Stabilization of Labile Grass-Fungal and Grass-Insect Extract Mixtures
During Storage at Sub-Zero (Conventional Freezer) Temperatures
Thomas J. Grier, Ph.D., Dawn M. Hall, B.S., Elizabeth A. Duncan, B.S., and Terrance C. Coyne, M.D.
Research & Development Laboratory
Greer Laboratories, Inc.
Lenoir, North Carolina
Abstract
Materials and Methods
Background
Preparation of optimal extract mixtures for allergen
immunotherapy can be restricted by changing
regulations or limits for reimbursement. Stabilization of
allergen combinations known to be incompatible under
conventional storage conditions, such as pollens mixed
with protease-rich fungi or insects, can provide viable
alternatives to help allergists administer the desired
immunotherapy formulations to their patients.
The following GREER® extract concentrates in 50%
glycerin, obtained from current released product
inventories, were used to prepare the test samples
examined in this study:
Timothy grass (100,000 BAU/mL)
Meadow fescue grass (100,000 BAU/mL)
Alternaria alternata (1:20 w/v)
Aspergillus fumigatus (1:20 w/v)
Penicillium chrysogenum/notatum (1:20 w/v)
American cockroach (1:20 w/v)
German cockroach (1:20 w/v)
Fire ant Solenopsis invicta (1:20 w/v)
Objectives
To examine the compatibilities of standardized timothy
and meadow fescue grass pollen extracts in mixtures
containing fungal or insect extracts at elevated
glycerin concentrations after storage for up to 12
months at refrigerator (2-8°C) or freezer temperatures
(-15 to -20°C).
Methods
Timothy and meadow fescue extracts were combined
with individual fungal (Alternaria, Aspergillus,
Penicillium) or insect (American cockroach, German
cockroach, fire ant) extracts at glycerin concentrations
ranging from 25-50%, in 5% increments. Extract
levels corresponded to those producing effective
maintenance doses for subcutaneous immunotherapy.
Grass extract potencies were determined by IgE ELISA
inhibition assay, the same method used to standardize
these products.
Results
Grass allergens in mixtures with fungal or insect
extracts were stabilized considerably by storage in a
conventional freezer. Several mixtures at 25-30%
glycerin were partially frozen, but those at 35-50%
glycerin remained in liquid phase. All combinations
exhibited significant improvements in grass allergen
potency relative to identical mixtures maintained at 28°C (up to 16-fold for timothy; up to 10-fold for
meadow fescue). Recoveries for most mixtures at 25%
glycerin stored in a freezer exceeded those at 50%
glycerin stored in a refrigerator.
Conclusions
Storage of glycerinated extract mixtures at freezer
temperatures stabilizes labile allergens, supporting
formulation of specific product combinations that must
currently be separated into different treatment vial
sets.
© GREER 2014 All Rights Reserved
MPN 091614HD1229
Two-part extract mixtures (10.0 mL volumes) were
prepared using 1.0 mL of each product concentrate
(final concentrations: 10,000 BAU/mL of each grass
extract, 1:200 w/v of each fungal or insect extract).
Test extract mixtures and single-extract grass controls
were prepared using defined volumes of normal saline
and 50% glycerin-saline diluents to produce solutions
with final glycerin concentrations ranging from 2550%, in 5% increments.
Samples (5.0 mL per condition) were stored for up to
12 months at refrigeration (2-8C) or conventional
freezer temperatures (-15 to -20C), and analyzed
after 3, 7 and 11 months for recoveries of multiple
allergens in each grass species by IgE ELISA inhibition
assay, the potency method currently used for lot
release and stability testing of standardized grass
extracts by all licensed U.S. allergen manufacturers.
Grass allergen potencies were assessed relative to
those of freshly-prepared control samples at identical
extract and glycerin concentrations.
Grass extract recoveries for test mixtures were
expressed relative to single-extract controls on graphs
as follows:
green shading
> 75% of control values
yellow shading
51-75% of control values
red shading
26-50% of control values
purple shading
0-25% of control values
Statistical analyses of allergen recoveries for extract
mixtures and controls were performed using twosample t tests assuming equal variances and means,
with significance achieved for data comparisons
yielding two-tailed (two-sided) P values below 0.05.
Physical Condition of Controls and
Mixtures at Freezer Temperatures
Stabilities of Meadow Fescue and
Timothy Grass Extract Controls
Compatibilities of Meadow Fescue
Grass Allergens in Extract Mixtures
Compatibilities of Timothy Grass
Allergens in Extract Mixtures
Storage of extract mixtures and controls at -15 to 20°C resulted in partial freezing (solid mass when
inverted, rapid thaw rates at 2-8°C or 20-25°C) for the
following samples:
Meadow fescue and timothy extract controls displayed
consistent and stable IgE-binding activities (relative to
freshly-prepared reference solutions of identical
compositions) during storage for up to 12 months at
either 2-8°C or -15 to -20°C (illustrated on top rows of
the two figure panels provided below).
