A Single Nutrient Feed Supports Both Chemically Defined NS0 and CHO

A Single Nutrient Feed Supports Both Chemically De?ned NS0and CHO Fed-Batch Processes:Improved Productivity and Lactate Metabolism Ningning Ma,JoAnn Ellet,Centy Okediadi,Paul Hermes,Ellen McCormick,and Susan Casnocha Bioprocess R&D,Global Biologics,P?zer Inc,Chester?eld,MO63017

DOI10.1002/btpr.238

Published online July27,2009in Wiley InterScience(https://www.sodocs.net/doc/0917191069.html,).

A chemically de?ned nutrient feed(CDF)coupled with basal medium preloading was

developed to replace a hydrolysate-containing feed(HCF)for a fed-batch NS0process.The

CDF not only enabled a completely chemically de?ned process but also increased recombi-

nant monoclonal antibody titer by115%.Subsequent tests of CDF in a CHO process indi-

cated that it could also replace the hydrolysate-containing nutrient feed in this expression

system as well as providing an80%increase in product titer.In both CDF NS0and CHO

processes,the peak lactate concentrations were lower and,more interestingly,lactate metab-

olism shifted markedly from net production to net consumption when cells transitioned from

exponential to stationary growth phase.Subsequent investigations of the lactate metabolic

shift in the CHO CDF process were carried out to identify the cause(s)of the metabolic

shift.These investigations revealed several metabolic features of the CHO cell line that we

studied.First,glucose consumption and lactate consumption are strictly complementary to

each other.The combined cell speci?c glucose and lactate consumption rate was a constant

across exponential and stationary growth phases.Second,Lactate dehydrogenase(LDH)ac-

tivity?uctuated during the fed-batch process.LDH activity was at the lowest when lactate

concentration started to decrease.Third,a steep cross plasma membrane glucose gradient

exists.Intracellular glucose concentration was more than two orders of magnitude lower

than that in the medium.Fourth,a large quantity of citrate was diverted out of mitochondria

to the medium,suggesting a partially truncated tricarboxylic acid(TCA)cycle in CHO cells.

Finally,other intermediates in or linked to the glycolytic pathway and the TCA cycle,which

include alanine,citrate,isocitrate,and succinate,demonstrated a metabolic shift similar to

that of lactate.Interestingly,all these metabolites are either in or linked to the pathway

downstream of pyruvate,but upstream of fumarate in glucose metabolism.Although the spe-

ci?c mechanisms for the metabolic shift of lactate and other metabolites remain to be eluci-

dated,the increased understanding of the metabolism of CHO cultures could lead to future

improvements in medium and process development.V C2009American Institute of Chemical

Engineers Biotechnol.Prog.,25:1353–1363,2009

Keywords:CHO,NS0,chemically de?ned process,lactate,metabolic shift,metabonomics,

metabolomics

Introduction

Hydrolysates,especially those of nonanimal origin,are widely used as serum replacements in therapeutic protein production processes using mammalian cells.Hydrolysates can improve cell growth and protein yield in serum-free processes(Burteau et al.,2003;Heidemann et al.,2000; Sung et al.,2004;Xie et al.,1997).However,there are a few limitations that complicate hydrolysates’utilization.First of all,lot-to-lot variation exists for hydrolysates(Luo and Chen,2007;Zhang et al.,2003).Prescreening using small-scale performance testing is typically required to eliminate underperforming lots.Second,the nutrient composition in hydrolysates is not balanced for cell consumption.Various components,such as ash and salts,are either not required by the cells or are required at much lower levels than supplied. Third,the chemically unde?ned nature of hydrolysates hin-ders the scientists’understanding process of cell metabolism. For instance,it is not well understood how mammalian cells metabolize amino acids in peptides,so that amino acid con-sumption analysis based on free amino acids measurements may or may not be accurate(Nyberg et al.,1999). Development of various chemically de?ned media for NS0cells and CHO cells has been reported(Chen et al., 2000;Hata et al.,1992;Huang et al.,2007;Zhang and Rob-inson,2005),and commercial chemically de?ned media are readily available from all major mammalian cell culture me-dium manufacturers.However,the development of chemi-cally de?ned fed-batch processes that support rapid cell proliferation,high cell density,and high production rate has been challenging.Chemically de?ned fed-batch processes have been reported recently.Gong et al.(2006)developed a chemically de?ned fed-batch process for hybridoma cells.

