High-throughput bioanalysis of bevacizumab in human plasma based

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High-throughput bioanalysis of bevacizumab in human plasma based on enzyme-linked aptamer assay using anti-idiotype DNA aptamer Tomohiro Yamada, Taro Saito, Yoshia Hill, Yutaka Shimizu, Kaori Tsukakoshi, Hajime Mizuno, Hideki Hayashi, Kazunori Ikebukuro, Toshimasa Toyo'Oka, and Kenichiro Todoroki Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05725 • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Analytical Chemistry

High-throughput bioanalysis of bevacizumab in human plasma based on enzyme-linked aptamer assay using anti-idiotype DNA aptamer Tomohiro Yamada1, Taro Saito2, Yoshia Hill1, Yutaka Shimizu2, Kaori Tsukakoshi2, Hajime Mizuno1, Hideki Hayashi3, Kazunori Ikebukuro2, Toshimasa Toyo’oka1, Kenichiro Todoroki1* Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, UniversitSy of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan 2 Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan 3 Laboratory of Pharmacy Practice and Social Science, Gifu Pharmaceutical University, Daigaku-nishi 1-25-4, Gifu, 5011196, Japan 1

ABSTRACT: We propose a highly selective, sensitive, accurate, and high-throughput bioanalysis method for bevacizumab utilizing an anti-idiotype DNA aptamer. With this method, bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A–HRP conjugate. The resulting HRP activity was colorimetrically detected using a microplate reader. The calibration curve of bevacizumab ranged from 0.05 to 5.0 μg/mL, and showed a good correlation coefficient (r2 =1.000). The limit of detection was 2.09 ng/mL. We also demonstrated both the possibility of highly sensitive detection using luminol chemiluminescence and the repeated use of affinity plates. The proposed method is applicable for planning optimal therapeutic programs, and for an evaluation of the biological equivalencies in the development of biosimilars.

Bevacizumab, developed by Genentech, is a humanized monoclonal antibody (mAb) against vascular endothelial growth factor A, and is used alone or with other drugs to treat metastatic colon cancer, lung cancer, renal cancer, ovarian cancer, and glioblastoma 1-6. Bevacizumab has been extremely successful, reaching fifth place in terms of global antibody sales in 2017 7, and the FDA approved its biosimilar for six cancer indications last year 8-10. To determine the therapeutic mAb concentrations in blood samples, a so-called bioanalysis has also been conducted to improve the therapeutic efficacy, reduce any side effects, and improve therapeutic activity 11-13. Pharmacokinetic (PK) behaviors are usually more complex with biopharmaceutical products than with conventional small-molecule drugs 14. In the bioanalyses of therapeutic mAbs, it is necessary to analyze the target drugs that differ only in the complementarity determining region (CDR) in structures as compared with their abundant immunoglobulins (IgGs) in blood samples. Therefore, the high selectivity of such methods is required. The popular choice in a bioanalysis of therapeutic mAbs has been the use of a ligand binding assay (LBA) such as an enzyme-linked immunosorbent assay (ELISA) 15, 16 or a chemiluminescent immunoassay (CLIA) 17 owing to its high sensitivity, high throughput, and low costs per sample once an assay is developed and validated. With these methods, the results may differ owing to the quality of the antibodies used 18, 19, the differences between lots, the preservation state, and other factors, and harmonization is essential.

