1
Introduction
2
Electrochemiluminescence (ECL) based ligand binding bridging assays to support
3
immunogenicity testing have been widely published (1-5). These assays typically require
4
biotherapeutic-specific (TS) anti-biotherapeutic or drug antibody (ADA) positive control
5
generation and labeling of a capture and detection reagent (ie, biotinylation and ruthenylation).
6
Preparation and characterization of these TS critical reagents can be time consuming and
7
costly, making their application limited for support of time sensitive preclinical studies during
8
early biotherapeutic development. The assay proposed in this manuscript eliminates the need
9
for TS reagents by implementing a universal positive control and antibody detection reagent.
10
Surface plasmon resonance (SPR)-based immunoassays utilizing Biacore technology are also
11
widely used in the industry (6, 7). Although this platform does not require labeled reagents, the
12
method typically has to be optimized for immobilization and regeneration of each biotherapeutic,
13
often not amenable to early research support. Up-front assay development time can be
14
eliminated by implementing a universal method applicable across programs. The universal
15
indirect species-specific assay (UNISA) supports utilization of one method across all
16
biotherapeutics/ species that require immunogenicity assessments while retaining the sensitivity
17
and dynamic range associated with the readout of the ECL based assay format on the MSD
18
Sector Imager 6000.
19
20
This supplemental text will highlight the methods and results for the qualification of the UNISA
21
across all species and the further robust validation performed for the cynomolgus monkey-
22
specific UNISA.
23
24
Materials and Methods
25
26
Chemical and Reagent Preparation:
27
Species-specific Isotype/ subclass antibodies: Mouse IgG1, IgG2a, IgG2b, and IgG3 isotype
28
controls (R&D Systems; catalog numbers MAB002, MAB003, MAB004, and MAB007
29
respectively), rat IgG1, IgG2a, and IgG2b isotype controls (R&D Systems; catalog numbers
30
MAB005, MAB006, and MAB0061 respectively), cynomolgus monkey IgG1, IgG2, IgG3, and
31
IgG4 isotype controls (Amgen Inc., described by Jacobsen et al. (8)) were utilized to determine
32
the specificity of the secondary detector. All other chemical and reagent preparation captured in
33
the materials and methods section of the main text of manuscript.
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35
ADA Immunoassay: Universal Indirect Species-specific Assay (UNISA)
36
The UNISA is a species-specific indirect sandwich assay utilizing the ECL technology as the
37
readout (Figure 1). Reagents were purchased from MSD Inc, Gaithersburg, MD (MSD Sulfo-
38
TAG NHS-Ester, standard-bind bare plate, and 4× read buffer T) and KPL, Gaithersburg, MD
39
(20× milk block/ diluent and 20× wash solution). Briefly, the MSD plate was coated overnight
40
with 35 µL of a biotherapeutic antibody candidate diluted to 1 µg/mL in PBS. The serum
41
samples were diluted to 1% in 5× KPL block, either untreated (screening test) or treated with
42
excess relevant or irrelevant biotherapeutic (specificity analysis or competitive binding test).
43
The coated and blocked plates (blocked with 5× KPL, 200 µL/well overnight) were washed on
44
Day 2 (wash procedure for all wash steps; 1× KPL wash, 3x300 µL/well), and 35 µL of 1%
45
serum sample was added to the plate well and incubated for approximately 1 hour. Plates were
46
washed and 35 µL of ruthenylated species-specific detection antibody at 0.5 µg/mL in 5xKPL
47
was added and incubated for approximately 30 minutes. Following another wash, 2× MSD T
48
read buffer was added (150 µL/well). The plates were finally read using the SECTOR® Imager
49
6000 Instrument (MSD, Gaithersburg, MD, USA) plate reader, where an electrical current was
50
placed across the plate-associated electrodes, resulting in a series of electrically induced
51
oxidation-reduction reactions involving ruthenium (from the bound secondary detector antibody)
52
and tripropylamine (from the MSD T read buffer). The resulting electrochemiluminescence was
53
measured. No acid dissociation was performed for this assay format.
54
55
56
57
58
59
60
Figure 1. Depiction of the potential binding events in the Universal Indirect Species-specific Assay
(UNISA). The stoichiometry of the biotherapeutic coated on the plate (MSD) allows the detection of both
anti-ID and anti-Fc or framework (non-ID) specific antibody detection. The signal of the sample over the
background response of the pooled species-specific serum (S/N) is proportional to the amount of antibiotherapeutic antibody (ADA) present in the sample.
