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lyophilizates at pH 5.5 (gray lines) and pH 3.5 (black lines) (A); intrinsic protein
fluorescence emission spectra of LDH reconstituted from collapsed (dashed lines) and
noncollapsed (solid lines) trehalose and PEG3350-(black lines), trehalose-(dark gray
lines) and sucrose and PEG3350-(light gray lines) based lyophilizates as compared to
standard (red line) and heat-denatured standard (gray shaded line) with the insert
showing a magnification of the emission maximum area (B); UV second derivative
spectra of PA01 prior to freeze-drying (C) and after reconstitution from collapsed (dashed
line) and noncollapsed (solid line) lyophilizates (D). FD: freeze-drying.
was observed that the excipient arginine overlaps structural information in the present study, as they
with the fluorescence emission of the protein. The were applied to qualitatively assess the preservation
position of peaks in the second derivative of UV of tertiary structure upon freeze-drying and upon the
absorption spectra is sensitive to the polarity of the occurrence of collapse by comparing spectra of
microenvironment of aromatic amino acids and thus solutions prior to freeze-drying and after reconstitu-
can also be used to assess the tertiary structure of tion from collapsed and noncollapsed lyophilizates.
proteins.98 In addition, the intensity of the peaks can Two major valleys were manifest in the derivative
give useful structural information as well. Fluores- spectra of PA01 samples, 1 at 284.5 nm arising from
cence spectroscopy is considered to be more sensitive both tryptophan and tyrosine absorbance and 1 at
towards subtle conformational changes,98 on the 292 m arising from tryptophan absorbance.52,53
other hand, 2DUV spectroscopy includes the amino Comparing PA01 reconstituted from collapsed and
acid phenylalanine to a higher degree. But the two noncollapsed cakes, the curves were congruent
techniques are considered to yield comparable with spectra recorded prior to freeze-drying
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
2274 SCHERSCH ET AL.
Figure 8. Recovery of LDH enzyme activity in the reconstituted solution after freeze-
drying to collapsed (gray bars) and noncollapsed (black bars) lyophilizates (A); activity of
PA01 after reconstitution from collapsed and noncollapsed lyophilizates as compared to
the PA01 enzyme activity prior to freeze-drying as determined by the clot lysis assay (B).
Activities are calculated average values of at least two independent measurements,
compared to the average activity prior to freeze-drying in the case of LDH.
(Fig. 7C and D). Thus, freeze-drying had no impact on precision of the method,56 the activity of the samples
PA01 tertiary structure, regardless of collapse. is not regarded as significantly different. Thus, it can
be concluded that collapse exerts no detrimental
effect on PA01 activity.
Biological Activity
The most important function of proteins is their
biological activity. Thus, a key analytical tool CONCLUSION
evaluating protein stability is the recovery of enzy-
matic activity. The goal of the present study was to investigate the
Figure 8A depicts the recovery of LDH activity in effect of lyophilizate collapse during freeze-drying on
the reconstituted solution after freeze-drying to protein stability. For this purpose, three structurally
collapsed (gray bars) and elegant (black bars) very different model proteins were analyzed in
lyophilizates. Most obviously LDH stability was well various formulations including both purely amor-
preserved in all the systems, as activities were not phous disaccharide- or amino acid-based formula-
strongly decreased as compared to the activities prior tions and partially crystalline mannitol- or PEG-
to freeze-drying. The preservation of bioactivity with based formulations, representing the most commonly
the onset of collapse has been recently observed by used freeze-drying formulations. Collapsed lyophili-
Luthra et al.99 as well. Interestingly, biological zates were intentionally generated in a reproducible
activities were higher in collapsed lyophilizates than manner by application of an aggressive freeze-drying
in elegant ones for two of the three investigated protocol, referred to as collapse-drying, and were
formulations (trehalose and sucrose and PEG 3350). compared to noncollapsed lyophilizates of identical
However, because of the relatively large standard formulation.
deviations, no significant differences between the two In order to clearly distinguish between the effect of
groups could be deduced. collapse and the effect of residual moisture, collapsed
Catalytic activity of the reconstituted PA01 lyo- and noncollapsed formulations of comparable resi-
philizates was assessed using the clot lysis assay dual moisture levels were produced. It was found that
described by Carlson et al.56 Figure 8B displays the reconstitution times of collapsed lyophilizates were
clot-lytic activities of PA01 after reconstitution from not significantly prolonged.
collapsed and noncollapsed lyophilizates, respec- In addition to the investigation of completely
tively. Freeze-drying slightly affected the enzyme collapsed and noncollapsed lyophilizates, various
stability, as the activity after freeze-drying was intermediate collapse states were analyzed as well.
reduced by 3% and 5% for not collapsed and This was achieved by stepwise variation of the ratio of
collapsed cakes, respectively. Given the described mannitol to sucrose, resulting in lyophilizates with
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 DOI 10.1002/jps
EFFECT OF LYOPHILIZATE COLLAPSE 2275
increasing degree of collapse upon decreasing amount ACKNOWLEDGMENTS
of mannitol.
Excipients were found to be insensitive towards the The authors thank Boehringer Ingelheim Pharma
onset of collapse: amorphous excipients remained GmbH & Co. KG for financial support and for provid- [ Pobierz całość w formacie PDF ]