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1.
J Pharm Sci ; 113(6): 1415-1425, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38373591

RESUMO

The comparability assessment of a biological product after implementing a manufacturing process change should involve a risk-based approach. Process changes may occur at any stage of the product lifecycle: early development, clinical manufacture for pivotal trials, or post-approval. The risk of the change to impact product quality varies. The design of the comparability assessment should be adapted accordingly. A working group reviewed and consolidated industry approaches to assess comparability of traditional protein-based biological products during clinical development and post-approval. The insights compiled in this review article encompass topics such as a risk-evaluation strategy, the design of comparability studies, definition of assessment criteria for comparability, holistic evaluation of data, and the regulatory submission strategy. These practices can be leveraged across the industry to help companies in design and execution of comparability assessments, and to inform discussions with global regulators.


Assuntos
Produtos Biológicos , Humanos , Medição de Risco/métodos , Aprovação de Drogas/métodos , Desenvolvimento de Medicamentos/métodos
2.
Front Immunol ; 14: 1119510, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37063860

RESUMO

Polatuzumab vedotin, marketed under the trade name POLIVY®, is a CD79b-targeted antibody-drug conjugate that preferentially delivers a potent anti-mitotic agent (monomethyl auristatin E) to B cells, resulting in anti-cancer activity against B-cell malignancies. In 2019, polatuzumab vedotin in combination with rituximab and bendamustine was approved by the United States Food and Drug Administration for the treatment of adult patients with diffuse large B-cell lymphoma who have received at least two prior therapies. Recent Health Authority guidance recommendations for submitting an Integrated Summary of Immunogenicity were followed including a comprehensive immunogenicity risk assessment, bioanalytical strategy, and immunogenicity data to support the registration of polatuzumab vedotin. Key components of the polatuzumab vedotin Integrated Summary of Immunogenicity and data are presented. Validated semi-homogeneous bridging enzyme-linked immunosorbent assays were used to detect anti-drug antibodies (ADA) to polatuzumab vedotin and characterize the immune response in patients with non-Hodgkin's lymphoma. The overall incidence of ADA observed for polatuzumab vedotin was low across seven clinical trials. The low incidence of ADA is likely due to the mechanism of action of polatuzumab vedotin that involves targeting and killing of B cells, thereby limiting the development to plasma cells and ADA secretion. Furthermore, patients are co-medicated with rituximab, which also targets B cells and results in B-cell depletion. Therefore, the immunogenicity risk is considered low and not expected to impact the polatuzumab vedotin benefit/risk profile.


Assuntos
Imunoconjugados , Linfoma Difuso de Grandes Células B , Linfoma não Hodgkin , Estados Unidos , Adulto , Humanos , Rituximab/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Imunoconjugados/efeitos adversos , Linfoma não Hodgkin/patologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico
3.
MAbs ; 8(2): 371-8, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26651858

RESUMO

Tryptic peptide mapping analysis of a Chinese hamster ovary (CHO)-expressed, recombinant IgG1 monoclonal antibody revealed a previously unreported +16 Da modification. Through a combination of MS(n) experiments, and preparation and analysis of known synthetic peptides, the possibility of a sequence variant (Ala to Ser) was ruled out and the presence of hydroxylysine was confirmed. Post-translational hydroxylation of lysine was found in a consensus sequence (XKG) known to be the site of modification in other proteins such as collagen, and was therefore presumed to result from the activity of the CHO homolog of the lysyl hydroxylase complex. Although this consensus sequence was present in several locations in the antibody sequence, only a single site on the heavy-chain Fab was found to be modified.


Assuntos
Anticorpos Monoclonais Humanizados/química , Hidroxilisina/química , Imunoglobulina G/química , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas Recombinantes/química
4.
J Chromatogr B Analyt Technol Biomed Life Sci ; 878(25): 2307-13, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20674519

RESUMO

High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0-3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.


Assuntos
Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas em Tandem/métodos , Misturas Complexas/química , Peptídeos/análise , Pressão , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
5.
Anal Chem ; 81(4): 1347-56, 2009 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-19140748

RESUMO

In recent years mass spectrometry based techniques have emerged as structural biology tools for the characterization of macromolecular, noncovalent assemblies. Many of these efforts involve preservation of intact protein complexes within the mass spectrometer, providing molecular weight measurements that allow the determination of subunit stoichiometry and real-time monitoring of protein interactions. Attempts have been made to further elucidate subunit architecture through the dissociation of subunits from the intact complex by colliding it into inert gas atoms such as argon or xenon. Unfortunately, the amount of structural information that can be derived from such strategies is limited by the nearly ubiquitous ejection of a single, unfolded subunit. Here, we present results from the gas-phase dissociation of protein-protein complexes upon collision into a surface. Dissociation of a series of tetrameric and pentameric proteins demonstrate that alternative subunit fragments, not observed through multiple collisions with gas atoms, can be generated through surface collision. Evidence is presented for the retention of individual subunit structure, and in some cases, retention of noncovalent interactions between subunits and ligands. We attribute these differences to the rapid large energy input of ion-surface collisions, which leads to the dissociation of subunits prior to the unfolding of individual monomers.


