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1.
Anal Biochem ; : 114505, 2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34856184

RESUMO

The charge heterogeneity of fusion proteins can vary dramatically compared with more traditional biopharmaceuticals like monoclonal antibodies, making the characterization of fusion proteins a challenge. A single platform method suitable for the analysis of multiple fusion proteins would reduce method development and streamline production workflows. Here, we develop a platform method to characterize the charge heterogeneity of a variety of fusion protein therapeutics using imaged capillary isoelectric focusing (icIEF). We describe the development of the platform method, and analyze 9 fusion protein therapeutics. The results are reproducible in peak group area percentage and apparent pI determination. We compare the platform icIEF method to traditional slab gel IEF, which is still used in many laboratories for the analysis of fusion proteins. The peak patterns obtained from the icIEF method is comparable to the band patterns of the gel IEF. The platform method can also be used as the starting point if further optimization is needed even when high resolution is required. The platform method described in this study can be applied as an identity and purity assay for fusion proteins in the biopharmaceutical industry.

2.
Int Immunopharmacol ; 101(Pt A): 108277, 2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34773758

RESUMO

CTLA-4 is an important immune checkpoint for the regulation of T cell activation, and anti-CTLA-4 monoclonal antibodies (mAbs) are being developed as mono- or combination therapy for various tumors with reliable clinical efficacy. Ipilimumab is the first approved inhibitor of immune checkpoint, and many other anti-CTLA-4 mAbs, including ipilimumab biosimilars, are in different stages of clinical trials. However, due to the immunomodulating nature of the mAbs targeting CTLA-4, mode of action (MoA) and cell-based bioassay to determine their bioactivities as the lot release or stability test has been a great challenge to quality control laboratories. In this study, we have developed and validated a reporter gene assay (RGA), in which two kinds of cell lines were engineered to measure the bioactivity of anti-CTLA-4 mAbs. Raji cells were stably transfected with the membrane-anchored anti-CD3 single chain antibody fragment (scFv) as antigen-presenting cells (APCs, Raji-CD3scFv cells), while Jurkat cells were stably transfected with CTLA-4 with Y201V mutation and NFAT controlled luciferase as the effector cells (Jurkat-CTLA-4-NFAT-luc cells). The ligation of CD80/CD86 on the APCs with CTLA-4 could reduce the luciferase expression accompanied with the activation of effector cells, while the anti-CTLA-4 mAb could reverse the reduction, which resulted in good dose response curve to determine its bioactivity. After optimizing various assay conditions, we performed full validation according to ICH-Q2 (R1), which demonstrated the excellent specificity, accuracy, precision, linearity, and the cell passage stability. The satisfied performance characteristics render the RGA a good bioassay in the bioactivity determination of anti-CTLA-4 mAbs, as applied in characterization, batch release control, stability study, and biosimilar assessment.

3.
J Pharm Sci ; 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34551350

RESUMO

In the study, subvisible particles in 205 samples from 17 commercial mAb drug products approved in China were analyzed using light obscuration (LO) and flow imaging microscopy (FIM) methods. For each method, a total 633 tests (runs) were performed. In the tests, samples in state of lyophilized powder or syringe package had significantly higher particle concentrations. It was confirmed by analyzing the 205 drug product samples that FIM particle counts are generally higher than LO counts. The cause of the higher counts of FIM method than LO counts was examined by looking into the contribution of proteinaceous, translucent particles in the samples. The data of the study showed that the number of proteinaceous, translucent particles was a factor in the elevated counts of FIM method compared to LO method.

