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1.
J Chromatogr A ; 1728: 464995, 2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-38805895

RESUMO

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal laser scanning microscopy (CLSM), but also as a tracer for determination of residence time distribution (RTD) in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. In this work, the site occupancy of two labeled antibodies, MAb1 (IgG1 subclass) and MAb2 (IgG2 subclass) conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the MAb1 and MAb2 molecules, in positions 188 and 189 respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling, despite the secondary region responsible for binding to the protein A was partly labeled. Overall, the fluorescent-labeled antibodies are a good compromise as an inert tracer in residence time distribution and chromatography studies because they are much cheaper than isotope-labeled antibodies; However, the differences between the labeled and unlabeled antibodies should be considered.


Assuntos
Anticorpos Monoclonais , Corantes Fluorescentes , Proteína Estafilocócica A , Corantes Fluorescentes/química , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Cromatografia de Afinidade/métodos , Sítios de Ligação , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Espectrometria de Massas em Tandem/métodos , Mapeamento de Peptídeos/métodos , Animais
2.
MAbs ; 16(1): 2341641, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38652517

RESUMO

Peptide mapping with mass spectrometry (MS) is an important tool for protein characterization in the biopharmaceutical industry. Historically, peptide mapping monitors post-translational modifications (PTMs) of protein products and process intermediates during development. Multi-attribute monitoring (MAM) methods have been used previously in commercial release and stability testing panels to ensure control of selected critical quality attributes (CQAs). Our goal is to use MAM methods as part of an overall analytical testing strategy specifically focused on CQAs, while removing or replacing historical separation methods that do not effectively distinguish CQAs from non-CQAs due to co-elution. For example, in this study, we developed a strategy to replace a profile-based ion-exchange chromatography (IEC) method using a MAM method in combination with traditional purity methods to ensure control of charge variant CQAs for a commercial antibody (mAb) drug product (DP). To support this change in commercial testing strategy, the charge variant CQAs were identified and characterized during development by high-resolution LC-MS and LC-MS/MS. The charge variant CQAs included PTMs, high molecular weight species, and low molecular weight species. Thus, removal of the IEC method from the DP specification was achieved using a validated LC-MS MAM method on a QDa system to directly measure the charge variant PTM CQAs in combination with size exclusion chromatography (SE-HPLC) and capillary electrophoresis (CE-SDS) to measure the non-PTM charge variant CQAs. Bridging data between the MAM, IEC, and SE-HPLC methods were included in the commercial marketing application to justify removing IEC from the DP specification. We have also used this MAM method as a test for identity to reduce the number of QC assays. This strategy has received approvals from several health authorities.


Assuntos
Anticorpos Monoclonais , Mapeamento de Peptídeos , Cromatografia por Troca Iônica/métodos , Anticorpos Monoclonais/química , Mapeamento de Peptídeos/métodos , Humanos , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem/métodos , Controle de Qualidade
3.
J Pharm Biomed Anal ; 243: 116124, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38520959

RESUMO

Peptide mapping is the key method for characterization of primary structure of biotherapeutic proteins. This method relies on digestion of proteins into peptides that are then analyzed for amino acid sequence and post-translational modifications. Owing to its high activity and cleavage specificity, trypsin is the protease of choice for peptide mapping. In this study, we investigated critical requirements of peptide mapping and how trypsin affects these requirements. We found that the commonly used MS-grade trypsins contained non-specific, chymotryptic-like cleavage activity causing generation of semi-tryptic peptides and degradation of tryptic-specific peptides. Furthermore, MS-grade trypsins contained pre-existing autoproteolytic peptides and, moreover, additional autoproteolytic peptides were resulting from prominent autoproteolysis during digestion. In our long-standing quest to improve trypsin performance, we developed novel recombinant trypsin and evaluated whether it could address major trypsin drawbacks in peptide mapping. The study showed that the novel trypsin was free of detectable non-specific cleavage activity, had negligible level of autoproteolysis and maintained high activity over the course of digestion reaction. Taking advantage of the novel trypsin advanced properties, especially high cleavage specificity, we established the application for use of large trypsin quantities to digest proteolytically resistant protein sites without negative side effects. We also tested trypsin/Lys-C mix comprising the novel trypsin and showed elimination of non-specific cleavages observed in the digests with the commonly used trypsins. In addition, the improved features of the novel trypsin allowed us to establish the method for accurate and efficient non-enzymatic PTM analysis in biotherapeutic proteins.


