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1.
J Pharm Sci ; 113(4): 1029-1037, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37839612

RESUMO

Monoclonal antibodies (mAbs) can be damaged during the aseptic compounding process, with aggregation being the most prevalent form of degradation. Protein aggregates represent one of several risk factors for undesired immunogenicity of mAbs, which can potentially lead to severe adverse drug reactions and less effective treatments. Since data on aggregate and particle formation by robotic compounding is missing, we aimed to compare the antibody stability between robotic- and manual compounding of mAbs with regard to formation of (sub)visible aggregates. Infliximab and trastuzumab were compounded into infusion bags with the APOTECAchemo robot or manually by nurses or pharmacy technicians. The products were analyzed by quantifying (sub)visible particles with nanoparticle tracking analysis, dynamic light scattering (DLS), light obscuration, micro-flow imaging, high pressure size exclusion chromatography (HP-SEC), and visual inspection. HP-SEC showed high percentages monomers in trastuzumab (99.4 % and 99.4 %) and infliximab (99.5 % and 99.6 %) infusion bags for both manual and robotic compounding, respectively. DLS indicated more consistent and reproducible results with robotic compounding, and confirmed monodisperse samples with a higher polydispersity index for manual compounding (0.16, interquartile range; IQR 0.14-0.18) compared to robotic compounding (0.12, IQR 0.11-0.15). This study shows that the studied compounding methods had a minor impact on the number of aggregates and particles, and that robotic compounding of mAbs provided at least similar quality as manual compounding.


Assuntos
Procedimentos Cirúrgicos Robóticos , Robótica , Humanos , Anticorpos Monoclonais/química , Infliximab/química , Robótica/métodos , Trastuzumab/química , Composição de Medicamentos/métodos
2.
J Pharm Biomed Anal ; 235: 115596, 2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-37540995

RESUMO

Monoclonal antibodies (mAbs) used as therapeutics need comprehensive characterization for appropriate quality assurance. For analysis, cost-effective methods are of high importance, especially when it comes to biosimilar development which is based on extended physicochemical characterization. The use of forced degradation to study the occurrence of modifications for analysis is well established in drug development and may be used for the evaluation of critical quality attributes (CQAs). For mAb analysis different procedures of liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses are commonly applied. In this study the middle-up approach is compared to the more expensive bottom-up analysis in a forced oxidation biosimilar comparability study. Bevacizumab and infliximab as well as biosimilar candidates for the two mAbs were forcefully oxidized by H2O2 for 24, 48 and 72 h. For bottom-up, the reduced and alkylated trypsin or Lys-C digested samples were analysed by LC-MS with quadrupole time-of-flight mass analyser (LC-QTOF-MS) to detect susceptible residues. By middle-up analysis several species of every subunit (Fc/2, light chain and Fd') were detected which differed in the number of oxidations. For the most abundant species, results from middle-up were in line with results from bottom-up analysis, confirming the strength of middle-up analysis. However, for less abundant species of some subunits, results differed between the two approaches. In both mAbs, the Fc was extensively oxidized. In infliximab, additional extensive oxidation was found in the Fab. Assignment to specific amino acid residues was finally possible using the results from bottom-up analyses. Interestingly, the C-terminal cysteine of the light chain was partially found triply oxidized in both mAbs. The comparison of susceptibility to oxidation showed high similarity between the reference products and their biosimilar candidates. It is suggested that the findings of middle-up experiments should be complemented by bottom-up analysis to confirm the assignments of the localization of modifications. Once the consistency of results has been established, middle-up analyses are sufficient in extended forced degradation biosimilar studies.


Assuntos
Medicamentos Biossimilares , Infliximab/química , Bevacizumab , Medicamentos Biossimilares/química , Peróxido de Hidrogênio , Anticorpos Monoclonais/química , Espectrometria de Massas/métodos
3.
J Pharm Biomed Anal ; 234: 115541, 2023 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-37399702

