Traces matter: Targeted optimization of monoclonal antibody N-glycosylation based on/by implementing automated high-throughput trace element screening

The use of high-throughput systems in cell culture process optimization offers various opportunities in biopharmaceutical process development. Here we describe the potential for acceleration and enhancement of product quality optimization and de novo bioprocess design regarding monoclonal antibody N-glycosylation by using an iterative statistical Design of Experiments (DoE) strategy based on our automated microtiter plate-based system for suspension cell culture. In our example, the combination of an initial screening of trace metal building blocks with a comprehensive DoE-based screening of 13 different trace elemental ions at three concentration levels in one run revealed most effective levers for N-glycan processing and biomass formation. Obtained results served to evaluate optimal concentration ranges and the right supplementation timing of relevant trace elements at shake flask and 2 L bioreactor scale. This setup identified manganese, copper, zinc, and iron as major factors. Manganese and copper acted as inverse key players in N-glycosylation, showing a positive effect of manganese and a negative effect of copper on glycan maturation in a zinc-dependent manner. Zinc and iron similarly improved cell growth and biomass formation. These findings allowed determining optimal concentration ranges for all four trace elements to establish control on desired product quality attributes regarding premature afucosylated and mature galactosylated glycan species. Our results demonstrates the power of combining robotics with DoE screening to enhance product quality optimization and to improve process understanding, thus, enabling targeted product quality control.     

Differential effects of zinc on amyloid precursor protein (APP) processing in copper-resistant variants of cultured Chinese hamster ovary cells.

Previous studies have demonstrated that adding copper to Chinese-hamster ovary (CHO) cells greatly reduced the levels of beta-amyloid (Abeta) peptide in parental CHO-K1 and in copper resistant CHO-CUR3 cells which have lower intracellular copper levels. In the current study, zinc, the zinc chelator 1,10-phenanthroline or copper chelators bathocuproine and D-penicillamine were added to the culture media of stably transfected CHO cells. The data show that zinc up to concentrations of 50 microM or the presence of 1,10-phenanthroline specifically increased the level of secreted APP in CHO-K1 cells. By contrast, the level of secreted APP in CHO-CUR3 cells remained unaffected. APP holoprotein increased dramatically in CHO-CUR3 cells compared with CHO-K1 cells. The large decrease of Abeta release seen in both cell lines at elevated extracellular zinc levels was due to specific inhibition of secretion. These results indicate that a disturbed zinc-homeostasis may be an important factor influencing APP production, transport and processing.     

Trace element uptake by L-cells as a function of trace elements in a synthetic growth medium

The concentration of trace elements in L-cells has been studied as a function of the trace metal content of the growth medium. Cells were cultured in synthetic media which contained varying trace amounts of the elements manganese, iron, cobalt, copper, zinc and molybdenum. The cellular concentration of the elements potassium, iron, copper and zinc were then determined. It was found that the cell accumulates trace metals at a different rate than they are made available. Deficiencies in zinc could be “induced” in the cell by increasing the concentration of iron, manganese and cobalt; cellular iron deficiencies were observed at larger medium concentrations of zinc, manganese, copper and cobalt. Trace metal uptake by the cell was seen to parallel the utilization by multicellular organisms.     

Effect of dietary copper and zinc levels on tissue copper,zinc, and iron in male rats

The interaction between dietary copper and zinc as determined by tissue concentrations of trace elements was investigated in male Sprague-Dawley rats. Animals were fed diets in a factorial design with two levels of copper (0.5, 5 μg/g) and five levels of zinc (1, 4.5, 10, 100, 1000 μg/g) for 42 d. In rats fed the low copper diet, as dietary zinc concentration increased, the level of copper decreased in brain, testis, spleen, heart, liver, and intestine. There was no significant effect of dietary copper on tissue zinc levels. In the zinc-deficient groups, the level of iron was higher in most tissues than in tissues from controls (5 μg Cu, 100 μg Zn/g diet). In the copper-deficient groups, iron concentration was higher than control values only in the liver. These data show that dietary zinc affected tissue copper levels primarily when dietary copper was deficient, that dietary copper had no effect on tissue zinc, and that both zinc deficiency and copper deficiency affected tissue iron levels.     