To solve the above problems, we recently developed an anti-idiotype DNA aptamer against bevacizumab 20. Aptamers are single-stranded DNA or RNA that bind to a wide range of molecules with high specificity and affinity 21, 22. DNA aptamers are chemically stable and can be supplied cheaply through a chemical synthesis. Aptamers that recognize the constant region of IgG antibodies have been reported thus far 23, 24, however, an antiidiotype aptamer that selectively recognizes the CDRs of therapeutic mAbs is the first such aptamer developed globally. The dissociation constant of this aptamer to bevacizumab is estimated to be 130 nM. From the results of an X-ray crystal structure analysis, this aptamer is bound to all three CDRs of bevacizumab, and the results suggest the high affinity and high selectivity of this aptamer toward bevacizumab. In this paper, we report the development of an enzymelinked aptamer assay (ELAA) 25–27 method, which is a highthroughput LBA method using an anti-idiotype DNA aptamer for bevacizumab. Unlike conventional LBAs such as ELISA or CLIA, an ELAA uses a chemically stable DNA aptamer as a capture molecule for bevacizumab. Therefore, an inexpensive, sensitive, reproducible, and robust high-throughput bioanalysis of therapeutic mAbs was realized. Moreover, an ELAA enables the construction of various detection formats by combining antibodies, antigens, ligation, or the hybridization of the aptamers. Figure 1 shows a schematic diagram of the bioanalysis of bevacizumab using the ELAA method. Here, a 5’-aminohexyl anti-bevacizumab DNA aptamer for bevacizumab was

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covalently bound to succinimidyl ester activated 96-well polystyrene plates. Bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A–HRP conjugate. The resulting HRP activity was colorimetrically detected using a microplate reader.

Figure 1. Schematic diagram of bioanalysis of bevacizumab using ELAA method. To establish the analytical method, we optimized the amount of immobilization of the DNA aptamer, the binding conditions of the aptamer and bevacizumab, the sample dilution, and the blocking conditions. After optimization, the method was validated according to the FDA’s LBA guidelines and further applied to a bevacizumab-spiked plasma sample analysis. Furthermore, we evaluated the repeated use of aptamer-modified microtiter plates utilizing the chemical stability of a DNA aptamer and chemiluminescent ELAA aimed at a further sensitive analysis. There have been no reports quantifying therapeutic mAbs by sandwiching with an antiidiotype DNA aptamer and an HRP–protein A conjugate. This methodology is quite reasonable and suitable for a highthroughput bioanalysis of therapeutic mAbs.

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approved by the Ethics Committees of the University of Shizuoka (Approved No. 26-49). All other chemicals were of the highest purity available and used as received. Preparation of aptamer-modified 96-well microtiter plates

A DNA-BIND surface 96-well plate (Corning, USA) modified with an active ester group on the bottom of the plate was used for all ELAA experiments. A 5’-aminohexyl antibevacizumab DNA aptamer (5’GCGGTTGGTGGTAGTTACGTTCGC-3’), synthesized by Integrated DNA Technologies (Coralville, USA), was dissolved in an oligo binding buffer composed of a 50 mM phosphate buffer (pH 8.5) containing 1 mM EDTA to adjust the concentration to 1 µM. A portion of this solution (100 μL) was added to each well of the plate and incubated for 120 min at room temperature. After removal of the reaction solution, 200 µL of PBST was added to each well and then washed by shaking for 30 s. After three iterations of this procedure, blocking was carried out by incubating each well with 200 µL of 5% (w/v) skimmed milk containing PBS for a 2 h period. The resulting plate was washed three times using PBST and stored at 4 °C. ELAA procedure

One hundred microliters of a bevacizumab-spiked plasma sample diluted 1,000 times with PBST containing 0.6% trehalose, 0.1% (w/v) skimmed milk was added to aptamermodified 96-well plates prepared according to the above procedure, and then incubated at room temperature for 2 h. After washing with PBST (200 µL), a protein A-HRP conjugate solution (100 µL) dissolved in PBST containing 1% (w/v) skimmed milk was added and then incubated at 37 °C for 1 h. After washing three times with PBST (200 µL), the TMB solution (100 µL) was added and then left to stand at room temperature and light-shielded for 5 min. The enzymatic reaction was stopped by adding 100 μL of 2 M sulfuric acid, and the absorbance at 450 nm of the resulting solution was determined using a microplate reader. Method validation

