Several reports on degradation of HA involving microwaves, UV or γ-rays have been published. Rheological studies also show that HA solutions degrade at high temperature: viscosity of the solutions decreases in time as a function of temperature. Therefore, the thermal sterilization treatment of HA solutions represents a great challenge. An optimized moist-heat sterilization cycle can minimize product degradation (and change of molecular weight) maintaining the required viscosity for the specific application.
1. Sodium hyaluronate PFS
The Sodium Hyaluronate used to prepare the batch had an average molecular weight equal to 1.6 x 106 Dalton. The formulations were prepared by hydrating the polysaccharide in a physiological phosphate buffer solution for 12 hours at 50°C. The bulk preparations were divided in pre-filled syringes.
2. Process selected
Samples were treated using an Air over Steam autoclave (Fedegari FOA); this process equipment works with counterpressure in the chamber to balance pressure increase inside the syringes during the sterilization and to avoid the risk of plunger movement.
The air initially contained in the chamber is not extracted – as in a pure saturated steam cycle – but it is only “controlled” by increasing or decreasing its quantity (i.e. the pressure) during the heating/sterilization/ cooling phase according to the characteristics of the load. The pressure of chamber air increases as it heats (with the relation of the absolute sterilization and cycle start temperatures). The total pressure in the chamber during the sterilization phase is given by the partial pressure of the heated air plus the partial steam pressure; the final pressure in the chamber will be therefore much higher than the one obtained with pure saturated steam autoclaves.
The counter-pressure inside the autoclave was adjusted by setting the air pressure correction coefficient at 1.2. This value is a little higher than the standard value (1.08) to avoid plunger expulsion. At 121°C, 115°C and 130°C, the pressure of the vessel was 3.56 abs, 3.18 abs, and 4.25 abs, respectively. These values were calculated by using the counter-pressure equation (equation 1) and also verifying the results on Thema 4 in the final phase data section.
4. F₀ calculation
The sterilization process was controlled by an F₀ target of 15’ using a Kaye validator. It was calculated in the sample with a thermocouple device (TC) inserted into a “reference syringe”.
The accumulation period started once the temperature reached 100°C. The sterilization phase was manually stopped at F₀ < 15’ so that at the end of the thermal process, the total F₀ accumulation – all cycle long – was roughly 15 minutes for each cycle.
5. Loads onfiguration
Load Type 1:
• 1 PFS was vertically attached to the side of the tray inside the machine using zip-ties. The thermocouple was placed inside the solution, ensuring that it was not touching any side of the glass syringe (Image 1).
• The autoclave temperature probes were free in the chamber.
Load Type 2:
• This load is the same as Type 1 but we also introduced 15 PFS placed vertically in a tray (Image 2).
6. Structure of the study
Fedegari R&D laboratory performed several thermal sterilization cycles on Sodium hyaluronate PFS:
a) at 121°C, with a F₀ = 15 minutes;
b) at a higher temperature (130°C) and a shorter dwell time, F₀ = 15 minutes;
c) at a lower temperature (115°C) and a longer exposure time, F₀ = 15 minutes.
7. Test performed
For each temperature tested:
a) a preliminary trial including a PFS with a thermocouple inserted to obtain the F₀ accumulated during each of the three phases: heating, sterilization and cooling.
b) at least, two replicates with a tray containing 15 PFS.
8. Viscosity analysis
All the samples were analyzed at IBSA laboratories and compared with a nontreated
8.1 Dynamic viscosity determination
A Rheological investigation was performed: a “Cone on Plate” rheomether was used to
determine the solution viscosity. In detail, the diameter of the plate was 50 mm and the cone angle was 1°. The dynamic viscosity of each formulation was evaluated carrying out a rotational test at controlled shear rate (from 0.01 to 10ˢ⁻¹).
Two main shear rate intervals were identified:
1) a low shear range (from 0.01 to aprox. 1ˢ⁻¹ ) with a plateau value of viscosity corresponding to a “zero shear rate”.
2) a medium shear range (from aprox. 1 to 10ˢ⁻¹) which corresponds to a “flow range” where the formulation starts to flow due to viscosity decrease.
During shearing, in fact, a certain number of macromolecules are oriented into shear
direction (dis – entanglements). As a consequence, the viscosity tends to decrease. Simultaneously, however, other molecules, previously dis-entangled, are recoiling and re- entangling because of their viscoelastic behavior. At low shear conditions, the overlapping caused by these effects (dis- entanglements and re –entanglements) zeros the shear rate.
On the other side, at increased shear rate, the number of dis-entanglements exceeds the number of re-entanglements: the curve of viscosity decreases continuously.
Sodium Hyaluronate formulation presented viscosity decrease during all the sterilization
cycles tested: the heat amount given to the specific formulation decreased its viscosity, independently of the sterilization temperature used. On the other side, the degree of damage and, as a consequence, of the viscosity change, was reduced during a peak cycle: the higher is the temperature and the lower is the exposure time, the lower is product degradation. In spite of the lower temperature, the worst cycle is the one run at 115°C for a long period. The long exposure time seems to be the cause of sample degradation.
By M. L. BERNUZZI and A. GIORI
Manager, R&D, Fedegari Group; Manager, R&D, IBSA Farmaceutici Italia
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as Starting Materials Intended for Use in Biomedical and Tissue Engineered Medical
M. B. Caspersen, J. P. Roubroeks, L. Qun, H. Shan, J. Fogh, Z. RuiDong, K. Tømmeraas,
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1. Technical details about trials and cycles can be provided by Fedegari R&D department upon request.
2. A special thanks to IBSA Farmaceutici for providing the PFS with Hyaluronic Sodium for this study and for making available their R&D laboratory for the additional rheological investigation.
3. A special thanks to Amphenol for making available their Kaye Validator AVS for process control.