不育男性患者和健康男性血液与精浆中微量元素含量的对比分析

目的:研究不育患者精浆和血液中微量元素的含量,为男性不育的诊断和治疗提供理论依据。方法:对73例正常生育组男性和265例男性不育患者的精浆和血液中的锌、铁、铜、钙、镁、镉进行检测分析,分析两组血液和精浆微量元素的差异。结果:不育组患者精浆和血液中锌的含量明显低于正常对照组,铜、镉离子含量明显高于正常对照组,与正常对照组比较均有显著性差异(P<0.01);而两组中的钙、铁、镁的含量接近,差异无统计学意义(P>0.05)。不育组患者精浆中的锌元素水平明显高于血液锌含量,而血液中的镉含量明显高于精浆中的镉含量,差异有明显的统计学意义(P<0.05)。结论:精浆和血液中锌、铜、镉的变化与男性不育密切相关。     

Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivation

Metallothionein (MT) is a small cysteine-rich protein thought to play a critical role in cellular detoxification of inorganic species by sequestering metal ions that are present in elevated concentrations. We demonstrate here that metallothionein can play an important role at the other end of the homeostatic spectrum by scavenging an essential metal in a mouse fibroblast cell line that has been cultured under conditions of extreme zinc deprivation (LZA-LTK-). These cells unexpectedly produce constitutively high levels of metallothionein mRNA; however, the MT protein accumulates only when high concentrations of zinc are provided in the media. Until this MT pool is saturated, no measurable zinc remains in the external media. In this case, zinc deprivation leads to amplification of the MT gene locus in the LZA-LTK- cell line. Furthermore, the intracellular zinc levels in the fully adapted cells remain at the normal level of 0.4 fmol zinc/cell, even when extracellular zinc concentration is decreased by 2 orders of magnitude relative to normal media.     

Association between concentration of trace elements in serum and bronchial asthma among Japanese general population

Background and aimsA number of previous studies had revealed the association between trace elements in serum and bronchial asthma. However, only a few researches had focused on serum concentration of trace elements in a general population. In this study, an association between trace elements in serum and bronchial asthma was investigated in a general population.MethodsSubjects were 1025 volunteers (385 males and 640 females between ages 19 and 82 years old) who had participated in the Iwaki Health Promotion Project 2005. Bronchial asthma was diagnosed based on the European Community Respiratory Health Survey II according to the self-questionnaires on health conditions of subjects. The serum concentrations of certain trace elements (manganese, copper, zinc, selenium and iron) were measured and compared. Also, an association between serum trace elements level and neutrophil-related functions (oxidative burst activity, phagocytic activity, serum opsonic activity) were determined.ResultsIn males, no significant differences were seen in any serum trace elements concentrations. In females, serum zinc level was significantly higher in bronchial asthma group than in control. A positive correlation was seen between serum concentration of zinc and serum opsonic activity in both genders.ConclusionsIn female asthmatics, increase of oxidative stress was suggested to be caused by superoxide dismutase pathway (elimination system of reactive oxygen species) rather than serum opsonic activity (production system of reactive oxygen species from neutrophils) pathway, as the zinc concentration in bronchial asthma group was higher than that in control.     

Optimization of an extracellular zinc-metalloprotease (SVP2) expression in Escherichia coli BL21 (DE3) using response surface methodology

In this work, SVP2 from Salinivibrio proteolyticus strain AF-2004, a zinc metalloprotease with suitable biotechnological applications, was cloned for expression at high levels in Escherichia coli with the intention of changing culture conditions to generate a stable extracellular enzyme extract. The complete ORF of SVP2 gene was heterologously expressed in E. coli BL21 (DE3) by using pQE-80L expression vector system. In initial step, the effect of seven factors include: incubation temperature, peptone and yeast extract concentration, cell density (OD600) before induction, inducer (IPTG) concentration, induction time, and Ca(2+) ion concentrations on extracellular recombinant SVP2 expression and stability were investigated. The primary results revealed that the IPTG concentration, Ca(2+) ion concentration and induction time are the most important effectors on protease secretion by recombinant E. coli BL21. Central composite design experiment in the following showed that the maximum protease activity (522 U/ml) was achieved in 0.0089 mM IPTG for 24h at 30 °C, an OD600 of 2, 0.5% of peptone and yeast extract, and a Ca(2+) ion concentration of 1.3 mM. The results exhibited that the minimum level of IPTG concentration along with high cell density and medium level of Ca(2+) with prolonged induction time provided the best culture condition for maximum extracellular production of heterologous protease SVP2 in E. coli expression system.     

Clioquinol mediates copper uptake and counteracts copper efflux activities of the amyloid precursor protein of Alzheimer's disease

The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels. A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques. Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol. Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels. This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels. The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels. The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold. These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease.     

