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Stability of anti-immunotherapeutic antibodies in frozen human serum samples
Background: To generate exhaustive data on the stability of human antiimmunotherapeutic antibodies. Materials & methods: Samples collected from over 100 different subjects at various timepoints were analyzed shortly after serum collection using specific ELISAs and re-analyzed after long-term storage or multiple cycles of freeze–thaw. The general acceptance criteria for incurred sample reanalysis for ligand-binding assays were applied, as well as alternative stricter acceptance criteria promoted by various white papers. Results: Anti-immunotherapeutic antibodies are stable in undiluted serum samples stored at -80°C for at least 3.5 years and 3–12 freeze–thaw cycles. Conclusion: Samples were selected to cover the heterogeneity of the polyclonal human immune response, therefore this stability data can be extended to all anti-vaccine and anti-drug antibodies.
During the course of clinical studies for vaccine or biological drug development, levels of anti-vaccine antibodies (AVAs) or antidrug antibodies (ADAs) are assessed at various time points after immunization or biological drug dosing. For better comparability of the titers, as well as for logistical reasons, it may be preferable to analyze all of the samples from the same patient or study at the same time. This requires the samples to be stored in a manner that preserves antibody reactivity until the time of testing. It is universally recognized that the use of serum samples spiked with a positive control is a poor surrogate for ADA stability [1] . Although reported in the respective individual immunogenicity method validation reports, no extensive data on the stability of human AVAs or ADAs in clinical serum samples is publicly available in the scientific literature. Since we do not have access to a significant number of drug-induced ADApositive samples for comprehensive stability studies, we took advantage of the fact that vaccine programs generate a large number of samples containing various amounts of antibodies coming from many differ-
10.4155/BIO.14.97 © 2014 Future Science Ltd
Lydia Michaut*,1, Nathalie Laurent1, Kerstin Kentsch1, Sebastian Spindeldreher1 & Fabienne Deckert-Salva1 Novartis Institutes for Biomedical Research, DMPK-Biologics, Bioanalytics, Klybeckstrasse 141, CH-4057 Basel, Switzerland *Author for correspondence: [email protected]
1
ent individuals. These samples have been used to extensively study the long-term and freeze–thaw (FT) stability of AVAs of the IgG and IgM isotypes. CAD106 is a second-generation active immunotherapeutic targeted against a small peptide fragment of amyloid-β (Aβ) [2,3] . The N-terminal hexapeptide of Aβ (Aβ1–6) is a B-cell epitope, which was coupled to an adjuvant carrier formed by multiple copies of the coat protein of bacteriophage Qβ. CAD106 was designed to ensure repetitive B-cell presentation and strong B-cell response, stimulating the generation of antiAβ-specific antibodies, while avoiding initiation of Aβ-specific T-cell response [2] . Following positive preclinical findings [2] , the first-in-human CAD106 study investigated safety, tolerability and anti-Aβ-specific antibody response following three subcutaneous injections of CAD106 or placebo in patients with mild-to-moderate Alzheimer’s disease. Findings from this study suggested that three injections of CAD106 (50 or 150 μg) have a favorable safety profile and are capable of eliciting promising antibody responses, without inducing autoimmune reactions [3] .
Bioanalysis (2014) 6(10), 1395–1407
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ISSN 1757-6180
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Research Article Michaut, Laurent, Kentsch, Spindeldreher & Deckert-Salva
Key terms Anti-vaccine antibody: Anti-vaccine antibodies induced upon vaccination with either a preventive or a therapeutic vaccine. Anti-drug antibody: Anti-drug antibodies induced by biological therapeutic molecules. Immunogenicity: The ability of a biological substance to provoke an immune response in the body of a human or animal. In the context of biopharmaceutical proteins, the term immunogenicity is, in general, used synonymously for the formation of anti-drug antibodies. Immunotherapeutics: Immunotherapeutic products can be classified under: active immunotherapy (therapeutic or preventive vaccines); passive immunotherapy (administration of antibody or receptor/ligand); and cellular immunotherapy (transfer of immune cells, of precursor cells or of gene-modified autologous or allogenic cells). Therapeutic vaccine: In contrast to preventive vaccines like those that protect against pathogens and which people receive before contracting an infection, therapeutic vaccines are administered to patients already suffering from a disease in order to trigger the patient’s immune system to specifically fight the disease. GLP: System promoting the quality and validity of test data, based on a set of principles that provides a framework within which laboratory activities are planned, performed, monitored, recorded, reported and archived. GLP helps assure regulatory authorities that the data submitted are a true reflection of the results obtained during the study and can therefore be relied upon when making risk/safety assessments. ELISA: Solid-phase assay that uses antibodies and color change triggered by the enzymatic transformation of a substrate to identify or quantify a substance in a liquid or wet sample.
In this paper, we report the stability testing of the endogenous anti-Aβ IgG and IgM antibodies induced upon administration of the CAD106 therapeutic vaccine. The stability of the AVAs was assessed in serum samples from three clinical studies submitted to various storage conditions using validated ligandbinding assays (LBAs) implemented in GLP-compliant laboratories. Materials & methods Assay format
Two sandwich ELISAs were developed to semiquantify the IgM or IgG AVAs generated in response to injection of a vaccine encompassing the first six N-terminal amino acids of a self-antigen coupled to a carrier. In both assays, serum samples were diluted 1/1000 and incubated on a microtiter plate where the whole antigen, Aβ1–40, was coated. AVAs of the IgM or IgG isotypes were detected using a horseradish peroxidase-coupled goat anti-human IgM or IgG antibody, respectively, allowing a colorimetric readout. Biacore™ (GE Healthcare Bio-Sciences AB, Sweden) analysis confirmed that the anti-human IgG detection antibody is capable of detecting all four IgG subclasses, with κ- and λ-light chains (Supplementary Figure 1) .
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To maximize sample analysis throughput, a calibrator curve format was developed such that samples could be screened and a titer assigned using a single sample dilution in one assay, rather than screening samples in one assay then running a second assay to calculate the titers of the samples. Positive controls which were used to prepare the calibrators (Cs) and quality control (QC) samples were obtained from rhesus monkeys injected with CAD106. Sera collected 8, 11 and 14 days after immunization of one monkey were pooled to obtain the IgM-enriched reference serum and sera collected 40, 50 and 60 days after immunization of a second monkey were pooled to generate the IgG-enriched reference serum. The positive controls were serially diluted to establish the calibration curves. Relative ‘titer’ units (ru) were defined as the reciprocal value of the dilution applied to the reference serum multiplied by 106. The LLOQ of the assays were 8.9 ru for IgG AVA and 66.7 ru for IgM AVA ELISA. Serum samples were analyzed in duplicate and could be further diluted if their value was above the assay ULOQ: 142.9 ru for IgG AVA and 666.7 ru for IgM AVA ELISA. A set of acceptance criteria was established for Cs and QCs on the basis of those used for pharmacokinetic (PK) LBAs [4] to ensure the inter-day reproducibility of the runs and hence the comparability of the results obtained on different runs over the years: a minimum signal intensity was defined for the highest calibrator; precision of both Cs and QCs had to be within 20% (25% at LLOQ and ULOQ); and accuracy of the back-calculated titers had to be within 20% of their nominal value (25% at LLOQ and ULOQ). Assay validation & transfer to third-party laboratories
The assays were initially developed and validated at Novartis (Basel, Switzerland) with a precision and accuracy of 20% (25% at LLOQ and ULOQ; Supplementary Table 1). During the assay validation, short-term and FT stability (three cycles) of the IgGand IgM-positive controls were demonstrated using reference sera-spiked QC samples. Both assays were then subsequently transferred to two GLP-compliant contract research organizations (CRO). In the first CRO, the assays were validated in triplicate (three wells per sample) with identical LOQs as those established at Novartis, and extensive long-term and FT stability studies were performed. For logistical reasons, the assays were later transferred to a second CRO. There, during assay validation, sample analysis in duplicate was implemented and the LOQs were slightly extended: 15–300 ru for IgG AVA ELISA and 60–1200 ru for IgM AVA ELISA (Supplementary Table 1) .
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Stability of anti-immunotherapeutic antibodies in frozen human serum samples
Acceptable precision and accuracy of the assays were demonstrated in both CROs and their comparability was further evaluated by reanalysis by the second CRO of 30 study samples that were previously analyzed in the first CRO. In a manner similar to incurred sample reanalysis (ISR) for LBAs [5] , the method was deemed to perform equivalently if twothirds of the reanalysis results were within 30% of the mean of both determinations [4] . This acceptance criterion was indeed perfectly met for 29 out of 30 of the IgG AVA and 27 out of 30 of the IgM AVA samples, demonstrating that the assays performed identically in the two laboratories.