RESUMO

Despite reports indicating the potential impact of post-translational modifications on the activity of a monoclonal antibody, their prediction or monitoring post-administration remains a challenge. In addition, with the expiration of patents concerning the early generation of mAbs, the production of biosimilars is constantly increasing. Structural differences of biosimilars compared to the innovator product are commonly evaluated for the formulated product in the context of biosimilarity assessment. However, estimating their structural outcome after administration is particularly difficult. Due to the complexity of in vivo studies, there is a need to develop analytical strategies to predict PTMs consequently to their administration and their impact on mAbs potency. Here, we identified and evaluated the modification kinetics of 4 asparagine deamidations and 2 aspartate isomerizations of infliximab innovator product (Remicade®) and two biosimilars (Inflectra® and Remsima®) in vitro using serum incubation at 37 °C. The methodology was based on a bottom-up approach with capillary electrophoresis hyphenated with mass spectrometry analysis for an unequivocal assignment of modified and unmodified forms. 2 asparagines demonstrated a gradual deamidation correlated with incubation time. The specific extraction efficiency was evaluated to determine possible changes in the antigen binding affinity of infliximab with the incubation. Results showed the possibility to achieve an additional aspect concerning biosimilarity assessment, oriented on the study of the structural stability after administration.


Assuntos
Medicamentos Biossimilares , Infliximab/química , Medicamentos Biossimilares/química , Espectrometria de Massas em Tandem/métodos , Cinética , Processamento de Proteína Pós-Traducional , Eletroforese Capilar/métodos , Asparagina
4.
Artigo em Inglês | MEDLINE | ID: mdl-35605365

RESUMO

Therapeutic monoclonal antibodies (mAbs) represent a very important class of the current biopharmaceutics. The great complexity of their structure made necessary the use of different analytical approaches for assessing different physico-chemical properties. In this work, weak cation exchange (WCX) high performance liquid chromatography with diode array detection ((WCX)HPLC/DAD) is used to assess the charge variant profile. The method here developed combined the effect of ionic strength and controlled pH gradient and allows for the charge variants analysis of the five mAbs studied, namely bevacizumab (BVZ), cetuximab (CTX) infliximab (INF), rituximab (RTX) and trastuzumab (TTZ), which are among the most used mAbs worldwide. The differences in the charge variants in the natural isoforms of the mAbs promoted characteristic WCX chromatograms for each of mAbs that can be also useful for identification purposes. These chromatograms have provided to be suitable for tracking changes in the charge variants of each mAb analyzed both in controlled degraded and in stabilities study along time of in-use samples solutions at 2 mg/mL in 0.9% NaCl stored refrigerated (at 4 °C) and frozen (-20 °C) for two months. The results obtained indicated different stabilities of these mAbs, all IgG1, against degradation by different stressed environmental conditions and in-use stability along two months.


Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/química , Cátions , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Infliximab/química , Trastuzumab
5.
Carbohydr Polym ; 273: 118556, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34560967

RESUMO

The anti-tumor necrosis factor-α (anti-TNF-α) blocker, has shown great efficacy for the treatment of inflammatory bowel disease (IBD). However, systemic exposure to it can cause considerable safety problems due to reduced suppression of the systemic immune response and loss of response to the production of anti-drug antibodies. Thus, we try to devise a targeted vehicle system for oral administration of anti-TNF-α antibodies for the treatment of IBD. In the present study, we developed an oral Infliximab (IFX) loaded nano-in-microparticles, based on chitosan (CS)/carboxymethyl chitosan (CMC) and alginate (Alg), which could protect IFX from the harsh environment of the gastrointestinal tract and produce targeted drug delivery to the inflamed intestine. In vivo studies demonstrated that the IFX loaded nano-in-micro vehicle can alleviate colitis by ameliorating inflammation and maintaining the intestinal epithelial barrier.


Assuntos
Alginatos/química , Quitosana/análogos & derivados , Doenças Inflamatórias Intestinais/tratamento farmacológico , Infliximab/administração & dosagem , Nanopartículas/química , Administração Oral , Animais , Quitosana/química , Colite/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Feminino , Células HT29 , Humanos , Inflamação/tratamento farmacológico , Doenças Inflamatórias Intestinais/metabolismo , Infliximab/química , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Inibidores do Fator de Necrose Tumoral/administração & dosagem , Fator de Necrose Tumoral alfa/metabolismo
6.
BioDrugs ; 35(1): 75-87, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33306186

