Drying of Problematic Purification Samples by Lyophilization in a Centrifugal Evaporator

The KuDOS case authors: Adrian Moore is an Analytical Group Leader at KuDOS Pharmaceuticals, Horsham, U.K.; Mandy Crowther is an Associate Principal Scientist at Astra Zeneca, Alderly Park, U.K.; and Rob Darrington is Product Manager at Genevac Ltd., Ipswich, U.K.

The Novartis case authors: Dr. Klemens Hoegenauer is a Senior Researcher at the Novartis Institute for BioMedical Research (NIBMR), Vienna, Austria. Dr. Induka Abeysena is an Applications Chemist at Genevac Ltd., Ipswich, U.K.

The U.K. Agrochemicals Research Centre case authors: Research team leaders; names withheld upon request.

Lyophilization is often the preferred method of sample drying because a high level of dryness is achieved with low residual solvent levels, and because of the light, powdery, “fluffy” finish of the sample, which enables it to be easily removed and weighed out. Nevertheless, there are many limitations of lyophilization.

This study shows how, by transferring the process to its centrifugal evaporators, Genevac (Ipswich, U.K.) has managed to change the rules and achieve the results that purification laboratories are searching for.

For many years, lyophilization, or freeze-drying, has been used to dry samples in the laboratory. The technique is well researched and has become the method of choice for many researchers with a few samples to dry. However, there are a number of potential drawbacks encountered with the conventional freeze-drying apparatus, including: 1) Samples must be prepared in a limited range of solvents (normally only water can be used), 2) volatile organic solvents and their mixtures cannot be used, and 3) the process is slow.

Figure 1 - Genevac HT-4X centrifugal evaporator.

Therefore, researchers with many samples to process, or mixtures of solvents, i.e., from preparative reversed-phase HPLC separations containing water and acetonitrile, have turned to centrifugal evaporators. In other laboratories—compound handling, for example—the aggressive nature of the organic solvents used makes a freeze-dryer unsuitable. Even in these environments, state-of-the-art centrifugal evaporators, such as the Genevac HT-4X, shown in Figure 1, have some limitations.

Problems with purification

Purification laboratories receive samples dissolved in water and acetonitrile, with a low level of a modifier present, normally 0.1% trifluoroacetic acid (TFA). Freeze-drying to remove these solvents is fraught with difficulties: 1) The acetonitrile requires either a very deep vacuum to freeze it, or a freeze-dryer that actively freezes the samples. Acetonitrile freezes at –65 °C. If it is not frozen, then bumping is inevitable, resulting in sample loss and cross-contamination. 2) Acetonitrile in the cold trap will spoil the vacuum, making lyophilization of the water almost impossible. 3) This slow process is incompatible with the reduced process times strived for. The centrifugal evaporator rapidly dries many different samples in parallel, without bumping.

However, users report difficulties with a few samples dried in a batch. Not all of the TFA is removed and may damage the sample when in storage; in addition, the compound may interact with the water, boosting the boiling point. Residual solvent appears in nuclear magnetic resonance (NMR) analysis. While occurring occasionally, the implications of selecting a few samples by hand are prohibitive for automated laboratories; therefore the whole sample rack is reprocessed.

Lyophilization in a centrifugal evaporator

Samples prepared in water can be lyophilized in a centrifugal evaporator by pulling the best vacuum available. In the Genevac HT-4X with solvent-resistant scroll pump, the ultimate vacuum is below 0.5 mbar, which is more than adequate for freezing water. This is like freeze-drying, and it is slow.

Genevac developed a process whereby users evaporate some solvent using the speed of a centrifugal evaporator, then switch to lyophilization mode when only a few milliliters of solvent are left. This reduced the process time for 96 × 30 mL fractions from 48 hr in a freeze-dryer to 16 hr (overnight) in a Genevac HT-12, equivalent to only 10 min per 30-mL sample sequentially. The effects of heating a sample during lyophilization were studied to determine if this gave a speed advantage.