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Results & discussion Long-term stability of IgG AVAs
Levels of specific IgG AVAs were monitored in clinical serum samples collected from 32 different subjects at various time points after vaccine administration (Figures 1 & 2) . IgG AVAs were quantified shortly (generally within 1 month) after serum collection and after 2.1–3.6 years of storage at a nominal temperature of -80°C. The samples were selected to cover the entire range of AVA titers observed during the studies and were reanalyzed at the same dilution as the original determination. We took advantage of the assay being validated in two different CROs to strengthen our results on the long-term stability (LTS) of IgG AVAs. Predefined acceptance criteria for stability A total of 34 samples were reanalyzed at the first CRO As predefined in the study plan, the reanalyzed sam- and 28 in the second CRO. This corresponds to a ples were considered to be stable if two-thirds of all total of 62 determinations from 58 samples; four samreanalysis values were within 30% of the mean of both ples were indeed analyzed in both test facilities, where the original and re-test determinations. This percent- different aliquots were stored for a different amount age difference (%DIFF) was calculated for each sample of time (Supplementary Figure 2) . The results from both laboratories (Supplementary Table 2) yielded using the following formula: almost identical statistical outputs (Figure 3) and were therefore merged for subsequent data representation. The mean percentage difference between the values initially determined and those reassessed after at least The rationale of using these acceptance criteria is 2.1 years of storage was -3% with a median of -5.6% discussed later in this article. (n = 62; range: -25.7–24.5%). All %DIFF values lay
Variability between two measurements performed >2.1 years apart
30.0
20.0
10.0
0.0
-10.0
-20.0
-30.0 Figure 1. IgG anti-vaccine antibodies long-term stability is demonstrated in all tested samples. One to four samples were collected from 32 subjects at various time points after vaccination: (A) 13 subjects gave one sample, (B) 11 subjects gave two samples, (C) five subjects gave three samples and (D) three subjects gave four samples (total = 62 samples from 32 subjects). Each vertical line corresponds to one subject and each data point represents the variability between the two IgG anti-vaccine antibody measurements performed on the same sample at least 2.1 years apart. The variability is expressed by calculating the percentage difference.
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Research Article Michaut, Laurent, Kentsch, Spindeldreher & Deckert-Salva
Numbers of subjects
15
14
10 8 6 5 2
3
2
1
4
6
5
2
2
2
1
0 2
4
6
12
After 1st injection
2
4
6
2
After 2nd injection
4
6
8
14
20
34
After 3rd injection
Sample collection time after vaccine administration (weeks) Figure 2. IgG anti-vaccine antibodies long-term stability is demonstrated in all tested subjects (n = 32) independently of the sample collection time point after vaccine administration. Collection time points of the IgG anti-vaccine antibodies samples after vaccine administration, from 2 weeks after the first vaccine injection (six subjects) to 34 weeks after the third vaccine injection (one subject).
inside the ±30% acceptance interval. No significant variability was observed between individual subjects (Figure 1) or between titer levels (Figure 4A–C) . This conclusion on LTS of AVA IgG applies to all four IgG isotypes since the detection antibody is capable of detecting all isotypes (Supplementary Figure 1) . In addition, the clinical samples were collected at various time points after vaccination, between two weeks after the first vaccine administration and 34 weeks after the third vaccine administration (Figure 2 & Supplementary Table 2) . This wide sampling schedule should cover the heterogeneity of the human humoral immune response to a protein antigen: according to the temporal model of human antibody function proposed by Collins and Jackson [6] , a representative panel of IgG isotype combinations is expected to be present in the samples studied here. LTS of IgM AVAs
Levels of specific anti-vaccine IgMs were monitored in 60 samples from 40 different subjects after 1–3.5 years of storage at a nominal temperature of -80°C. The mean percentage difference between the titers initially determined and those reassessed after longterm storage was 14.0% with a median of 14.1% (Figure 5) . A total of 15% (9/60) of the values lay outside of the ±30% acceptance interval and a clear positive bias was observed. This tendency to overrecovery, as exemplified by a trend towards positive %DIFF values, was also observed in a previous stability study where 16 samples from 12 subjects were
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reanalyzed after 1.1–1.4 years of storage at -80°C: 12.5% of the samples exhibited a %DIFF >30% (Figure 5) , indicating that the tendency to overrecovery already occurred after a shorter storage period. This observation is not due to a shift in the assay performance, since all predefined acceptance criteria for assay accuracy, precision and reproducibility described in the ‘Materials & methods’ section were met. IgG AVA assay QC performance and IgM AVA assay QC performance are described in Supplementary Figures 3 & 4 , respectively. No trend towards a positive bias was observed. The observed trend to over-recovery is more consistent with the known tendency of IgMs to aggregate. The LBA used here relies on a precise stoichiometry of the coating antigen: IgM: detection antibody for IgM quantification (see ‘Materials & methods’ section). In the case of IgM aggregation, several goat anti-human IgM detection antibodies could bind to one coated antigen, leading to an artificially elevated amount. However, the observed trend remains very limited, since the general predefined acceptance criteria of two-thirds of the reanalysis values within ±30% of the mean concentration were met for 85% of the values (Figure 5) . In addition, samples with values below the assay LOQ remained negative after long-term storage, low values remained in the low range (but still quantifiable), moderate values remained in the moderate range and high values remained in the high range (Figure 6A–C) . In addition, no significant variability linked neither to the subject, nor to the length of storage was observed (Supplementary Table 3) .
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Stability of anti-immunotherapeutic antibodies in frozen human serum samples
FT stability of IgG AVAs
Levels of specific anti-vaccine IgGs were also monitored in 16 clinical samples from 16 different subjects shortly after blood collection and after 3, 6, 9 and 12 FT cycles. The selected samples contained various IgG AVA amounts spanning the whole ‘concentration’ range observed in the clinical studies and were within their stability period (5.3–8.8 months between collection and measurement after the 12th FT cycle). Samples were allowed to thaw unassisted for approximately 1 h at room temperature and were refrozen for approximately 24 h at -80°C. The percentage difference from the mean of both determinations (original
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and reanalysis after 3, 6, 9 or 12 FT cycles) was determined (Supplementary Table 4) . Comparison of the original and reanalysis values showed perfect sample stability for up to six FT cycles (Figure 7) . After nine and 12 cycles, three and one samples, respectively, were outside the ±30% acceptable stability criteria. Therefore, the predefined stability acceptance criteria were still met up to 12 FT cycles, by 13 (81%) of the samples submitted to nine FT cycles and 14 (93%) of the samples submitted to 12 FT cycles. There was no influence of the IgG AVA amount nor of the sample origin (16 different subjects) on the FT stability of the analyte (Supplementary Figure 5) .
Variability between the two measurements (%DIFF)
30
20
10
0
-10
-20
-30 %DIFF
Lab 1
Lab 2
Labs 1 + 2
n
34
28
62
Mean
-1.7
-4.6
-3.0
Median
-0.4
-6.7
-5.6
Max
24.5
22.7
24.5
Min
-25.7
-21.5
-25.7
n outside ±30%
0
0
0
Figure 3. Influence of long-term storage on the quantification of IgG anti-vaccine antibodies. Descriptive statistics and box-plot representation of the variability (%DIFF) between the original and reassay results. Lab 1: n = 34 samples stored for 2.2–3.6 years; lab 2: n = 28 samples stored for 2.1 years; labs 1 + 2: cumulative results obtained in the two labs (n = 62 samples). The boundary of the box closest to zero indicates the 25th percentile, the line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. In addition, the mean is displayed as a dotted line. (Software: SigmaPlot 12.5; Systat Software, Germany). %DIFF: Percentage difference.
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Research Article Michaut, Laurent, Kentsch, Spindeldreher & Deckert-Salva
Original value (ru)
A
Reassay value (ru)
Amount of IgG AVAs (ru)
40 35 30 25 20 15
**
10 5 2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.7
Years of storage at -80˚C before reassay B
Amount of IgG AVAs (ru)
500 450 400 350 300 250 200 150 2.1
2.1
2.1
2.1
2.1
2.1
2.4
2.5
3.5
Years of storage at -80˚C before reassay
Amount of IgG AVAs (ru)
C 125 115 105 95 85 75 65 55 45 35
2.1 2.1 2.1 2.1 2.1
2.1 2.1 2.1 2.1 2.1 2.3
2.5 2.6 2.7 2.8 2.8
2.8 3.0 3.1 3.2 3.2
3.2 3.4
Years of storage at -80˚C before reassay Figure 4. Influence of long-term storage on the quantification of IgG anti-vaccine antibodies. (A) Low amounts (n =3 0); (B) moderate amounts (n = 23); (C) high amounts (n = 9) of IgG AVAs. The amounts of AVAs of the IgG isotype are displayed in ru as determined in each sample shortly after serum collection (original value, dark fill) and after 2.1–3.6 years storage at a nominal temperature of -80°C (reassay value, lighter fill). The asterisks (*) mark the values below LOQ (15 ru in lab 2), which were substituted to 14 ru for graphical display. AVA: Anti-vaccine antibody; ru: Relative units.