Assuntos
Multimerização Proteica , Proteínas/química , Argônio/química , Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Humanos , Espectrometria de Massas , Pré-Albumina/química , Pré-Albumina/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteínas/metabolismo , Propriedades de Superfície
6.
Anal Chem ; 80(5): 1425-36, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18247517

RESUMO

A novel in-line surface-induced dissociation (SID) device was designed and implemented in a commercial QTOF instrument (Waters/Micromass QTOF II). This new setup allows efficient SID for a broad range of molecules. It also allows direct comparison with conventional collision-induced dissociation (CID) on the same instrument, taking advantage of the characteristics of QTOF instrumentation, including extended mass range, improved sensitivity, and better resolution compared with quadrupole analyzers and ion traps. Various peptides and a noncovalent protein complex have been electrosprayed and analyzed with the new SID setup. Here we present SID of leucine enkephalin, fibrinopeptide A, melittin, insulin chain-B, and a noncovalent protein complex from wheat, heat shock protein 16.9. The SID spectra were also compared to CID spectra. With the SID setup installed, ion transmission proved to be efficient. SID fragmentation patterns of peptides are, in general, similar to CID, with differences in the relative intensities of some peaks such as immonium ions, backbone cleavage b- versus y-type ions, and y- versus y-NH3 ions, suggesting enhanced accessibility to high-energy/secondary fragmentation channels with SID. Furthermore, these results demonstrate that the in-line SID setup is a valid substitute for CID, with potential advantages for activation of singly/multiply charged peptides and larger species such as noncovalent protein complexes.


Assuntos
Espectrometria de Massas/instrumentação , Complexos Multiproteicos/química , Peptídeos/química , Animais , Bovinos , Encefalina Leucina/química , Fibrinopeptídeo A/química , Insulina/química , Meliteno/química , Propriedades de Superfície
7.
J Am Soc Mass Spectrom ; 19(2): 190-208, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18191578

RESUMO

This article provides a perspective on collisions of ions with surfaces, including surface-induced dissociation (SID) and reactive ion scattering spectrometry (RISS). The content is organized into sections on surface-induced dissociation of small ions, surface characterization of organic thin films by collision of well-characterized ions into surfaces, the use of SID to probe peptide fragmentation, and the dissociation of large non-covalent complexes by SID. Examples are given from the literature with a focus on experiments from the authors' laboratory. The article is not a comprehensive review but is designed to provide the reader with an overview of the types of results possible by collisions of ions into surfaces.


Assuntos
Espectrometria de Massas , Peptídeos/análise , Peptídeos/química , Proteínas/análise , Proteínas/química , Distinções e Prêmios , Peso Molecular , Propriedades de Superfície
8.
J Am Chem Soc ; 128(47): 15044-5, 2006 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-17117828

RESUMO

Previous gas-phase dissociation experiments of protein-protein complexes have resulted in product ion distributions that are asymmetric by charge and mass, providing limited insight into the chemical nature of subunit organization and interaction. In these experiments, a symmetric charge distribution results from an "energy sudden" collision of protein-protein complexes with a surface, indicating that it may be possible to probe the suboligomeric structure of noncovalent complexes in the gas phase. It is proposed that energy sudden surface activation of cytochrome C homodimers results in dissociation without significant unfolding of one of the monomeric subunits. Previously proposed mechanisms for the dissociation of protein-protein complexes are discussed in the context of these results. These experiments demonstrate the potential to preserve the structural details of subunit interaction within a protein-protein complex and help elucidate the asymmetric nature of macromolecular dissociation in the gas phase.


Assuntos
Citocromos c/química , Espectrometria de Massas em Tandem/métodos , Gases , Termodinâmica
9.
J Proteome Res ; 5(11): 2935-46, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17081045

RESUMO

The ribosomal proteins of Caulobacter crescentus were amidinated before and after disassembly of the organelle and the results analyzed by mass spectrometry. Comparison with structural information from previous X-ray crystal studies of other bacterial ribosomes provides insight about the C. crescentus ribosome. In total, 47 of the 54 proteins present in the ribosome of C. crescentus were detected after labeling. The extent of derivatization for each protein is strongly dependent on the solvent accessibility of its target residues. Proteins of the ribosome stalk, which are known to be largely solvent-accessible, were labeled quite extensively. In striking contrast, other proteins that are known to be highly shielded in their subunits were labeled at very few of their potential sites. Furthermore, evidence that protein L12 binds to the ribosome via its N-terminal domain is consistent with previous findings.