RESUMO

BACKGROUND: Regions within England, Scotland and Wales show variation in rate of adoption of biosimilar infliximab and etanercept. OBJECTIVES: This study aims to examine how local decisions and practices in regions within England, Scotland and Wales might explain initial variation in market dynamics of biosimilar and originator infliximab and etanercept. METHODS: Market data provided by the National Health Service (NHS) on biosimilar and originator infliximab and etanercept uptake were analysed for the 10 historical regions of England, 14 health boards in Scotland and 7 health boards in Wales (2015-2018). Findings were discussed in ten semi-structured interviews: on a national level with an industry representative (1), on a regional level with NHS employees in England (6), Scotland (1) and Wales (1), and on a local level with a representative of a clinical commissioning group in England (1). RESULTS: Tenders for infliximab and etanercept in England, Scotland and Wales have consistently resulted in a biosimilar as the best value biological. Early and late biosimilar adopters are seen, with overall convergence towards high biosimilar market shares over time. Qualitative results suggest that biosimilar adoption was positively influenced by (a) a price difference between biosimilar and originator product making it worthwhile to switch patients; (b) a good relationship between commissioner and provider in England resulting in gain share agreements; (c) leadership on biosimilars in regional NHS offices in England or Scottish and Welsh health boards; (d) key opinion leaders or leading hospitals that start using biosimilars early and gain experience. CONCLUSIONS: This study has shown that the savings potential drives biosimilar use. Regions with a proactive attitude, good stakeholder relationships, and clinician engagement were identified as early adopters.


Assuntos
Medicamentos Biossimilares , Infliximab/química , Inglaterra , Etanercepte/química , Etanercepte/metabolismo , Humanos , Infliximab/farmacologia , Escócia , Medicina Estatal , País de Gales
7.
Artigo em Inglês | MEDLINE | ID: mdl-33360415

RESUMO

Charge variants are the most commonly observed sources of heterogeneity in the routine manufacturing of monoclonal antibodies. To gain further insight into the structural foundation of charge heterogeneity and its influence on biological functions, an infliximab biosimilar HS626 from a biopharmaceutical facility was isolated by semipreparative cation exchange chromatography (CEX) to obtain fractions of acidic and basic charge variants and determine the main species. It was assessed again by CEX to ensure purities. Through a series of structural and physicochemical characterizations, we concluded that the acidic variants were caused by fragments, Met oxidation, Asn deamidation, higher levels of sialylation and galactosylation of N-linked glycans, and less high mannose. The basic variants resulted mainly from aggregates, fragments, and Met oxidation. Through further analysis of antigen binding affinity, cell death inhibitory activity, ADCC, and CDC, as well as FcRn, FcγRIIIa, and C1q affinity, we demonstrated that the charge heterogeneity did not affect biological functions. This research enhances the understanding of charge variants, which are usually effective components that should not be intentionally reduced unless biological functions are affected.


Assuntos
Medicamentos Biossimilares , Infliximab , Sequência de Aminoácidos , Animais , Medicamentos Biossimilares/análise , Medicamentos Biossimilares/química , Medicamentos Biossimilares/isolamento & purificação , Células CHO , Linhagem Celular , Cromatografia por Troca Iônica/métodos , Cricetinae , Cricetulus , Glicosilação , Infliximab/análise , Infliximab/química , Infliximab/isolamento & purificação , Camundongos
8.
Bioconjug Chem ; 31(8): 1960-1970, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32609496

RESUMO

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in synovial joints and protease-induced cartilage degradation. Current biologic treatments for RA can effectively reduce symptoms, primarily by neutralizing the proinflammatory cytokine TNFα; however, continued, indiscriminate overinhibition of inflammatory factors can significantly weaken the host immune system, leading to opportunistic infections and interrupting treatment. We hypothesize that localizing anti-TNFα therapeutics to denatured collagen (dCol) present at arthritic joints, via conjugation with collagen-hybridizing peptides (CHPs), will reduce off-site antigen binding and maintain local immunosuppression. We isolated the antigen-binding fragment of the clinically approved anti-TNFα therapeutic infliximab (iFab) and prepared iFab-CHP conjugates via lysine-based conjugation with an SMCC linker. After successful conjugation, confirmed by LC-MS, the binding affinity of iFab-CHP was characterized by ELISA-like assays, which showed comparable antigen binding relative to infliximab, comparable dCol binding relative to CHP, and the hybrid ability to bind both dCol and TNFα simultaneously. We further demonstrated localization of Fab-CHP to areas of high dCol in vivo and promising therapeutic efficacy, assessed by histological staining (Safranin-O and H&E), in a pilot mouse study.