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Stability of anti-immunotherapeutic antibodies in frozen human serum samples
FT stability of IgM AVAs
Levels of specific anti-vaccine IgMs were monitored in nine samples from nine different subjects shortly after blood collection and after three and 12 cycles of FT. The testing strategy was slightly different from the one described in the previous section: the samples used in the IgM AVA FT stability assessment were indeed stored between 1.6 and 2.2 years at -80°C and at that time, the LTS assessment of IgM AVAs was ongoing. Therefore, the back-up samples (that were never submitted to any FT cycle) were first reanalyzed at the beginning of the FT stability assessment to establish the reference value. In all cases, this reference value was within 30% of the original determination, supporting the IgM
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AVA LTS results obtained later on and reported above. The samples were then aliquoted, submitted to the FT cycles, and reanalyzed after the third and the 12th cycle. After three FT cycles, eight of the nine samples (89%) returned values within 30% of the mean value and seven (78%) after 12 FT cycles (Table 1). Taken together, the mean %DIFF between the titers initially determined and those reassessed after three and 12 FT cycles were -0.6 and -2.8%, respectively, with respective median values of 0.8 and 3.8% (Table 1). There was no influence of the IgM AVA amount nor of the sample origin (nine different subjects) on the FT stability of the analyte (Supplementary Figure 6). In contrast to what was observed in the LTS stability assessment of IgM AVAs,
50
Variability between the two measurements (%DIFF)
40
30
20
10
0
-10
-20
-30 %DIFF
Storage
1.6 to 3.5 years
1 to 1.4 years
1 to 3.5 years
n
44
16
60
Mean
14.6
12.2
14.0
Median
14.8
10.3
14.1
Min
-21.7
-9.6
-21.7
Max
44.3
38.9
44.3
n outside ±30%
7 (16)%
2 (12.5%)
9 (15%)
Figure 5. Influence of long-term storage on the quantification of IgM anti-vaccine antibodies. Descriptive statistics and box-plot representation of the variability (%DIFF) between the original and reassay results. (A) Results obtained for 44 samples stored 1.6–3.5 years; (B) results obtained for 16 samples stored 1–1.4 years; (C) cumulative box plot (n = 60 samples). The boundary of the box closest to zero indicates the 25th percentile, the line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. In addition, the mean is displayed as a dotted line. (Software: SigmaPlot 12.5; Systat Software, Germany).
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Research Article Michaut, Laurent, Kentsch, Spindeldreher & Deckert-Salva
A
Original value (ru)
Reassay value *
Amount of IgM AVAs (ru)
260 210
*
160
*
*
* 110 # 60
#
#
#
1.3 1.3 1.3 1.3 1.4 1.4 1.4 1.4 1.6 1.8 1.9 2.5 2.5 2.5 2.6 2.6 2.7 2.7 2.8 2.8 2.8 2.8 2.8 2.9 2.9 2.9 3.2 3.2 3.3 3.5 3.5 3.5 3.5 Years of storage at -80˚C before reassay
Amount of IgM AVAs (ru)
B *
850 650
*
450 250
1.4
1.4
1.4
1.4
1.6
2.0
2.0
2.5
2.6
2.6
2.6
2.6
2.7
2.7
2.8
2.8
2.8
3.3
Years of storage at -80˚C before reassay C
Amount of IgM AVAs (ru)
4700 3700 2700 1700 700
1.0
1.1
1.1
1.1
2.2
2.2
2.4
*
*
2.6
2.6
Years of storage at -80˚C before reassay Figure 6. Influence of long-term storage on the quantification of IgM anti-vaccine antibodies. (A) Low amounts (n = 33); (B) moderate amounts (n = 18); (C) high amounts (n = 9) of IgM AVAs. The amounts of AVAs of the IgM isotype are displayed in ru as determined in each sample shortly after serum collection (original value, dark fill) and after 2.1–3.5 years storage at a nominal temperature of -80°C (reassay value, lighter fill). The asterisks (*) mark the values with variability >30%. The hashes (#) mark the values below LOQ (66.7 ru), which were substituted to 66 ru for graphical display. AVA: Anti-vaccine antibody; ru: Relative units.
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Stability of anti-immunotherapeutic antibodies in frozen human serum samples
no bias towards over-recovery (i.e., positive %DIFF values) was observed. IgM AVA seemed to be slightly more sensitive than IgG AVAs to more than three FT cycles, but the predefined stability acceptance criteria were still met for at least 78% of the IgM AVA samples after 12 FT cycles.
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ISR. To assess assay reproducibility, ISR must indeed be performed during the analyte stability period. Consequently, when the reproducibility of the method was already established (as it is the case here), the success of an ISR demonstrates that the samples were analyzed within their stability period. Here, the samples were considered to be stable at the tested condition if two-thirds of the reanalysis results were within 30% of the mean of both determinations. This approach is recommended by the EMA Guideline on Bioanalytical Method Validation [4] and by the report and follow-up of the American Association of Pharmaceutical Scientists workshop on assay reproducibility
Alternative & stricter acceptance criteria
The outcome of a stability study is based on a set of predefined acceptance criteria that have to be met. Since a large number of clinical samples and a calibration curve-based assay were used in this study, we decided to apply the same acceptance criteria as used for PK 60
Variability between the two measurements (%DIFF)
40 20 0 -20 -40 -60 -80 -100 -120 -140
%DIFF
3 FT cycles
6 FT cycles
9 FT cycles
12 FT cycles 15
n
16
16
16
Mean
2.9
-0.4
-15.3
13.0 11.2
Median
5.5
2.2
-9.35
Min
-18.8
-21.5
-116.3
-1.9
Max
15.2
18.2
32.7
39
n outside ±30%
0
0
3 (18.8%)
1 (6.7%)
Figure 7. Influence of freeze–thaw cycles on the quantification of IgG anti-vaccine antibodies (n = 16 samples). Summary statistics and box-plot representation of the variability (expressed as %DIFF) between the original and reassay results after three, six, nine and 12 FT cycles. The boundary of the box closest to zero indicates the 25th percentile, the line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. In addition, the mean is displayed as a dotted line. (Software: SigmaPlot 12.5; Systat Software, Germany). %DIFF: Percentage difference; FT: Freeze–thaw.
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Table 1. Influence of freeze–thaw cycles on the quantification of IgM anti-vaccine antibodies: results, normalized difference and bias calculation. Subject Value (ru) before FT
After 3 FT cycles
After 12 FT cycles
Value (ru) after %DIFF %Bias towards 3 FT cycles towards mean original value‡ value†
Value (ru) after %DIFF towards Bias (%) 12 FT cycles mean value† towards original value‡
R
87.7
112
24.3
27.7
106
18.9
20.9
S
92.9
99.9
7.3
7.5
111
17.8
19.5
T
120
121
0.8
0.8
168
33.3
40.0
U
309
326
5.4
5.5
321
3.8
3.9
V
417
332
-22.7
-20.4
386
-7.7
-7.4
W
479
481
0.4
0.4
419
-13.4
-12.5
X
1550
1850
17.6
19.4
1440
-7.4
-7.1
Y
2320
2210
-4.9
-4.7
2470
6.3
6.5
Z
4450
3160
-33.9
-29.0
1980
-76.8
-55.5
n
9
9
9
9
Mean (%)
-0.6
0.8
-2.8
0.9
Median (%)
0.8
0.8
3.8
3.9
Min (%)
-33.9
-29.0
-76.8
-55.5
Max (%)
24.3
27.7
33.3
40.0
n outside ±30%
1 (11%)
0
2 (22%)
2 (22%)
%DIFF = 100 × (reanalysis value – original value)/(mean of reanalysis and original values), as promoted by the EMA Guideline on Bioanalytical Method validation [4]. Bias = 100 × (reanalysis value – original value)/original value, as promoted by Timmerman et al. [7]. %DIFF: Percentage difference; FT: Freeze–thaw; ru: Relative units. †
‡
for incurred samples [5] and is implemented in Novartis Standard Operation Procedures for LBAs. Alternatively, the variability of the measurements could be compared with the first determination, as promoted by the European Bioanalysis Forum in 2009 [7] . This was calculated for all analytes in Table 2, and AVA stability was demonstrated in all the tested conditions: less than a third of the samples were outside the ±30% bias acceptance range. However, when stability of QC samples is assessed during assay validation, the acceptance criteria
are based on the mean precision and accuracy at each tested level, which must be within the acceptance range determined during assay validation (here ±20%) [4] . Owing to the small number of clinical samples tested, this stricter approach appears to be more appropriate also for the FT stability assessment (Tables 3 & 4). Mean biases remained within 20% for moderate and high AVA levels up to 12 FT cycles. Although mean biases of 21.2 and 26.8% were observed for low levels of IgG and IgM AVAs, respectively, these values were only slightly out of
Table 2. Bias of the IgG and IgM anti-vaccine antibody stability assessments. Bias from original value
LTS
3 FT cycles
IgG 12 FT cycles
LTS
3 FT cycles
IgM 12 FT cycles
n
62
16
15
60
9
9
Mean (%)
-2.3
3.4
14.6
16.4
5.9
8.2
Median (%)
-5.4
5.6
11.8
15.1
5.5
3.9
Min (%)
27.9
-17.2
-1.8
-19.5
-29.0
-55.5
Max (%)
-22.8
16.4
48.5
56.9
27.7
40.0
n outside ±30%
0
0
1 (6.7%)
14 (23%)
0
2 (22%)
The variability between the two measurements was determined as promoted by the EMA Guideline on Bioanalytical Method validation [4] (i.e., by calculating the bias from the original value: 100 × [reanalysis value – original value]/original value). FT: Freeze–thaw; LTS: Long-term stability.
