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1.
Biotechnol Bioeng ; 113(10): 2100-6, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-26989081

RESUMEN

Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Carboxipeptidasas/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Lisina/metabolismo , Ingeniería de Proteínas/métodos , Animales , Células CHO/enzimología , Carboxipeptidasas/genética , Cricetulus , Lisina/genética
2.
Biotechnol Bioeng ; 108(11): 2600-10, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21618472

RESUMEN

Protein glycation is a non-enzymatic glycosylation that can occur to proteins in the human body, and it is implicated in the pathogenesis of multiple chronic diseases. Glycation can also occur to recombinant antibodies during cell culture, which generates structural heterogeneity in the product. In a previous study, we discovered unusually high levels of glycation (>50%) in a recombinant monoclonal antibody (rhuMAb) produced by CHO cells. Prior to that discovery, we had not encountered such high levels of glycation in other in-house therapeutic antibodies. Our goal here is to develop cell culture strategies to decrease rhuMAb glycation in a reliable, reproducible, and scalable manner. Because glycation is a post-translational chemical reaction between a reducing sugar and a protein amine group, we hypothesized that lowering the concentration of glucose--the only source of reducing sugar in our fed-batch cultures--would lower the extent of rhuMAb glycation. When we decreased the supply of glucose to bioreactors from bolus nutrient and glucose feeds, rhuMAb glycation decreased to below 20% at both 2-L and 400-L scales. When we maintained glucose concentrations at lower levels in bioreactors with continuous feeds, we could further decrease rhuMAb glycation levels to below 10%. These results show that we can control glycation of secreted proteins by controlling the glucose concentration in the cell culture. In addition, our data suggest that rhuMAb glycation occurring during the cell culture process may be approximated as a second-order chemical reaction that is first order with respect to both glucose and non-glycated rhuMAb. The basic principles of this glycation model should apply to other recombinant proteins secreted during cell culture.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Glicoproteínas/metabolismo , Animales , Células CHO , Técnicas de Cultivo de Célula , Cricetinae , Glicosilación , Humanos , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo
3.
Biotechnol Prog ; 34(3): 584-592, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-28960825

RESUMEN

In the development of biopharmaceutical products, the expectation of regulatory agencies is that the recombinant proteins are produced from a cell line derived from a single progenitor cell. A single limiting dilution step followed by direct imaging, as supplemental information, provides direct evidence that a cell line originated from a single progenitor cell. To obtain this evidence, a high-throughput automated imaging system was developed and characterized to consistently ensure that cell lines used for therapeutic protein production are clonally-derived. Fluorescent cell mixing studies determined that the automated imaging workflow and analysis provide ∼95% confidence in accurately and precisely identifying one cell in a well. Manual inspection of the images increases the confidence that the cell line was derived from a single-cell to >99.9%. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:584-592, 2018.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Automatización , Técnicas de Cultivo de Célula , Células Clonales/citología , Células Clonales/metabolismo , Procesamiento de Imagen Asistido por Computador , Proteínas Recombinantes/biosíntesis , Animales , Anticuerpos Monoclonales/uso terapéutico , Células CHO , Cricetulus , Ensayos Analíticos de Alto Rendimiento , Proteínas Recombinantes/uso terapéutico
4.
Biotechnol Prog ; 31(1): 226-38, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25311542

RESUMEN

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cobre/farmacología , Animales , Células CHO , Cobre/química , Cobre/metabolismo , Cricetinae , Cricetulus , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Medios de Cultivo/farmacología , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo
5.
Biotechnol Prog ; 29(4): 980-5, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23606666

RESUMEN

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass.


Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Tetrahidrofolato Deshidrogenasa/deficiencia , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Células CHO , Células Cultivadas , Cricetulus , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Mitocondrias/metabolismo , Tetrahidrofolato Deshidrogenasa/genética
6.
Appl Environ Microbiol ; 71(4): 1717-28, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15811994

RESUMEN

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Fragmentos de Inmunoglobulinas/biosíntesis , Proteoma , Proteínas Recombinantes/metabolismo , Proteínas Bacterianas/metabolismo , Antígenos CD18/inmunología , Electroforesis en Gel Bidimensional , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Fermentación , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Procesamiento de Imagen Asistido por Computador , Fragmentos de Inmunoglobulinas/genética , Microbiología Industrial/métodos , Proteínas Recombinantes/genética
7.
Biotechnol Bioeng ; 85(5): 463-74, 2004 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-14760686

RESUMEN

During production of a humanized antibody fragment secreted into the periplasm of Escherichia coli, proteolytic degradation of the light chain was observed. In order to determine which protease(s) were responsible for this degradation, we compared expression of the F(ab')(2) antibody fragment in several E. coli strains carrying mutations in genes encoding periplasmic proteases. Analysis of strains cultured in high cell density fermentations showed that the combination of mutations in degP prc spr was necessary for the cells to produce high levels of the desired recombinant antibody fragment. In order to eliminate the possible effects of mutations in other genes, we constructed E. coli strains with protease mutations in isogenic backgrounds and repeated the studies in high cell density fermentations. Extensive light chain proteolysis persisted in degP strains. However, light chain proteolysis was substantially decreased in prc and prc spr strains, and was further decreased with the introduction of a degP mutation in prc and prc spr mutant strains. These results show that the periplasmic protease Prc (Tsp) is primarily responsible for proteolytic degradation of the light chain during expression of a recombinant antibody fragment in E. coli, and that DegP (HtrA) makes a minor contribution to this degradation as well. The results also show that spr, a suppressor of growth defects in prc strains, is required for a prc mutant to survive throughout high cell density fermentations.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Endopeptidasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Ingeniería de Proteínas/métodos , Antígenos CD18/inmunología , División Celular , Supervivencia Celular , Endopeptidasas/genética , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/inmunología , Mutagénesis Sitio-Dirigida , Periplasma/metabolismo , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Proteínas Recombinantes/biosíntesis , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Especificidad de la Especie
8.
Microbiology (Reading) ; 147(Pt 7): 1887-1896, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11429465

RESUMEN

Two mutational mechanisms, both supported by experimental studies, have been proposed for the evolution of new or improved enzyme specificities in bacteria. One mechanism involves point mutation(s) in a gene conferring novel substrate specificity with partial or complete loss of the original (wild-type) activity of the encoded product. The second mechanism involves gene duplication followed by silencing (inactivation) of one of these duplicates. Some of these 'silent genes' may still be transcribed and translated but produce greatly reduced levels of functional protein; gene silencing, in this context, is distinct from the more common associations with bacterial partitioning sequences, and with genes which are no longer transcribed or translated. Whereas most Salmonella enterica strains are ushA(+), encoding an active 5'-nucleotidase (UDP-sugar hydrolase), some natural isolates, including most genetically related strains of serotype Typhimurium, have an ushA allele (designated ushA(c)) which produces a protein with, comparatively, very low 5'-nucleotidase activity. Previous sequence analysis of cloned ushA(c) and ushA(+) genes from serotype Typhimurium strain LT2 and Escherichia coli, respectively, did not reveal any changes which might account for the significantly different 5'-nucleotidase activities. The mechanism responsible for this reduced activity of UshA(c) has hitherto not been known. Sequence analysis of Salmonella ushA(+) and ushA(c) alleles indicated that the relative inactivity of UshA(c) may be due to one, or more, of four amino acid substitutions. One of these changes (S139Y) is in a sequence motif that is conserved in 5'-nucleotidases across a range of diverse prokaryotic and eukaryotic species. Site-directed mutagenesis confirmed that a Tyr substitution of Ser-139 in Salmonella UshA(+) was solely responsible for loss of 5'-nucleotidase activity. It is concluded that the corresponding single missense mutation is the cause of the UshA(c) phenotype. This is the first reported instance of gene inactivation in natural isolates of bacteria via a missense mutation. These results support a model of evolution of new enzymes involving a 'silent gene' which produces an inactive, or relatively inactive, product, and are also consistent with the evolution of a novel, but unknown, enzyme specificity by a single amino acid change.


Asunto(s)
Proteínas de Escherichia coli , Mutación Missense , Hidrolasas Diéster Fosfóricas/genética , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Evolución Biológica , Duplicación de Gen , Silenciador del Gen , Genes Bacterianos , Immunoblotting , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Análisis de Secuencia de ADN
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