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Tests and reporting criteria: Samples were reported based on the testing strategy summarized
62
in Figure 2.
63
64
65
66
67
68
Figure 2. UNISA result flow diagram. Note: Diagram does not necessarily reflect the assay flow as
detailed in the method.
69
The assay cut point (ACP) is the S/N value that is used to differentiate between negative and
70
potentially ADA containing samples. The depletion cut point (DCP) is the percent S/N reduction
71
that is used to define the specificity of samples. A combination of the 2 cut points identifies a
72
positive ADA sample.
73
Qualification Across all Species: To be consistent across programs and aid the rapid
74
turnaround of results required during early discovery support, universal cut points were applied
75
after evaluating a subset of animals in the UNISA specificity test across 1 fully human
76
monoclonal antibody (hMab1) based biotherapeutic as follows: ACP (S/N > 1.5); DCP
77
(%Depletion > 50). These universal cut points were then applied to all study support and
78
monitored to ensure sensitivity in detecting potential ADA positive animals. Subsequent to the
79
case studies shown in this manuscript, a retrospective analysis of the historical data led to the
80
DCP creation of 20%. This value was in better alignment with the assay cut point and enabled
81
a more conservative assessment of the response specificity in determining the final antibody
Cut Point Determination
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conclusion status. All current UNISA support across species utilizes an ACP of 1.5 and a DCP
83
of 20%.
84
85
Validation of the Cynomolgus Monkey-specific UNISA: Twenty eight normal cynomolgus
86
monkeys were split into 2 group sequences with gender as a stratification factor. Group
87
sequence 1 (S1) contained 9 male and 9 female animals. Group sequence 2 (S2) contained 5
88
male and 5 female animals plus an 8-point ADA titration curve (see sensitivity section below for
89
further details). All samples were then tested according to a statistically derived experimental
90
design model to evaluate the assay cut point (ACP) and depletion cut point (DCP) for 4 hMabs
91
(A, B, C, and D) in the UNISA specificity test (Table I). In addition, ruggedness of the UNISA
92
across all 4 hMabs could be explored using a mixed effect model applied to the S/N to study the
93
effect of analyst, secondary detector, plate lot, and plate coating and their interactions on the
94
assay performance with all sample types and assay controls. To determine the cut points, the
95
following statistical methods were employed:
96
a) ACP was calculated as the upper bound of a one-sided 99% prediction interval for the
97
distribution of the assay values (S/N). The form of the equation utilized was:
98
U99 = LS-mean + TINV(.99, DF)*SQRT(Variancetotal + VarianceLS-mean)
99
b) DCP was calculated using equation: 100% - L99 of %T/U, where L99 is the lower bound
100
of a one-sided 99% prediction interval for the distribution of the %T/U values. The form
101
of the equations utilized are:
102
%T/U: (S/N of untreated/ S/N of treated )*100
103
104
105
L99 = LS-mean - TINV(.99, DF)*SQRT(Variancetotal + VarianceLS-mean)
106
107
108
109
110
111
112
113
Table I. Cynomolgus Monkey Specific UNISA Validation Design of Experimentsa
Assay Run
1
Analyst
1
Secondary
Detector
1
Plate Lot
1
2
1
2
2
3
2
2
1
4
2
1
2
Plate Coating
1
2
1
2
1
2
1
2
Group
Sequence
S1
S2
S2
S1
S2
S1
S1
S2
aDesign
of experiments utilized to validate UNISA across 4 biotherapeutics while studying the ruggedness
of the assay. All 4 assay runs were repeated across each biotherapeutic in the specificity analysis. Plate
coating refers to 1 MSD 6000 bare plate lot that was coated with the appropriate biotherapeutic in 2
different preparations by 2 different analysts. Group sequence refers to a unique combination of
cynomologus monkey animals and pooled cynomolgus monkey serum spiked with cyno-specific positive
control antibody.
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Assay Sensitivity
115
The assay sensitivity is defined as the lowest ADA concentration that gives a S/N response
116
equivalent to the ACP.
117
118
Qualified Across all Species: The species-specific (SS) positive control was titrated from 7.8 to
119
1000 ng/mL in SS-pooled serum and analyzed once against hMab1. The S/N values were then
120
analyzed against the SS-positive control concentrations in GraphPad Prism v5.04 using the
121
following 4PL regression model: log(agonist) vs. normalized response -- Variable slope. The
122
interpolated concentration equal to the universal ACP (S/N of 1.5) was captured as the assay
123
sensitivity (Table I, main manuscript).