Assuntos
Fragmentos de Peptídeos , Proteínas , Mapeamento de Peptídeos/métodos , Tripsina/química , Fragmentos de Peptídeos/química , Peptídeos/análise
4.
J Pharm Biomed Anal ; 243: 116094, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38479303

RESUMO

BACKGROUND: Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic peptide drugs, and peptide drug products including side chain modifications in the Peptide drugs. This article explains a step-by-step guide to developing a high-throughput method using high resolution mass spectrometry for characterization of Calcitonin Salmon injection containing high proportion of UV-active excipients. METHODS: The major challenge in the method development of Amino acid sequencing and Peptide mapping was presence of phenol in drug product. Phenol is a UV-active excipient and reacts with both Dithiothreitol (DTT) and Trypsin. Hence Calcitonin Salmon was extracted from the Calcitonin Salmon injection using solid phase extraction after the extraction, Amino acid sequencing and peptide mapping study was performed. Upon incubation of Calcitonin Salmon with Trypsin and DTT, digested fragments were generated which were separated by mass compatible reverse phase chromatography and the molecular mass of each fragment was determined using HRMS. RESULTS: A reverse phase chromatographic method was developed using UHPLC-HRMS for the determination of direct mass, peptide mapping and to determine the amino acid sequencing in the Calcitonin Salmon injection. The method was found Specific and fragments after trypsin digest are well resolved from each other and the molecular mass of each fragment was determined using HRMS. Sequencing was performed using automated identification of b and y ions annotation and identifications based on MS/MS spectra using Biopharma finder and Proteome discoverer software. CONCLUSION: Using this approach 100% protein coverage was obtained and protein was identified as Calcitonin Salmon and the observed masses of tryptic digest of peptide was found similar with theoretical masses. The method can be used for both UV and MS based Peptide mapping and whereas the UV based peptide mapping method can be used as identification test for Calcitonin Salmon drug substance and drug product in quality control.


Assuntos
Calcitonina , Peptídeos , Espectrometria de Massas em Tandem , Mapeamento de Peptídeos , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Tripsina/metabolismo , Análise de Sequência de Proteína , Proteoma , Fenóis
5.
J Pharm Biomed Anal ; 243: 116098, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38493753

RESUMO

Antibody-drug conjugates (ADCs) are a heterogeneous mixture of conjugated species with varied drug loadings. Depending on conjugation sites, linkers and drugs can exhibit different stability as influenced by the solvent-accessibility and local charge, resulting in different ADC efficacy, pharmacokinetics, and toxicity. Conjugation site analysis is critical for ADC structural characterization to assure product quality and consistency. It enables early conjugation studies at site-specific levels, confirms the absence of unexpected products to support conjugation process development, and aids in ensuring lot-to-lot consistency for comparability studies. Peptide mapping using liquid chromatography-tandem mass spectrometry is the industry standard method for analyzing conjugation sites. However, some concerns remain for this approach as the large and hydrophobic drug moieties often result in poor MS/MS fragmentation quality and impede the identification of conjugation sites. Additionally, the ionization discrepancy between conjugated and unconjugated peptides can lead to a relatively large bias for site occupancy calculation. In this work, we present a simple drug deconjugation-assisted peptide mapping method to identify and quantify the drug conjugation for ADCs with protease-cleavable linkers. Papain-based drug deconjugation was used to remove the highly hydrophobic drug moiety, which significantly improved the quantitation accuracy of conjugation level and the fragmentation quality. Sample preparation conditions were optimized to avoid introducing artificial modifications, allowing the tracking of initial sample status and subsequent changes of quality attributes during process development and stability assessment. This method was applied to analyze thermally-stressed ADC samples to monitor changes of site-specific conjugation levels, DAR, succinimide hydrolysis of the linker, and various PTMs. We believe this is an effective and straightforward tool for conjugation site analysis while simultaneously monitoring multiple quality attributes for ADCs with protease-cleavable linkers.


