IntroductionElectrochemiluminescence (ECL) based ligand binding bridging assays to support

immunogenicity testing have been widely published (1-5). These assays typically require

biotherapeutic-specific (TS) anti-biotherapeutic or drug antibody (ADA) positive control

generation and labeling of a capture and detection reagent (ie, biotinylation and ruthenylation).

Preparation and characterization of these TS critical reagents can be time consuming and

costly, making their application limited for support of time sensitive preclinical studies during

early biotherapeutic development. The assay proposed in this manuscript eliminates the need

for TS reagents by implementing a universal positive control and antibody detection reagent.

Surface plasmon resonance (SPR)-based immunoassays utilizing Biacore technology are also

widely used in the industry (6, 7). Although this platform does not require labeled reagents, the

method typically has to be optimized for immobilization and regeneration of each biotherapeutic,

often not amenable to early research support. Up-front assay development time can be

eliminated by implementing a universal method applicable across programs. The universal

indirect species-specific assay (UNISA) supports utilization of one method across all

biotherapeutics/ species that require immunogenicity assessments while retaining the sensitivity

and dynamic range associated with the readout of the ECL based assay format on the MSD

Sector Imager 6000.

This supplemental text will highlight the methods and results for the qualification of the UNISA

across all species and the further robust validation performed for the cynomolgus monkey-

specific UNISA.

Materials and Methods

Chemical and Reagent Preparation: Species-specific Isotype/ subclass antibodies: Mouse IgG1, IgG2a, IgG2b, and IgG3 isotype

controls (R&D Systems; catalog numbers MAB002, MAB003, MAB004, and MAB007

respectively), rat IgG1, IgG2a, and IgG2b isotype controls (R&D Systems; catalog numbers

MAB005, MAB006, and MAB0061 respectively), cynomolgus monkey IgG1, IgG2, IgG3, and

IgG4 isotype controls (Amgen Inc., described by Jacobsen et al. (8)) were utilized to determine

the specificity of the secondary detector. All other chemical and reagent preparation captured in

the materials and methods section of the main text of manuscript.

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ADA Immunoassay: Universal Indirect Species-specific Assay (UNISA)The UNISA is a species-specific indirect sandwich assay utilizing the ECL technology as the

readout (Figure 1). Reagents were purchased from MSD Inc, Gaithersburg, MD (MSD Sulfo-

TAG NHS-Ester, standard-bind bare plate, and 4× read buffer T) and KPL, Gaithersburg, MD

(20× milk block/ diluent and 20× wash solution). Briefly, the MSD plate was coated overnight

with 35 µL of a biotherapeutic antibody candidate diluted to 1 µg/mL in PBS. The serum

samples were diluted to 1% in 5× KPL block, either untreated (screening test) or treated with

excess relevant or irrelevant biotherapeutic (specificity analysis or competitive binding test).

The coated and blocked plates (blocked with 5× KPL, 200 µL/well overnight) were washed on

Day 2 (wash procedure for all wash steps; 1× KPL wash, 3x300 µL/well), and 35 µL of 1%

serum sample was added to the plate well and incubated for approximately 1 hour. Plates were

washed and 35 µL of ruthenylated species-specific detection antibody at 0.5 µg/mL in 5xKPL

was added and incubated for approximately 30 minutes. Following another wash, 2× MSD T

read buffer was added (150 µL/well). The plates were finally read using the SECTOR® Imager

6000 Instrument (MSD, Gaithersburg, MD, USA) plate reader, where an electrical current was

placed across the plate-associated electrodes, resulting in a series of electrically induced

oxidation-reduction reactions involving ruthenium (from the bound secondary detector antibody)

and tripropylamine (from the MSD T read buffer). The resulting electrochemiluminescence was

measured. No acid dissociation was performed for this assay format.

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 anti-biotherapeutic antibody (ADA) present in the sample.

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Tests and reporting criteria: Samples were reported based on the testing strategy summarized

in Figure 2.

Figure 2. UNISA result flow diagram. Note: Diagram does not necessarily reflect the assay flow as detailed in the method.