Correspondence concerning this article should be addressed to N.Ma

at ningning.ma@p?https://www.sodocs.net/doc/0917191069.html,.

V C2009American Institute of Chemical Engineers1353

Peak viable cell density reached about8?106cells/mL,but viable cells dropped sharply after a5-day culture.Burky et al.(2006)developed a generic chemically de?ned fed-batch process for NS0cells.The process supported peak via-ble cell density around1?107cells/mL and product titer of 2.64g/L on Day13.

Lactic acid,or lactate,has long been recognized as a major by-product in cell culture processes(Hu et al.,1987; Miller et al.,1989;Ozturk and Palsson,1992).Base addition to neutralize lactic acid elevates medium osmolarity,which, in turn,inhibits cell growth and causes viability to drop. Reducing lactic acid production had been shown to increase peak cell density,extend process duration,and increase recombinant protein yield.Four strategies had been reported in the literature to reduce lactate production.The?rst one is to reduce medium glucose concentration to limit glucose supply(Cruz et al.,1999;Zhou et al.,1995,1997b).Using an online dynamic feeding protocol,Zhou et al.(1995, 1997b)successfully maintained glucose at a low level of about0.5mM.As a result,lactate production was reduced and higher peak cell densities and higher viability were achieved.A second strategy is to use alternative,slow-con-suming carbon sources(Altamirano et al.,2004).Altamirano et al.(2004)used a feeding strategy in which glucose and galactose feeds were alternated.This strategy forced CHO cells to consume more residual lactate than the culture fed with glucose only.As a result,harvest viability was mod-estly improved.The third strategy is to downregulate lactate dehydrogenase-A(LDH-A)(the LDH subunit that effectively catalyzes pyruvate to lactate conversion)using a wide vari-ety of techniques including homologous recombination (Chen et al.,2001),antisense mRNA(Jeong et al.,2001; Paredes et al.,1999),and small interfering RNA(Kim and Lee,2007a).Chen et al.(2001)compared a LDH-A downre-gulated hybridoma clone to a control clone.In the culture utilizing the LDH-A downregulated cell line,lactate concen-tration was about35%lower and monoclonal antibody titer was about200%higher.However,although Kim and Lee (2007a)reduced lactate production rate even further(by45–79%),the recombinant thrombopoietin product rates of three LDH-A downregulated clones were not statistically better than the control clone.A fourth strategy is to enhance the ?ow of glucose carbon into tricarboxylic acid(TCA)cycle.

A focus area has been the overexpression of pyruvate car-boxylase of yeast(Irani et al.,1999,2002)or human origin (Kim and Lee,2007b).In a perfusion culture,Irani et al. (2002)demonstrated that pyruvate carboxylase-transfected BHK-21cells produced half of the lactate when compared with the control cells.Cell density was about50%higher and recombinant erythropoietin production rate was about 100%higher.More recently,it was reported that the expres-sion of antiapoptosis genes,E1B-19K and Aven(EA167), could reduce lactate production of CHO cells(Dorai et al., in press).In batch cultures with or without glucose limita-tion,a CHO clone transfected with antiapoptotic genes gave lower lactate concentration compared with the control cell line at corresponding conditions.

In this study,we developed a chemically de?ned nutrient feed(CDF)with companion production medium preloading to replace a hydrolysate-containing feed(HCF)for a fed-batch NS0process.The CDF was later investigated in a CHO process.In both NS0and CHO processes,CDF resulted in higher product titer and lower peak lactate con-centration.The CDF processes also exhibited a dramatic lac-tate metabolic shift from net production to net consumption. At the end of the process,lactate concentration in the CDF processes was almost undetectable.Subsequent investigations were carried out to better understand this metabolic shift in the CDF CHO process.