EXPERIMENTAL SECTION Materials, chemicals, and apparatus

Deionized and distilled water, purified using the ELGA Purelab Flex system (ELGA, Marlow, UK), was applied to prepare all aqueous solutions. Bevacizumab (Avastin, 400 mg/16 mL for intravenous infusion) was produced by Chugai Pharmaceutical (Tokyo, Japan). Tocilizumab (Actemra, 80 mg for intravenous infusion) and trastuzumab (Herceptin, 150 mg/7.2 mL for intravenous infusion) were produced by Chugai Pharmaceutical (Tokyo, Japan). Nivolumab (Opdivo, 20 mg/2 mL for injection) and infliximab (Remicade, 100 mg for intravenous infusion) were produced by Ono Pharmaceutical (Osaka, Japan) and Mitsubishi Tanabe Pharma (Osaka, Japan), respectively. Each therapeutic mAbs formulation was diluted to the desired concentration using 0.05% (w/v) tween 20 containing phosphate buffered saline (PBST, pH 7.4) with 0.1% (w/v) skim milk. Colorimetric assays were conducted using a Wallac ARVO SX 1420 Multilabel Counter (PerkinElmer, Waltham, USA) and its detection main wavelength 450 nm and sub-wavelength were set at 630 nm, respectively. A protein A– HRP conjugate solution was obtained from ThermoFisher Scientific (Waltham, USA) and diluted 8,000-fold with 1% (w/v) BSA containing PBS. An ELISA substrate solution of HRP (POD substrate TMB kit) containing tetramethylbenzidine (TMB) was purchased from Nakalai Tesque (Kyoto, Japan). Human plasma as a control was obtained from healthy volunteers at the University of Shizuoka. This study was

The proposed analytical method was partially followed by a Bioanalytical Method Validation 28. The calibration curve was fitted to four-parameter logistics regression. The precision of the assay was determined through the repeated measurements of five samples (colorimetric detection, 0.05, 0.1, 0.5, 1.0, and 5.0 μg/mL; chemiluminescence detection, 0.005, 0.01, 0.05, 0.1, and 0.5; n = 6). A level of precision of between 80–120%, with the exception of 75–125% for LLOQ and ULOQ, was considered to be acceptable. The accuracy was determined as the relative difference between the nominal input concentration and the measured concentration. A level of accuracy of between 80–120%, with the exception of 75–125% for LLOQ and ULOQ, was considered to be acceptable. The limit of detection (LOD) was individually calculated based on a value of 3-times the standard deviation of the blank value. Evaluation of the repeated use of aptamer-modified microtiter plates

A 3 M aqueous sodium chloride solution containing 8 M urea (200 μL) was added to an aptamer-immobilized plate after the ELAA experiment 26. The plate was then incubated at 37 °C for 1 h to remove the adsorbed contaminants, dissociate the aptamer–bevacizumab binding, and unfold the aptamer DNA. Thereafter, by adding an oligo binding buffer to the plate, the unfold DNA aptamer could be refolded to the original threedimensional structure. The resulting plates were used for a repeated analysis.


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Analytical Chemistry Chemiluminescent ELAA procedure

Preparation of aptamer-modified 96-well microtiter plates for chemiluminescent ELAA is as follows. A maleic anhydride activated 96-well plate (ThermoFisher Scientific) was used for all chemiluminescent ELAA experiments. A 5’-aminohexyl anti-bevacizumab DNA aptamer (5’GCGGTTGGTGGTAGTTACGTTCGC-3’) synthesized by Integrated DNA Technologies was used. This aptamer was dissolved in the oligo binding buffer to adjust the concentration to 1 µM. A portion of this solution (100 μL) was added to each well of the plate and incubated overnight at room temperature. After removal of the reaction solution, 200 µL of a washing buffer was added to each well and followed by shaking for 30 s. After three iterations of this procedure, blocking was carried out by incubating each well with 200 µL of 5% (w/v) skimmed milk containing an oligo binding buffer for a 2 h period. The resulting plate was washed three times using PBST and stored at 4 °C. One hundred microliters of a bevacizumab-spiked plasma sample diluted 1,000 times with a 0.1 M phosphate buffer (pH 7.4) containing 0.6% trehalose, 0.04% tween 200, and 1% (w/v) skimmed milk was added to the aptamer-modified 96-well plates and incubated at 37 °C for 1 h. After washing with PBST (200 µL), a protein A–HRP conjugate solution (100 µL) was added followed by incubation at 37 °C for 1 h. After washing with PBST (200 µL), a BM chemiluminescence ELISA substrate (POD) solution (100 µL, Sigma-Aldrich) was then added. The solution was then left to stand at room temperature and light-shielded for 3 min, and the chemiluminescence of the resulting solution was determined using a microplate reader.