Cut Point DeterminationThe assay cut point (ACP) is the S/N value that is used to differentiate between negative and

potentially ADA containing samples. The depletion cut point (DCP) is the percent S/N reduction

that is used to define the specificity of samples. A combination of the 2 cut points identifies a

positive ADA sample.

Qualification Across all Species: To be consistent across programs and aid the rapid

turnaround of results required during early discovery support, universal cut points were applied

after evaluating a subset of animals in the UNISA specificity test across 1 fully human

monoclonal antibody (hMab1) based biotherapeutic as follows: ACP (S/N > 1.5); DCP

(%Depletion > 50). These universal cut points were then applied to all study support and

monitored to ensure sensitivity in detecting potential ADA positive animals. Subsequent to the

case studies shown in this manuscript, a retrospective analysis of the historical data led to the

DCP creation of 20%. This value was in better alignment with the assay cut point and enabled

a more conservative assessment of the response specificity in determining the final antibody

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conclusion status. All current UNISA support across species utilizes an ACP of 1.5 and a DCP

of 20%.

Validation of the Cynomolgus Monkey-specific UNISA: Twenty eight normal cynomolgus

monkeys were split into 2 group sequences with gender as a stratification factor. Group

sequence 1 (S1) contained 9 male and 9 female animals. Group sequence 2 (S2) contained 5

male and 5 female animals plus an 8-point ADA titration curve (see sensitivity section below for

further details). All samples were then tested according to a statistically derived experimental

design model to evaluate the assay cut point (ACP) and depletion cut point (DCP) for 4 hMabs

(A, B, C, and D) in the UNISA specificity test (Table I). In addition, ruggedness of the UNISA

across all 4 hMabs could be explored using a mixed effect model applied to the S/N to study the

effect of analyst, secondary detector, plate lot, and plate coating and their interactions on the

assay performance with all sample types and assay controls. To determine the cut points, the

following statistical methods were employed:

a) ACP was calculated as the upper bound of a one-sided 99% prediction interval for the

distribution of the assay values (S/N). The form of the equation utilized was:

U99 = LS-mean + TINV(.99, DF)*SQRT(Variancetotal + VarianceLS-mean)

b) DCP was calculated using equation: 100% - L99 of %T/U, where L99 is the lower bound

of a one-sided 99% prediction interval for the distribution of the %T/U values. The form

of the equations utilized are:

%T/U: (S/N of untreated/ S/N of treated )*100

L99 = LS-mean - TINV(.99, DF)*SQRT(Variancetotal + VarianceLS-mean)

Table I. Cynomolgus Monkey Specific UNISA Validation Design of Experimentsa

Assay Run AnalystSecondary Detector Plate Lot Plate Coating

Group Sequence

1 1 1 1 1 S12 S2

2 1 2 2 1 S22 S1

3 2 2 1 1 S22 S1

4 2 1 2 1 S12 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 SensitivityThe assay sensitivity is defined as the lowest ADA concentration that gives a S/N response

equivalent to the ACP.

Qualified Across all Species: The species-specific (SS) positive control was titrated from 7.8 to

1000 ng/mL in SS-pooled serum and analyzed once against hMab1. The S/N values were then

analyzed against the SS-positive control concentrations in GraphPad Prism v5.04 using the

following 4PL regression model: log(agonist) vs. normalized response -- Variable slope. The

interpolated concentration equal to the universal ACP (S/N of 1.5) was captured as the assay

sensitivity (Table I, main manuscript).

Validation of the Cynomolgus Monkey-specific UNISA: The cyno-specific UNISA universal

positive control (mouse anti-human IgG/cynomolgus monkey Fc chimeric antibody or cyno-

ADA) was titrated from 1000 to 0.0078 ng/mL (2-fold dilution) in cynomolgus monkey pooled

serum (PNCS). This titration curve was then analyzed following the design model captured in

supplemental table 1 against all 4 hMabs as part of group sequence 2. The S/N values were

then analyzed against the cyno-ADA concentrations by a biostatistician. Whenever the non-

linear regression model, such as 4PL, was not well defined for back-calculating the assay

sensitivity, the first degree polynomial regression was applied to the log transformed S/N ratios

vs. log transformed cyno-ADA concentration using the data in the range as noted.