124
125
Validation of the Cynomolgus Monkey-specific UNISA: The cyno-specific UNISA universal
126
positive control (mouse anti-human IgG/cynomolgus monkey Fc chimeric antibody or cyno-
127
ADA) was titrated from 1000 to 0.0078 ng/mL (2-fold dilution) in cynomolgus monkey pooled
128
serum (PNCS). This titration curve was then analyzed following the design model captured in
129
supplemental table 1 against all 4 hMabs as part of group sequence 2. The S/N values were
130
then analyzed against the cyno-ADA concentrations by a biostatistician. Whenever the non-
131
linear regression model, such as 4PL, was not well defined for back-calculating the assay
132
sensitivity, the first degree polynomial regression was applied to the log transformed S/N ratios
133
vs. log transformed cyno-ADA concentration using the data in the range as noted.
134
135
Biotherapeutic tolerance
136
Biotherapeutic tolerance is defined as the highest drug concentration that still gives a S/N
137
response above the ACP for each ADA concentration evaluated.
138
139
Qualified Across all Species: The hMAb1 was titrated from 1000 to 10 µg/mL in SS-pooled
140
serum containing 500 ng/mL of the SS-positive control and analyzed once against hMab1. The
141
S/N values for each hMab were then analyzed in GraphPad Prism v5.04 using the following 4PL
142
regression model: log(inhibitor) vs. normalized response -- Variable slope. The interpolated
143
concentration equal to the universal ACP (S/N of 1.5) was captured as the biotherapeutic
144
tolerance level of the assay. In addition, once the tolerance level was determined, the molar
145
ratio (ADA to biotherapeutic) could be determined (Table I, main manuscript).
146
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Validation of the Cynomolgus Monkey-specific UNISA: The 4 hMabs (A, B, C, and D) were
148
titrated from 1000 to 15.625 µg/mL (2-fold dilution) in PNCS containing 500 ng/mL of the cyno-
149
ADA and analyzed once against their respective hMab. The S/N values for each hMab were
150
then analyzed in GraphPad Prism v5.04 using the following 4PL regression model: log(inhibitor)
151
vs. normalized response -- Variable slope. The interpolated concentration equivalent to the
152
statistically derived ACP for each hMAb was captured as the biotherapeutic-specific tolerance
153
level of the assay. In addition, once the tolerance level was determined, the molar ratio (ADA to
154
biotherapeutic) was expressed.
155
156
Specificity
157
158
Qualified Across all Species - Secondary detector specificity: A test was performed to examine
159
specificity of the ruthenylated SS-secondary detectors for their ability to bind to different SS-IgG
160
isotype subclasses. MSD 6000 bare plates were coated with either mouse-IgG1, IgG2a, IgG2b
161
or IgG3 (mouse-UNISA test) or rat-IgG1, IgG2a or IgG2b (rat-UNISA test) each at 0.5 and 1.0
162
µg/mL and processed per the method for incubating, blocking and washing. No samples were
163
added to the plates. After washing the coating material, SS-detector was added and tested at
164
concentrations of 250 and 500 ng/mL. Plates were processed per the method for detector
165
incubation, washing and plate reading. ECL signals from a minimum of four wells were
166
averaged for each condition. For purposes of this assessment, the cynomolgus monkey
167
specific subclasses were tested using the Biacore 3000 by immobilizing the UNISA cyno-
168
detector on a biosensor chip (immobilization range aim for 5000 response units) then passing
169
each subclass (at 5 µg/mL) over the immobilized surface (flow rate of 5 µL/ min; 3 minutes,
170
100 mM HCL regeneration solution between cycles). The response units for each subclass (1
171
per cycle) were then captured.
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174
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Figure 3. Evaluation of the subclass specificity of the UNISA secondary antibody detectors. Specificity
against IgG4 and IgG3/ IgG4 not evaluated for the mouse and rat detection antibodies respectively. a For
the anti-cyno IgG2 subclass, a or b was not specified.