Assuntos
Imunoconjugados , Imunoconjugados/química , Cromatografia Líquida/métodos , Espectrometria de Massa com Cromatografia Líquida , Espectrometria de Massas em Tandem , Mapeamento de Peptídeos , Papaína
6.
J Pharm Sci ; 113(6): 1498-1505, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38342339

RESUMO

Stop codon readthroughs were examined in 48 recombinant therapeutic protein candidates produced from multiple clones of Chinese hamster ovary cells, using peptide mapping with LC-MS/MS detection. We found that stop codon readthrough is a common phenomenon occurring in most of these candidates, with levels varying from below the detection limit of ∼0.001 % to ∼1 %. The readthrough propensity depends on the stop codon being used, as well as the nucleotides surrounding it. The amino acids misincorporated into the stop position can be well-predicted by a third-base wobble mismatch and a first-base U/G mismatch during codon recognition, i.e., tyrosine or glutamine insertion for the UAA and UAG stop codons, and tryptophan, cysteine or arginine insertion for the UGA stop codon. Data shown in this report demonstrate the importance of optimizing the DNA sequence near the stop codon, and the importance of detecting stop codon readthroughs during the development of a therapeutic product.


Assuntos
Códon de Terminação , Cricetulus , Proteínas Recombinantes , Células CHO , Animais , Códon de Terminação/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Cricetinae , Mapeamento de Peptídeos/métodos , Biossíntese de Proteínas/genética
7.
Biotechnol Appl Biochem ; 71(3): 627-640, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38311972

RESUMO

Parmotrema perlatum, a lichen belonging to the family Parmeliaceae, is well known for its culinary benefits and aroma used as a condiment in Indian homes is also known as the "black stone flower" or "kalpasi" in India. This research intends to analyze the antioxidant power of the crude extracts using four pH-based buffers solubilized proteins/peptides and RP-HPLC fractions of P. perlatum obtained by purification. The proteins that were extracted from the four different buffers were examined using LC-MS/MS-based peptide mass fingerprinting. When compared to the other buffers, the 0.1 M of Tris-HCl buffer pH 8.0 solubilized proteins/peptides had the strongest antioxidant capacity. The sequential purification of the peptide was carried out by using a 3-kDa cut-off membrane filter and semipreparative RP-HPLC. Additionally, the purified fractions of the peptide's antioxidant activity were assessed, and effects were compared with those of the crude and 3 kDa cut--off membrane filtrates. The peptide fractions were sequenced by LC-MS/MS, which reveals that fraction 2 from RP-HPLC with the sequence LSWFMVVAP has shown the highest antioxidant potential in comparison with other fractions which can serve as the potential natural antioxidant drug. Further, fraction 2 also showed antibacterial activity against the selected microorganisms.


Assuntos
Antibacterianos , Antioxidantes , Espectrometria de Massas em Tandem , Antioxidantes/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Mapeamento de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/isolamento & purificação , Líquens/química , Parmeliaceae/química , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos/isolamento & purificação , Espectrometria de Massa com Cromatografia Líquida
8.
Anal Biochem ; 688: 115475, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38336012

RESUMO

Biosimilars are a cost-effective alternative to biopharmaceuticals, necessitating rigorous analytical methods for consistency and compliance. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is a versatile tool for assessing key attributes, encompassing molecular mass, primary structure, and post-translational modifications (PTMs). Adhering to ICH Q2R1, we validated an LC-HRMS based peptide mapping method using NISTmab as a reference. The method validation parameters, covering system suitability, specificity, accuracy, precision, robustness, and carryover, were comprehensively assessed. The method effectively differentiated the NISTmab from similar counterparts as well as from artificially introduced spiked conditions. Notably, the accuracy of mass error for NISTmab specific complementarity determining region peptides was within a maximum of 2.42 parts per million (ppm) from theoretical and the highest percent relative standard deviation (%RSD) observed for precision was 0.000219 %. It demonstrates precision in sequence coverage and PTM detection, with a visual inspection of total ion chromatogram approach for variability assessment. The method maintains robustness when subjected to diverse storage conditions, encompassing variations in column temperature and mobile phase composition. Negligible carryover was noted during the carryover analysis. In summary, this method serves as a versatile platform for multiple biosimilar development by effectively characterizing and identifying monoclonal antibodies, ultimately ensuring product quality.


