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1.
Bioengineering (Basel) ; 9(4)2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35447733

RESUMO

Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process.

2.
Biotechnol Prog ; 33(6): 1456-1462, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28393481

RESUMO

The long journey of developing a drug from initial discovery target identification to regulatory approval often leaves many patients with missed window of opportunities. Both regulatory agencies and biopharmaceutical industry continue to develop creative approaches to shorten the time of new drug development in order to deliver life-saving medicine to patients. Generally, drug substance materials to support the toxicology and early phase clinical study can only be manufactured after creating the final Master Cell Bank (MCB) of the clonally derived cell line, which normally takes 1-2 years. With recent advances in cell line development, cell culture process and analytical technologies, generating more homogeneous bulk/mini-pool population with higher productivity and acceptable quality attributes has become a norm, thereby making it possible to shorten the timeline to initiate First in Human (FIH) trial by using bulk/mini-pool generated materials to support toxicology and FIH studies. In this study, two monoclonal antibodies of different subclasses (IgG1 and IgG4) were expressed from the mini-pool cells as well as clonally derived cell lines generated from the same mini-pool. Cell growth, productivity, and product quality were compared between the materials generated from the mini-pool and clonally derived cell line. The results demonstrate the similarity of the antibody products generated from mini-pool cells and clonally derived cell lines from the same mini-pool, and strongly support the concept and feasibility of using antibody materials produced from mini-pool cultures for toxicology and FIH studies. The strategy to potentially shorten the FIH timeline is discussed. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1456-1462, 2017.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Reatores Biológicos , Células Clonais/efeitos dos fármacos , Toxicologia/métodos , Animais , Anticorpos Monoclonais/imunologia , Técnicas de Cultura Celular por Lotes/tendências , Células CHO/efeitos dos fármacos , Cricetinae , Cricetulus , Humanos
3.
Rapid Commun Mass Spectrom ; 26(2): 101-8, 2012 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-22173797

RESUMO

Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 µL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.


Assuntos
Apolipoproteína B-100/sangue , Apolipoproteína B-48/sangue , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Apolipoproteína A-I/sangue , Apolipoproteína B-100/genética , Apolipoproteína B-48/genética , Doenças Cardiovasculares/sangue , Simulação por Computador , Cricetinae , Cães , Técnicas de Silenciamento de Genes , Humanos , Modelos Lineares , Macaca mulatta , Camundongos , Fragmentos de Peptídeos/análise , RNA Interferente Pequeno/genética , Ratos , Especificidade da Espécie , Tripsina/química , Tripsina/metabolismo
4.
J Lipid Res ; 51(9): 2611-8, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20453200

RESUMO

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates hepatic low-density lipoprotein receptor (LDLR) levels in humans. PCSK9 has also been shown to regulate the levels of additional membrane-bound proteins in vitro, including the very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (ApoER2) and the beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), which are all highly expressed in the CNS and have been implicated in Alzheimer's disease. To better understand the role of PCSK9 in regulating these additional target proteins in vivo, their steady-state levels were measured in the brain of wild-type, PCSK9-deficient, and human PCSK9 overexpressing transgenic mice. We found that while PCSK9 directly bound to recombinant LDLR, VLDLR, and apoER2 protein in vitro, changes in PCSK9 expression did not alter the level of these receptors in the mouse brain. In addition, we found no evidence that PCSK9 regulates BACE1 levels or APP processing in the mouse brain. In conclusion, our results suggest that while PCSK9 plays an important role in regulating circulating LDL cholesterol levels by reducing the number of hepatic LDLRs, it does not appear to modulate the levels of LDLR and other membrane-bound proteins in the adult mouse brain.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/anatomia & histologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Ligação Proteica , Serina Endopeptidases/genética
5.
J Lipid Res ; 51(9): 2739-52, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20458119

RESUMO

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.


Assuntos
Anticolesterolemiantes/química , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Oxazolidinonas/química , Quinolinas/química , Compostos de Sulfidrila/química , Amidas , Animais , Anticolesterolemiantes/metabolismo , Proteínas Sanguíneas/metabolismo , Ésteres , Humanos , Camundongos , Estrutura Molecular , Oxazolidinonas/metabolismo , Quinolinas/metabolismo , Compostos de Sulfidrila/metabolismo
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