Purity

Botulinum toxin is produced by anaerobic fermentation of the bacterium Clostridium botulinum type A (Hall strain). During the manufacture of incobotulinumtoxinA, following fermentation and extraction of the toxin, the complexing proteins are removed by chromatography. Thus incobotulinumtoxinA contains markedly less clostridial protein than do onabotulinumtoxinA and abobotulinumtoxinA (Fig. 6.1). In addition, incobotulinumtoxinA has the highest specific biological activity of all three products. A high-sensitivity sandwich ELISA was used in a study by Frevert to measure the amount of botulinum toxin type A per 100 U of onabotulinumtoxinA, incobotulinumtoxinA and abobotulinumtoxinA. The results were 0.73 ng, 0.44 ng and 0.65 ng, respectively, giving incobotulinumtoxinA the highest specific biological activity (U/ng neurotoxin) at 227 U/ng compared with 137 U/ng for onabotulinumtoxinA and 154 U/ng for abobotulinumtoxinA (but it must be noted that the units of abobotulinumtoxinA are different from those of onabotulinumtoxinA and incobotulinumtoxinA). This suggests that, in addition to containing complexing proteins, onabotulinumtoxinA may also contain denatured / inactivated neurotoxin, unlike incobotulinumtoxinA (see Fig. 6.1). Like the other botulinum toxin type A products, incobotulinumtoxinA contains human serum albumin as an excipient but, whereas incobotulinumtoxinA contains sucrose, abobotulinumtoxinA contains lactose, and onabotulinumtoxinA contains neither of these sugars (Table 6.1).

Figure 6.1 The amount of clostridial protein and neurotoxin protein contained in incobotulinumtoxinA, onabotulinumtoxinA and abobotulinumtoxinA per dose approved for the treatment of glabellar frown lines.

Stability

It was assumed that complexing proteins are required to stabilize the neurotoxin, but incobotulinumtoxinA can be stored at room temperature and without refrigeration for 4 years. In addition to the practical advantage of not requiring cold storage, incobotulinumtoxinA did not lose activity in a temperature stress stability study by Grein & Fink. Furthermore, its quality did not decrease following storage at 60°C for 1 month. In accordance with the prescribing information, once reconstituted, incobotulinumtoxinA should be stored at 2–8°C and used within 24 hours to limit the risk of bacterial growth.

Diffusion

It is important to understand the difference between diffusion and spread of the neurotoxin. Diffusion is the passive movement of the toxin down a concentration gradient within the medium. In contrast, spread occurs when the injected molecule travels from the site of injection. The latter is usually a result of the injection technique, skills of the injector, injected volume, and needle size and the consistency of the treated tissue and can cause undesirable effects in adjacent muscles.

Under acidic conditions the complexing proteins form a high-molecular-weight complex (900 kDa) with the neurotoxin. Accordingly, it was speculated that complexing proteins limit the diffusion of the active neurotoxin within the target muscle. However, experiments in animals and humans suggest otherwise. The diffusion of onabotulinumtoxinA, abobotulinumtoxinA, and incobotulinumtoxinA was investigated by Carli and colleagues in a mouse model using staining for neural cell adhesion molecule (N-CAM) as a marker. N-CAM is expressed only by denervated muscle cells and so is suitable for testing for muscles affected by botulinum toxin type A. Results indicated that, following injection into the tibialis anterior (TA) muscle of the mouse leg, there was limited diffusion into tissue that was adjacent to the site of injection and no difference in diffusion was observed between incobotulinumtoxinA, onabotulinumtoxinA, and abobotulinumtoxinA using a 1 : 1 : 4 dose conversion ratio.

A recent clinical study by Kerscher and colleagues showed no difference in the size of the anhidrotic area produced following injection of 5 U of incobotulinumtoxinA (free from complexing proteins) compared with 5 U of onabotulinumtoxinA (containing complexing proteins) on either side of the forehead after 6 weeks and 6 months.

These results suggest that complexing proteins have no effect on diffusion. The reason for this was revealed by a recent study by Eisele and co-workers on the stability of the complexes and the kinetics of their dissociation. This report showed that the high molecular weight complexes presumably present in onabotulinumtoxinA and abobotulinumtoxinA are dissociated by reconstitution and physiological pH. Reconstitution of the commercially available complexing-protein-containing products, onabotulinumtoxinA and abobotulinumtoxinA, with saline (as in the clinic) led to a complete dissociation of the 900 kDa complexes and resulted in at least 85% of the neurotoxin being in its free form. In addition, it was shown that more than 80% of the 150 kDa neurotoxin was released from complexes in under a minute at physiological pH. Therefore, following injection into target tissue, where the pH is usually close to neutral, any remaining complexes dissociate immediately, making the presence or absence of complexing proteins in the original preparation irrelevant in terms of diffusion. Complexing proteins do not contribute to the therapeutic effect and are therefore only clostridial impurities.

Immunogenicity

Botulinum toxin is a foreign protein and is therefore antigenic. Immunogenicity remains a controversial subject in the field of botulinum toxin type A treatment in aesthetics. While the generation of neutralizing antibodies is more common in therapeutic indications, where dosages tend to be much larger, cases of secondary non-responsiveness have been reported in aesthetics. In a study by Blümel and colleagues investigating the generation of neutralizing antibodies following repeated injections of onabotulinumtoxinA, abobotulinumtoxinA or incobotulinumtoxinA into New Zealand white rabbits, neutralizing antibodies were detected after both abobotulinumtoxinA and onabotulinumtoxinA treatment. In contrast, incobotulinumtoxinA treatment did not induce the production of neutralizing antibodies. This suggests that the presence of complexing proteins might be clinically relevant.

Introduction