RESULTS AND DISCUSSION Optimization of anti-idiotype DNA aptamer immobilization and sample dilution conditions

The concentration of the DNA aptamer solution to be immobilized on microtiter plates was examined at three concentrations of 1, 5, and 10 μM (Table S1). A small increase in absorbance was observed as the concentration of the aptamer solution increased; because its difference was small, however, the concentration was set at 1 μM. When the quantification was conducted using a phosphate buffer as a dilution solvent of the bevacizumab solution, the resulting absorbance was markedly decreased. This phenomenon is thought to be from the nonspecific adsorption of bevacizumab onto the inner walls of the pipette tips or tubes used in the preparation 29. To prevent this, we examined the use of two types of sample diluents containing an adsorption inhibitor (Fig. 2). These diluent solutions were used for commercial therapeutic mAb formulations as additives. In both diluents, an increase in absorbance was observed at each concentration as compared with dilution with purified water, and in particular, the former diluent (phosphate buffer containing 0.6% trehalose and 0.04% tween 20) provided better results. Thus, this diluent was used in the following experiments. Through the use of these conditions,

a calibration curve of the bevacizumab can be drawn within the range of 0.005–1 μg/mL with good linearity (r2 = 0.994). The LLOQ, ULOQ, and precision and accuracy were 0.755 ng/mL, 2.50 ng/mL, and less than 19.8%, respectively (Table S2). Optimization of blocking and dilution conditions for plasma sample analysis

When analyzing bevacizumab-spiked plasma samples according to the analytical protocol for standard bevacizumab solutions, intense absorbance was observed within the overall concentration ranges examined. This phenomenon is likely derived from the nonspecific adsorption of large amounts of IgGs in human blood, which causes an increase in the colorimetric enzyme reaction product through a binding with the protein A–HRP conjugate. Therefore, we examined whether the nonspecific adsorption of contaminant IgGs can be reduced by increasing the sample dilution ratio. Bevacizumab-spiked plasma samples were analyzed after a 400-, 800-, 1,000-, and 1,200-fold dilution with the sample preparation solution (Table S3).

Figure 2. The effects of the sample diluent for bevacizumab on the resulting absorbance. Legend (left to right): 0.6% trehalose and 0.04% tween 20 (blue), 0.02% SDS and 0.1% N-lauroylsarcosine (orange), and water (gray). A slight reduction of the background absorbance can be confirmed as compared with prior to the dilution, however, it cannot be quantitatively improved. Next, comparing the results of three types of blocking agents (BSA, skimmed milk, and peptone), the order of the blocking ability was shown to be skim milk > BSA > peptone (Table 1). Furthermore, both the dilution ratio of the plasma sample containing bevacizumab spiked at 0.001–10 μg/mL and the concentration of skim milk as a blocking agent were optimized (Fig. S1). The validity was quantitatively evaluated from a determination of the coefficient using a linear approximation method. The 500- and 1,000-fold dilution ratios and concentrations of skimmed milk were examined within the range of 0.1 to 0.6%. One-thousand-fold dilution with 0.1% skimmed milk gave the best result quantitatively (r2 = 0.944), and thus this condition was determined as optimal.



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Table 1. Effects of the different types of blocking agents on the absorbance produced. Bevacizumab (μg/mL)

Blocking agent BSA

Peptone

Skimmed milk

0.001

0.398

0.600

0.208

0.005

0.369

0.612

0.196

0.01

0.402

0.627

0.281

0.05

0.377

0.643

0.347

0.1

0.439

0.671

0.370

0.5

0.520

0.721

0.561

1

0.467

0.689

0.496

5

0.660

0.746

0.640

Table 2. LLOQ, ULOQ, linearity, precision, and accuracy of the colorimetric ELAA in plasma sample analysis. LLOQ (μg/mL) 0.05

ULOQ (μg/mL) 5.0

Spiked concentration (μg/mL)

