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1.
J Chromatogr A ; 1732: 465202, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39079362

RESUMO

Despite advancements in therapeutic monoclonal antibodies (mAbs) and cell line engineering, separating host cell proteins (HCPs) from mAbs during downstream purification remains challenging. Therefore, in this study, we developed a novel multimodal chromatography (MMC) resin to enhance HCP removal during mAb polishing processes. We evaluated the impact of both ligand structure and pore size of the MMC resin by purifying a post-protein A chromatography solution in flow-through mode. We observed that the efficiency of HCP clearance depended on the hydrophobic moiety structure of the ligand and predicted the mAb purification capability of MMC through linear salt-gradient elution experiments involving a mixture of transferrin, bovine serum albumin (BSA), and pepsin. Our findings revealed that the prototype immobilized 1,12-dodecanediamine via the formyl group exhibited the best performance attributed to its long alkyl chain. Furthermore, an investigation of effects of base bead pore size on HCP capacity using cellulose base beads of five different pore sizes showed that larger pore resin base beads had the highest HCP removal capacity. Specifically, MMC resins with a pore diameter exceeding 440 nm reduced the HCP level by three orders of magnitude under high mAb loading conditions (> 1000 mg/mL-resin). The MMC resin developed in this study, along with the insights gained into ligand structure and pore size, not only enhances mAb polishing efficiency but also contributes to improving downstream processes in mAb biopharmaceutical production.


Assuntos
Anticorpos Monoclonais , Cricetulus , Soroalbumina Bovina , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Animais , Células CHO , Ligantes , Soroalbumina Bovina/química , Porosidade , Cromatografia de Afinidade/métodos , Proteína Estafilocócica A/química , Transferrina/química , Transferrina/isolamento & purificação , Pepsina A/química , Pepsina A/metabolismo , Proteínas/isolamento & purificação , Proteínas/química , Resinas Sintéticas/química , Interações Hidrofóbicas e Hidrofílicas
2.
Artigo em Inglês | MEDLINE | ID: mdl-12659730

RESUMO

The early applications of microarrays and detection technologies have been centered on DNA-based applications. The application of array technologies to proteomics is now occurring at a rapid rate. Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools. The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology. To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips. Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules. A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection. Other approaches include the creation of intact protein microarrays directly on glass slides or chips. Although many of the proteins may likely be denatured, successful screening has been demonstrated. The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid. In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains. Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins. In an innovative departure from the traditional concept of protein chips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes. Other researchers are using in-jet printing technology to create protein microarrays on chips. The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future.


Assuntos
Análise Serial de Proteínas , Proteoma
3.
Proteomics ; 2(2): 145-50, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11840560

RESUMO

In-gel peptide digestion has become a widely used technique for characterizing proteins resolved by two-dimensional gel electrophoresis. Peptides generated from gel pieces are frequently contaminated with detergent and salts. Prior to matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, these contaminants are removed using micro scale C18 sample preparation columns. In this paper, data are presented to demonstrate the application of a solvent resistant MultiScreen 96-well plate with a low peptide binding membrane and ZipTip micropipette based sample preparation. Recoveries of peptides (m/z of 1000 to 5000 Da) derived from standard protein protease digests, were estimated at various stages of the analytical process. An optimized protocol has been established and all the reagents and consumables have been packaged in a ready to use commercial kit. Data will be presented to show the application of this technology package to accelerate the throughput of protein characterization by protease fragmentation.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Eletroforese das Proteínas Sanguíneas/métodos , Eletroforese das Proteínas Sanguíneas/estatística & dados numéricos , Proteínas Sanguíneas/isolamento & purificação , Eletroforese em Gel Bidimensional/estatística & dados numéricos , Humanos , Peptídeos/isolamento & purificação , Proteoma/isolamento & purificação , Sensibilidade e Especificidade
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