Development of an Enhanced Methodology for Safe, Rapid Freeze Drying of HPLC Purification Fractions

In most laboratories, it is a routine as well as essential task to dry HPLC purification fractions, especially those consisting of acetonitrile and water, into a powder form, for accurate weighing, sub-sampling, and re-dissolving of samples. Although the preferred technique is freeze drying, processing organic solvents is a challenge for large-scale conventional freeze driers due to the possibility of boiling out of these solvents, which can cause damage to the vacuum pump.

Actelion Pharmaceuticals adopted the fast lyophilisation (LyoSpeed™) methodology developed by Genevac1, and implemented this using the Genevac HT-12 sii centrifugal evaporation system. Organic solvents are removed and the volume concentrated by centrifugal evaporation. The organic solvent is thendrained out from the condenser and the remaining water is lyophilised by the system.

This method is extremely effective in the case of hydrophilic samples, but issues may come when it is not possible to dissolve the sample only in water. Samples may crash out or form an oil subsequent to the removal of the organic solvent. In such cases, further processing is required to obtain the required dry powder form.

Limiting Factors

Lyophilisation with acetonitrile still present in the samples is the simple way to increase the success rate. However, existing equipment could not perform this task, temperature performance of the HT-12 sii evaporator’s VC3000 solvent condenser being limiting factor. The minimum temperature achievable by a VC3000 is -50°C. This in turn limits the vacuum level that can be achieved in the evaporation system.

Having a very low vacuum (below 0.1mbar) is ideal for good lyophilisation. True lyophilisation as in the case of vaccine production is not the aim of the LyoSpeed methodology, but targeting the formation of a dry, amorphous powder instead of oil. In the case of true lyophilisation, the result will be superior with better vacuum.

Developing an Superior Solvent Condenser

A colder condenser capable of condensing and holding acetonitrile at a low vacuum could address these challenges. A battery of trials were carried out in collaboration with Genevac engineering team in order to compare the performance of the VC3000 with a VirTis Benchtop K -85°C freeze drier (BTK), coupled to the HT-12 evaporator. This led to the development of the VC6000 condenser for the HT-12 sii, and also the VC7000 integral condenser now available on the Series 3 HT-12 (Figure 1.)

Genevac Series 3 HT-12 with integral VC7000 condenser.

Figure 1. Genevac Series 3 HT-12 with integral VC7000 condenser. Image credit: SP Scientific

Materials & Methods

Samples were processed using the Lyospeed methodology in a Genevac HT-12 sii evaporator, coupled to the VC3000, a BTK freeze drier, or the new VC6000 condenser. Glass vials of 100mm height, 16mm diameter, and 1.2mm wall thickness were used to prepare samples consisting of 10ml of a 50:50 mix of water and acetonitrile.

The method utilised for all samples was the same in the case of each condenser:

1. Dri-Pure® vacuum ramp over 1 hour to control boiling / bumping

2. Full vacuum without heat for 3 hours

3. Full vacuum with heat to the sample holders for 4 hours, at 40°C

4. Full vacuum without heat for 18 hours

[NB Stage 4 was considerably longer than actually required to provide for a standardised comparison].

Results and Discussion

The compound in each tube was tested subsequent to lyophilisation to determine whether the lyophilisation process successfully formed a diffuse powder, or if an oily sample had formed. The residual solvent volume in each tube was also estimated.

Total drying time, determined as the time taken for temperature convergence of the sample and the sample holder, was comparted.

While processing the samples, the success rate for the VC3000 was the lowest when compared to the Benchtop K and the VC6000. The VC6000 met expectations, while the Benchtop K faced challenges in lyophilising 288 vials due to smaller inner volume.

Conclusion

The results have demonstrated the superior freeze drying performance of the VC6000 over other systems, especially under high load. To achieve good results, the vacuum performance of a system becomes critical. An adequately cold, adequately powerful condenser along with a vacuum tight system can deliver good results.

The implementation of the VC6000 condenser into the daily workflow allowed routine observation of similar evaporation results for “real libraries” of samples consisting of various compounds with highly variable solubility patterns. Identical results are achieved by processing some samples in tubes secured in aluminium sample holders.

Genevac Series 3 HT Evaporators, launched in 2015, feature the VC7000 -75°C condenser and LyoSpeed methodology as standard.

Acknowledgements

Produced from materials originally authored by Rob Darrington, Genevac Ltd., Ipswich, UK.

Reference

Developments in Laboratory Scale Lyophilisation for Purification Laboratories. Dr Induka Abeysena & Rob Darrington, 2006, available via www.Genevac.com

This information has been sourced, reviewed and adapted from materials provided by Genevac.

For more information on this source, please visit Genevac.

 

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