Linearity 1.000

0.05

0.1

0.5

1.0

5.0

Precision

3.2

6.8

5.4

5.6

3.1

Accuracy

-4.2

4.5

1.3

-0.3

-1.1

Method validation for bioanalysis of bevacizumab

Bevacizumab was added to plasma within the range of 0.05–5.0 μg/mL, and the LLOQ, ULOQ, linearity, precision, and accuracy of the proposed method were evaluated (Table 2). By adopting the four-parameter logistics regression model, the calibration curve showed good linearity with a determination coefficient of 1.000 (Fig. 3). The calculated LOD value giving 3-times the standard deviation of the blank value was 2.06 ng/mL. This provides a comparable quantification range to the previous sandwich ELISA method for bevacizumab in plasma samples (5–500 ng/mL) using an anti-human IgG antibody and VEGF 15, or for trastuzumab in the plasma samples (1.6 ng/mL to 1.6 μg/mL) using two anti-idiotype antibodies against trastuzumab 16. The precision of the assay ranged from 3.1% to 6.8%, and the bias was -4.2–4.5%, which are within the acceptable limits. We used a commercially available protein A– HRP conjugate for an enzymatic amplification reaction. Because protein A can bind the contaminant IgGs derived plasma samples during the ELAA reaction procedure, the low sensitivity and precision based on high background signals were of concern as compared with the results in Table S2. However, owing to the high selectivity of the aptamer, the proposed method showed sufficient sensitivity and precision even in a plasma sample analysis. The ranges of concentrations tested were chosen based on the inserts of the drug packages; the effective blood concentrations of the drugs ranged from 50 to 500 μg/mL 30, and showed insufficient sensitivity for a bioanalysis of bevacizumab. Selectivity of the proposed method toward other commercial therapeutic mAbs

The proposed method was applied to aqueous solutions (1 ng/mL to 5 μg/mL) of five types of therapeutic mAbs (bevacizumab, infliximab, trastuzumab, tocilizumab, and nivolumab) (Fig. 4). In other mAbs, with the exception of bevacizumab, the quantitatively could not be confirmed. In particular, the absorbance for three types of mAbs (infliximab,

trastuzumab, and nivolumab) showed the background levels even at 5 μg/mL; it was found that these mAbs could not bind to the aptamer.

Figure 3. Calibration curve of colorimetric ELAA for bevacizumab in plasma samples. Curve fitting was applied according to the four-parameter logistic model


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Analytical Chemistry Figure 4 Photographs of aptamer-modified 96-well plates after ELAA reaction with a) bevacizumab (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 μg/mL; n = 6) and b) five therapeutic mAbs (1, bevacizumab; 2, infliximab; 3, trastuzumab; 4, tocilizumab; 5, nivolumab; 5 μg/mL each) spiked in human plasma.

Table 3. LLOQ, ULOQ, linearity, precision, and accuracy of the chemiluminescence ELAA in plasma sample analysis. LLOQ (μg/mL)

ULOQ (μg/mL)

Linearity

0.005

0.5

0.998

Spiked concentration (μg/mL) 0.005

0.01

Precision

1.8

3.2

Accuracy

10.7

1.4

0.05

0.1

0.5

5.4

1.9

15.4

-15.8

-7.4

14.3

Evaluation of the repeated use of aptamer-modified microtiter plates

Unlike the mAbs used in ELISA methods, the DNA is highly stable against heat, organic solvents, and acid. In addition, the chemical modification of the DNA aptamer onto a microplate surface is also stable, and thus if the washing protocol is appropriately applied, and the three-dimensional structure of the DNA aptamer is preserved, a repeated assay using the same plate will be possible. Therefore, we examined the linearity and quantitative range when ELAA was repeated five times using the same plates (Fig. 5, Table S4). Even when the assay plate was reused five times, the acceptable linearity (r2> 0.968) of the calibration curves was obtained. In contrast, the repeated use of assay plates decreased the gradient of the calibration curves and increased the difference in the absorbance between the bevacizumab spiked plasma samples and the standard solution. Both of these phenomena could have been caused by the non-specific adsorption of endogenous human IgG or other components onto the plate surface, and by the aptamer itself. However, even when the assay plate was used five times, the CV values were 18.3% or less under all concentrations, and the accuracy was maintained. Such a repeated assay is unrealistic for conventional LBAs utilizing mAbs and is a unique advantage of the ELAA method. Figure 5. Calibration curves of standard bevacizumab and bevacizumab-spiked plasma samples analyzed using