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Table 3. Alternative acceptance criteria for IgG anti-vaccine antibodies freeze–thaw stability assessment. Bias level
3 FT cycles
6 FT cycles
9 FT cycles
12 FT cycles
Low
Moderate
High
Low
Moderate
High
Low
Moderate
High
Low
Moderate
High
n
6
6
4
6
6
4
6
6
4
6
6
3†
Mean (%)
3.2
-0.1
9.2
0.8
-4.3
6.3
-17.0
-11.5
-1.9
21.2
9.8
11.1
Median (%)
5.6
-3.0
9.1
4.8
-7.2
5.4
-12.2
-9.3
-1.3
16.8
11.3
11.8
Min (%)
-11.2
-17.2
5.0
-19.4
-16.3
0.0
-56.0
-73.6
-8.7
4.1
-1.8
3.3
Max (%) 16.4
14.7
13.8
20.0
16.4
14.5
3.5
39.0
3.4
48.5
18.3
18.1
The variability between the two measurements was determined at each level of IgG anti-vaccine antibodies by calculating the bias from the original value (considered as nominal value) = 100 × (reanalysis value – original value)/original value. † Exclusion of one technical outlier after 12 FT cycles. ‡ Value does not meet the acceptance criterion on the mean bias (n = 3). FT: Freeze–thaw.
the acceptability range. Using this stricter acceptance criterion, the AVA stability was therefore demonstrated at each level (low, moderate and high) up to nine and three FT cycles for IgG and IgM AVAs, respectively. Conclusion Here, we used a high number of clinical samples collected from over 100 different subjects to provide solid evidence for the long-term and FT stability of AVAs. The format of the assays used in this GLP-compliant study allowed demonstrating both the AVA ability to bind to their target and their ability to be recognized by immunoglobulins raised against their Fc portion. We showed that AVAs of the IgG isotype are stable in undiluted human serum at least 3.6 years after collection when stored at a nominal temperature of -80°C in a temperature-monitored freezer. Similarly, our data showed that AVAs of the IgM isotype are stable in undiluted human serum at least 3.5 years after collection. A slight over-recovery of IgM after long-term storage was observed but was very limited and had neither qualitative nor quantitative impact on the ability of the assay to identify IgM AVA-positive
samples. The same applies to our FT stability assessment, where results obtained after up to three cycles adequately reflected those obtained shortly after sampling of IgG and IgM AVAs. These conclusions hold true for any of the stringency of the various acceptance criteria mentioned in the guidelines and white papers published to date. Similar conclusions were obtained by Hendriks et al. (this issue of Bioanalysis) [8] , who also demonstrated AVA stability upon long-term storage and FT cycling for a completely different preventive vaccine project. This GLP-compliant study therefore provides a strong experimental demonstration to the USP Guidance statement [9] , that it is generally accepted, that immunoglobulins stored in 100% serum at -80°C are stable and therefore stability assessment is not required. Future perspective The antibody response is the response that confers protection in most, if not all vaccines, making the AVA response wanted and beneficial. By contrast, the ADA response is unwanted because it can negatively impact the PKs, pharmacodynamics and efficacy of the drug,
Table 4. Alternative acceptance criteria for IgM anti-vaccine antibodies freeze–thaw stability assessment. Bias level
3 FT cycles
12 FT cycles
Low
Moderate
High
Low
Moderate
High
n
3
3
3
3
3
3
Mean (%)
12.0
-4.8
-4.8
26.8
-5.4
-18.7
Median (%)
7.5
0.4
-4.7
20.9
-7.4
-7.1
Min (%)
0.8
-20.4
-29.0
19.5
-12.5
-55.5
Max (%)
27.7
5.5
19.4
40
3.9
6.5
†
The variability between the two measurements was determined at each level of IgM anti-vaccine antibodies by calculating the bias from the original value (considered as nominal value) = 100 × (reanalysis value – original value)/original value. † Value does not meet the acceptance criterion on the mean bias. FT: Freeze–thaw.
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Research Article Michaut, Laurent, Kentsch, Spindeldreher & Deckert-Salva and possibly induce adverse events. In spite of these differences in the frequency and magnitude of the immune response, AVA and ADA responses are identical in nature. It has been proposed that the stability of ADAs is the same independently of the biological drug they are directed to [10] . If this is the case, it is possible to extend the conclusion of our study to all AVAs and ADAs, making it unnecessary to test LTS for each new biological therapeutics or vaccine program. This proposal is supported by the member companies of the European Bioanalysis Forum (this issue of Bioanalysis) [11] . Supplementary data To view the supplementary data that accompany this paper please visit the journal website at: www.future- science. com/doi/suppl/10.4155/BIO.14.97
Financial & competing interests disclosure The authors were Novartis employees at the time of the study and have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary Experimental, assay format • Validated ligand-binding assays (ELISA) implemented in GLP-compliant laboratories. • The stability assessments were conducted using a large number of samples coming from different subjects and collected at various time points after immunotherapeutic administration, reflecting the large individual variability of the humoral immune response. • Long-term stability of IgG anti-vaccine antibodies (AVAs) was tested on 62 occasions in samples from 32 subjects and was demonstrated for at least 3.6 years at -80°C. • Long-term stability of IgM AVAs was tested on 60 samples from 40 subjects and was demonstrated for at least 3.5 years at -80°C. • Freeze–thaw (FT) stability of IgG AVAs was tested on samples from 16 subjects and was demonstrated for at least nine FT cycles. • FT stability of IgM AVAs was tested on samples from nine subjects and was demonstrated for at least three FT cycles.
Alternative & stricter acceptance criteria • Long-term and freeze–thaw stability conclusions remained valid independently of the stringency of the acceptance criteria.
Conclusion • These data demonstrate the stability of anti-immunotherapeutic antibodies in frozen human samples stored at -80°C.
Future perspective • The nature of the humoral immune response allows extending the conclusion of this study to all AVAs and ADAs, making unnecessary to test long-term stability of frozen serum samples for each new biological therapeutics or vaccine program, consistently with the recommendation of the European Bioanalytical Forum. immunotherapy with CAD106 in patients with Alzheimer’s disease: randomised, double-blind, placebo-controlled, firstin-human study. Lancet Neurol. 11(7), 597–604 (2012).
References Papers of special note have been highlighted as: • of interest; •• of considerable interest 1
Gorovitz B. Antidrug antibody assay validation: industry survey results. Meeting report. AAPS J. 11(1) 133–138 (2009).
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Active immunotherapy targeting the amyloid-β (Aβ) peptide designed to induce N-terminal Aβ-specific antibodies without an Aβ-specific T-cell response.
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Wiessner C, Wiederhold KH, Tissot AC et al. The second-generation active Abeta immunotherapy CAD106 reduces amyloid accumulation in APP transgenic mice while minimizing potential side effects. J. Neurosci. 31(25), 9323–9331 (2011).
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EMA. Committee for Medical Products for Human Use. Guideline on Bioanalytical Method Validation. EMA, London, UK (2011).
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Fast DM, Kelley M, Viswanathan CT et al. Workshop report and follow-up – AAPS workshop on current topics
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and validation of immunoassays to detect anti-drug antibodies. First supplement to USP 36- NF 31 (2012). www.usp.org/usp-nf/pharmacopeial-forum
in GLP bioanalysis: assay reproducibility for incurred samples – Implications of Crystal City recommendations. AAPS J. 11(2), 238–241 (2009). 10
Collins AM, Jackson KJL. A temporal model of human IgE and IgG antibody function. Front. Immunol. 4, 235 (2013).