Cadmium-induced alterations in essential trace element homoeostasis in the tissues of scallop Mizuhopecten yessoensis

Studies were performed regarding the effect of cadmium accumulation on the levels of essential elements (copper, zinc and iron) in the tissues of a marine bivalve mollusc, Mizuhopecten yessoensis, exposed to cadmium at 250 ppb during 2 weeks. It was found that the concentration of cadmium in the tissues increased in the order gonads < gills < hepatopancreas < kidney during exposure time. However, the highest value of concentration factor was recorded in the gills. Our data demonstrate that cadmium accumulation in all mollusc tissues is followed by the alterations in copper, zinc and iron concentration, but that the pattern of these changes varies with each tissue. Cadmium had the most pronounced effects on essential trace element homoeostasis in the kidney. The present study suggests that levels of the essential metals in a particular tissue can be modified depending on the level of cadmium accumulation. The possible mechanisms of the effects of cadmium on the essential trace elements are discussed.     

Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture

A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture. The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed-batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level.     

Characterization of soy protein hydrolysates and influence of its iron content on monoclonal antibody production by a murine hybridoma cell line

A challenging aspect with the use of protein hydrolysates in commercial manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.     

Trace elements in diabetes mellitus. Peculiarities and clinical validity of determinations in blood cells

Significantly more information about trace element status can be obtained by investigating concentrations in blood cells instead of only evaluating the concentrations in plasma. This can be explained by the fact that essential trace elements such as zinc, copper, chromium and selenium take part in a variety of enzymatic processes on a molecular cellular level. Ignoring these important biochemical roles, trace element concentrations determined in whole blood or plasma very often lead to conclusions contrary to the actual intracellular concentration. Especially in metabolic diseases like diabetes mellitus, conclusions drawn from trace element concentrations in blood cells usually offer more valuable clinical information about the metabolic state than trace element concentrations in plasma or whole blood. In the present investigation copper and zinc concentrations were increased in all blood fractions of diabetic patients (IDDM). In insulin-dependent diabetic children significantly higher values of zinc in erythrocytes were also found, and they were higher in patients with poor metabolic control (HbA1c>9%). When different blood fractions in diabetic patients (NIDDM) were compared with a control group, chromium was significantly increased in plasma and polymorphonuclear cells. Patients with IDDM had pronounced decreased selenium concentrations in erythrocytes as compared to controls.     

Regulation of copper uptake and transport in intestinal cell monolayers by acute and chronic copper exposure

Adaptation to high and low copper intake in mammals depends on the cellular control of influx, efflux and storage mechanisms of cellular copper concentrations. In the present study, we used an intestinal cell line (Caco-2), grown in bicameral chambers to study the effect of equilibrium loading with copper. We analyzed (64)Cu uptake from the apical surface, intracellular metal (Cu, Zn, Fe) content, (64)Cu transport into the basal chamber, and total copper, zinc and iron in the basal chamber. We found that the (64)Cu uptake is saturable, shows a linear response phase up to 1.5 microM reaching a plateau at 4-6 microM extracellular Cu. Intracellular copper increased 21.6-fold, from 1.5 to 32.4 mM (at 0.2-20.2 microM extracellular copper respectively). The time course for (64)Cu uptake and transport was linear when the cells were incubated with different copper concentrations. Uptake increased 10-fold when intracellular copper concentration was raised. Fluxes were lowest at 1.5 mM and highest at 32.4 mM Cu intracellular copper (2.03 and 20. 98 pmole (64)Cu insert(-1) h(-1), respectively). The apical-to-basolateral copper transfer rate was lower at 32.4 mM as compared to 1.5 mM intracellular copper (0.55-1.95 pmole (64)Cu insert(-1) h(-1), respectively). The total copper in the basal chamber increased 4.2-fold (from 3.04 to 12.85 pmole Cu insert(-1) h(-1)) when the intracellular copper concentration was raised. If cells are preincubated in a low copper medium most of the newly incorporated copper (64%) is transferred to the basolateral compartment. In contrast, under preloading with high copper concentration, only 4% of the fresh copper is transferred to the basal chamber; however, the intracellular copper contribution to this chamber increases by 4.2-fold. Thus, the process results in an increase in both storage and intracellular-to-basolateral flux of copper. In summary, our results indicate that copper fluxes from apical-to-cell and apical-to-basolateral domains are affected by intracellular copper concentration suggesting that mechanisms of copper transport involved in cellular adaptation to low and high copper exposure are different.     