4.
Int Immunopharmacol ; 100: 108112, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34521023

RESUMO

More than 100 monoclonal antibodies (mAbs) have been approved by FDA. The mechanism of action (MoA) involves in neutralization of a specific target via the Fab region and Fc effector functions through Fc region, while the latter include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCP has been recognized one of the most important MoAs, especially for anti-cancer mAbs in recent years. However, traditional bioassays measuring ADCP always introduced primary macrophages and flow cytometry, which are difficult to handle and highly variable. In this study, we engineered a monoclonal Jurkat/NFAT/CD32a-FcεRIγ effector cell line that stably expresses CD32a-FcεRIγ chimeric receptor and NFAT-controlled luciferase. The corresponding mAb could bind with the membrane antigens on the target cells with its Fab fragment and CD32a-FcεRIγ on the effector cells with its Fc fragment, leading to the crosslinking of CD32a-FcεRIγ and the resultant expression of subsequent NFAT-controlled luciferase, which represents the bioactivity of ADCP based on the MoA of the mAb. With rituximab as the model mAb, Raji cells as the target cells, and Jurkat/NFAT/CD32a-FcεRIγ cells as the effector cells, we adopted the strategy of Design of Experiment (DoE) to optimize the bioassay. Then we fully validated the established bioassay according to ICH-Q2(R1), which proved the good assay performance characteristics of the bioassay, including specificity, accuracy, precision, linearity, stability and robustness. This RGA can be applied to evaluate the -ADCP bioactivity for anti-CD20 mAbs in lot release, stability testing as well as biosimilar comparability. The engineered cells may also potentially be used to evaluate the ADCP bioactivity of mAbs with other targets.

5.
Emerg Microbes Infect ; 10(1): 1519-1529, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34278967

RESUMO

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutated continuously and newly emerging variants escape from antibody-mediated neutralization raised great concern. S protein is heavily glycosylated and the glycosylation sites are relatively conserved, thus glycans on S protein surface could be a target for the development of anti-SARS-CoV-2 strategies against variants. Here, we collected 12 plant-derived lectins with different carbohydrate specificity and evaluated their anti-SARS-CoV-2 activity against mutant strains and epidemic variants using a pseudovirus-based neutralization assay. The Lens culinaris-derived lentil lectin which specifically bind to oligomannose-type glycans and GlcNAc at the non-reducing end terminus showed most potent and broad antiviral activity against a panel of mutant strains and variants, including the artificial mutants at N-/O-linked glycosylation site, natural existed amino acid mutants, as well as the epidemic variants B.1.1.7, B.1.351, and P.1. Lentil lectin also showed antiviral activity against SARS-CoV and MERS-CoV. We found lentil lectin could block the binding of ACE2 to S trimer and inhibit SARS-CoV-2 at the early steps of infection. Using structural information and determined N-glycan profile of S trimer, taking together with the carbohydrate specificity of lentil lectin, we provide a basis for the observed broad spectrum anti-SARS-CoV-2 activity. Lentil lectin showed weak haemagglutination activity at 1 mg/mL and no cytotoxicity activity, and no weight loss was found in single injection mouse experiment. This report provides the first evidence that lentil lectin strongly inhibit infection of SARS-COV-2 variants, which should provide valuable insights for developing future anti-SARS-CoV-2 strategies.


Assuntos
Antivirais/farmacologia , Lens (Planta)/química , Extratos Vegetais/farmacologia , Lectinas de Plantas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/química , Lectinas de Plantas/química , SARS-CoV-2/crescimento & desenvolvimento , Sementes/química
6.
Electrophoresis ; 42(19): 1900-1913, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34240427

RESUMO

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is an analytical method to assess the purity of proteins, commonly applied to monoclonal antibodies (mAbs) in the biopharmaceutical industry. To address the need to standardize the CE-SDS method in the pharmaceutical industry and to enhance the confidence in method transfer between laboratories operating different commercial capillary electrophoresis (CE) instrument platforms, an interlaboratory CE-SDS method validation was organized involving 13 laboratories in 13 companies on four different types of commercial capillary electrophoresis instruments. In the validation, a commercial mAb therapeutic was used as the sample. The validation process followed the analytical guidelines set by the ICH guidelines (International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). The method's precision, accuracy, linearity and range, and limit of quantitation (LOQ) were validated in the study. Variations of all the parameters validated in the study passed the pre-set criteria defined at the beginning of the study. The definition was based on previously published works and the intended application purpose of the CE-SDS method for mAbs. The study proved that the CE-SDS method fits its intended application purpose as a size impurity assay and size heterogeneity characterization assay for mAb therapeutic products. This study is the first time a CE-SDS method is validated by multiple laboratories using different commercial CE instrument platforms and on a commercial mAb therapeutic. Its results will enhance the confidence of the biopharmaceutical industry to develop CE-SDS methods and transfer CE-SDS methods between different laboratories.

