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
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
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.
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
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:
Concentration of the bulk of the solvent
using fast evaporation
- Cooling of the samples and sample
holders in preparation for
- Freezing of the sample using deep vacuum
- 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.
Dri-Pure® (Genevac)—vacuum ramping and high
rotor speed prevent bumping.
- Concentration—a 40-mbar stage that removes
the acetonitrile without freezing the water (here
acetonitrile boils at +2 °C).
- 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.
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.
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.
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:
Dried fractions were redissolved in minimal
water and methanol
- Fractions were pooled into 20-mL scintillation
vials and dried
- Samples were dissolved in minimal
1,4-dioxane; some stubborn samples
present as gums or oils
- Fractions were pooled into a bar-coded,
- Vials were placed in sample holders for
evaporation and frozen in a –20 °C freezer
- Vials were lyophilized using a Genevac
HT-24 evaporator at full vacuum, normally
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
The case shows an easy way to protect samples from cross-contamination
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.
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:
- 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.
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.
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.
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.
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: email@example.com.