Initially, just water was used to develop the optimum conditions, and then water and acetonitrile were used to simulate samples taken from HPLC.

Lyophilization of water

Figure 2 - Twenty-milliliter scintillation vial holder.

A stock solution of 0.01 M ibuprofen sodium salt in water was used as the standard sample; 15 mL of solution was loaded into each of 48 20-mL scintillation vials (see Figure 2) and placed dry in a Genevac HT-4X evaporator to dry under various conditions.

The actual evaporation method usually comprises up to four stages:

  1. Concentration of the bulk of the solvent using fast evaporation
  2. Cooling of the samples and sample holders in preparation for
  3. Freezing of the sample using deep vacuum
  4. Lyophilizing the residual solvent, with or without heat.

In fact, the freezing stage achieves cooling as well as freezing; therefore a separate stage 2 is not necessary. However, the freezing stage with no heat appears to be essential. Without it, the samples dried normally and did not lyophilize; thus it was evident that the sample had not frozen at all. Careful adjustment of heating time permitted a reduction in concentration time from 8 hr to 5.5 hr.

Lyophilization of water and acetonitrile

While the time savings for water is welcome, the issue remains of how to deal with solvent mixtures. An earlier stage—stage 0, in three parts—is introduced to remove the acetonitrile before concentration of the water.

  1. Dri-Pure® (Genevac)—vacuum ramping and high rotor speed prevent bumping.
  2. Concentration—a 40-mbar stage that removes the acetonitrile without freezing the water (here acetonitrile boils at +2 °C).
  3. Draining the condenser—or residual acetonitrile will spoil the vacuum in later stages.

Figure 3 - Samples of the same mass prepared via different methods in scintillation vials—left: traditionally evaporated result (overdried), and right: lyophilized result.

A 0.01 M solution of ibuprofen sodium salt was prepared in a 60:40 mixture of water and acetonitrile. Trials showed that the cooling stage was not required. With adjustment to achieve the correct balance of concentration and lyophilization, 48 × 15 mL samples dried in 5 hr, equivalent to 6.25 min of sequential drying. Figure 3 shows the difference between a lyophilized result and a traditional, centrifugally evaporated sample. The difference is stark, and the ease of resuspension is greater with the lyophilized sample, whereas the dried sample does not fully dissolve.

Discussion

Lyophilization of the samples made it easier to redissolve the sample postdrying. The addition of heat during the lyophilization stage reduced the lyophilization time considerably. The cooling stage is not required, but the freezing stage is essential. When evaporating water and acetonitrile mixtures, it is necessary to drain the condenser following acetonitrile evaporation and before evaporation and lyophilization of the water.

The Genevac HT-4X system had to be drained manually at the end of stage 1. Some Genevac systems automate this facility, eliminating the need for user intervention. Today, Genevac HT systems incorporate Auto Defrost and Drain.

A full set of original trial data on the above plus the specific customer applications below are available upon request from Genevac.

Case 1: KuDOS Pharmaceuticals

Preparation of weighable samples from difficult-to-dry HPLC purification fractions is shown.

Problem

Following purification, sample fractions are normally dried and resuspended in minimal solvent, and the fractions are pooled into a (small) sample container for storage. Analytical methods are often included, such as dry weight determination of yield and subsampling for NMR analysis. Until recently, scientists at KuDOS Pharmaceuticals had been experiencing difficulty drying samples after purification. For KuDOS, typical stubborn samples form a gum or an oil that creates handling problems, e.g., incompletely dried samples, resulting in the inability to determine the true weight, and residual solvent, promoting faster compound degradation.

Solution

A new sample drying methodology was developed by KuDOS/Astra Zeneca that overcomes both residual solvent and fraction pooling problems.

Fraction processing and pooling flow:

  1. Dried fractions were redissolved in minimal water and methanol
  2. Fractions were pooled into 20-mL scintillation vials and dried
  3. Samples were dissolved in minimal 1,4-dioxane; some stubborn samples present as gums or oils
  4. Fractions were pooled into a bar-coded, tared vial
  5. Vials were placed in sample holders for evaporation and frozen in a –20 °C freezer
  6. Vials were lyophilized using a Genevac HT-24 evaporator at full vacuum, normally overnight.