Materials and Methods

Cells

NS0and CHO cells producing test recombinant monoclo-nal antibodies were used in this study.Both cell types used glutamine synthatase as the transfection selection marker. Process

NS0and CHO cells were routinely subcultured in250mL shake?asks(Corning,Acton,MA)in chemically de?ned hy-bridoma and CHO media,respectively.Both hybridoma and CHO media are commercially available.The CHO medium was supplemented with25mM methionine sulfoximine(MSX). Two-liter Applikon bioreactors(Applikon,Foster city, CA)with a1-L working volume were used in this study.In these bioreactors,two pitched blade impellers provided mixing and gas bubble dispersion.Temperature was controlled at 36.5 C using a heating blanket.pH was controlled at7.0by carbon dioxide sparging through a ring sparger and by adding either0.5N sodium hydroxide or7.5%sodium bicarbonate. Dissolved oxygen(DO)was controlled at30%air saturation using air overlay and oxygen sparging.The fed-batch process for NS0and CHO cells was very similar.Cells are seeded at 2.5?105viable cells/mL in corresponding NS0or CHO pro-duction media.Starting from Day3,a nutrient feed was added into the bioreactors at28mL/L/day and16mL/L/day for NS0 and CHO cells,respectively,till the end of the process.Glucose was fed separately to control glucose concentration around2g/ L.Bioreactors were sampled daily for of?ine measurements. Routine bioreactors of?ine measurements

Cell density and viability were measured using a Cedex cell counter(Innovatis,Bielefeld,Germany).Of?ine DO, pH,glucose concentration,and lactate concentration were measured with a BioPro?le chemical analyzer(Nova Bio-medical,Waltham,MA).A portion of the broth from each daily sampling was?ltered through a0.2l m polyethersul-fone?lter(Pall,Ease Hills,NY)for product titer and amino acid measurements.

Monoclonal antibody titer was measured using an Agilent HPLC(Hewlett Packard,Waldbronn,Germany)with a POROS protein-A af?nity column(Applied Biosystems,Fos-ter City,CA).The column was calibrated using correspond-ing reference standards before titer quanti?cation. Intracellular LDH activity measurement

About2–4mL of broth was taken out of bioreactors and transferred to a centrifuge tube chilled on ice.The cells were washed twice with cold phosphate buffered saline(PBS).In each wash,the cells were pelletted in a refrigerated centri-fuge at150g for5min,the supernatant was discarded,and the cell pellet was gently resuspended in4mL of cold PBS. After the second wash,cells were diluted to5?105viable cells/mL and lysed using freeze-thaw.In the freeze-thaw procedure,cells were?rst?ash frozen in liquid nitrogen and

then thawed overnight at2–8 C.LDH activity was quanti?ed using a colorimetric LDH assay kit(CytoTox96nonradioac-tive cytotoxicity assay,Promega,Madison,WI)and a spec-trophotometer(Molecular Devices,Sunnyvale,CA).Sample preparation and measurement followed manufacturers’instructions.The LDH assay kit quanti?es LDH activity based on the conversion rate of lactate and NADtto pyru-vate and NADH.

Sample collection for metabonomics assay

Both cell and spent medium samples were collected for metabonomics analysis.To collect cell samples,cell culture broth containing2?107viable cells was removed from the bioreactor and immediately chilled in an ice bucket.The cells were pelleted at1,000g for3min in a refrigerated cen-trifuge(Bechman Coulter,Fullerton,CA)and then rinsed twice using cold PBS.After two washes,the cell pellet was ?ash frozen in liquid nitrogen and stored atà80 C.A2mL spent medium sample was collected where cell broth was ?rst centrifuged at1,000g for3min and then the supernatant was?ltered through a0.2l m polyethersulfone?lter.Spent medium samples were also stored atà80 C.After all sam-ples were collected,they were sent on dry ice to the analysis provider.