Assuntos
Caulobacter crescentus/ultraestrutura , Proteínas Ribossômicas/química , Ribossomos/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Cristalografia por Raios X , Indicadores e Reagentes , Espectrometria de Massas/métodos , Modelos Moleculares , Peso Molecular , Conformação Proteica , Proteínas Ribossômicas/isolamento & purificação
10.
Anal Chem ; 77(22): 7274-81, 2005 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16285675

RESUMO

A chemical derivatization method, amidination, that has recently been effectively employed in peptide mass spectrometry experiments is used to covalently modify lysines in several standard proteins. Protein and peptide mass spectra identify sites at which the reaction does or does not occur. This is therefore a rapid approach to elucidate solvent-accessible regions of folded proteins.


Assuntos
Proteínas/química , Proteínas/metabolismo , Aminação , Aminas/química , Sequência de Aminoácidos , Animais , Anidrase Carbônica II/química , Anidrase Carbônica II/metabolismo , Bovinos , Cristalografia por Raios X , Cisteína/química , Hemoglobinas/química , Hemoglobinas/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Solventes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ubiquitina/química , Ubiquitina/metabolismo
11.
Anal Chem ; 77(19): 6300-9, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16194092

RESUMO

A novel peptide derivatization strategy based on guanidination and amidination is presented. Mass-coded labels help distinguish N- and C-terminal fragment ions produced by collision-induced dissociation and are of general utility since peptide N-termini are coded. The amidine labels also promote specific fragmentation pathways that elucidate N-terminal residues and provide valuable internal calibrants. This strategy is demonstrated with the tryptic peptides of several model proteins, including two that are phosphorylated. Additionally, interpreted peptide sequences are matched against a database of over 80,000 proteins to assess the selectivity of this sequencing approach.


Assuntos
Peptídeos/química , Sequência de Aminoácidos , Animais , Calibragem , Bovinos , Guanidina/química , Humanos , Íons/química , Lisina/química , Dados de Sequência Molecular , Fosforilação , Proteínas/química , Espectrometria de Massas em Tandem
12.
J Am Soc Mass Spectrom ; 15(2): 158-67, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14766283

RESUMO

The collision-induced dissociation characteristics of amidinated and unmodified tryptic peptides are compared using an ion trap mass spectrometer with both electrospray ionization and matrix-assisted laser/desorption ionization (MALDI). Several fragmentation pathways in a number of tryptic peptides of various precursor charge states are found to be enhanced. The additional information conveyed by the observed fragment ions should facilitate protein identifications.


Assuntos
Amidas/química , Peptídeos/química , Sequência de Aminoácidos , Íons/química , Dados de Sequência Molecular , Estrutura Molecular , Fragmentos de Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
J Proteome Res ; 2(1): 15-21, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12643539

RESUMO

Differential amidination of N-termini and lysine residues provides the basis for a novel approach to protein quantitation using MALDI mass spectrometry. Because the amidination of lysines increases their basicity and therefore MALDI ionization yields, the method is called quantitation using enhanced signal tags (QUEST). Amidine labels differ by methylene groups, leading to 14 Da mass differentials. The utility of QUEST is demonstrated while analyzing the digests of two model proteins using MALDI-TOF mass spectrometry.


Assuntos
Proteínas/química , Proteoma , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Amidinas/química , Animais , Cromatografia , Grupo dos Citocromos c/análise , Grupo dos Citocromos c/química , Hemoglobinas/análise , Hemoglobinas/química , Íons , Lisina/química , Modelos Químicos , Peptídeos/química , Estrutura Terciária de Proteína , Proteínas/análise , Fatores de Tempo , Tripsina/farmacologia
14.
Anal Chem ; 74(8): 1884-90, 2002 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11985322

RESUMO

Improved procedures for guanidination of lysine-containing peptides, a derivatization that results in increased MALDI mass spectral signal intensities are presented. The complete conversion of lysines to homoarginines can be accomplished in as little as 5 min. The method is demonstrated on a model peptide and on tryptic digests of three proteins. To demonstrate the applicability to proteomics samples, it is successfully applied to the digest of 50 fmol of a protein. Approaches for concentrating and purifying low-quantity protein digests following guanidination are evaluated. Experiments with the model peptide GRGDSPK enable investigation of the specificity of the guanidination reaction.


Assuntos
Guanidinas/química , Mapeamento de Peptídeos/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Grupo dos Citocromos c/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Lisina/química , Mioglobina/química
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