Shankar G, Devanarayan V, Amaravadi L et al. Recommendation for the validation of immunoassays used for detection of host antibodies against biotechnology products. J. Pharm. Biomed. Anal. 48(5), 1267–1281 (2008).
••
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Timmerman P, Luedtke S, van Amsterdam P et al. Incurred sample reproducibility: views and recommendations by the European Bioanalysis Forum. Bioanalysis 1(6), 1049–1056 (2009).
Provides scientific recommendations for the validation of anti-drug antibody immunoassays to foster a more unified approach to antibody testing across the biopharmaceutical industry.
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Hendriks J, Stals C, Versteilen A et al. Stability studies of binding and functional anti-vaccine antibodies. Bioanalysis 6(10), 1385–1393 (2014).
Pihl S, Michaut L, Hendriks J et al. EBF recommendation for stability testing of anti-drug antibodies; lessons learned from anti-vaccine antibody stability studies. Bioanalysis 6(10), 1409–1413 (2014).
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USP Immunogenicity Testing Expert Panel. US Pharmacopeial Guidance on Immunogenicity assays – design
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Strategies for conducting incurred sample reanalysis are addressed to assist the bioanalytical scientist in establishing a robust incurred sample reanalysis program.
6
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For reprint orders, please contact [email protected]
Stability of anti-immunotherapeutic antibodies in frozen human serum samples
Background: To generate exhaustive data on the stability of human antiimmunotherapeutic antibodies. Materials & methods: Samples collected from over 100 different subjects at various timepoints were analyzed shortly after serum collection using specific ELISAs and re-analyzed after long-term storage or multiple cycles of freeze–thaw. The general acceptance criteria for incurred sample reanalysis for ligand-binding assays were applied, as well as alternative stricter acceptance criteria promoted by various white papers. Results: Anti-immunotherapeutic antibodies are stable in undiluted serum samples stored at -80°C for at least 3.5 years and 3–12 freeze–thaw cycles. Conclusion: Samples were selected to cover the heterogeneity of the polyclonal human immune response, therefore this stability data can be extended to all anti-vaccine and anti-drug antibodies.
During the course of clinical studies for vaccine or biological drug development, levels of anti-vaccine antibodies (AVAs) or antidrug antibodies (ADAs) are assessed at various time points after immunization or biological drug dosing. For better comparability of the titers, as well as for logistical reasons, it may be preferable to analyze all of the samples from the same patient or study at the same time. This requires the samples to be stored in a manner that preserves antibody reactivity until the time of testing. It is universally recognized that the use of serum samples spiked with a positive control is a poor surrogate for ADA stability [1] . Although reported in the respective individual immunogenicity method validation reports, no extensive data on the stability of human AVAs or ADAs in clinical serum samples is publicly available in the scientific literature. Since we do not have access to a significant number of drug-induced ADApositive samples for comprehensive stability studies, we took advantage of the fact that vaccine programs generate a large number of samples containing various amounts of antibodies coming from many differ-
10.4155/BIO.14.97 © 2014 Future Science Ltd
Lydia Michaut*,1, Nathalie Laurent1, Kerstin Kentsch1, Sebastian Spindeldreher1 & Fabienne Deckert-Salva1 Novartis Institutes for Biomedical Research, DMPK-Biologics, Bioanalytics, Klybeckstrasse 141, CH-4057 Basel, Switzerland *Author for correspondence: [email protected]
1
ent individuals. These samples have been used to extensively study the long-term and freeze–thaw (FT) stability of AVAs of the IgG and IgM isotypes. CAD106 is a second-generation active immunotherapeutic targeted against a small peptide fragment of amyloid-β (Aβ) [2,3] . The N-terminal hexapeptide of Aβ (Aβ1–6) is a B-cell epitope, which was coupled to an adjuvant carrier formed by multiple copies of the coat protein of bacteriophage Qβ. CAD106 was designed to ensure repetitive B-cell presentation and strong B-cell response, stimulating the generation of antiAβ-specific antibodies, while avoiding initiation of Aβ-specific T-cell response [2] . Following positive preclinical findings [2] , the first-in-human CAD106 study investigated safety, tolerability and anti-Aβ-specific antibody response following three subcutaneous injections of CAD106 or placebo in patients with mild-to-moderate Alzheimer’s disease. Findings from this study suggested that three injections of CAD106 (50 or 150 μg) have a favorable safety profile and are capable of eliciting promising antibody responses, without inducing autoimmune reactions [3] .
Bioanalysis (2014) 6(10), 1395–1407
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Research Article Michaut, Laurent, Kentsch, Spindeldreher & Deckert-Salva
Key terms Anti-vaccine antibody: Anti-vaccine antibodies induced upon vaccination with either a preventive or a therapeutic vaccine. Anti-drug antibody: Anti-drug antibodies induced by biological therapeutic molecules. Immunogenicity: The ability of a biological substance to provoke an immune response in the body of a human or animal. In the context of biopharmaceutical proteins, the term immunogenicity is, in general, used synonymously for the formation of anti-drug antibodies. Immunotherapeutics: Immunotherapeutic products can be classified under: active immunotherapy (therapeutic or preventive vaccines); passive immunotherapy (administration of antibody or receptor/ligand); and cellular immunotherapy (transfer of immune cells, of precursor cells or of gene-modified autologous or allogenic cells). Therapeutic vaccine: In contrast to preventive vaccines like those that protect against pathogens and which people receive before contracting an infection, therapeutic vaccines are administered to patients already suffering from a disease in order to trigger the patient’s immune system to specifically fight the disease. GLP: System promoting the quality and validity of test data, based on a set of principles that provides a framework within which laboratory activities are planned, performed, monitored, recorded, reported and archived. GLP helps assure regulatory authorities that the data submitted are a true reflection of the results obtained during the study and can therefore be relied upon when making risk/safety assessments. ELISA: Solid-phase assay that uses antibodies and color change triggered by the enzymatic transformation of a substrate to identify or quantify a substance in a liquid or wet sample.
In this paper, we report the stability testing of the endogenous anti-Aβ IgG and IgM antibodies induced upon administration of the CAD106 therapeutic vaccine. The stability of the AVAs was assessed in serum samples from three clinical studies submitted to various storage conditions using validated ligandbinding assays (LBAs) implemented in GLP-compliant laboratories. Materials & methods Assay format
Two sandwich ELISAs were developed to semiquantify the IgM or IgG AVAs generated in response to injection of a vaccine encompassing the first six N-terminal amino acids of a self-antigen coupled to a carrier. In both assays, serum samples were diluted 1/1000 and incubated on a microtiter plate where the whole antigen, Aβ1–40, was coated. AVAs of the IgM or IgG isotypes were detected using a horseradish peroxidase-coupled goat anti-human IgM or IgG antibody, respectively, allowing a colorimetric readout. Biacore™ (GE Healthcare Bio-Sciences AB, Sweden) analysis confirmed that the anti-human IgG detection antibody is capable of detecting all four IgG subclasses, with κ- and λ-light chains (Supplementary Figure 1) .
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To maximize sample analysis throughput, a calibrator curve format was developed such that samples could be screened and a titer assigned using a single sample dilution in one assay, rather than screening samples in one assay then running a second assay to calculate the titers of the samples. Positive controls which were used to prepare the calibrators (Cs) and quality control (QC) samples were obtained from rhesus monkeys injected with CAD106. Sera collected 8, 11 and 14 days after immunization of one monkey were pooled to obtain the IgM-enriched reference serum and sera collected 40, 50 and 60 days after immunization of a second monkey were pooled to generate the IgG-enriched reference serum. The positive controls were serially diluted to establish the calibration curves. Relative ‘titer’ units (ru) were defined as the reciprocal value of the dilution applied to the reference serum multiplied by 106. The LLOQ of the assays were 8.9 ru for IgG AVA and 66.7 ru for IgM AVA ELISA. Serum samples were analyzed in duplicate and could be further diluted if their value was above the assay ULOQ: 142.9 ru for IgG AVA and 666.7 ru for IgM AVA ELISA. A set of acceptance criteria was established for Cs and QCs on the basis of those used for pharmacokinetic (PK) LBAs [4] to ensure the inter-day reproducibility of the runs and hence the comparability of the results obtained on different runs over the years: a minimum signal intensity was defined for the highest calibrator; precision of both Cs and QCs had to be within 20% (25% at LLOQ and ULOQ); and accuracy of the back-calculated titers had to be within 20% of their nominal value (25% at LLOQ and ULOQ). Assay validation & transfer to third-party laboratories
The assays were initially developed and validated at Novartis (Basel, Switzerland) with a precision and accuracy of 20% (25% at LLOQ and ULOQ; Supplementary Table 1). During the assay validation, short-term and FT stability (three cycles) of the IgGand IgM-positive controls were demonstrated using reference sera-spiked QC samples. Both assays were then subsequently transferred to two GLP-compliant contract research organizations (CRO). In the first CRO, the assays were validated in triplicate (three wells per sample) with identical LOQs as those established at Novartis, and extensive long-term and FT stability studies were performed. For logistical reasons, the assays were later transferred to a second CRO. There, during assay validation, sample analysis in duplicate was implemented and the LOQs were slightly extended: 15–300 ru for IgG AVA ELISA and 60–1200 ru for IgM AVA ELISA (Supplementary Table 1) .