7.
Anal Biochem ; 634: 114291, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34161831

RESUMO

Calcitonin gene-related peptide (CGRP) is critical for the pathophysiology of migraine, and four therapeutic antibodies targeting CGRP and its corresponding receptors have been approved by the Food and Drug Administration (FDA), while many others are in the different stages of clinical trials. Bioactivity determination is essential for the quality control and clinical application of therapeutic monoclonal antibodies (mAbs). However, no bioassay has been reported to date. In this study, we developed a reporter gene assay (RGA) based on SK-N-MC cells stably expressing firefly luciferase driven by cAMP response element (CRE). The key assay parameters were optimized according to signal-to-noise (SNR), the response value, and the fitted dose-response curve. Validation of the RGA in accordance with ICH-Q2 guidelines showed that the method had good specificity, accuracy, linearity, and precision. The established RGA can be utilized as a reference method for release testing and stability studies of relevant antibodies.

8.
J Pharm Biomed Anal ; 199: 114033, 2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-33774455

RESUMO

Type 2 inflammatory cytokines, including IL-4, IL-5 and IL-13, contribute considerably to the pathogenesis of asthma. Anti-IL-4R monoclonal antibody (mAb) has been approved for the therapeutic treatment of asthma, and many mAbs with the same target are in the different stages of R&D and clinical trials. Bioactivity determination is required to ensure the quality control of mAbs. However, current ELISA and SPR assays or cell-based anti-proliferation assays for IL-4R mAbs are either not mechanism-of-action (MOA) representative or tedious and time consuming. Therefore, we developed a reporter gene assay (RGA) based on the HEK-293 cell line that stably expressed signal transducer and activator of transcription 6 (STAT6) and the luciferase reporter controlled by STAT6 binding elements. Anti-4R mAb could bind to IL-4R, and block the interaction between IL-4 and IL-4R, resulting in the reduction of IL-4 induced STAT6 controlled luciferase expression. After careful optimization of the experiment parameters, the RGA method demonstrated optimal dose-response curve between anti-IL-4R mAb concentration and luciferase expression level. Validation according ICH-Q2 proved the excellent assay performance characteristics of the established RGA, including specificity, accuracy, precision, linearity and range. The established transgenic cell line was stable for the bioactivity determination of anti-IL-4R mAb up to 46 generations, and the RGA was also suitable for the bioactivity determination of anti-IL-4 mAbs, and potentially of anti-IL-13 mAbs. The established RGA could be adopted to determine the bioactivity during the development, characterization, lot release, stability, and comparability studies of anti-IL-4R mAbs.


Assuntos
Anticorpos Monoclonais , Bioensaio , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética
9.
Int Immunopharmacol ; 93: 107418, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33540248