Assuntos
Medicamentos Biossimilares , Medicamentos Biossimilares/análise , Medicamentos Biossimilares/química , Anticorpos Monoclonais/química , Espectrometria de Massa com Cromatografia Líquida , Mapeamento de Peptídeos/métodos , Peptídeos
9.
Nat Nanotechnol ; 19(5): 652-659, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38351230

RESUMO

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Intrinsicamente Desordenadas/química , Imagem Individual de Molécula/métodos , Aprendizado de Máquina , Mapeamento de Peptídeos/métodos
10.
Anal Chim Acta ; 1288: 342162, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38220294

RESUMO

BACKGROUND: In "shotgun" approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered as an indispensable tool. Despite the fact that CZE offers a high tolerance towards salts, most publications reported on the use of desalting. There seems to be no clear consensus on the utilization of desalting in the CZE-MS community, most probably due to the absence of works addressing the comparison of desalted and non-desalted digests. Our aim was to fill this research gap using protein samples of varying complexity in different sample matrices. RESULTS: First, standard protein digests were analyzed to build the knowledge on the effect of sample clean-up by solid-phase extraction (SPE) pipette tips and the possible stacking phenomena induced by different sample matrices. Desalting led to a somewhat altered peptide profile, the procedure affected mostly the hydrophilic peptides (although not to a devastating extent). Nevertheless, desalting samples allowed remarkable stacking efficiency owing to their low-conductivity sample background, enabling a so-called field-amplified sample stacking phenomenon. Non-desalted samples also produced a stacking event, the mechanism of which is based on transient-isotachophoresis due to the presence of high-mobility ions in the digestion buffer itself. Adding either extra ammonium ions or acetonitrile into the non-desalted digests enhanced the stacking efficiency. A complex sample (yeast cell lysate) was also analyzed with the optimal conditions, which yielded similar tendencies. SIGNIFICANCE: Based on these results, we propose that sample clean-up in the bottom-up sample preparation process prior to CZE-MS analysis can be omitted. The preclusion of desalting can even enhance detection sensitivity, separation efficiency or sequence coverage.


Assuntos
Proteômica , Espectrometria de Massas em Tandem , Mapeamento de Peptídeos , Espectrometria de Massas em Tandem/métodos , Proteômica/métodos , Eletroforese Capilar/métodos , Peptídeos/química , Íons
11.
Anal Chem ; 96(6): 2491-2499, 2024 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-38294207

RESUMO

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are important large biotherapeutics (∼150 kDa) and high structural complexity that require extensive sequence and structure characterization. Middle-down mass spectrometry (MD-MS) is an emerging technique that sequences and maps subunits larger than those released by trypsinolysis. It avoids potentially introducing artifactual modifications that may occur in bottom-up MS while achieving higher sequence coverage compared to top-down MS. However, returning complete sequence information by MD-MS is still challenging. Here, we show that assigning internal fragments in direct infusion MD-MS of a mAb and an ADC substantially improves their structural characterization. For MD-MS of the reduced NIST mAb, including internal fragments recovers nearly 100% of the sequence by accessing the middle sequence region that is inaccessible by terminal fragments. The identification of important glycosylations can also be improved after the inclusion of internal fragments. For the reduced lysine-linked IgG1-DM1 ADC, we show that considering internal fragments increases the DM1 conjugation sites coverage to 80%, comparable to the reported 83% coverage achieved by peptide mapping on the same ADC (Luo et al. Anal. Chem. 2016, 88, 695-702). This study expands our work on the application of internal fragment assignments in top-down MS of mAbs and ADCs and can be extended to other heterogeneous therapeutic molecules such as multispecifics and fusion proteins for more widespread applications.