Sample preparation and metabonomics assay

Sample preparation and metabonomics analysis were con-ducted at Metabolon(Durham,NC).The metabonomics methodology is detailed elsewhere(Lawton et al.,2008)but brie?y,the samples were thawed,small molecules were extracted,and the reconstituted extracts were split for analy-sis by GC and LC mass spectrometry.Chromatographic sep-aration of all ions in each sample was followed by library matching of these ions to Metabolon’s reference library of standards.The identity of metabolites was determined by matching the combination of chromatographic retention index and mass spectra signatures compared with the refer-ence library entries.

Results and Discussion

Development of a chemically de?ned nutrient

feed for NS0process

A CDF was developed to replace a hydrolysate-containing

nutrient feed(HCF)that was previously used for a fed-batch NS0process.In the hydrolysate-containing fed-batch pro-cess,the basal medium is chemically de?ned,but the nutri-ent feed contains a nonanimal-derived hydrolysate.The limitations of hydrolysates,such as lot-to-lot variation, prompted the development of the CDF to achieve a fully chemically de?ned process.

The development took a top-down approach,as illustrated in Figure1.There were three major development steps:(1) develop an over-rich nutrient feed,(2)simplify formula by removing unnecessary components and reduce the concentra-tion of overfeed components,and(3)polishing and formula-tion.The plan for the over-rich solution was that it should contain all nutrients that NS0cells require and all required nutrients should be at suf?ciently high concentrations.To ensure that all necessary components were included in the over-rich solution,over90components were incorporated. These components came from the formula of a P?zer propri-etary chemically de?ned NS0medium and a previously developed CDF.To ensure that all necessary components were in ample supply,most components were added at a high concentration.In the next step,nutrients in the over-rich solution were divided into eight rough groups to balance their ratio.Grouping was based on nutritional category(trace elements,nucleotides,etc.)or by solubility(low pH,ethanol, etc.).In the subsequent formula simpli?cation steps,compo-nents were screened in groups for two cycles,?rst in rough groups and then in re?ned groups.In the?rst cycle,two of eight rough groups were found unnecessary where their exclusion rendered no detectable difference in process per-formance(cell density,viability,and product titer).Compo-nents in these two subgroups were subsequently removed from the formula.The remaining components were divided into13re?ned groups again based on nutritional category and solubility.Most groups contained less than?ve compo-nents.In the second cycle of screening,?ve re?ned

groups Figure1.The top-down approach used in the chemically de?ned nutrient feed development.

The development included three major steps:(I)obtain an over

rich nutrient feed,(II)formula simpli?cation,and(III)polish-

ing and formulation.

were found to offer no detectable bene?cial (or detrimental)effect on the process performance and were removed.In the ?nal step,the components in the simpli?ed formula,with less than 40components,were balanced for optimization.Amino acids were optimized based on spent medium analy-sis,whereas the other components were optimized relied on re?ned group titration study.Finally,all components were formulated into a single pH neutral nutrient feed.For com-ponents with low solubility in the feed,the portion over the solubility limit was preloaded into the basal medium.All de-velopment studies were conducted in fed-batch shake ?asks.At each milestone,the performance was con?rmed in 2L bench-scale bioreactors.The whole development took $9months,with each of three major steps for about 3months.A comparison of the CDF process and HCF process in 2L bioreactors is presented in Figure 2,where results are the average of three bioreactor runs and error bars indicate 1SD.Cell growth phase was extended in the CDF process where the peak viable cell density reached 15?106cells/mL,$50%higher than that in the HCF process.Viability was modestly improved in the CDF process,the harvest via-bility was about 10%higher in the CDF process.Product ti-ter was much higher in the CDF process after Day 5.At harvest on Day 11,product titer in the CDF process was 115%higher than that in the HCF process.This 115%increase was from a 33%increase in the integral viable cell

concentration and a 62%increase in cell speci?c productiv-ity (Q p ).These results demonstrate that CDF not only allows for removing hydrolysate from the process but also delivers better cell growth and productivity.Another bene?t of the CDF process is the lower lactate concentration.As shown in Figure 2b,lactate in the CDF process was lower throughout the whole process,and the peak concentration was only half of that in the HCF process.In both processes,cells switched from net lactate production to net consumption around Day 5.In the CDF process,residual lactate was consumed in 2days and remained lower than the detection limit until har-vest.In the HCF process,the lactate concentration was gen-erally higher and net lactate production resumed,although at a very slow rate,after Day 8.