RESUMO

The tumor necrosis factor alpha (TNF-α)/nuclear factor-kappa B (NF-κB) signaling pathway plays a crucial role in the pathogenesis of inflammatory diseases. Several therapeutic monoclonal antibodies (mAbs) and biosimilars against TNF-α have been developed to treat patients who suffer from inflammatory diseases caused by disordered expression of TNF-α. Hence, quality control of biopharmaceuticals is crucial during research and development. The high-order structure of these complex molecules cannot be entirely identified by physiochemical attributes; however, they can be inferred by observing biological activities. Thus, we developed a U937-based bioassay to determine the biological activities of mAbs and biosimilars against TNF-α using a low-basal NF-κB-inducible lentiviral reporter gene. The reporter gene assay (RGA) can be induced with a high signal-to-noise ratio (SNR) in a short time by TNF-α. Validation of the RGA showed accuracy (% relative standard deviation [RSD] = 4.64%), linearity (r2 = 0.9856), and precision (Interday RSD = 4.6%, between analysts RSD = 3.51%) as well as reasonable specificity and robustness. The measured potency values of a biosimilar to adalimumab were between 90% and 110%. Results showed our RGA is suitable for mAb quality control and lot release, and for evaluation of the biological activity similarity of the biosimilar.


Assuntos
Anticorpos Monoclonais/farmacologia , Bioensaio/métodos , Medicamentos Biossimilares/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Medicamentos Biossimilares/metabolismo , Genes Reporter/genética , Humanos , Lentivirus/genética , Camundongos , NF-kappa B/metabolismo , Controle de Qualidade , Fator de Necrose Tumoral alfa/imunologia , Células U937
10.
Luminescence ; 36(4): 885-893, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33382183

RESUMO

OX40 plays a prominent role in the onset and development of solid tumors, and OX40-targeted monoclonal antibodies (mAbs) have entered clinical trials for various tumors. Bioactivity determination of therapeutic mAbs is of great significance in product quality, however, mechanism of action-based bioassays to determine the bioactivity of anti-OX40 mAbs is still lacking. Here, we established a reporter gene assay system based on two cell lines, namely Jurkat-OX40-NFκB-Luc which stably expresses NFκB-controlled luciferase, and Raji cells which inherently express FcγRs. In the model, FcγRs on Raji cells could crosslink the Fc of anti-OX40 mAbs, which leads to the further crosslinking between Fab of anti-OX40 mAbs and OX40 on Jurkat-OX40-NFκB-Luc cells. OX40 crosslinking could activate Jurkat-OX40-NFκB-Luc cells, and induce the expression of NFκB-controlled luciferase, the extent of which could reflect the bioactivity of anti-OX40 mAbs in a dose-dependent manner. After the optimization of various assay conditions, the validation of the cell-based bioassay showed good assay performance characteristics, including specificity, accuracy, precision, linearity, and stability. This innovative assay that is based on the OX40-NFκB pathway can be a powerful pool to measure the bioactivity of OX40-targeted mAbs.


Assuntos
Anticorpos Monoclonais , Bioensaio , Linhagem Celular , Genes Reporter , Luciferases/genética
11.
Sci Rep ; 10(1): 16615, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024203

RESUMO

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.


Assuntos
Anticorpos Antivirais/análise , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Infecções por Coronavirus/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/virologia , Genes Reporter , Células HEK293 , Humanos , Células Jurkat , Luciferases/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Fatores de Transcrição NFATC/genética , Receptores de IgG/genética , Receptores de IgG/imunologia , Elementos de Resposta , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção
12.
Int Immunopharmacol ; 85: 106647, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32504997

RESUMO

Environmental disturbances may result in dysregulation of interleukin-23 (IL-23), which is a crucial modulator of immunity. Several therapeutic monoclonal antibodies (mAbs) have been developed for treating IL-23-related autoimmune inflammation, such as ustekinumab, guselkumab, tildrakizumab, and risankizumab. Accurate bioactivity determination of therapeutic mAbs is essential for their quality control and clinical application. However, the current methods are tedious and complicated. In the present study, we employed low-background lentivirus carrying sis-inducible element (SIE)-driven firefly luciferase to generate a stable DB-SIE-Luc cell line that expresses endogenous IL-23 receptors and developed a sensitive and straightforward reporter gene assay (RGA) based on DB-SIE-Luc cells. After the optimization of various assay parameters, we set up a bioassay with the best fit of a four-parameter model and an appropriate signal-to-noise ratio (SNR) for bioactivity determination of guselkumab. We further verified the excellent assay performance characteristics of our RGA, including specificity, linearity, accuracy, precision, and stability, according to ICH-Q2. Taken together, we established a reliable and robust cell-based RGA, which potentially serves as a valubale alternative bioactivity determination assay for the release control and stability study of anti-IL-23 mAbs.