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Stability of anti-immunotherapeutic antibodies in frozen human serum samples
Acceptable precision and accuracy of the assays were demonstrated in both CROs and their comparability was further evaluated by reanalysis by the second CRO of 30 study samples that were previously analyzed in the first CRO. In a manner similar to incurred sample reanalysis (ISR) for LBAs [5] , the method was deemed to perform equivalently if twothirds of the reanalysis results were within 30% of the mean of both determinations [4] . This acceptance criterion was indeed perfectly met for 29 out of 30 of the IgG AVA and 27 out of 30 of the IgM AVA samples, demonstrating that the assays performed identically in the two laboratories.
Research Article
Results & discussion Long-term stability of IgG AVAs
Levels of specific IgG AVAs were monitored in clinical serum samples collected from 32 different subjects at various time points after vaccine administration (Figures 1 & 2) . IgG AVAs were quantified shortly (generally within 1 month) after serum collection and after 2.1–3.6 years of storage at a nominal temperature of -80°C. The samples were selected to cover the entire range of AVA titers observed during the studies and were reanalyzed at the same dilution as the original determination. We took advantage of the assay being validated in two different CROs to strengthen our results on the long-term stability (LTS) of IgG AVAs. Predefined acceptance criteria for stability A total of 34 samples were reanalyzed at the first CRO As predefined in the study plan, the reanalyzed sam- and 28 in the second CRO. This corresponds to a ples were considered to be stable if two-thirds of all total of 62 determinations from 58 samples; four samreanalysis values were within 30% of the mean of both ples were indeed analyzed in both test facilities, where the original and re-test determinations. This percent- different aliquots were stored for a different amount age difference (%DIFF) was calculated for each sample of time (Supplementary Figure 2) . The results from both laboratories (Supplementary Table 2) yielded using the following formula: almost identical statistical outputs (Figure 3) and were therefore merged for subsequent data representation. The mean percentage difference between the values initially determined and those reassessed after at least The rationale of using these acceptance criteria is 2.1 years of storage was -3% with a median of -5.6% discussed later in this article. (n = 62; range: -25.7–24.5%). All %DIFF values lay
Variability between two measurements performed >2.1 years apart
30.0
20.0
10.0
0.0
-10.0
-20.0
-30.0 Figure 1. IgG anti-vaccine antibodies long-term stability is demonstrated in all tested samples. One to four samples were collected from 32 subjects at various time points after vaccination: (A) 13 subjects gave one sample, (B) 11 subjects gave two samples, (C) five subjects gave three samples and (D) three subjects gave four samples (total = 62 samples from 32 subjects). Each vertical line corresponds to one subject and each data point represents the variability between the two IgG anti-vaccine antibody measurements performed on the same sample at least 2.1 years apart. The variability is expressed by calculating the percentage difference.
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Research Article Michaut, Laurent, Kentsch, Spindeldreher & Deckert-Salva
Numbers of subjects
15
14
10 8 6 5 2
3
2
1
4
6
5
2
2
2
1
0 2
4
6
12
After 1st injection
2
4
6
2
After 2nd injection
4
6
8
14
20
34
After 3rd injection
Sample collection time after vaccine administration (weeks) Figure 2. IgG anti-vaccine antibodies long-term stability is demonstrated in all tested subjects (n = 32) independently of the sample collection time point after vaccine administration. Collection time points of the IgG anti-vaccine antibodies samples after vaccine administration, from 2 weeks after the first vaccine injection (six subjects) to 34 weeks after the third vaccine injection (one subject).
inside the ±30% acceptance interval. No significant variability was observed between individual subjects (Figure 1) or between titer levels (Figure 4A–C) . This conclusion on LTS of AVA IgG applies to all four IgG isotypes since the detection antibody is capable of detecting all isotypes (Supplementary Figure 1) . In addition, the clinical samples were collected at various time points after vaccination, between two weeks after the first vaccine administration and 34 weeks after the third vaccine administration (Figure 2 & Supplementary Table 2) . This wide sampling schedule should cover the heterogeneity of the human humoral immune response to a protein antigen: according to the temporal model of human antibody function proposed by Collins and Jackson [6] , a representative panel of IgG isotype combinations is expected to be present in the samples studied here. LTS of IgM AVAs
Levels of specific anti-vaccine IgMs were monitored in 60 samples from 40 different subjects after 1–3.5 years of storage at a nominal temperature of -80°C. The mean percentage difference between the titers initially determined and those reassessed after longterm storage was 14.0% with a median of 14.1% (Figure 5) . A total of 15% (9/60) of the values lay outside of the ±30% acceptance interval and a clear positive bias was observed. This tendency to overrecovery, as exemplified by a trend towards positive %DIFF values, was also observed in a previous stability study where 16 samples from 12 subjects were
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reanalyzed after 1.1–1.4 years of storage at -80°C: 12.5% of the samples exhibited a %DIFF >30% (Figure 5) , indicating that the tendency to overrecovery already occurred after a shorter storage period. This observation is not due to a shift in the assay performance, since all predefined acceptance criteria for assay accuracy, precision and reproducibility described in the ‘Materials & methods’ section were met. IgG AVA assay QC performance and IgM AVA assay QC performance are described in Supplementary Figures 3 & 4 , respectively. No trend towards a positive bias was observed. The observed trend to over-recovery is more consistent with the known tendency of IgMs to aggregate. The LBA used here relies on a precise stoichiometry of the coating antigen: IgM: detection antibody for IgM quantification (see ‘Materials & methods’ section). In the case of IgM aggregation, several goat anti-human IgM detection antibodies could bind to one coated antigen, leading to an artificially elevated amount. However, the observed trend remains very limited, since the general predefined acceptance criteria of two-thirds of the reanalysis values within ±30% of the mean concentration were met for 85% of the values (Figure 5) . In addition, samples with values below the assay LOQ remained negative after long-term storage, low values remained in the low range (but still quantifiable), moderate values remained in the moderate range and high values remained in the high range (Figure 6A–C) . In addition, no significant variability linked neither to the subject, nor to the length of storage was observed (Supplementary Table 3) .
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Stability of anti-immunotherapeutic antibodies in frozen human serum samples
FT stability of IgG AVAs
Levels of specific anti-vaccine IgGs were also monitored in 16 clinical samples from 16 different subjects shortly after blood collection and after 3, 6, 9 and 12 FT cycles. The selected samples contained various IgG AVA amounts spanning the whole ‘concentration’ range observed in the clinical studies and were within their stability period (5.3–8.8 months between collection and measurement after the 12th FT cycle). Samples were allowed to thaw unassisted for approximately 1 h at room temperature and were refrozen for approximately 24 h at -80°C. The percentage difference from the mean of both determinations (original
Research Article
and reanalysis after 3, 6, 9 or 12 FT cycles) was determined (Supplementary Table 4) . Comparison of the original and reanalysis values showed perfect sample stability for up to six FT cycles (Figure 7) . After nine and 12 cycles, three and one samples, respectively, were outside the ±30% acceptable stability criteria. Therefore, the predefined stability acceptance criteria were still met up to 12 FT cycles, by 13 (81%) of the samples submitted to nine FT cycles and 14 (93%) of the samples submitted to 12 FT cycles. There was no influence of the IgG AVA amount nor of the sample origin (16 different subjects) on the FT stability of the analyte (Supplementary Figure 5) .
Variability between the two measurements (%DIFF)
30
20
10
0
-10
-20
-30 %DIFF
Lab 1
Lab 2
Labs 1 + 2
n
34
28
62
Mean
-1.7
-4.6
-3.0
Median
-0.4
-6.7
-5.6
Max
24.5
22.7
24.5
Min
-25.7
-21.5
-25.7
n outside ±30%
0
0
0
Figure 3. Influence of long-term storage on the quantification of IgG anti-vaccine antibodies. Descriptive statistics and box-plot representation of the variability (%DIFF) between the original and reassay results. Lab 1: n = 34 samples stored for 2.2–3.6 years; lab 2: n = 28 samples stored for 2.1 years; labs 1 + 2: cumulative results obtained in the two labs (n = 62 samples). The boundary of the box closest to zero indicates the 25th percentile, the line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. In addition, the mean is displayed as a dotted line. (Software: SigmaPlot 12.5; Systat Software, Germany). %DIFF: Percentage difference.