Assuntos
Anticorpos Monoclonais , Imunoconjugados , Anticorpos Monoclonais/química , Imunoconjugados/química , Espectrometria de Massas/métodos , Mapeamento de Peptídeos , Lisina/química
12.
J AOAC Int ; 107(1): 177-188, 2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-37606972

RESUMO

BACKGROUND: Biologics are essential in cancer treatment because they stimulate the body's natural response to fight cancer, but they are expensive. Biosimilars are more affordable compared to patent biologicals, but it must be verified that they are as effective as their innovators. Characterization of biosimilars and assessment of interchangeability requires many data points for verification. OBJECTIVE: The proposed study provides a quality assessment of two new bevacizumab (BVZ) biosimilars, produced by Amgen and Biocad, Inc., through the development and greenness assessment of an orthogonal testing protocol and purity indicating assay, including size-exclusion (SE-HPLC), reversed-phase (RP-HPLC), and cation exchange chromatography (CEX-HPLC) in addition to dynamic light scattering (DLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). METHODS: SE-HPLC method was performed and validated to screen the BVZ monomer and its aggregates and/or fragments. Peak purity and system suitability parameters were calculated. Results indicate that the orthogonal protocol is a useful tool for assessing monoclonal antibody stability. It is a key criterion for biosimilarity assessment. DLS and SDS-PAGE results were compared to each other to reveal close retention times and banding patterns between BVZ innovator and its biosimilars. These results showed that Avastin® and the investigated biosimilars have the same profile in terms of peak area of related compounds within the acceptance limit and apparent molecular weight, and the SDS-PAGE technique was found to be the most eco-friendly technique among others. CONCLUSIONS: The results obtained highlighted the importance of assessing similarities and differences in ensuring the biosimilarity and interchangeability of the studied products. HIGHLIGHTS: BVZ is one of the essential monoclonal antibodies in the treatment of colorectal cancer (CRC). BVZ biosimilars were evaluated by developing an orthogonal testing protocol and a purity-indicating assay. The size-exclusion (SE)-HPLC method was applied and validated to monitor the BVZ monomer and its aggregates. The results demonstrated the importance of assessing the stability and biosimilarity of BVZ.


Assuntos
Anticorpos Monoclonais , Medicamentos Biossimilares , Anticorpos Monoclonais/química , Bevacizumab , Mapeamento de Peptídeos/métodos , Análise de Componente Principal
13.
Molecules ; 28(22)2023 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-38005254

RESUMO

To enhance the understanding of enzymatic hydrolysis and to accelerate the discovery of key bioactive peptides within enzymatic products, this research focused on elastin as the substrate and investigated the variations in peptide profiles and the production of key bioactive peptides (those exceeding 5% of the total) and their impacts on the biological activity of the hydrolysates. Through the application of advanced analytical techniques, such as stop-flow two-dimensional liquid chromatography and ultra-high-performance liquid chromatography-tandem mass spectrometry, the research tracks the release and profiles of peptides within elastin hydrolysates (EHs). Despite uniform peptide compositions, significant disparities in peptide concentrations were detected across the hydrolysates, hinting at varying levels of bioactive efficacy. A comprehensive identification process pinpointed 403 peptides within the EHs, with 18 peptides surpassing 5% in theoretical maximum content, signaling their crucial role in the hydrolysate's bioactivity. Of particular interest, certain peptides containing sequences of alanine, valine, and glycine were released in higher quantities, suggesting Alcalase® 2.4L's preference for these residues. The analysis not only confirms the peptides' dose-responsive elastase inhibitory potential but also underscores the nuanced interplay between peptide content, biological function, and their collective synergy. The study sets the stage for future research aimed at refining enzymatic treatments to fully exploit the bioactive properties of elastin.


Assuntos
Elastina , Peptídeos , Animais , Bovinos , Hidrólise , Mapeamento de Peptídeos , Elastina/química , Peptídeos/química , Elastase Pancreática , Hidrolisados de Proteína
14.
Anal Chem ; 95(42): 15514-15521, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37816151

RESUMO

LC-MS based peptide mapping, i.e., proteolytic digestion followed by LC-MS/MS analysis, is the method of choice for protein primary structural characterization. Manual proteolytic digestion is usually a labor-intensive procedure. In this work, a novel method was developed for fully automated online protein digestion and LC-MS peptide mapping. The method generates LC-MS data from undigested protein samples without user intervention by utilizing the same HPLC system that performs the chromatographic separation with some additional modules. Each sample is rapidly digested immediately prior to its LC-MS analysis, minimizing artifacts that can grow over longer digestion times or digest storage times as in manual or automated offline digestion methods. In this report, we implemented the method on an Agilent 1290 Infinity II LC system equipped with a Multisampler. The system performs a complete digestion workflow including denaturation, disulfide reduction, cysteine alkylation, buffer exchange, and tryptic digestion. We demonstrated that the system is capable of digesting monoclonal antibodies and other proteins with excellent efficiency and is robust and reproducible and produces fewer artifacts than manually prepared digests. In addition, it consumes only a few micrograms of material as most of the digested sample protein is subjected to LC-MS analysis.