Assuntos
Anticorpos Monoclonais/farmacologia , Bioensaio , Genes Reporter , Interleucina-23/imunologia , Linhagem Celular , Humanos , Lentivirus/genética , Luciferases de Vaga-Lume/genética
13.
Luminescence ; 35(8): 1408-1415, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32598535

RESUMO

Although enormous success has been achieved with anti-PD-1/PD-L1 and anti-CTLA-4 monoclonal antibodies (mAbs), their unsatisfactory response rate in cancer patients has been driving the research and development of novel immune checkpoint inhibitors (ICIs). Anti-LAG-3 mAbs, as one of the most promising candidates, are now being tested for various human cancers at different stages of clinical trials. Here, we describe the development and validation of a reporter gene assay (RGA) to measure the bioactivity of anti-LAG-3 mAbs. We established the bioassay based on parental Raji cells and a Jurkat cell line stably transfected with human LAG-3 gene and luciferase reporter elements controlled by nuclear factor of activated T cell (NFAT) from the IL-2 promoter. After optimization of key parameters, the established RGA showed excellent precision, specificity, accuracy, and stability. The mechanism of action (MOA) relatedness and the excellent assay performance make the RGA suitable for the characterization, lot release, and stability test of anti-LAG-3 mAbs.


Assuntos
Anticorpos Monoclonais , Bioensaio , Linhagem Celular , Genes Reporter , Humanos , Luciferases/genética
14.
Anal Bioanal Chem ; 412(8): 1901-1914, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32030491

RESUMO

Immunoglobin E (IgE)-related allergy constitutes a high proportion in allergic diseases. The production of specific IgE is key to evoking serial cascades and pathological reactions. Thus, targeting IgE is a different therapeutic approach from symptomatic treatments. Monoclonal antibodies (mAbs) against IgE were developed and a humanized antibody, omalizumab, was approved by five countries. It could inhibit the binding of IgE with epsilon receptor I of crystallizable fragment (FcεRI), thus preventing anaphylactic reactions. However, no bioactivity assay, which is the critical quality attribute and should thoroughly reflect the clinical mechanism, has been established to date. In commercial lot release, only the enzyme-linked immunosorbent assay (ELISA) method was applied, which only reflects the binding of omalizumab to IgE but not the subsequent reaction. In scientific research works, human FcεRI-transfected RBL-2H3 cells were used to indicate degranulation based on the detection of ß-hexosaminidase. Nevertheless, this method needs much work to stabilize the response and, hence, is not suitable for routine usage in commercial production and control of antibodies. To evaluate the bioactivity of anti-IgE antibodies including omalizumab using a simple assay that reflects the following mechanism of actions (MOA) after binding, we established an RBL-2H3 cell line transfected with both the α subunit of human FcεRI and nuclear factor-activated T cell (NFAT) response elements, the latter is conjugated with a luciferase gene, which could shed luminescence when substrates exist. The method was proven to possess good specificity, accuracy, linearity, and precision and may be utilized as a supplement to anti-IgE antibody bioactivity assays in terms of development, lot release, stability, and comparability studies. Graphical abstract The mechanism sketch of reporter gene assay for bioactivity determination of anti-IgE antibodies by RBL-2H3/FcεRIα/NFAT-Luc cells (left) and representative curves generated by the reporter gene assay (right).