Assuntos
Colágeno/química , Fragmentos Fab das Imunoglobulinas/química , Peptídeos/química , Animais , Anticorpos , Antígenos , Antirreumáticos/química , Antirreumáticos/farmacologia , Cromatografia Líquida , Feminino , Fragmentos Fab das Imunoglobulinas/imunologia , Infliximab/química , Infliximab/farmacologia , Espectrometria de Massas , Camundongos , Camundongos Nus , Camundongos Transgênicos , Ligação Proteica , Fator de Necrose Tumoral alfa
9.
BioDrugs ; 34(4): 529-539, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32696266

RESUMO

BACKGROUND: Long-term, real-world data are required to support the use of CT-P13 in chronic conditions such as ankylosing spondylitis. However, real-world evidence may be influenced by selection bias, which can confound outcomes. The aim of the current analysis was to confirm the long-term comparability of CT-P13 and reference infliximab treatment in patients with ankylosing spondylitis, using propensity score matching to adjust for baseline differences between groups. METHODS: A propensity score-matching analysis was conducted on data from patients with ankylosing spondylitis in the Korean College of Rheumatology Biologics registry who received CT-P13 or reference infliximab. Drug retention, reasons for biologic therapy changes or discontinuation, and efficacy parameters were analyzed overall and by treatment line with up to 4 years of follow-up. Adverse events were recorded for each treatment group. RESULTS: Propensity score matching was effective in matching 124 CT-P13-treated and 124 reference infliximab-treated patients. Median treatment duration and drug retention were similar between CT-P13 and reference infliximab. Three-year retention rates (95% confidence interval [CI]) were 64.2% (53.5-73.0) for CT-P13 and 55.6% (42.9-66.6) for reference infliximab. Overall, 17.1% (CT-P13) and 29.3% (reference infliximab) of patients discontinued biologic therapy, and 20.0% (CT-P13) and 15.2% (reference infliximab) changed biologic therapy. Efficacy assessments were generally similar between groups; both treatments were well tolerated. CONCLUSIONS: Propensity score-matching analysis confirmed that CT-P13 treatment was not associated with significant differences in drug retention, treatment duration, most efficacy parameters, or safety versus reference infliximab in Korean patients with ankylosing spondylitis, building evidence for the long-term comparability of these treatments. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01965132.


Assuntos
Anticorpos Monoclonais/química , Antirreumáticos , Medicamentos Biossimilares , Reumatologia , Espondilite Anquilosante , Anticorpos Monoclonais/farmacologia , Antirreumáticos/uso terapêutico , Medicamentos Biossimilares/efeitos adversos , Humanos , Infliximab/efeitos adversos , Infliximab/química , Pontuação de Propensão , Sistema de Registros , República da Coreia , Espondilite Anquilosante/tratamento farmacológico , Resultado do Tratamento
10.
Pharm Res ; 37(6): 114, 2020 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-32476063

RESUMO

PURPOSE: ABP 710 has been developed as a biosimilar to infliximab reference product (RP). The objective of this study was to assess analytical similarity (structural and functional) between ABP 710 and infliximab RP licensed by the United States Food and Drug Administration (infliximab [US]) and the European Union (infliximab [EU]), using sensitive, state-of-the-art analytical methods capable of detecting minor differences in product quality attributes. METHODS: Comprehensive analytical characterization utilizing orthogonal techniques was performed with 14 to 28 unique lots of ABP 710 or infliximab RP, depending on the assay. Comparisons were used to investigate the primary structure related to amino acid sequence; post-translational modifications (PTMs) including glycans; higher order structure; particles and aggregates; primary biological properties mediated by target and receptor binding; product-related substances and impurities; and general properties. RESULTS: ABP 710 had the same amino acid sequence, primary structure, higher order structure, PTM profiles and biological activities as infliximab RP. The finished drug product had the same strength (protein content and concentration) as infliximab RP. CONCLUSIONS: Based on the comprehensive analytical similarity assessment, ABP 710 was found to be highly analytically similar to infliximab RP for all biological activities relevant for clinical efficacy and safety.