aptamer-modified plates after (a) initial and (b) five uses. Closed circle, plasma samples; open circle, standard samples. Demonstration of chemiluminescent ELAA for higher sensitive bioanalysis of bevacizumab

The proposed colorimetric detection method showed adequate sensitivity for the application of a bioanalysis of bevacizumab. However, to apply this method to a quantification using a trace amount of blood samples and next-generation drugs with low-dose administration, such as Potelligent antibodies 31 and recycling antibodies 32, we investigated the use of a higher sensitive chemiluminescence detection that uses an HRP-luminol reaction. The quantitative range was 0.005–0.5 μg/mL and the LOD value was 1.96 ng/mL. Because chemiluminescence detection showed higher background signals than those in colorimetric detection, both LODs showed similar values. However, the calibration range of the method was lowered 10-fold. Although a direct comparison of the detection methods was difficult owing to the difference in aptamer-immobilized plates applied, we proved that chemiluminescent detection is a useful option for a highly sensitive ELAA.

CONCLUSIONS We developed a highly selective, sensitive, accurate, and rapid bioanalysis method for bevacizumab that uses an antiidiotype DNA aptamer. With this method, bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A–HRP conjugate. The resulting HRP activity can be colorimetrically detected using a microplate reader. This method enables not only a high-throughput analysis comparable to that of a conventional ELISA method, but also achieves a robust analysis using a chemically stable DNA aptamer. Furthermore, we also demonstrated the possibility of highly sensitive detection using luminol chemiluminescence and the possibility of the repeated use of the affinity plates. In the future, the acquisition of DNA aptamers that recognize other therapeutic mAbs will allow high throughput bioanalyses to be conducted. Unlike anti-idiotype antibodies, chemically synthesized DNA aptamers are readily available as low batch-to-batch variation products, and various homogeneous bioanalyses using these aptamers can be conducted at any location in the world. The method described herein is applicable not only to the planning of optimal therapeutic programs, and to an evaluation of biological equivalencies in the development of biosimilars, but also for simple production quantification during the manufacturing processes.

ASSOCIATED CONTENT The supporting information is available free of charge on the ACS Publications website at DOI: Effect of DNA aptamer concentration used for microplate immobilization on the developed absorbance (Table S1), LLOQ, ULOQ, linearity, precision and accuracy of the colorimetric method in standard solution analysis (Table S2), Effect of dilution ratio of bevacizumab-spiked plasma samples on the produced absorbance (Table S3), Changes in coefficient of variation (CV, %) of absorbance due to repeated use of assay plates (Table S4), Optimization of sample dilution ratio of the plasma sample spiked at 0.001-10 μg/mL and the concentration of skimmed milk (Figure S1). (PDF)

Author Contributions Corresponding Author * Kenichiro Todoroki. Tel: +81-54-264-5656, Fax: +81-54-2645654, E-mail: [email protected]. The manuscript was written with the contributions of all authors. T.Y.* and K.T. conceived the study. T.Y.*, T.S., Y.H., H.S., K.T., H.M., K. I., T.T., and K.T. conducted the experiments, data analysis, and interpretation. H.H. considered the results from a clinical perspective.

ORCID Hajime Mizuno: 0000-0001-6715-7748 Kazunori Ikebukuro: 0000-0003-2838-0562 Kenichiro Todoroki: 0000-0002-9714-2659

ACKNOWLEDGMENT This work was supported by JSPS KAKENHI, Grant Nos. 25460040 and 16K08200. We would like to thank Editage (www.editage.jp) for English language editing. The authors declare no competing financial interest.

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High-throughput bioanalysis of bevacizumab in human plasma based

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