Assuntos
Anticorpos Anti-Idiotípicos/análise , Bioensaio/métodos , Genes Reporter , Animais , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos
15.
Anal Chem ; 92(4): 3161-3170, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31983199

RESUMO

BVZ-BC (bevacizumab-biosimilar candidate) is a proposed biosimilar to bevacizumab. Bevacizumab binds to vascular endothelial growth factor (VEGF) type A and prevents the interaction of VEGF with its receptors on the surface of endothelial cells, neutralizing angiogenesis required for the growth, persistence, and metastases of solid tumors. An analytical comparison of BVZ-BC and bevacizumab was performed using state-of-the-art analytical techniques, including biochemical and biophysical characterization, biological activity, and immunological properties. Multiple attributes of the molecules were evaluated, including amino acid sequence, disulfide structure, glycan profiles, free thiol content, isoelectric point, protein content, subvisible particles, higher-order structure such as near- and far-ultraviolet circular dichroism and differential scanning calorimetry, product purity and product-related impurities, and process-related impurities. Biological activity assessment employed orthogonal assays such as the VEGF cell-based bioassay and the VEGF enzyme-linked immunosorbent assay, and Fc functional assays to interrogate all expected biological activities. An accumulation of 18 batches of bevacizumab (sourced in China, manufactured in Europe, Roche) and 10 batches of BVZ-BC representing unique drug product lots from each individual drug substance lot were utilized in this study. The analytical similarity between BVZ-BC and bevacizumab was assessed and demonstrated the similarities of all of the quality attributes between BVZ-BC and bevacizumab.


Assuntos
Bevacizumab , Medicamentos Biossimilares/química , Medicamentos Biossimilares/farmacologia , Bioensaio , Medicamentos Biossimilares/metabolismo , Polissacarídeos/química , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Biotechnol Adv ; 39: 107466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31697994

RESUMO

Complex structure and structure-function relationship of biopharmaceuticals require extensive analytical characterization and appropriate quality control of the products. Despite rapid development of sophisticated physicochemical techniques, biological activity measurement remains the critical role in inferring the high-order structure of biopharmaceuticals. Cell-based biological assays are mostly applied to determine the biological activity of biopharmaceuticals, however, refined biological assays are continually needed to increase their robustness. Reporter gene assays (RGAs) which are mechanism of action (MOA) related, less variable, accurate, precise, and labor-saving are becoming more and more recognized and adopted in the quality control. Here we discuss the importance of bioactivity determination, the strength and weakness of various assay formats with RGAs. We also introduce the mechanism of RGAs, and present a number of examples for RGAs to determine the bioactivity of various biopharmaceuticals, which indicate their extensive use in the screening, characterization, quality control, stability and biosimilarity study. We believe that with the rapid development of biotechnology, new strategies of bioassays based on RGAs will be more widely applied in various fields of biopharmaceuticals.


Assuntos
Bioensaio , Produtos Biológicos , Biotecnologia , Genes Reporter , Controle de Qualidade
17.
Jpn J Infect Dis ; 73(2): 111-118, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-31666494

RESUMO

Fever is a systemic inflammatory response of the body to pyrogens. Nuclear factor κB (NF-κB) is a central signaling molecule that causes the excessive secretion of various pyrogen-induced pro-inflammatory factors. This study explored the feasibility of a novel reporter gene assay (RGA) for pyrogen detection using RAW264.7 cells stably transfected with the NF-κB reporter gene as a pyrogenic marker. The RGA could detect different types of pyrogens, including the lipopolysaccharide of gram-negative bacteria, the lipoteichoic acid of gram-positive bacteria, and the zymosan of fungi, and a good dose-effect relationship was observed in terms of NF-κB activity. The limits of detection of the RGA to those pyrogens were 0.03 EU/ml, 0.001 µg/ml, and 1 µg/ml, respectively. The method had good precision and accuracy and could be applied to many molecules (e.g., nivolumab, rituximab, bevacizumab, etanercept, basiliximab, Haemophilus influenzae type b conjugate vaccine, 23-valent pneumococcal polysaccharide vaccine, group A and group C meningococcal conjugate vaccine, diphtheria, tetanus, pertussis [acellular, component], poliomyelitis [inactivated] vaccine, and imject alum adjuvant). The results of this study suggest that the novel RGA has a wide pyrogen detection spectrum and is sufficiently sensitive, stable, and accurate for various applications.