178
Validation of the Cynomolgus Monkey-specific UNISA - Biotherapeutic Target Interference
179
Assessment: To evaluate the specificity of this assay format for antibody detection, impact from
180
excess soluble target was assessed in hMab C. A titration curve (0 to 10 µg/mL) of hMab C
181
soluble ligand and soluble target was spiked into PNCS with and without the universal cyno-
182
ADA (0, 100 and 500 ng/mL, levels 1-3 respectively) and tested once in the UNISA screening
183
assay against hMab C. For comparison purposes, the same soluble ligand/ receptor was
184
spiked into PNCS with 0, 250, and 1000 ng/mL (levels 1, 4, and 5 respectively) of TS-positive
185
control antibody (rabbit anti-hMab C polyclonal antibody) and tested once in the validated TS-
186
bridging immunoassay. Level 1 (0 ng/mL of ADA) should produce a response less than or
187
equal to the ACP, regardless of soluble ligand or soluble target concentration. For the PNCS
188
spiked with ADA (levels 2-5), all samples should produce a response greater than the ACP,
189
regardless of soluble target/ ligand concentration.
190
191
Results
192
193
Qualified Parameters
194
A summary of the qualified parameters are provided as Table I in the main manuscript text. In
195
addition, Figure 3 represents the specificity of the detector across the subclass evaluated. As
196
all combinations of isotype control coating (0.5 or 1 µg/mL) and ruthenylated detector (0.25 and
7|Page
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0.5 µg/mL) yielded similar results, only results for 0.5 µg/mL of both coating and detector are
198
shown. The mouse-specific detector demonstrated different reactivity for the isotypes tested
199
with the strongest specificity to the IgG2b subclass. Overall, the detector was sensitive enough
200
to detect acceptable levels of IgG1, IgG2a and IgG2b; however, the detector demonstrated
201
weak specificity to IgG3. As a result, IgG3 may not be detected in an antibody assessment
202
using the current detector and conditions. The rat-specific detector had similar reactivity for
203
both IgG2a and IgG2b, but was about 50% less reactive to IgG1. Overall, the detector was
204
sensitive enough in detecting all isotypes and subclasses evaluated. The cyno-specific
205
detector had similar reactivity for both IgG1 and IgG2, was about 50% less reactive to IgG3 and
206
demonstrated week specificity for IgG4. As a result, IgG4 may not be detected in an antibody
207
assessment using the current detector and conditions. At this time, IgM, IgE, and IgA were not
208
evaluated for specificity, as the scope for this assay is to detect high affinity IgG responses.
209
210
Cynomolgus Monkey-specific UNISA Validation
211
The UNISA validation results for hMab A-D showed the following: ACP range of 1.25 to 1.38
212
(99% prediction limit), DCP range of 23 to 45% (99% prediction limit), a sensitivity range of 6 to
213
8 ng/mL and a biotherapeutic tolerance ranging from 272 to 403 µg/mL at 500 ng/mL of cyno-
214
ADA (Table II, Figure 4).
215
216
217
218
219
220
221
222
223
224
225
Figure 4. Assay sensitivity and biotherapeutic tolerance levels evaluated across 4 hMabs. Assay
sensitivity utilized the cyno-specific UNISA positive control (cyno-ADA) titrated in pooled normal
cynomolgus monkey serum (PNCS) from 7.8 to 1000 ng/mL; Biotherapeutic tolerance utilized the cynoADA spiked at 500 ng/mL in PNCS with an 8 point dose-response curve of each individual hMab from
15.625 to 1000 µg/mL. Responses from the screening UNISA test (signal to noise or S/N) were plotted
against the ADA (ng/mL, solid line) or biotherapeutic (µg/mL, dashed line) concentration to evaluate the
variability between the 4 hMAbs and interpolate the biotherapeutic tolerance levels. Observe that the
slope and range of the ADA dose-response between all 4 hMabs is consistent and the slope of the
biotherapeutic tolerance dose-response curve is similar for hMabs B-D.
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Table II. Summary of Universal Validation Parametersb
Fully Human Monoclonal Antibody
Biotherapeutic:
A
B
C
D
Assay Cut Point (ACP); Response: Signal to Noise (S/N)
Number of Donors (Used in Analysis*)
Number of Samples (Used in Analysis*)
Min, Max*
Upper Bound on:
95% Prediction Limit
99% Prediction Limit
28 (26)
112 (103)
0.75,1.49
28 (27)
112 (107)
0.77, 1.35
28 (28)
112 (111)
0.77, 1.36
28 (27)
112 (108)
0.80, 1.35
1.23
1.35
1.20
1.30
1.23
1.32
1.15
1.25
Depletion Cut Point (DCP); Response: %
Number of Donors (Used in Analysis*)
Number of Samples (Used in Analysis*)
Min, Max*
Lower Bound on:
95% Prediction Limit
99% Prediction Limit
28 (26)
112 (103)
-36, 31
28 (27)
112 (103)
-27, 13
28 (28)
112 (110)
-20, 22
28 (27)
112 (108)
-29, 20
21%
30%
14%
20%
16%
21%
10%
16%
7.41
4.37, 12.55
6.52
4.24, 10.02
Sensitivity (ng/mL) at 99% ACP
Model: Linear Regression (7.8125 – 125
ng/mL); N=4
L95, U95
8.06
4.95, 13.11
6.73
4.70, 9.65
Biotherapeutic Tolerance (µg/mL) with 500 ng/mL ADA at 99% ACP
Model: 4PL Regression; N=1
ADA to Biotherapeutic Molar Ratio
403
1:806
302
1:604
272
1:544
311
1:622
227
228
229
230
231
232
233
234
235
236
*After outlier removal.