Biotherapeutic toleranceBiotherapeutic tolerance is defined as the highest drug concentration that still gives a S/N

response above the ACP for each ADA concentration evaluated.

Qualified Across all Species: The hMAb1 was titrated from 1000 to 10 µg/mL in SS-pooled

serum containing 500 ng/mL of the SS-positive control and analyzed once against hMab1. The

S/N values for each hMab were then analyzed in GraphPad Prism v5.04 using the following 4PL

regression model: log(inhibitor) vs. normalized response -- Variable slope. The interpolated

concentration equal to the universal ACP (S/N of 1.5) was captured as the biotherapeutic

tolerance level of the assay. In addition, once the tolerance level was determined, the molar

ratio (ADA to biotherapeutic) could be determined (Table I, main manuscript).

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Validation of the Cynomolgus Monkey-specific UNISA: The 4 hMabs (A, B, C, and D) were

titrated from 1000 to 15.625 µg/mL (2-fold dilution) in PNCS containing 500 ng/mL of the cyno-

ADA and analyzed once against their respective hMab. The S/N values for each hMab were

then analyzed in GraphPad Prism v5.04 using the following 4PL regression model: log(inhibitor)

vs. normalized response -- Variable slope. The interpolated concentration equivalent to the

statistically derived ACP for each hMAb was captured as the biotherapeutic-specific tolerance

level of the assay. In addition, once the tolerance level was determined, the molar ratio (ADA to

biotherapeutic) was expressed.

Specificity

Qualified Across all Species - Secondary detector specificity: A test was performed to examine

specificity of the ruthenylated SS-secondary detectors for their ability to bind to different SS-IgG

isotype subclasses. MSD 6000 bare plates were coated with either mouse-IgG1, IgG2a, IgG2b

or IgG3 (mouse-UNISA test) or rat-IgG1, IgG2a or IgG2b (rat-UNISA test) each at 0.5 and 1.0

µg/mL and processed per the method for incubating, blocking and washing. No samples were

added to the plates. After washing the coating material, SS-detector was added and tested at

concentrations of 250 and 500 ng/mL. Plates were processed per the method for detector

incubation, washing and plate reading. ECL signals from a minimum of four wells were

averaged for each condition. For purposes of this assessment, the cynomolgus monkey

specific subclasses were tested using the Biacore 3000 by immobilizing the UNISA cyno-

detector on a biosensor chip (immobilization range aim for 5000 response units) then passing

each subclass (at 5 µg/mL) over the immobilized surface (flow rate of 5 µL/ min; 3 minutes,

100 mM HCL regeneration solution between cycles). The response units for each subclass (1

per cycle) were then captured.

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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.

Validation of the Cynomolgus Monkey-specific UNISA - Biotherapeutic Target Interference

Assessment: To evaluate the specificity of this assay format for antibody detection, impact from

excess soluble target was assessed in hMab C. A titration curve (0 to 10 µg/mL) of hMab C

soluble ligand and soluble target was spiked into PNCS with and without the universal cyno-

ADA (0, 100 and 500 ng/mL, levels 1-3 respectively) and tested once in the UNISA screening

assay against hMab C. For comparison purposes, the same soluble ligand/ receptor was

spiked into PNCS with 0, 250, and 1000 ng/mL (levels 1, 4, and 5 respectively) of TS-positive

control antibody (rabbit anti-hMab C polyclonal antibody) and tested once in the validated TS-

bridging immunoassay. Level 1 (0 ng/mL of ADA) should produce a response less than or

equal to the ACP, regardless of soluble ligand or soluble target concentration. For the PNCS

spiked with ADA (levels 2-5), all samples should produce a response greater than the ACP,

regardless of soluble target/ ligand concentration.