Assuntos
Genes Reporter , NF-kappa B/genética , Pirogênios/análise , Animais , Bioensaio/métodos , Febre/diagnóstico , Febre/etiologia , Limite de Detecção , Camundongos , Pirogênios/química , Células RAW 264.7 , Sensibilidade e Especificidade
18.
Emerg Microbes Infect ; 8(1): 1584-1592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31682199

RESUMO

The genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened.


Assuntos
Variação Antigênica , Vírus da Raiva/imunologia , Raiva/virologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Feminino , Cobaias , Humanos , Testes de Neutralização , Raiva/imunologia , Raiva/prevenção & controle , Vacinas Antirrábicas/administração & dosagem , Vacinas Antirrábicas/genética , Vacinas Antirrábicas/imunologia , Vírus da Raiva/química , Vírus da Raiva/genética , Vírus da Raiva/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
19.
Anal Bioanal Chem ; 410(27): 7067-7075, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30178083

RESUMO

IL-6 has an important role in the pathogenesis of autoimmunity and chronic inflammation. Several mAbs that target IL-6 or the IL-6 receptor (IL-6R) have been established and approved for the treatment of various diseases such as multicentric Castleman's disease and rheumatoid arthritis. Quality control of therapeutic antibodies requires accurate determination of bioactivity. However, current cell-based anti-proliferation assays are tedious, time consuming, and result in high variation. We therefore developed a reporter gene assay (RGA) based on an IL-6-dependent DS-1 cell line that stably expressed the reporter luciferase controlled by the serum-induced element (SIE) response element, which was a key element located downstream of the IL-6 signaling pathway. The RGA method demonstrated good performance characteristics after careful optimization, including high specificity, stability, accuracy, precision, and robustness. It also had superior precision and sensitivity. The assay is simple compared with the traditional anti-proliferation assay. This novel RGA based on the IL-6-IL-6R-STAT3 pathway can be useful, in conjunction with the anti-proliferation bioassay, to determine the bioactivity of anti-IL-6/anti-IL-6R therapeutic mAbs. Graphical abstract The mechanism sketch of the reporter gene assay for the bioactivity determination of anti-IL-6/anti-IL-6Rα mAbs.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Avaliação Pré-Clínica de Medicamentos , Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/imunologia , Engenharia Celular , Linhagem Celular , Proliferação de Células , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Humanos , Interleucina-6/imunologia , Luciferases/genética , Luciferases/imunologia , Receptores de Interleucina-6/imunologia , Proteínas Recombinantes/imunologia
20.
Electrophoresis ; 39(16): 2091-2098, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29797663

RESUMO

CE is central to the analysis, process development and approval of therapeutic monoclonal antibodies (mAbs). Recently, imaged capillary isoelectric focusing (icIEF) has emerged as a powerful technique for quantitative protein charge heterogeneity monitoring and characterization, particularly for mAbs. However, icIEF has yet to be validated for therapeutically relevant mAbs adhering to the ICH guideline (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use). Here, for the first time, icIEF technology was validated by 10 laboratories across 8 independent companies using a therapeutic mAb. The parameters of this method validation strictly follow the guideline of the ICH. This guideline includes specificity, precision, accuracy, linearity, range, LOQ and robustness. These results represent a significant step forward in standardizing the use of icIEF methods for the clinical approval of therapeutic mAbs.


Assuntos
Anticorpos Monoclonais/análise , Focalização Isoelétrica/métodos , Eletroforese Capilar/métodos , Eletroforese Capilar/normas , Guias como Assunto , Focalização Isoelétrica/normas , Laboratórios/normas , Reprodutibilidade dos Testes
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