Traces of copper ions deplete glutathione in human hepatoma cell cultures with low cysteine content

BACKGROUND: Cell death induced by intracellular glutathione depletion has been reported to be dependent on the presence of trace amounts of extracellular copper ions. Since little is known about the relationship between glutathione depletion and copper homeostasis, we have in the present study further investigated the role of low amounts of copper ions in glutathione depletion. METHODS: Glutathione turnover was investigated in HeLa and hepatoma cell cultures with normal and low cysteine content in the presence of copper ions (1 and 10micromol/L) and two other glutathione-stimulating agents (lipoic acid and mercury ions). RESULTS: Copper ions (10micromol/L) caused relatively small increases in total amount of glutathione (the sum of the intracellular and the extracellular amount of glutathione) in HeLa and hepatoma cell cultures with normal cysteine levels (420nmol/mL) compared to control cell cultures, whereas lipoic acid and mercury ions strongly increased total glutathione in both types of cell cultures. Lower amount of total glutathione was observed in cell cultures with a lower cysteine levels (84nmol/mL), which is similar to that in human plasma. A strongly decreased total amount of glutathione in the presence of copper ions was observed in hepatoma cell cultures with lower cysteine levels, whereas the other agents showed effects similar to those described for cell cultures with normal cysteine levels. CONCLUSION: Glutathione synthesis in hepatoma cell cultures is probably more sensitive to a low cysteine level than HeLa cell cultures, and the presence of copper ions further decreases the availability of cysteine probably by increasing the disulfide binding to cysteine residues in extracellular proteins, which causes a further decrease of total glutathione.     

Selenium, iron, copper, and zinc levels and copper-to-zinc ratios in serum of patients at different stages of viral hepatic diseases

Viral hepatic diseases, especially those induced by the hepatitis B virus, can progress into more serious pathological outcomes and eventually to hepatocellular carcinoma. A growing body of evidence indicates that many trace elements play important roles in a number of carcinogenic processes that proceed through various mechanisms. To examine the status of trace elements during the development of hepatic carcinoma, we determined the selenium, iron, copper, and zinc levels and copper-to-zinc ratios in the serum of patients at different stages of viral hepatic disease. We observed significant changes in the selenium, iron copper, and zinc levels in the serum of patients having hepatocellular carcinoma, relative to those of healthy controls (p<0.05). The mean serum copper level in patients with hepatocellular carcinoma was significantly higher than that of the control group. In contrast, the mean selenium, iron, and zinc levels in patients having hepatocellular carcinoma were significantly lower than those of the control group. In addition, the mean zinc level in the serum of patients with hepatic cirrhosis was significantly lower than that of the control group (p<0.05). Moreover, we found markedly elevated Cu: Zn ratios (p<0.05) in patients having hepatic cirrhosis or hepatocellular carcinoma. Our findings imply that the levels of some trace elements, such as selenium, iron, copper, and zinc, and Cu:Zn ratios, might serve as biomarkers for the increased severity of viral hepatic damage.     

Improvement of the production of GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 fusion protein using a molecular chaperone-assisted insect-cell-based expression system

A stable Tn-5B1-4 insect cell line co-expressing the recombinant GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 (GFPuv-beta3GnT2) protein fused to a melittin signal sequence with a lectin-like molecular chaperone, human calnexin (hCNX) or human calreticulin (hCRT), was constructed. The expression of either of these molecular chaperones is under the control of a weak promoter, OpMNPV IE2, while that of GFPuv-beta3GnT2 is under the control of Bombyx mori actin promoter. This co-expression system was compared between two different insect cell-baculovirus expression systems: (1) co-infection of the recombinant baculovirus containing a molecular chaperone (AcNPV-hCNX or -hCRT) with a recombinant baculovirus containing GFPuv-beta3GnT2 fused with the melittin signal sequence (AcNPV-me-GGT); (2) infection of AcNPV-me-GGT to a stably expressing cell line for either hCNX or hCRT. In the co-infection system, the intracellular GFPuv-beta3GnT2 expression level was low because of the improved secretion level ratio of the fusion protein, due to the chaperone expression. In the case of infection to the stably expressing cell line for a chaperone, the extracellular GFPuv-beta3GnT2 expression level was similar to the intracellular expression level. This suggests that the amount of expressed chaperone is not sufficient to process beta3GnT2. On the other hand, the co-expression system produced an extracellular beta3GnT activity of 22-23 mU/mL, which was approximately 3.5- and 11-fold higher than those of the stable expression of the fusion gene without the chaperone and the conventional BES with the addition of protease, respectively. The secretion level ratio of the fusion protein of this system increased to 82%, which was approximately 1.5-fold that of any other expression system investigated thus far. These results indicate that the ratio of the expression level of the target gene to that of the chaperone gene may be an important factor in maximizing the production of a target protein. The molecular-chaperone-assisted expression system using a stably transformed insect cell line offers promising prospects for the efficient production of recombinant secretory proteins in insect cells.