Results

Figure 4 - Genevac HT-24 evaporator.

More than 90% of previously intractable samples are friable, “fluffy” solids that can be easily handled and are fully dried. For the few samples that do not dissolve well in 1,4-dioxane, two methods have been used successfully: 1) Samples are redissolved in a small amount of methanol (2–3 mL); then water (approx. 10 mL) is added, and the sample is dried in a Genevac HT-24 evaporator (Figure 4) using vacuum ramping (Dri-Pure) to prevent bumping plus full vacuum to freeze the water. 2) Samples are redissolved in a small volume of tertiary butanol (freezing at +25 °C, which is ideal for lyophilization) and are then dried at full vacuum. The result is “fluffy” solid or crystalline sample (dry and weighable).

Case 2: Novartis Institute for BioMedical Research (NIBMR)

The case shows an easy way to protect samples from cross-contamination during lyophilization.

Problem

At NIBMR, samples must be prepared as a dry, weighable solid for the Compound Storage Department. The production of perfectly lyophilized, “fluffy” samples, free of residual solvent and cross-contamination, was not easily achieved with current techniques.

Solution

The amino acids L-Asn, L-Asp, and L-Arg and one Macrolide xy were selected as model compounds for the lyophilization trials. The following range of solvent mixtures and volumes was developed and selected for samples based on the researched knowledge of sample solubility:

  • 1,4-Dioxane—2–3 mL
  • Tertiary butanol—2–3 mL
  • Water—between 1 and 1.5 mL
  • 1,4-Dioxane and water (1:1)—approx. 2 mL
  • Tertiary butanol and water (1:1)—approx. 2 mL.

Solvent volumes were measured using a 1.0-mL syringe. Sufficient solvent must be used to enable good freezing, since samples are being frozen using vacuum alone. Sample volumes were halved under vacuum and samples were completely frozen after 20 min. The samples were lyophilized in 4-mL vials held in microplate holders that insulated the samples from too much heat.

Results

Trials were carried out with and without a heating stage. Both settings produced lyophilized samples, but occasionally some powder was seen to drift out of the tubes and into the evaporation chamber. Cross-contamination by fluffy samples that could have been blown around was avoided by restricting the vent inlet and covering the samples with pierced plastic paraffin film. Samples were tested using proton NMR and were found to be free of residual solvent and cross-contamination. Care is required when removing the film. Cross-contamination can also be eliminated by applying a higher g-force.

Case 3: U.K. Agrochemicals Research Centre

A method to dry stubborn samples from dimethylsulfoxide (DMSO) solutions is shown.

Problem

When handling new chemical entity (NCE) drug or agrochemical libraries and arrays, it is common practice to dissolve each NCE in DMSO, making it possible to process them with liquid handling automation. Many samples form a gum, oil, or waxy solid and do not truly dry, although complete drying is essential or inaccurate conclusions may be drawn. Ideally, a method to freeze-dry DMSO was required so as not to disrupt the sample work flow. Any work done outside of this work flow would be highly disruptive.

Solution

At the stage at which samples are redissolved, a mixture of DMSO and water was added (water helps to keep the DMSO frozen during evaporation). The holder was then frozen in a bath of liquid nitrogen and transferred to the Genevac HT-24 evaporator for drying under vacuum. Samples were dried at full vacuum for 7 hr.

Results

Seven out of nine samples dried fully, although two only dried to a wax. The powdered nature of the resulting samples enables safe storage. Analysis by proton NMR showed samples to be free of residual DMSO and other solvents.

Dr. Abeysena is Applications Chemist, and Mr. Darrington is Product Manager, Genevac Ltd., The Sovereign Center, Farthing Rd., Ipswich, Suffolk IPI 5AP, U.K.; tel.: +44 1473 240000; fax: +44 1473 461176; e-mail: rob.darrington@genevac.co.uk.

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