Assuntos
Anticorpos Monoclonais/análise , Medicamentos Biossimilares/análise , Infliximab/análise , Sequência de Aminoácidos , Medicamentos Biossimilares/química , Dicroísmo Circular , Humanos , Infliximab/química , Processamento de Proteína Pós-Traducional , Espectroscopia de Infravermelho com Transformada de Fourier
11.
SLAS Technol ; 25(4): 380-387, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32458729

RESUMO

Protein glycosylation can impact the efficacy and safety of biotherapeutics and therefore needs to be well characterized and monitored throughout the drug product life cycle. Glycosylation is commonly assessed by fluorescent labeling of released glycans, which provides comprehensive information of the glycoprofile but can be resource-intensive regarding sample preparation, data acquisition, and data analysis. In this work, we evaluate a comprehensive solution from sample preparation to data reporting using a liquid chromatography-mass spectrometry (LC-MS)-based analytical platform for increased productivity in released glycan analysis. To minimize user intervention and improve assay robustness, a robotic liquid handling platform was used to automate the release and labeling of N-glycans within 2 h. To further increase the throughput, a 5 min method was developed on a liquid chromatography-fluorescence-mass spectrometry (LC-FLR-MS) system using an integrated glycan library based on retention time and accurate mass. The optimized method was then applied to 48 released glycan samples derived from six batches of infliximab to mimic comparability testing encountered in the development of biopharmaceuticals. Consistent relative abundance of critical species such as high mannose and sialylated glycans was obtained for samples within the same batch (mean percent relative standard deviation [RSD] = 5.3%) with data being acquired, processed, and reported in an automated manner. The data acquisition and analysis of the 48 samples were completed within 6 h, which represents a 90% improvement in throughput compared with conventional LC-FLR-based methods. Together, this workflow facilitates the rapid screening of glycans, which can be deployed at various stages of drug development such as process optimization, bioreactor monitoring, and clone selections, where high-throughput and improved productivity are particularly desired.


Assuntos
Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/métodos , Polissacarídeos/análise , Coloração e Rotulagem , Espectrometria de Massas em Tandem , Automação , Cromatografia Líquida , Infliximab/química , Soluções
12.
J Pharm Biomed Anal ; 185: 113252, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32193039

RESUMO

Immunogenicity related to the degradation of therapeutic monoclonal antibodies (mAbs) remains a major concern for their therapeutic efficacy and safety. Therefore, an analytical method allowing characterization and detection of mAbs degradation is mandatory. In this study, a simultaneous coupling of size exclusion chromatography (SEC) to native mass spectrometry (MS) and fluorescence detection (FLD) is proposed to detect degraded therapeutic mAbs and biases of structural changes (e.g. dimerization, denaturation) that may occur during native MS. A comprehensive study on infliximab behaviors have been performed under different mobile phase conditions (e.g. composition, pH, organic solvent, etc.) and MS parameters (e.g. gas temperatures, CID energies, etc.). Experimental conditions avoiding artificial denaturation and/ or dimerization have been defined. We have also demonstrated that under the developed conditions infliximab affinity towards its biological target TNFα is preserved. In addition, using this method dimers, denatured monomers and fragments could be detected in trastuzmab samples stressed by a long-term storage. These results were confirmed by using SEC coupled to ion mobility mass spectrometry as an orthogonal method for the detection of denatured monomer.


Assuntos
Anticorpos Monoclonais/análise , Controle de Qualidade , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Química Farmacêutica/métodos , Cromatografia em Gel/métodos , Armazenamento de Medicamentos , Estudos de Viabilidade , Infliximab/análise , Infliximab/química , Infliximab/uso terapêutico , Espectrometria de Massas/métodos , Conformação Proteica , Proteólise , Trastuzumab/análise , Trastuzumab/química , Trastuzumab/uso terapêutico
13.
BioDrugs ; 34(2): 225-233, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31925703

RESUMO

BACKGROUND: Biosimilars must meet stringent regulatory requirements, both at the time of authorization and during their lifecycle. Yet it has been suggested that divergence in quality attributes over time may lead to clinically meaningful differences between two versions of a biologic. Therefore, this study investigated the batch-to-batch consistency across a range of parameters for released batches of the etanercept biosimilar (SB4) and infliximab biosimilar (SB2). METHODS: SB4 (Benepali®) and SB2 (Flixabi®) were both developed by Samsung Bioepis and are manufactured in Europe by Biogen at their facility in Hillerød, Denmark. A total of 120 batches of SB4 and 25 batches of SB2 were assessed for consistency and compliance with specified release parameters, including purity, post-translational glycosylation (SB4 only), protein concentration, and biological activity. RESULTS: The protein concentration, purity, tumor necrosis factor-α (TNF-α) binding, and TNF-α neutralization of all batches of SB4 and SB2 were within the strict specification limits set by regulatory agencies, as was the total sialic acid (TSA) content of all batches of SB4. CONCLUSIONS: Quality attributes of SB4 and SB2 batches showed little variation and were consistently within the rigorous specifications defined by regulatory agencies.