Results

Qualified ParametersA summary of the qualified parameters are provided as Table I in the main manuscript text. In

addition, Figure 3 represents the specificity of the detector across the subclass evaluated. As

all combinations of isotype control coating (0.5 or 1 µg/mL) and ruthenylated detector (0.25 and

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0.5 µg/mL) yielded similar results, only results for 0.5 µg/mL of both coating and detector are

shown. The mouse-specific detector demonstrated different reactivity for the isotypes tested

with the strongest specificity to the IgG2b subclass. Overall, the detector was sensitive enough

to detect acceptable levels of IgG1, IgG2a and IgG2b; however, the detector demonstrated

weak specificity to IgG3. As a result, IgG3 may not be detected in an antibody assessment

using the current detector and conditions. The rat-specific detector had similar reactivity for

both IgG2a and IgG2b, but was about 50% less reactive to IgG1. Overall, the detector was

sensitive enough in detecting all isotypes and subclasses evaluated. The cyno-specific

detector had similar reactivity for both IgG1 and IgG2, was about 50% less reactive to IgG3 and

demonstrated week specificity for IgG4. As a result, IgG4 may not be detected in an antibody

assessment using the current detector and conditions. At this time, IgM, IgE, and IgA were not

evaluated for specificity, as the scope for this assay is to detect high affinity IgG responses.

Cynomolgus Monkey-specific UNISA ValidationThe UNISA validation results for hMab A-D showed the following: ACP range of 1.25 to 1.38

(99% prediction limit), DCP range of 23 to 45% (99% prediction limit), a sensitivity range of 6 to

8 ng/mL and a biotherapeutic tolerance ranging from 272 to 403 µg/mL at 500 ng/mL of cyno-

ADA (Table II, Figure 4).

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 cyno-ADA 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. Table II. Summary of Universal Validation Parametersb

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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*) 28 (26) 28 (27) 28 (28) 28 (27)Number of Samples (Used in Analysis*) 112 (103) 112 (107) 112 (111) 112 (108)Min, Max* 0.75,1.49 0.77, 1.35 0.77, 1.36 0.80, 1.35Upper Bound on:95% Prediction Limit 1.23 1.20 1.23 1.1599% Prediction Limit 1.35 1.30 1.32 1.25

Depletion Cut Point (DCP); Response: %Number of Donors (Used in Analysis*) 28 (26) 28 (27) 28 (28) 28 (27)Number of Samples (Used in Analysis*) 112 (103) 112 (103) 112 (110) 112 (108)Min, Max* -36, 31 -27, 13 -20, 22 -29, 20Lower Bound on:95% Prediction Limit 21% 14% 16% 10%99% Prediction Limit 30% 20% 21% 16%

Sensitivity (ng/mL) at 99% ACPModel: Linear Regression (7.8125 – 125 ng/mL); N=4 8.06 6.73 7.41 6.52L95, U95 4.95, 13.11 4.70, 9.65 4.37, 12.55 4.24, 10.02

Biotherapeutic Tolerance (µg/mL) with 500 ng/mL ADA at 99% ACPModel: 4PL Regression; N=1 403 302 272 311ADA to Biotherapeutic Molar Ratio 1:806 1:604 1:544 1:622

*After outlier removal.

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

that the animal ID was the highest contributor to variability across all variables tested, which is

due to biological variability and has no relevance on assay variability. Additionally, for hMab A,

22% of the variability could be attributed to analyst and plate lot, compared to negligible

contribution for all variables against hMab B, C, and D (Table IIIa). For PNCS spiked with

increasing concentrations of the cyno-ADA, percent contribution to total assay variability was

evaluated for the combined data set (all curves across all hMab products; N=16) and does not

take into account impact of the hMAb on all interactions. Thus, for the combined cyno-ADA

titration curves, the main contributor to assay variability was observed for analyst to analyst,

where each analyst consistently and purposefully used different incubation parameters to

include flexibility in the final validated method (i.e. room temperature controlled incubator vs

benchtop; orbital vs micromix shaking) (Table IIIb). For both the animals and the cyno-ADA

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titration curve, the total %CV never exceeded 20%, thus demonstrating that the assay was in

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 BiotherapeuticsVariance Components A B C DAnalyst 22 0 0 1Secondary Detector 0 4 0 4Plate Lot 12 0 3 1Coating 1 1 0 4Analyst*Coating 2 7 4 1Secondary*Coating 0 9 0 0Plate Lot*Coating 5 0 0 0Animal ID 33 56 58 72Residual 24 22 34 18Total % CV 14 12 9 11

b) Percent Contribution to Total Assay Variability Across ADA Concentrations (ng/mL)Variance Components 7.8125 15.625 31.25 62.5 125 250 500 1000Analyst 49 71 73 71 56 68 50 43Secondary Detector 22 0 6 2 5 1 8 0Plate Lot 0 7 5 11 21 16 23 27hMab Biotherapeutic 2 0 0 2 8 4 9 11Residual 26 22 16 14 11 11 10 18Total % CV 11 12 15 18 16 18 19 14

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.