237
that the animal ID was the highest contributor to variability across all variables tested, which is
238
due to biological variability and has no relevance on assay variability. Additionally, for hMab A,
239
22% of the variability could be attributed to analyst and plate lot, compared to negligible
240
contribution for all variables against hMab B, C, and D (Table IIIa). For PNCS spiked with
241
increasing concentrations of the cyno-ADA, percent contribution to total assay variability was
242
evaluated for the combined data set (all curves across all hMab products; N=16) and does not
243
take into account impact of the hMAb on all interactions. Thus, for the combined cyno-ADA
244
titration curves, the main contributor to assay variability was observed for analyst to analyst,
245
where each analyst consistently and purposefully used different incubation parameters to
246
include flexibility in the final validated method (i.e. room temperature controlled incubator vs
247
benchtop; orbital vs micromix shaking) (Table IIIb). For both the animals and the cyno-ADA
bThe
ACP, DCP, and assay sensitivity were established based on the data generated following the design
of experiments in supplemental table I and analyzed by a biostatistician according to the appropriate
methods section. Biotherapeutic tolerance was determined by running an 8 point dose-response curve
(15.625 to 1000 µg/mL) of each hMab in pooled cynomolgus monkey serum containing 500 ng/mL of
cyno-specific UNISA positive control antibody. The concentration corresponding to the ACP at a 99%
prediction limit for each biotherapeutic was then interpolated on GraphPad Prism v5.04.
The variability assessment across the 4 hMabs for normal cynomologus monkeys demonstrated
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titration curve, the total %CV never exceeded 20%, thus demonstrating that the assay was in
249
250
control and robust for the variance components evaluated during validation.
Table III. Ruggedness Demonstrated Across Variables and Biotherapeuticsc
a)
Percent Contribution to Total Assay Variability Across 4 hMab Biotherapeutics
Variance Components
A
B
C
22
Analyst
Secondary Detector
Plate Lot
Coating
Analyst*Coating
Secondary*Coating
Plate Lot*Coating
Animal ID
Residual
Total % CV
0
12
1
2
0
5
33
24
0
4
0
1
7
9
0
56
22
0
0
3
0
4
0
0
58
34
14
12
9
D
1
4
1
4
1
0
0
72
18
11
b)
Percent Contribution to Total Assay Variability Across ADA Concentrations (ng/mL)
Variance Components
7.8125
15.625
31.25
62.5
125
250
Analyst
Secondary Detector
Plate Lot
hMab Biotherapeutic
Residual
Total % CV
251
252
253
254
255
256
257
49
22
500
1000
71
73
71
56
68
50
43
0
2
26
0
7
0
22
1
16
4
11
18
8
23
0
27
12
2
11
2
14
18
5
21
11
6
5
0
16
15
9
10
19
11
18
14
8
11
16
c Percent
contribution to total assay variability for identified critical variables evaluated in the cyno-specific
UNISA specificity test following the design of experiments in Table I. a Normal animals assessed across
4 hMabs, total % CV ≤ 14%, thus assay is in control. b Pooled normal cynomologus monkey serum
(PNCS) spiked with increasing concentrations of the cyno-specific UNISA positive control, total % CV ≤
19%, thus assay is in control.
258
In addition, the cyno-specific UNISA demonstrated no cross reactivity or interference from
259
excess soluble target or ligand for hMab C up to 10 µg/mL for all ADA sample levels (Figure 5a).
260
In contrast to the UNISA results, the same test performed in the bridging assay format for hMab
261
C demonstrates cross-reactivity at ≥ 5 µg/mL of receptor, where the negative sample (level 1)
262
becomes positive (ie, false positive detection) and the magnitude of level 4 (250 ng/mL rabbit
263
anti-hMab C antibody) is increased above the 0 µg/mL receptor concentration, or expected
264
magnitude (Figure 5b).