Assuntos
Anti-Inflamatórios não Esteroides/normas , Antirreumáticos/normas , Medicamentos Biossimilares/normas , Etanercepte/normas , Tecnologia Farmacêutica/normas , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/imunologia , Anti-Inflamatórios não Esteroides/farmacologia , Antirreumáticos/química , Antirreumáticos/farmacologia , Medicamentos Biossimilares/química , Medicamentos Biossimilares/farmacologia , Etanercepte/química , Etanercepte/farmacologia , Europa (Continente) , Glicosilação , Humanos , Infliximab/química , Infliximab/farmacologia , Ácido N-Acetilneuramínico , Controle de Qualidade , Tecnologia Farmacêutica/métodos , Fator de Necrose Tumoral alfa
14.
Biologicals ; 63: 39-47, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31883741

RESUMO

The generation of anti-drug antibodies (ADAs) is one of the most serious problems in therapy using monoclonal antibodies (mAbs), because ADAs can impact the pharmacokinetics, efficacy, and safety of mAbs. It is therefore important to detect the generated ADAs in patients. For the appropriate detection of ADAs, methods that detect various types of ADAs (e.g., low- and high-affinity ADAs) are needed, but since there are no adequate reference preparations of ADAs relevant to human ADAs in most cases, it is difficult to determine whether or not the developed methods have enough analytical performance. Here, we developed human-rat chimeric ADA panels against the anti-TNF-α therapeutic antibodies infliximab and adalimumab. The developed ADA panels consist of 7 (for infliximab) and 11 (for adalimumab) ADAs with various binding characters, and most of the ADAs are neutralizing antibodies. Using these ADA panels, we compared the detectability of model methods, i.e., binding assays using SPR, BLI, and ECL, and a cell-based assay to detect neutralization activity. Since we obtained ADAs showing low and high responses with the various methods, the ADA panels we developed were shown to be useful for the development of ADA assays.


Assuntos
Adalimumab , Anticorpos Neutralizantes , Infliximab , Adalimumab/química , Adalimumab/imunologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Células HEK293 , Humanos , Infliximab/química , Infliximab/imunologia , Ratos
15.
BioDrugs ; 34(1): 77-87, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31650490

RESUMO

BACKGROUND: Higher-order structure (HOS) assessment is an important component of biosimilarity evaluations. While established spectroscopic methods are routinely used to characterize structure and evaluate similarity, the addition of X-ray crystallographic analysis to these biophysical methods enables orthogonal elucidation of HOS at higher resolution. METHODS: Crystal structures of the infliximab biosimilar PF-06438179/GP1111 and the reference product Remicade®, sourced from US and European Union markets, were determined and compared to evaluate HOS similarity. Analytical ultracentrifugation studies were conducted to understand reversible self-association. RESULTS: In contrast to more routine spectroscopic methods, the crystal structures enable three-dimensional assessment of complementarity-determining regions and other local regions at near-atomic resolution. The biosimilar structures are highly similar to those of the reference product, as demonstrated visually and though all-atom root-mean-squared deviation measurements. CONCLUSION: The structures provide new insights into the physicochemical properties of the proposed biosimilar and the reference product, further strengthening the 'totality of evidence' in the evaluation of similarity.


Assuntos
Medicamentos Biossimilares/química , Infliximab/química , União Europeia , Humanos
16.
Mikrochim Acta ; 186(12): 780, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729556

RESUMO

Infliximab (INF) is a chimeric monoclonal immunoglobulin acting against tumor necrosis factor-alpha (TNF-α). The drug is used for the treatment of chronic autoimmune and inflammatory diseases. A target-specific nanomaterial is presented for the extraction of INF from human plasma along with a label-free surface enhanced Raman spectroscopy (SERS) method for its determination using a handheld device. A gold-coated copper oxide chip was functionalized with TNF-α and used to extract the drug from plasma. INF was recovered from the extractor by lowering the pH value to 2.5. The disulfide bond structure of the drug was then reduced and used for its oriented chemisorption onto a gold-coated copper oxide substrate for SERS measurements using the INF-specific band at 936 cm-1. The working range of the SERS method was between 10-7 and 10-14 M of reduced INF. The relative standard deviation (RSD), between three different measurements was 4.2% (intra-day) and 7.1% (inter-day). The quantification and detection limits of the assay (LOQ, LOD) were 0.01 pM and 1.4 fM respectively. The SERS detection was cross-validated against ELISA where 99% agreement was found between the two methods. Graphical abstractSchematic representation of the determination of Infliximab (INF) in blood. A gold coated copper oxide chip was functionalised with tumor necrosis factor (TNF-α) and used to extract INF from blood plasma. The captured INF was released, reduced, chemisorbed onto a second gold-coated copper oxide substrate and screened by surface-enhanced Raman spectroscopy (SERS) using a handheld device.