Assuntos
Peptídeo Hidrolases , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Mapeamento de Peptídeos/métodos , Proteólise , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos
15.
Artigo em Inglês | MEDLINE | ID: mdl-37776677

RESUMO

The antiangiogenic drug bevacizumab is a blockbuster therapeutic pharmaceutical product that is used to treat many different types of cancer including kidney, colon, rectum, lung, and breast cancer. As a result, multiple biosimilars have been approved across the various regulatory jurisdictions in India (>20 in number till date). The rapidly growing market and acceptance of biosimilars was the motivation to perform comparability study of bevacizumab biosimilars that are presently available in the Indian market. A comprehensive analytical and functional biosimilarity assessment has been performed to examine and compare innovator product of bevacizumab (Avastin-innovator product, Roche Products (India) Pvt Ltd) and six biosimilars that are being marketed in India (Abevmy from Mylan Pharmaceuticals Pvt Ltd, Bevazza from Lupin Ltd, Bryxta from Zydus Cadila, Krabeva from Biocon, Ivzumab from RPG Life Sciences Ltd, and Advamab from Alkem Laboratories Ltd). Physiochemical characterization of drug products was performed with respect to their primary structure (intact mass, reduced mass, peptide mapping by LC-MS), higher order structure (secondary structure by FTIR, Far-UV-CD, and tertiary structure by Near-UV-CD, intrinsic fluorescence spectroscopy), impurity profile (SE-HPLC, SEC-MALS, extrinsic fluorescence: size heterogenicity, degradation, stability; DLS: hydrodynamic radius; WCX-HPLC: charge variants analysis) and post-translational modifications by measuring reduced glycans through fluorescence dye analysis. Functional characterization was performed by SPR and cell proliferation assay. Further, chemometrics based quantitative evaluation of biosimilarity has been performed by combining the data obtained from analytical characterization platform. The analysis of the analytical, functional and chemometric results revealed significant levels of similarity, with biosimilar4 being the sole exception. Despite being within product specifications, Biosimilar4 displayed significant deviations with respect to critical quality attributes, including a lower proportion of monomer content, a larger percentage of basic charge variant species, and a lower proportion of aglycosylated glycoform.


Assuntos
Medicamentos Biossimilares , Bevacizumab , Medicamentos Biossimilares/análise , Processamento de Proteína Pós-Traducional , Inibidores da Angiogênese , Mapeamento de Peptídeos/métodos
16.
Biochemistry ; 62(18): 2700-2709, 2023 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-37622182

RESUMO

As protein therapeutics play an important role in almost all medical fields, numerous studies have been conducted on proteins using artificial intelligence. Artificial intelligence has enabled data-driven predictions without the need for expensive experiments. Nevertheless, unlike the various molecular fingerprint algorithms that have been developed, protein fingerprint algorithms have rarely been studied. In this study, we proposed the amino acid molecular fingerprints repurposing-based protein (AmorProt) fingerprint, a protein sequence representation method that effectively uses the molecular fingerprints corresponding to 20 amino acids. Subsequently, the performances of the tree-based machine learning and artificial neural network models were compared using (1) amyloid classification and (2) isoelectric point regression. Finally, the applicability and advantages of the developed platform were demonstrated through a case study and the following experiments: (3) comparison of dataset dependence with feature-based methods, (4) feature importance analysis, and (5) protein space analysis. Consequently, the significantly improved model performance and data-set-independent versatility of the AmorProt fingerprint were verified. The results revealed that the current protein representation method can be applied to various fields related to proteins, such as predicting their fundamental properties or interaction with ligands.