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Figure 5. The UNISA demonstrates no interference from soluble ligand or receptor up to 10 µg/mL for
hMab C. a Levels 1, 2, and 3 corresponding to 0, 100 and 500 ng/mL cyno-ADA respectively, spiked into
PNCS with increasing concentrations of soluble target/ receptor – response is consistent in UNISA, thus
no interference observed. b Levels 1, 4, and 5 corresponding to 0, 250 and 1000 ng/mL rabbit anti-hMab
C ADA respectively, spiked into PNCS with increasing concentrations of soluble target/ receptor –
concentrations of soluble receptor ≥ 5 µg/mL cause an increase in the response, demonstrating the
potential to cross-react in the assay and elevate arbitrarily the ADA measurement.
Discussion
276
277
Immunogenicity testing to detect ADA during biotherapeutic development is commonly
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supported through bridging assays using ECL technology or SPR-based immunoassays (1, 3-
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5). During early biotherapeutic development, impact on resources (immunoassay development
280
time, critical reagent cost, analyst effort, etc) is important to consider. The UNISA was
281
developed to facilitate immunogenicity assessment requiring minimal resources. The assay has
282
several advantages over conventional immunoassays, which include a) universal positive
283
control, b) universal analytical method, c) elimination of biotherapeutic-specific conjugate, and
284
d) antibody specific detector.
285
286
Due to the stoichiometry of the biotherapeutic binding interaction with the bare plate (MSD),
287
UNISA can measure both ID and non-ID specific antibodies in a similar manner. Our lab has
288
confirmed this by evaluating TS-ADA (ID) titration curves against SS-ADA (non-ID or Fc)
289
titration curves where there was minimal difference observed in the slope and dynamic range for
290
2 hMAbs (data not shown). Thus, by utilizing monoclonal SS-positive controls directed against
291
the CH2 domain of human IgG1, 2, and 4 antibodies, this assay can be universally applied to all
292
biotherapeutics and variants of a biotherapeutic in mouse, rat, and cynomolgus monkey studies.
293
For conventional immunoassays, specific ADA is required in order to develop and optimize
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assay performance. A considerable material investment, time to immunize and generate sera,
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and perform purification and characterization was avoided by removing the requirement for TS
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positive control antibodies (9-11). Instead, the UNISA monoclonal controls are generated in
297
bulk and characterized for stability and function once and can be used indefinitely. In addition, a
298
universally applied analytical method along with the positive control has eliminated the up-front
299
lead time typically required for TS assay development and allows direct comparison of
300
performance attributes (i.e. assay sensitivity, biotherapeutic tolerance etc.) between
301
biotherapeutics. It should be noted that the universal positive control antibodies were
302
developed internally by Amgen and are not commercially available.
303
304
Moreover, by removing use of biotherapeutic-specific conjugated material as capture or
305
detection reagents, as is the norm for ECL based bridging assays, the requirement for large
306
amounts of biotherapeutic and cumbersome conjugation and qualification of these reagents,
307
has been eliminated in the UNISA. Instead, the biotherapeutic in its naive state is directly
308
coated on the surface of a bare MSD plate and a universal ruthenylated detector employed.
309
This ruthenylated detector is a generic anti-IgG Fc antibody, conjugated with ruthenium and
310
qualified only once in bulk per species. As it is specific for binding to species ADA, it confirms
311
the signal is due to an antibody, unlike other assay formats where soluble receptors and
312
proteins can also form the bridge and provide false positive results (2, 12, 13). This generic SS
313
detector can be commercially obtained and commercially ruthenylated in bulk quantities. Thus,
314
significant reduction in resources (time and raw biotherapeutic material) and increased
315
specificity can be achieved by utilizing this testing strategy.
316
317
Conclusion
318
319
The universal indirect species specific assay (UNISA) is a rapid means to assess
320
immunogenicity throughout the early biotherapeutic development process. The universality
321
across species and reagents has led to feasible options for immune response monitoring and
322
comparison across biotherapeutic candidates. The ability to overcome time and critical reagent
323
associated resources makes the UNISA particularly attractive. Apart from the ease of use and
324
lack of complicated validation and reagent qualification, UNISA can screen multiple antibody
325
candidates within the same test and map the antibody specificity to the idiotypic (CDR) or non-
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idiotypic (Fc) region of the biotherapeutic, thus supporting a quality by design early development
327
approach.
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