Factorial Analysis of Allergenic Extract Compatibilities using a
Fractional 6-Factor, 2-Level Experimental Design Matrix
TJ Grier, P h.D., DM Hall, B.S., EA Duncan, B.S., RE Esch, P h.D. and TC Coyne, M .D.
Research & Developm ent Laboratory
Greer Laboratories, I nc.
Lenoir, North Carolina USA
Abstract
Materials and Methods
Background
Immunotherapy vaccines frequently contain 3 or more allergenic extracts from diverse
source materials.
The following Greer extract concentrates and diluent in 50% glycerin were employed for
this study: Timothy grass, 100,000 BAU/mL; Bermuda grass, 10,000 BAU/mL; Johnson
grass, 1:20 w/v; D. farinae, 10,000 AU/mL; D. pteronyssinus, 10,000 AU/mL; Cat hair,
10,000 BAU/mL; Dog epithelia, 1:20 w/v; Eastern 7 Tree mix, 1:20 w/v; Mold mix 1, 1:20
w/v; Short ragweed, 1:20 w/v; 50% glycero-saline. Extracts were stored at 2-8°C, and all
product and diluent lots were mixed and tested within their expiration dates.
Studies examining the immunochemical compatibilities of allergens in these mixtures are
limited by the large number of test samples needed to cover all multi-extract combinations
and single-extract controls.
Objectives
To examine the ability of a fractional experimental design matrix to assess the contributions
of individual extracts on allergen compatibilities in complex mixtures.
Methods
Glycerinated extract mixtures containing timothy grass and various combinations of 9 other
products (Bermuda grass, johnson grass, D. farinae, D. pteronyssinus, cat hair, dog
epithelia, 7-tree mix, 5-mold mix, short ragweed) were prepared based on a common 6factor (extracts), 2-level (presence or absence), 8-run (mixtures) factorial design matrix.
Recoveries of timothy extract potency in these mixtures (vs. timothy-only control sample)
were determined by multi-allergen IgE ELISA inhibition analysis after storage of all test
samples for up to 12 months at 2-8°C or up to 6 months at 20-25°C.
Factorial analysis was conducted using Minitab 14 (design of experiments module).
Results
Timothy extract recoveries ranged from 31-146% at 2-8°C and from 4-140% at 20-25°C.
All mixtures containing mold mix displayed significant reductions of timothy grass extract
potency during storage at both 2-8°C and 20-25°C.
Tree mix also produced low but detectable reductions at both temperatures.
As expected, the effects of mixing with most extracts were more pronounced after elevated
temperature exposures.
Conclusions
Factorial design matrices are convenient tools for assessing allergen compatibilities in a
wide variety of complex extract mixtures.
© GREER 2013 All Rights Reserved.
MPN 101012H716
Experimental designs are convenient tools for predictive modeling of complex physical and
chemical processes involving numerous interactive factors present in varying qualitative
types or at different quantitative levels.
A common practical application of these designs is to reduce the total number of
experimental conditions examined to a manageable number of runs to identify, and
subsequently validate, the conditions yielding optimal or desired outcomes.
In this study, extract mixtures were prepared by combining companion products in
10
25
50
accordance with a 6-factor, 2-level, 8-run experimental design
matrix,
in which each factor
represented a specific extract or extract pair, each level indicated either the presence (+)
or absence (-) of these extracts in a particular mixture.
Each run (mixture) contained 3-10 extracts, with each extract or extract pair present in 4
different runs.
A ninth run was added to serve as the single-extract control sample.
Timothy grass was selected as the target allergen to be included in all mixtures and
control at the same final concentration as all other companion extracts (one-tenth of
concentrate levels).
All samples (20.0 mL volumes at 50% glycerin) were split into two 10.0 mL volumes and
analyzed after storage for up to 12 months at 2-8°C or for up to 6 months at 20-25°C.
The stabilities of multiple Timothy grass allergens in test samples were measured by IgE
ELISA inhibition analyses using allergic human serum pool S5-Grass, the same serum pool
and analytical procedure used to standardize northern pasture grass extracts in BAU/mL
concentrations, as regulated by FDA.
Relative IgE-binding potencies were calculated from % inhibition values of test and
reference samples, with validity assessed by parallel line bioassay with paired t tests.
Recoveries for test extract mixtures were expressed relative to single-extract controls on
graphs as follows:
green shading
≥ 70% of control values
yellow shading
50-70% of control values
red shading
< 50% of control values
Factorial Analysis of Allergenic Extract Compatibilities using a
Fractional 6-Factor, 2-Level Experimental Design Matrix
TJ Grier, P h.D., DM Hall, B.S., EA Duncan, B.S., RE Esch, P h.D. and TC Coyne, M .D.
Research & Developm ent Laboratory
Greer Laboratories, I nc.
Lenoir, North Carolina USA
6-Factor, 2-Level, 8-Run Matrix + Control Run 9
Timothy Extract Compatibilities at 2-8°C
The 6-factor, 2-level sample matrix shown in Table 1 below was selected for analysis of
Timothy grass allergens in mixtures with up to 9 companion extracts frequently included in
immunotherapy vaccines. This fractional array specifies 8 runs that model 64 possible test
extract combinations.
IgE-binding potencies of Timothy grass allergens were assessed in extract mixtures and
control stored for 3, 6 and 12 months at 2-8°C. Recoveries ranged from 31-146% of
control run values, with progressive losses observed for runs 5-8 (Table 3). Mean
recoveries in the presence and absence of each factor were calculated, with mean
differences (∆) between values from + and − conditions evaluated for significance based
on probability (Table 4). Mold extract was the only factor producing significant changes in
Timothy allergen potencies after refrigerated storage for up to 12 months.
