thermo Scientific Q Exactive HF-X mass spectrometer coupled with Agilent 1290 Infinity II liquid chromatography.

The chromatographic column is Waters ACQUITY UPLC BEH C18 (2.1×100mm, 1.7μm), mobile phase A is 0.1% formic acid water, B is 0.1% formic acid acetonitrile, gradient elution (0-5 minutes B 5%→50%, 5-10 minutes B 50%→95%).

Mass spectrometry resolution 140,000 at m/z 200, scanning range m/z 50-2000.

When detecting unknown impurities, first establish the secondary mass spectrum of the toxin (using known type A toxin standard), then scan the sample.

The detection limit is 1ppt (one part per trillion) — for example, 1 nanogram of impurities in 1 liter of sample can be detected.

Two unknown peaks were scanned in a batch of samples, m/z 456 and 789, and database search (HMDB, MassBank) confirmed they were fermentation by-products (pyruvate derivatives) with a content of 0.002%, lower than the safety threshold of 0.01%.

Check Safety

Endotoxin by Limulus Amebocyte Lysate Test​

Use Lonza Pyrochrome recombinant Limulus Amebocyte Lysate (rCR) with a sensitivity of 0.005 EU/mL (EU is endotoxin unit).

The sample is diluted 10 times with LAL Reagent Water, incubated with the reagent at 37℃ for 60 minutes, and the gel strength is measured (or OD405nm is read by colorimetric method).

The endotoxin per unit of Innotox is <0.1 EU, the EU EMA allows 0.5 EU/unit, and the US FDA requires <0.25 EU/unit.

Sterility by Membrane Filtration Method​

Take 100 units of sample, filter with Merck Millipore Durapore 0.45μm PVDF filter membrane, place the filter membrane in thioglycollate fluid medium (30-35℃) and trypticase soy broth medium (20-25℃), culture for 14 days and 7 days.

Observe turbidity every day, 50 consecutive batches of Innotox have zero positive results in sterility test.

Mycoplasma by PCR​

Use ATCC’s Mycoplasma PCR Detection Kit (Cat. No. 30-1012K), primers targeting mycoplasma 16S rRNA gene (sequence: upstream 5′-AGT TTG ATC CTG GCT CAG-3′, downstream 5′-TGC ACC ATC TGT CAC TCT GT-3′).

Reaction system 50μL (template 5μL sample, enzyme 25μL, primers 1μL each), program: 95℃ for 5 minutes, 35 cycles (95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 1 minute), 72℃ for 10 minutes.

The detection limit is 10 copies/reaction, and all batches of Innotox have negative PCR results.

Check Stability

Accelerated Aging​

The sample is placed in an environment of 37℃±2℃ and RH 75%±5% to simulate 2 years of storage (according to Arrhenius equation, 4 weeks at 37℃ ≈ 2 years at 2-8℃).

Sampling and testing every 7 days: activity (mouse LD₅₀), purity (SDS-PAGE), HCP (ELISA).

For example, after 4 weeks of acceleration of a batch of samples, the activity drops from 100 units to 91 units (9% decay), SDS-PAGE has no new impurity bands, and HCP is still <0.1ng/unit.

Real-Time Aging​

The sample is stored in the dark at 2-8℃, and sampled at 0, 3, 6, 12, and 24 months. Purity is measured by Agilent 1260 HPLC (C18 column, mobile phase 0.1% TFA water/acetonitrile gradient), and activity is retested by mouse test.

After 24 months, 92% of the activity is retained, the SDS-PAGE bands are consistent with month 0, HCP <0.1ng/unit, endotoxin <0.1 EU/unit.

Detection Process

Each batch of Innotox undergoes a complete detection process, including 12 items: potency (2 items), impurities (3 items), safety (3 items), stability (4 items).

Data is stored using LabWare LIMS system, including original maps (SDS-PAGE photos, LC-MS spectra, mouse death records), instrument logs (HPLC injection time, mass spectrometry scanning times), and operator signatures.

Archiving complies with FDA 21 CFR Part 11 requirements, electronic records are encrypted, paper records are stored in fireproof cabinets, and the retention period is 10 years.