Assuntos
Algoritmos , Inteligência Artificial , Mapeamento de Peptídeos , Aminoácidos , Proteínas Amiloidogênicas
17.
J Pharm Sci ; 112(10): 2637-2643, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37595748

RESUMO

Trisulfide is a post-translational modification (PTM) commonly found in recombinant antibodies. It has been demonstrated that trisulfide had no impact on the bioactivity of mono-specific antibodies (MsAbs). However, the impact of trisulfide on multi-specific antibodies has not been evaluated. In this study, two mass spectrometric methods were developed for comprehensive trisulfide characterization. The non-reduced peptide mapping method combined with the unique electron activated dissociation (EAD) provided signature fragments for confident trisulfide identification as well as trisulfide quantitation at individual sites. A higher throughput method using Fab mass analysis was also developed and qualified to support routine monitoring of trisulfide during process development. Fab mass analysis features simpler sample preparation and shorter analysis time but provides comparable results to the non-reduced peptide mapping method. In this study, a bi-specific (BsAb) and a tri-specific antibody (TsAb) were compared side-by-side with a MsAb to evaluate the impact of trisulfide on the structure and function of multi-specific antibodies. Results indicated that trisulfide dominantly formed at similar locations across different antibody constructs and had no impact on the size heterogeneity, charge heterogeneity, or bioactivities of any assessed antibodies. Together with the in vitro stability under heat stress (25 °C and 40 °C for up to four weeks) and rapid conversion from trisulfide to disulfide during in vivo circulation, trisulfide could be categorized as a non-critical quality attribute (non-CQA) for antibody products.


Assuntos
Anticorpos , Dissulfetos , Espectrometria de Massas , Mapeamento de Peptídeos , Processamento de Proteína Pós-Traducional
18.
J Am Soc Mass Spectrom ; 34(9): 1837-1846, 2023 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-37478497

RESUMO

LC-MS analysis of therapeutic antibodies and other biotherapeutics from in-life studies (e.g., serum/plasma) has evolved from simple peptide digestion to peptide mapping and intact mass monitoring. From more advanced analytical approaches, a deeper understanding as to the fate of the biotherapeutic in vivo is gained. Here, we examine the next generation of approaches to facilitate the most comprehensive understanding of large molecule drug fate in circulation. Three case studies are presented: (1) use of relative and absolute calibration curves for biotherapeutic quantitation from the same sample set; (2) top-down mass spectrometry applied to bioanalytical assays; (3) biotherapeutic protein complexes from serum analyzed by native protein MS. We anticipate that these approaches will be further adapted and applied by other research groups.


Assuntos
Proteínas , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Proteínas/análise , Peptídeos , Mapeamento de Peptídeos/métodos
19.
Sci Rep ; 13(1): 9038, 2023 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-37270636

RESUMO

Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world's first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5' terminus capping and 3' terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.


Assuntos
COVID-19 , Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , SARS-CoV-2/genética , Vacinas contra COVID-19 , Oligonucleotídeos/genética , COVID-19/prevenção & controle , Vacinas de mRNA , Mapeamento de Peptídeos/métodos , RNA Mensageiro/genética
20.
Anal Biochem ; 674: 115211, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37302778

RESUMO

LC-MS based multi-attribute methods (MAM) have drawn substantial attention due to their capability of simultaneously monitoring a large number of quality attributes of a biopharmaceutical product. For successful implementation of MAM, it is usually considered a requirement that the method is capable of detecting any new or missing peaks in the sample when compared to a control. Comparing a sample to a control for rare differences is also commonly practiced in many fields for investigational purpose. Because MS signal variability differs greatly between signals of different intensities, this type of comparison is often challenging, especially when the comparison is made without enough replicates. In this report we describe a statistical method for detecting rare differences between two very similar samples without replicate analyses. The method assumes that an overwhelming majority of components have equivalent abundance between the two samples, and signals with similar intensities have similar relative variability. By analyzing several monoclonal antibody peptide mapping datasets, we demonstrated that the method is suitable for new-peak detection for MAM as well as for other applications when rare differences between two samples need to be detected. The method greatly reduced false positive rate without a significant increase of false negative rate.


Assuntos
Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Mapeamento de Peptídeos/métodos
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