To incorporate an analogous Timothy extract control into this matrix, a ninth run was
added, and extract volumes were combined as illustrated in Table 2 below. The contents of
each sample were vialed into equivalent 10 mL volumes that were incubated at either 2-8°C
or 20-25°C. Interactions between 2 factors are also embedded, or aliased, in this matrix, as
follows: D=A∗B, E=A∗C, F=B∗C.
Table 1: 6-Factor (A-F), 2-Level (+,−), 8-Run Matrix
Analyte = Timothy, Present in all samples
Test
samples
(Runs)
A
Mold
B
Tree
C
Mite
D
Grass
E
Cat/Dog
F
SRag
1
−
−
−
+
+
+
2
−
+
+
−
−
+
3
−
−
+
+
−
−
4
−
+
−
−
+
−
5
+
+
+
+
+
+
6
+
+
−
+
−
−
7
+
−
+
−
+
−
8
+
−
−
−
−
+
Test
samples
(Runs)
Table 2: Extract Volumes Combined in Each 20 mL Test Sample (mL)
Test
samples
(Runs)
Tim
Mold
mix
Tree
mix
1
2
0
2
2
0
3
2
4
2
5
Df
+
Dp
Ber
+
John
Cat
+
Dog
Short
Rag
0
0
2+2
2+2
2
8
2
2+2
0
0
2
10
0
0
2+2
2+2
0
0
10
0
2
0
0
2+2
0
12
2
2
2
2+2
2+2
2+2
2
0
6
2
2
2
0
2+2
0
0
10
7
2
2
0
2+2
0
2+2
0
8
8
2
2
0
0
0
0
2
14
9
2
0
0
0
0
0
0
18
50%
GS
Table 3: Recoveries at 2-8°C
(% of control run 9)
Test
samples
(Runs)
3 mo.
6 mo.
12 mo.
1
118
114
103
2
102
95
101
3
121
146
135
4
114
112
5
89
77
68
6
89
87
35
7
83
41
52
8
77
34
31
10
25
91
50
Table 4: Mean +, − and ∆ Recoveries after 12 mo. at 2-8°C
A
Mold
B
Tree
C
Mite
D
Grass
E
Cat/Dog
F
SRag
+
46
74
89
85
79
76
−
108
80
65
69
76
79
∆
−61
−6
+24
+16
+3
−3
Factorial Analysis of Allergenic Extract Compatibilities using a
Fractional 6-Factor, 2-Level Experimental Design Matrix
TJ Grier, P h.D., DM Hall, B.S., EA Duncan, B.S., RE Esch, P h.D. and TC Coyne, M .D.
Research & Developm ent Laboratory
Greer Laboratories, I nc.
Lenoir, North Carolina USA
Timothy Extract Compatibilities at 20-25°C
Discussion and Conclusion
The compatibilities of Timothy grass allergen activities in diverse multi-extract mixtures
were also examined after storage for 1, 3 and 6 months at 20-25°C. As expected, changes
in Timothy extract potencies at ambient temperatures were accelerated compared to
refrigerated storage, with major losses in activity noted for numerous samples after storage
for up to 1 month. Progressive reductions were found for most mixtures (Table 5). Factorial
analysis of data obtained at the 6 month time point showed that, similar to refrigerated
storage conditions, the presence of mold extract in a mixture was the only significant
contributor to Timothy extract instability (Table 6).
Extract compatibility studies are designed to identify companion extracts that compromise
target allergen activities.
Test
samples
(Runs)
+
Fractional designs provide effective working models for defining the importance of
different experimental factors and their interactions from a limited set of conditions both
representative and predictive of the full sample matrix.
Table 5: Recoveries at 20-25°C
(% of control run 9)
Test
samples
(Runs)
1 mo.
3 mo.
6 mo.
1
99
92
91
2
67
62
43
3
100
140
118
4
106
82
51
5
55
49
16
6
35
25
6
7
40
23
9
8
28
14
4
Sample mixtures in most studies consist of 2 products, a target extract and a series of
individual companion extracts. Mixtures of 3 or more extracts are typical of those used for
immunotherapy in the United States but are not often represented in compatibility studies
because the exponentially higher number of samples required to maintain a full matrix
design (all combinations included) is either impractical or unmanageable logistically.
In this study, complex mixtures of up to 9 extracts frequently included in immunotherapy
vaccines were examined using a common 6-factor, 2-level fractional design that reduced
64 possible test extract combinations to a convenient set of 8 conditions (plus 1 target
extract-only control) to identify those factors (companion extracts) that can destabilize
Timothy grass allergens in these samples.
10
25
50
Timothy allergens were destabilized significantly when mixed with mold extract and stored
at either 2-8°C or 20-25°C.
The presence of other extracts provided partial protection for some (but not all) mixtures
containing mold extract at 2-8°C, but was not as influential at 20-25°C.
Other individual extracts did not significantly impact Timothy extract potencies in mixtures
relative to Timothy-only controls, consistent with several published reports.
Table 6: Mean +, − and ∆ Recoveries after 6 mo. at 20-25°C
A
Mold
B
Tree
C
Mite
D
Grass
E
Cat/Dog
F
SRag
9
29
46
58
42
39
−
76
56
38
27
43
46
∆
−67
−27
+8
+31
−1
−7
The predictive value and diagnostic power of fractional design matrixes provides
researchers a useful tool for assessing extract compatibilities in diverse allergen mixtures
administered to patients during immunotherapy.
© GREER 2013 All Rights Reserved.
MPN 101012H716