Assuntos
Infliximab/sangue , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Fator de Necrose Tumoral alfa/química , Cobre/química , Ouro/química , Humanos , Infliximab/química , Infliximab/isolamento & purificação , Limite de Detecção , Oxirredução , Óxidos/química , Estudo de Prova de Conceito , Extração em Fase Sólida/métodos
17.
Anal Chem ; 91(24): 15769-15776, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31663332

RESUMO

We developed Pyr1-infliximab: a two-photon probe for TNF-α. Pyr1-infliximab showed absorption maxima at 280 and 438 nm and an emission maximum at 610 nm in an aqueous buffer and effective two-photon action cross-section values of (520-2830) × 10-50 cm4s/photon in RAW 264.7 cells. After this probe was labeled, it was possible to detect Pyr1-infliximab-transmembrane TNF-α complexes in a live cell and to determine the relative proportion of these complexes in human colon tissues. This proportion among healthy, possibly inflamed, and inflamed tissues of patients with ulcerative colitis was found to be 1.0/4.5/10. This probe may find useful applications for selective detection of transmembrane TNF-α in a live cell or tissue, for quantification of inflammation in human colon tissue or of antidrug antibodies in patients who stop responding to anti-TNF therapy, and for monitoring of the response to this therapy.


Assuntos
Colo/metabolismo , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Carbazóis/química , Sobrevivência Celular/efeitos dos fármacos , Colo/patologia , Corantes Fluorescentes/toxicidade , Humanos , Concentração de Íons de Hidrogênio , Infliximab/química , Infliximab/imunologia , Camundongos , Fotólise , Células RAW 264.7 , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/imunologia
18.
Anal Chim Acta ; 1089: 1-18, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31627805

RESUMO

Over the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of biosimilar products. As a result, multiple biosimilar mAbs are becoming available for a single originator drug. As opposed to small-molecular drugs, protein biopharmaceuticals do not have fully defined and reproducible structures, making it impossible to create identical copies. Therefore, regulators demand biosimilar sponsors to demonstrate similarity with the reference product to prevent safety and efficacy issues with the proposed product. Protein glycosylation is considered a crucially important quality attribute, because of its major role in immunogenicity and clinical efficacy of therapeutic proteins. However, the intrinsic biological variability of glycan structures creates a significant challenge for the current analytical platforms. In this review, we discuss the importance of glycan characterization on therapeutic proteins, with a particular focus on the analytical techniques applied for glycan profiling of biosimilar mAb products. In addition, we present a case study on infliximab biosimilars to illustrate the potential clinical implications of differences in glycan profile between originator and biosimilar mAb products.


Assuntos
Anticorpos Monoclonais/análise , Medicamentos Biossimilares/análise , Glicoproteínas/análise , Polissacarídeos/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Medicamentos Biossimilares/química , Medicamentos Biossimilares/metabolismo , Cromatografia Líquida , Glicoproteínas/química , Glicosilação , Humanos , Imunoglobulina G/análise , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Infliximab/análise , Infliximab/química , Infliximab/metabolismo , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem
19.
Eur J Pharm Sci ; 137: 104993, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31302214

