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1.
Pharm Res ; 40(12): 3087-3098, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37936013

RESUMO

PURPOSE: Monoclonal antibodies (mAbs), like other protein therapeutics, are prone to various forms of degradation, some of which are difficult to distinguish from the native form yet may alter potency. A generalizable LC-MS approach was developed to enable quantitative analysis of isoAsp. In-depth understanding of product quality attributes (PQAs) enables optimization of the manufacturing process, better formulation selection, and decreases risk associated with product handling in the clinic or during shipment. METHODS: Reversed-phase chromatographic peak splitting was observed when a mAb was exposed to elevated temperatures. Multiple LC-MS based methods were applied to identify the reason for peak splitting. The approach involved the use of complementary HPLC columns, multiple enzymatic digestions and different MS/MS ion dissociation methods. In addition, mAb potency was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The split peaks had identical masses, and the root cause of the peak splitting was identified as isomerization of an aspartic acid located in the complementarity-determining region (CDR) of the light chain. And the early eluting and late eluting peaks were collected and performed enzymatic digestion to confirm the isoAsp enrichment in the early eluting peak. In addition, decreased potency was observed in the same heat-stressed sample, and the increased isoAsp levels in the CDR correlate well with a decrease of potency. CONCLUSION: Liquid chromatography-mass spectrometry (LC-MS) has been utilized extensively to assess PQAs of biological therapeutics. In this study, a generalizable LC-MS-based approach was developed to enable identification and quantitation of the isoAsp-containing peptides.


Assuntos
Anticorpos Monoclonais , Espectrometria de Massas em Tandem , Anticorpos Monoclonais/química , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massa com Cromatografia Líquida , Cromatografia Líquida de Alta Pressão/métodos , Regiões Determinantes de Complementaridade/química
2.
J Pharm Sci ; 111(5): 1486-1496, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34717953

RESUMO

The protein complex of recombinant human insulin-like growth factor-1 and insulin­like growth factor binding protein­3 (rhIGF-1/rhIGFBP-3; mecasermin rinfabate), is an investigational product for the prevention of complications of prematurity. Delivery of rhIGF-1/rhIGFBP-3 is by continuous central line intravenous infusion in preterm infants until endogenous IGF-1 production begins. Protein-specific analytical methodologies were developed to evaluate the compatibility of rhIGF-1/rhIGFBP-3 at low protein concentrations (∼2.5-10 µg/mL) expected when co-administered with other required medications in the NICU. Highly sensitive detection of the biologic potential degradants (fragments) and/or molecular modifications (oxidized species, aggregates) required the use of reversed-phase high-performance liquid chromatography and size-exclusion ultra-performance liquid chromatography coupled with mass spectrometric detection. We report on the quantification of rhIGF-1/rhIGFBP-3, its components and degradants, to a limit of quantitation of 3.1 µg/mL upon mixing with 24 commonly administered neonatal medications. Methods developed for the rhIGF-1/rhIGFBP-3 admixtures, optimized in studies with furosemide, caffeine citrate and ampicillin, demonstrated good reproducibility, linearity, and limit of detection/quantitation. Using these methods, no increase in degradation of rhIGF-1/rhIGFBP-3 components and no increase in oxidation or aggregation level was observed with caffeine citrate, while admixtures of rhIGF-1/rhIGFBP-3 with ampicillin yielded lower mass recovery of rhIGF-1/rhIGFBP-3 components, which likely resulted from adduct formation. Furosemide was found to be physically incompatible with rhIGF-1/rhIGFBP-3. Our findings support the use of these methodologies for detection of protein modifications under various clinical administration conditions, and additionally supplement physical compatibility data studies of ultra-low concentrations of rhIGF-1/rhIGFBP-3 post co-administration to preterm infants with other medications (manuscript in-preparation).


Assuntos
Furosemida , Fator de Crescimento Insulin-Like I , Ampicilina , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro , Proteínas Recombinantes , Reprodutibilidade dos Testes
3.
J Pharm Pharmacol ; 70(5): 609-624, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-27861887

RESUMO

OBJECTIVES: Protein-based therapeutics garner significant attention because of exquisite specificity and limited side effects and are now being used to accomplish targeted delivery of small-molecule drugs. This review identifies and highlights individual chemical attributes and categorizes how site-specific changes affect protein stability based on published high-resolution molecular analyses. KEY FINDINGS: Because it is challenging to determine the mechanisms by which the stability of large, complex molecules is altered and data are sparse, smaller, therapeutic proteins (insulin, erythropoietin, interferons) are examined alongside antibody data. Integrating this large pool of information with the limited available studies on antibodies reveals common mechanisms by which specific alterations affect protein structure and stability. SUMMARY: Physical and chemical stability of therapeutic proteins and antibody drug conjugates (ADCs) is of critical importance because insufficient stability prevents molecules from making it to market. Individual moieties on/near the surface of proteins have substantial influence on structure and stability. Seemingly small, superficial modification may have far-reaching consequences on structure, conformational dynamics, and solubility of the protein, and hence physical stability of the molecule. Chemical modifications, whether spontaneous (e.g. oxidation, deamidation) or intentional, as with ADCs, may adversely impact stability by disrupting local surface properties or higher order protein structure.


Assuntos
Imunoconjugados/química , Estabilidade Proteica , Proteínas/química , Anticorpos/administração & dosagem , Anticorpos/química , Humanos , Imunoconjugados/administração & dosagem , Proteínas/administração & dosagem , Solubilidade , Propriedades de Superfície
4.
Protein Eng Des Sel ; 30(6): 467-475, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28541524

RESUMO

Biologics can be an improvement to small molecule drugs, providing high specificity for an identified target, lowering toxicity and limiting side effects. To achieve effective delivery, the biologic must have sufficient time to reach the target tissue. A prolonged half-life in the circulating environment is desired, but often serum stability is limited by proteases. Proteolysis in the serum causes degradation and inactivation as the biologic is fragmented and more rapidly cleared from the body. To improve the circulating half-life, large, hydrophilic polymers may be conjugated or stable fusion tags may be engineered to increase the effective size of the peptide and to hinder degradation by proteases. Improved resistance to proteases is essential for effective delivery. Here, a proof of concept study is presented using a metal-binding tripeptide tag known as the claMP Tag to create an inline conjugate and the ability of the tag to inhibit proteolysis was examined.


Assuntos
Oligopeptídeos/química , Oligopeptídeos/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Densitometria , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Níquel/química , Níquel/metabolismo , Oligopeptídeos/genética , Estabilidade Proteica , Proteólise , Proteínas Recombinantes de Fusão/genética
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