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Original value (ru)
A
Reassay value (ru)
Amount of IgG AVAs (ru)
40 35 30 25 20 15
**
10 5 2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.7
Years of storage at -80˚C before reassay B
Amount of IgG AVAs (ru)
500 450 400 350 300 250 200 150 2.1
2.1
2.1
2.1
2.1
2.1
2.4
2.5
3.5
Years of storage at -80˚C before reassay
Amount of IgG AVAs (ru)
C 125 115 105 95 85 75 65 55 45 35
2.1 2.1 2.1 2.1 2.1
2.1 2.1 2.1 2.1 2.1 2.3
2.5 2.6 2.7 2.8 2.8
2.8 3.0 3.1 3.2 3.2
3.2 3.4
Years of storage at -80˚C before reassay Figure 4. Influence of long-term storage on the quantification of IgG anti-vaccine antibodies. (A) Low amounts (n =3 0); (B) moderate amounts (n = 23); (C) high amounts (n = 9) of IgG AVAs. The amounts of AVAs of the IgG isotype are displayed in ru as determined in each sample shortly after serum collection (original value, dark fill) and after 2.1–3.6 years storage at a nominal temperature of -80°C (reassay value, lighter fill). The asterisks (*) mark the values below LOQ (15 ru in lab 2), which were substituted to 14 ru for graphical display. AVA: Anti-vaccine antibody; ru: Relative units.
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FT stability of IgM AVAs
Levels of specific anti-vaccine IgMs were monitored in nine samples from nine different subjects shortly after blood collection and after three and 12 cycles of FT. The testing strategy was slightly different from the one described in the previous section: the samples used in the IgM AVA FT stability assessment were indeed stored between 1.6 and 2.2 years at -80°C and at that time, the LTS assessment of IgM AVAs was ongoing. Therefore, the back-up samples (that were never submitted to any FT cycle) were first reanalyzed at the beginning of the FT stability assessment to establish the reference value. In all cases, this reference value was within 30% of the original determination, supporting the IgM
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AVA LTS results obtained later on and reported above. The samples were then aliquoted, submitted to the FT cycles, and reanalyzed after the third and the 12th cycle. After three FT cycles, eight of the nine samples (89%) returned values within 30% of the mean value and seven (78%) after 12 FT cycles (Table 1). Taken together, the mean %DIFF between the titers initially determined and those reassessed after three and 12 FT cycles were -0.6 and -2.8%, respectively, with respective median values of 0.8 and 3.8% (Table 1). There was no influence of the IgM AVA amount nor of the sample origin (nine different subjects) on the FT stability of the analyte (Supplementary Figure 6). In contrast to what was observed in the LTS stability assessment of IgM AVAs,
50
Variability between the two measurements (%DIFF)
40
30
20
10
0
-10
-20
-30 %DIFF
Storage
1.6 to 3.5 years
1 to 1.4 years
1 to 3.5 years
n
44
16
60
Mean
14.6
12.2
14.0
Median
14.8
10.3
14.1
Min
-21.7
-9.6
-21.7
Max
44.3
38.9
44.3
n outside ±30%
7 (16)%
2 (12.5%)
9 (15%)
Figure 5. Influence of long-term storage on the quantification of IgM anti-vaccine antibodies. Descriptive statistics and box-plot representation of the variability (%DIFF) between the original and reassay results. (A) Results obtained for 44 samples stored 1.6–3.5 years; (B) results obtained for 16 samples stored 1–1.4 years; (C) cumulative box plot (n = 60 samples). The boundary of the box closest to zero indicates the 25th percentile, the line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. In addition, the mean is displayed as a dotted line. (Software: SigmaPlot 12.5; Systat Software, Germany).
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A
Original value (ru)
Reassay value *
Amount of IgM AVAs (ru)
260 210
*
160
*
*
* 110 # 60
#
#
#
1.3 1.3 1.3 1.3 1.4 1.4 1.4 1.4 1.6 1.8 1.9 2.5 2.5 2.5 2.6 2.6 2.7 2.7 2.8 2.8 2.8 2.8 2.8 2.9 2.9 2.9 3.2 3.2 3.3 3.5 3.5 3.5 3.5 Years of storage at -80˚C before reassay
Amount of IgM AVAs (ru)
B *
850 650
*
450 250
1.4
1.4
1.4
1.4
1.6
2.0
2.0
2.5
2.6
2.6
2.6
2.6
2.7
2.7
2.8
2.8
2.8
3.3
Years of storage at -80˚C before reassay C
Amount of IgM AVAs (ru)
4700 3700 2700 1700 700
1.0
1.1
1.1
1.1
2.2
2.2
2.4
*
*
2.6
2.6
Years of storage at -80˚C before reassay Figure 6. Influence of long-term storage on the quantification of IgM anti-vaccine antibodies. (A) Low amounts (n = 33); (B) moderate amounts (n = 18); (C) high amounts (n = 9) of IgM AVAs. The amounts of AVAs of the IgM isotype are displayed in ru as determined in each sample shortly after serum collection (original value, dark fill) and after 2.1–3.5 years storage at a nominal temperature of -80°C (reassay value, lighter fill). The asterisks (*) mark the values with variability >30%. The hashes (#) mark the values below LOQ (66.7 ru), which were substituted to 66 ru for graphical display. AVA: Anti-vaccine antibody; ru: Relative units.
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no bias towards over-recovery (i.e., positive %DIFF values) was observed. IgM AVA seemed to be slightly more sensitive than IgG AVAs to more than three FT cycles, but the predefined stability acceptance criteria were still met for at least 78% of the IgM AVA samples after 12 FT cycles.
Research Article
ISR. To assess assay reproducibility, ISR must indeed be performed during the analyte stability period. Consequently, when the reproducibility of the method was already established (as it is the case here), the success of an ISR demonstrates that the samples were analyzed within their stability period. Here, the samples were considered to be stable at the tested condition if two-thirds of the reanalysis results were within 30% of the mean of both determinations. This approach is recommended by the EMA Guideline on Bioanalytical Method Validation [4] and by the report and follow-up of the American Association of Pharmaceutical Scientists workshop on assay reproducibility
Alternative & stricter acceptance criteria
The outcome of a stability study is based on a set of predefined acceptance criteria that have to be met. Since a large number of clinical samples and a calibration curve-based assay were used in this study, we decided to apply the same acceptance criteria as used for PK 60
Variability between the two measurements (%DIFF)
40 20 0 -20 -40 -60 -80 -100 -120 -140
%DIFF
3 FT cycles
6 FT cycles
9 FT cycles
12 FT cycles 15
n
16
16
16
Mean
2.9
-0.4
-15.3
13.0 11.2
Median
5.5
2.2
-9.35
Min
-18.8
-21.5
-116.3
-1.9
Max
15.2
18.2
32.7
39
n outside ±30%
0
0
3 (18.8%)
1 (6.7%)
Figure 7. Influence of freeze–thaw cycles on the quantification of IgG anti-vaccine antibodies (n = 16 samples). Summary statistics and box-plot representation of the variability (expressed as %DIFF) between the original and reassay results after three, six, nine and 12 FT cycles. The boundary of the box closest to zero indicates the 25th percentile, the line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. In addition, the mean is displayed as a dotted line. (Software: SigmaPlot 12.5; Systat Software, Germany). %DIFF: Percentage difference; FT: Freeze–thaw.