RESUMO

Therapeutic protein medicines have transformed the treatment of blinding diseases (e.g. age-related macular degeneration, AMD) during the last 1-2 decades. Many blinding conditions such as AMD are chronic; and require multiple intravitreal injections over a long period to achieve a high and reproducible dose needed for clinical benefit. Prolonging the duration of action of ophthalmic drugs is critical to reduce the frequency of injections. Thermoresponsive hydrogels (e.g. N-isopropylacrylamide, NIPAAM) that collapse in physiological conditions can entrap and sustain the release of a therapeutic protein. However, most NIPAAM hydrogels are not biodegradable and often requires invasive surgery to remove the depot. Here, we report the preparation of a hydrogel derived from NIPAAM and acrylated hyaluronic acid (Ac-HA) as a biodegradable, macromolecular crosslinker. Ac-HA was prepared by the acrylation of hyaluronic acid (HA). Antibody (infliximab (INF), 5.0 mg/mL or bevacizumab (BEVA), 12.5 mg/mL), NIPAAM (0.35 mmol) and Ac-HA (2.0-10.0 mg/mL, 40.0-200.0 nmol) were first mixed prior to redox polymerisation to ensure maximal protein mixing and to shorten the burst release. Hydrogels with lower amounts of Ac-HA (2.0-4.0 mg/mL, 40.0-80.0 nmol) showed favourable lower critical solution temperature (LCST) values and injectability (27-29G) than higher amounts of Ac-HA (>4.0 mg/mL, >80.0 nmol). These hydrogels were further characterised (swelling ratio (SR), water retention (WR) and rheology). All hydrogels degraded in presence of bovine testes hyaluronidase (0-50 U/mL, 37 °C, 100 rpm). Release studies of BEVA-loaded hydrogels were investigated in vitro using the PK-Eye™ model, which estimates the human clearance times of proteins from the back of the eye. Phosphate buffered saline (PBS, pH 7.4, 37 °C) was used rather than simulated vitreous to more effectively map trends between the formulations. A zero-order release profile was observed between days 5 to 50 with 43.3 ±â€¯9.5% protein released at day 50. Determining protein binding and functionality from a formulation is crucial to determine the optimal formulation prior to more detailed studies that might be necessary. BEVA showed binding to human vascular growth endothelial factor (VEGF165) throughout the study (two months) while still maintaining a therapeutic dose (123.5 ±â€¯45.6 ng) in the posterior cavity of the PK-Eye™ model. These encouraging results suggest that extended release of proteins in the vitreous can be achieved using injectable hydrogels derived from NIPAAM and HA.


Assuntos
Acrilamidas/administração & dosagem , Inibidores da Angiogênese/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Bevacizumab/administração & dosagem , Ácido Hialurônico/administração & dosagem , Hidrogéis/administração & dosagem , Infliximab/administração & dosagem , Acrilamidas/química , Anti-Inflamatórios/química , Bevacizumab/química , Olho/metabolismo , Humanos , Ácido Hialurônico/química , Hialuronoglucosaminidase/química , Hidrogéis/química , Infliximab/química , Injeções Intravítreas , Modelos Biológicos
20.
Drug Des Devel Ther ; 13: 791-805, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30880912

RESUMO

BACKGROUND: Infliximab (Remicade), a chimeric monoclonal antibody against human TNFα, will inevitably face competition from biosimilar products, because of its effectiveness in autoimmune diseases and rapidly increasing market demand. According to guidelines for biosimilar development, the "biosimilar-expression system" may differ from that of the innovator, but more appropriate studies should be carried out to demonstrate the comparability between biosimilar and innovator. CMAB008 is an infliximab biosimilar candidate developed by the State Key Laboratory of Antibody Medicine and Targeted Therapy of China. Infliximab was expressed in SP2/0 cells, while CMAB008 was produced in a CHO-expression system. METHODS: In this study, infliximab and CMAB008 were compared on physicochemical and biological characterizations, including protein content, activity, physiochemical integrity, impurities, additives, and immunogenicity. RESULTS: The results showed that they were highly similar and comparable, except some differences in glycosylation. As glycosylation profiles can influence immunogenicity and occurrence of allergy or other adverse reactions of antibody therapeutics, primary tolerability and pharmacokinetics of CMAB008 were evaluated. In the phase I clinical trial, plasma concentration of CMAB008 and antidrug antibodies were also measured using ELISA and bridging ELISA, respectively. CMAB008 exhibited favorable clinical tolerability, no adverse events in the 3 mg/kg single-dose group (recommended therapeutic dosage), and no serious adverse events in the multiple-dose group. Also, no injection-site reactions were observed in the experiment. CONCLUSION: In summary, CMAB008 might have the potential to be an effective drug compared with infliximab.


Assuntos
Infliximab/química , Infliximab/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Físico-Química , Voluntários Saudáveis , Humanos , Infliximab/efeitos adversos , Infliximab/farmacocinética , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos , Modelos Animais
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