In addition, the cyno-specific UNISA demonstrated no cross reactivity or interference from

excess soluble target or ligand for hMab C up to 10 µg/mL for all ADA sample levels (Figure 5a).

In contrast to the UNISA results, the same test performed in the bridging assay format for hMab

C demonstrates cross-reactivity at ≥ 5 µg/mL of receptor, where the negative sample (level 1)

becomes positive (ie, false positive detection) and the magnitude of level 4 (250 ng/mL rabbit

anti-hMab C antibody) is increased above the 0 µg/mL receptor concentration, or expected

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

Immunogenicity testing to detect ADA during biotherapeutic development is commonly

supported through bridging assays using ECL technology or SPR-based immunoassays (1, 3-

5). During early biotherapeutic development, impact on resources (immunoassay development

time, critical reagent cost, analyst effort, etc) is important to consider. The UNISA was

developed to facilitate immunogenicity assessment requiring minimal resources. The assay has

several advantages over conventional immunoassays, which include a) universal positive

control, b) universal analytical method, c) elimination of biotherapeutic-specific conjugate, and

d) antibody specific detector.

Due to the stoichiometry of the biotherapeutic binding interaction with the bare plate (MSD),

UNISA can measure both ID and non-ID specific antibodies in a similar manner. Our lab has

confirmed this by evaluating TS-ADA (ID) titration curves against SS-ADA (non-ID or Fc)

titration curves where there was minimal difference observed in the slope and dynamic range for

2 hMAbs (data not shown). Thus, by utilizing monoclonal SS-positive controls directed against

the CH2 domain of human IgG1, 2, and 4 antibodies, this assay can be universally applied to all

biotherapeutics and variants of a biotherapeutic in mouse, rat, and cynomolgus monkey studies.

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,

and perform purification and characterization was avoided by removing the requirement for TS

positive control antibodies (9-11). Instead, the UNISA monoclonal controls are generated in

bulk and characterized for stability and function once and can be used indefinitely. In addition, a

universally applied analytical method along with the positive control has eliminated the up-front

lead time typically required for TS assay development and allows direct comparison of

performance attributes (i.e. assay sensitivity, biotherapeutic tolerance etc.) between

biotherapeutics. It should be noted that the universal positive control antibodies were

developed internally by Amgen and are not commercially available.

Moreover, by removing use of biotherapeutic-specific conjugated material as capture or

detection reagents, as is the norm for ECL based bridging assays, the requirement for large

amounts of biotherapeutic and cumbersome conjugation and qualification of these reagents,

has been eliminated in the UNISA. Instead, the biotherapeutic in its naive state is directly

coated on the surface of a bare MSD plate and a universal ruthenylated detector employed.

This ruthenylated detector is a generic anti-IgG Fc antibody, conjugated with ruthenium and

qualified only once in bulk per species. As it is specific for binding to species ADA, it confirms

the signal is due to an antibody, unlike other assay formats where soluble receptors and

proteins can also form the bridge and provide false positive results (2, 12, 13). This generic SS

detector can be commercially obtained and commercially ruthenylated in bulk quantities. Thus,

significant reduction in resources (time and raw biotherapeutic material) and increased

specificity can be achieved by utilizing this testing strategy.

Conclusion

The universal indirect species specific assay (UNISA) is a rapid means to assess

immunogenicity throughout the early biotherapeutic development process. The universality

across species and reagents has led to feasible options for immune response monitoring and

comparison across biotherapeutic candidates. The ability to overcome time and critical reagent

associated resources makes the UNISA particularly attractive. Apart from the ease of use and

lack of complicated validation and reagent qualification, UNISA can screen multiple antibody

candidates within the same test and map the antibody specificity to the idiotypic (CDR) or non-

idiotypic (Fc) region of the biotherapeutic, thus supporting a quality by design early development

approach.

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