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Research Article Michaut, Laurent, Kentsch, Spindeldreher & Deckert-Salva
Table 1. Influence of freeze–thaw cycles on the quantification of IgM anti-vaccine antibodies: results, normalized difference and bias calculation. Subject Value (ru) before FT
After 3 FT cycles
After 12 FT cycles
Value (ru) after %DIFF %Bias towards 3 FT cycles towards mean original value‡ value†
Value (ru) after %DIFF towards Bias (%) 12 FT cycles mean value† towards original value‡
R
87.7
112
24.3
27.7
106
18.9
20.9
S
92.9
99.9
7.3
7.5
111
17.8
19.5
T
120
121
0.8
0.8
168
33.3
40.0
U
309
326
5.4
5.5
321
3.8
3.9
V
417
332
-22.7
-20.4
386
-7.7
-7.4
W
479
481
0.4
0.4
419
-13.4
-12.5
X
1550
1850
17.6
19.4
1440
-7.4
-7.1
Y
2320
2210
-4.9
-4.7
2470
6.3
6.5
Z
4450
3160
-33.9
-29.0
1980
-76.8
-55.5
n
9
9
9
9
Mean (%)
-0.6
0.8
-2.8
0.9
Median (%)
0.8
0.8
3.8
3.9
Min (%)
-33.9
-29.0
-76.8
-55.5
Max (%)
24.3
27.7
33.3
40.0
n outside ±30%
1 (11%)
0
2 (22%)
2 (22%)
%DIFF = 100 × (reanalysis value – original value)/(mean of reanalysis and original values), as promoted by the EMA Guideline on Bioanalytical Method validation [4]. Bias = 100 × (reanalysis value – original value)/original value, as promoted by Timmerman et al. [7]. %DIFF: Percentage difference; FT: Freeze–thaw; ru: Relative units. †
‡
for incurred samples [5] and is implemented in Novartis Standard Operation Procedures for LBAs. Alternatively, the variability of the measurements could be compared with the first determination, as promoted by the European Bioanalysis Forum in 2009 [7] . This was calculated for all analytes in Table 2, and AVA stability was demonstrated in all the tested conditions: less than a third of the samples were outside the ±30% bias acceptance range. However, when stability of QC samples is assessed during assay validation, the acceptance criteria
are based on the mean precision and accuracy at each tested level, which must be within the acceptance range determined during assay validation (here ±20%) [4] . Owing to the small number of clinical samples tested, this stricter approach appears to be more appropriate also for the FT stability assessment (Tables 3 & 4). Mean biases remained within 20% for moderate and high AVA levels up to 12 FT cycles. Although mean biases of 21.2 and 26.8% were observed for low levels of IgG and IgM AVAs, respectively, these values were only slightly out of
Table 2. Bias of the IgG and IgM anti-vaccine antibody stability assessments. Bias from original value
LTS
3 FT cycles
IgG 12 FT cycles
LTS
3 FT cycles
IgM 12 FT cycles
n
62
16
15
60
9
9
Mean (%)
-2.3
3.4
14.6
16.4
5.9
8.2
Median (%)
-5.4
5.6
11.8
15.1
5.5
3.9
Min (%)
27.9
-17.2
-1.8
-19.5
-29.0
-55.5
Max (%)
-22.8
16.4
48.5
56.9
27.7
40.0
n outside ±30%
0
0
1 (6.7%)
14 (23%)
0
2 (22%)
The variability between the two measurements was determined as promoted by the EMA Guideline on Bioanalytical Method validation [4] (i.e., by calculating the bias from the original value: 100 × [reanalysis value – original value]/original value). FT: Freeze–thaw; LTS: Long-term stability.
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Table 3. Alternative acceptance criteria for IgG anti-vaccine antibodies freeze–thaw stability assessment. Bias level
3 FT cycles
6 FT cycles
9 FT cycles
12 FT cycles
Low
Moderate
High
Low
Moderate
High
Low
Moderate
High
Low
Moderate
High
n
6
6
4
6
6
4
6
6
4
6
6
3†
Mean (%)
3.2
-0.1
9.2
0.8
-4.3
6.3
-17.0
-11.5
-1.9
21.2
9.8
11.1
Median (%)
5.6
-3.0
9.1
4.8
-7.2
5.4
-12.2
-9.3
-1.3
16.8
11.3
11.8
Min (%)
-11.2
-17.2
5.0
-19.4
-16.3
0.0
-56.0
-73.6
-8.7
4.1
-1.8
3.3
Max (%) 16.4
14.7
13.8
20.0
16.4
14.5
3.5
39.0
3.4
48.5
18.3
18.1
The variability between the two measurements was determined at each level of IgG anti-vaccine antibodies by calculating the bias from the original value (considered as nominal value) = 100 × (reanalysis value – original value)/original value. † Exclusion of one technical outlier after 12 FT cycles. ‡ Value does not meet the acceptance criterion on the mean bias (n = 3). FT: Freeze–thaw.
the acceptability range. Using this stricter acceptance criterion, the AVA stability was therefore demonstrated at each level (low, moderate and high) up to nine and three FT cycles for IgG and IgM AVAs, respectively. Conclusion Here, we used a high number of clinical samples collected from over 100 different subjects to provide solid evidence for the long-term and FT stability of AVAs. The format of the assays used in this GLP-compliant study allowed demonstrating both the AVA ability to bind to their target and their ability to be recognized by immunoglobulins raised against their Fc portion. We showed that AVAs of the IgG isotype are stable in undiluted human serum at least 3.6 years after collection when stored at a nominal temperature of -80°C in a temperature-monitored freezer. Similarly, our data showed that AVAs of the IgM isotype are stable in undiluted human serum at least 3.5 years after collection. A slight over-recovery of IgM after long-term storage was observed but was very limited and had neither qualitative nor quantitative impact on the ability of the assay to identify IgM AVA-positive
samples. The same applies to our FT stability assessment, where results obtained after up to three cycles adequately reflected those obtained shortly after sampling of IgG and IgM AVAs. These conclusions hold true for any of the stringency of the various acceptance criteria mentioned in the guidelines and white papers published to date. Similar conclusions were obtained by Hendriks et al. (this issue of Bioanalysis) [8] , who also demonstrated AVA stability upon long-term storage and FT cycling for a completely different preventive vaccine project. This GLP-compliant study therefore provides a strong experimental demonstration to the USP Guidance statement [9] , that it is generally accepted, that immunoglobulins stored in 100% serum at -80°C are stable and therefore stability assessment is not required. Future perspective The antibody response is the response that confers protection in most, if not all vaccines, making the AVA response wanted and beneficial. By contrast, the ADA response is unwanted because it can negatively impact the PKs, pharmacodynamics and efficacy of the drug,
Table 4. Alternative acceptance criteria for IgM anti-vaccine antibodies freeze–thaw stability assessment. Bias level
3 FT cycles
12 FT cycles
Low
Moderate
High
Low
Moderate
High
n
3
3
3
3
3
3
Mean (%)
12.0
-4.8
-4.8
26.8
-5.4
-18.7
Median (%)
7.5
0.4
-4.7
20.9
-7.4
-7.1
Min (%)
0.8
-20.4
-29.0
19.5
-12.5
-55.5
Max (%)
27.7
5.5
19.4
40
3.9
6.5
†
The variability between the two measurements was determined at each level of IgM anti-vaccine antibodies by calculating the bias from the original value (considered as nominal value) = 100 × (reanalysis value – original value)/original value. † Value does not meet the acceptance criterion on the mean bias. FT: Freeze–thaw.
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Research Article Michaut, Laurent, Kentsch, Spindeldreher & Deckert-Salva and possibly induce adverse events. In spite of these differences in the frequency and magnitude of the immune response, AVA and ADA responses are identical in nature. It has been proposed that the stability of ADAs is the same independently of the biological drug they are directed to [10] . If this is the case, it is possible to extend the conclusion of our study to all AVAs and ADAs, making it unnecessary to test LTS for each new biological therapeutics or vaccine program. This proposal is supported by the member companies of the European Bioanalysis Forum (this issue of Bioanalysis) [11] . Supplementary data To view the supplementary data that accompany this paper please visit the journal website at: www.future- science. com/doi/suppl/10.4155/BIO.14.97
Financial & competing interests disclosure The authors were Novartis employees at the time of the study and have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Executive summary Experimental, assay format • Validated ligand-binding assays (ELISA) implemented in GLP-compliant laboratories. • The stability assessments were conducted using a large number of samples coming from different subjects and collected at various time points after immunotherapeutic administration, reflecting the large individual variability of the humoral immune response. • Long-term stability of IgG anti-vaccine antibodies (AVAs) was tested on 62 occasions in samples from 32 subjects and was demonstrated for at least 3.6 years at -80°C. • Long-term stability of IgM AVAs was tested on 60 samples from 40 subjects and was demonstrated for at least 3.5 years at -80°C. • Freeze–thaw (FT) stability of IgG AVAs was tested on samples from 16 subjects and was demonstrated for at least nine FT cycles. • FT stability of IgM AVAs was tested on samples from nine subjects and was demonstrated for at least three FT cycles.
Alternative & stricter acceptance criteria • Long-term and freeze–thaw stability conclusions remained valid independently of the stringency of the acceptance criteria.
Conclusion • These data demonstrate the stability of anti-immunotherapeutic antibodies in frozen human samples stored at -80°C.
Future perspective • The nature of the humoral immune response allows extending the conclusion of this study to all AVAs and ADAs, making unnecessary to test long-term stability of frozen serum samples for each new biological therapeutics or vaccine program, consistently with the recommendation of the European Bioanalytical Forum. immunotherapy with CAD106 in patients with Alzheimer’s disease: randomised, double-blind, placebo-controlled, firstin-human study. Lancet Neurol. 11(7), 597–604 (2012).
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Research Article
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