Improved recoveries of meadow fescue extract
potencies were observed in mixtures with highprotease fungal or insect extracts stored for 3, 7 or 11
months at -15 to -20°C, compared to 2-8°C storage of
replicate samples.
Timothy allergen stabilities in mixtures with most highprotease fungal or insect extracts were also found to
be significantly higher when stored at -15 to -20°C for
3, 7, or 11 months, relative to refrigerated storage of
analogous solution formulations.
Meadow fescue allergen activities in mixtures with
fungal extracts increased significantly under 10 of the
12 conditions (p = 0.002-0.047), with degrees of
stabilization ranging from 1.2-3.4 (+ Penicillium) to
1.6-9.5 (+ Alternaria). Increases in meadow fescue
extract potencies in mixtures with insect extracts were
significant for 8 of the 12 conditions (p = 0.0010.045), with degrees of improvement ranging from
1.2-3.0 (+ Fire ant) to 1.4-9.9 (+ German cockroach).
Mixtures with fungal extracts were improved under 11
of the 12 conditions (p = 0.00001-0.031), with
degrees of stabilization of timothy allergens ranging
from 1.2-2.0 (+ Penicillium) to 1.4-9.0 (+ Alternaria).
Increases in timothy extract potencies in mixtures with
insect extracts were significant for 10 of the 12
conditions (p = 0.0009-0.034), with degrees of
improvement ranging from 1.1-3.4 (+ Fire ant) to 1.415.8 (+ German cockroach).
In all cases, statistically-significant increases in
meadow fescue grass allergen activity at -15 to -20°C
were found for mixtures that displayed recoveries in
different quartiles at the two storage temperatures.
Similar to meadow fescue, all data sets producing
different quartile recoveries at the two storage
temperatures (illustrated as different colors for the 3
refrigerated vs. 3 freezer time points of each test
mixture and graph) yielded significant improvements
in timothy allergen activity, and temperature–related
differences were lower at the elevated glycerin levels.
Meadow fescue
Control and all mixtures at 25% glycerin
Control and all mixtures except Meadow-fescueAspergillus mix at 30% glycerin
Timothy
Control and all mixtures at 25% glycerin
Timothy-German cockroach mix at 30% glycerin
All other samples at 30% glycerin, and all controls and
mixtures containing 35-50% glycerin, remained in
liquid phase with no evidence of freezing throughout
the 12 month storage period at -15 to -20°C.
Most samples at 25% glycerin required 1-2 days in
freezer to convert the vial contents to a solid phase.
Slightly longer storage times (up to 6 days) were
needed to freeze most samples at 30% glycerin.
*
Recoveries for meadow fescue controls ranged from
74-117%, while timothy controls exhibited 71-105%
recoveries at these temperatures.
No statistically-significant potency differences were
observed between fresh grass extract references and
control samples stored either in a refrigerator for 3, 7
or 11 months (shown on bar graphs below as 3R, 7R,
and 11R), or in a freezer for 3, 7, or 11 months
(indicated on bar graphs below as 3F, 7F, and 11F).
No major changes in allergenic activities were found
for control samples that were stored in a solid (frozen)
state for up to 12 months (meadow fescue at 25-30%
glycerin, timothy at 25% glycerin), as compared to
fresh, unfrozen reference preparations.
P values ranging from .010 to .049
**
P values below .010
The differences in IgE-binding potencies for analogous
samples at the two temperatures were reduced with
ach incremental increase in glycerin concentration of
the test mixtures, consistent with the known inhibitory
properties
of
glycerin
on
protease-catalyzed
degradation reactions.
Conclusions
In this study, storage of labile grass-fungal and grassinsect extract mixtures at conventional freezer
temperatures provided significant improvements in
grass allergen stabilities at several glycerin
concentrations, compared to refrigeration. Up to 16fold improvements were observed for timothy extracts,
and up to 10-fold increases were found for meadow
fescue extracts.
All control samples at 25-50% glycerin retained
favorable and comparable IgE-binding activities at the
two storage temperatures. For most pairs of extract
mixtures evaluated side-by-side for allergenic potency,
grass allergen recoveries in 25% glycerin solutions at 15 to -20°C were similar to or exceeded those in 50%
glycerin solutions at 2-8°C. Most mixtures and controls
at 25% glycerin and several at 30% glycerin exhibited
at least partial freezing, but as noted above, retained
high levels of IgE-binding activity after thawing.
Freezer storage of these unstable extract mixtures
lowers the glycerin levels required to stabilize
prominent allergenic components, and supports the
clinical use of specific extract combinations that must
currently be administered in separate treatment vials.