The bene?t of the top-down approach to nutrient feed de-velopment is that it eliminates nutrient limitation up front,thereby allowing the development to focus on nutrient bal-ancing and simpli?cation.The risk of this strategy is the inclusion of unnecessary components and overfeeding,which could be toxic or inhibitory to cell growth or production.The successful development of a CDF demonstrates that the risk is manageable.During the development,many compo-nents,such as vitamins and minerals,were found to be non-toxic even at a concentration one or two orders of magnitude higher than what the cells needed.This observation agrees with Xie and Wang (1994),who found that a high concentra-tion of vitamins was not harmful to animal cells.Amino acids have the potential to be inhibitory to cell growth at high concentrations.Fortunately,amino acid overfeeding can be avoided using spent medium analysis.

Evaluation of the CDF in CHO processes

The CDF developed for NS0cells was later tested on CHO cells and compared with the CHO HCF process.The medium used in both processes is a commercially available chemically de?ned CHO medium.As shown in Figure 3,the CDF process offered clear advantages over the HCF process.Results shown in Figure 3were the average of two duplicate bioreactors.Peak viable cell density reached $18?106cells/mL in both processes.However,viability was main-tained better in the CDF process.When the processes were terminated on Day 15,viability for the CDF process was around 80%,whereas that for the HCF process had declined to 50%.Product titer was similar in both processes till Day 10,after which titer started to level off in the HCF process,whereas that in the CDF process kept rising linearly till har-vest.By Day 15,product titer in the CDF process was 82%higher than that in the HCF https://www.sodocs.net/doc/0917191069.html,ctate concentration was much lower in the CDF process after Day 7.By Day 10,lactate concentration in the CDF process became close to undetectable,whereas that in the HCF process stayed at 1.8–2.6g/L.

Lactate metabolic shift in the CDF processes

A major metabolic feature of the CDF processes is the dramatic shift of lactate metabolism from net production to net consumption.On one hand,the metabolic shift is bene?-cial for the process as it signi?cantly reduced the concentra-tion of a major cellular metabolic by-product.At the end of the process,lactate concentration was almost undetectable.On the other hand,the shift could be due to a glucose carbon limitation,which may have a detrimental effect on the

cell

Figure https://www.sodocs.net/doc/0917191069.html,parison of the CDF (solid lines)and HCF (dot-ted lines)NS0processes.

(a)Viable cell density and viability.(b)Product titer and lac-tate concentration.The results shown are the average of three replicate batches.Error bar indicates 1SD.

growth and productivity.Although glucose supply was not limited in both CDF NS0and CDF CHO processes (glucose concentration [1g/L),possible limitations could still exist in the glucose transportation step or the ?ux through the gly-colytic pathway.To better understand and to identify the possible mechanism(s)for the lactate metabolic shift,we examined carbon metabolisms in the CDF CHO process.The lactate metabolic shift in fed-batch processes has been previ-ously reported with different cell expression systems,includ-ing DHFR-CHO (Tsao et al.,2005),GS-NS0(Zhou et al.,1997a),and a non-GS-NS0(Burkey et al.,2006).However,no thorough investigation of this phenomenon has been pub-lished.We focused our investigation on CHO cells,where the cell line used was a subclone of the CHO cell line used in the initial CDF evaluation (Figure 3).The CDF fed-batch process was kept the same.

As indicated in Figure 4a,the lactate metabolic shift took place in the same period when cells were transitioning from exponential to stationary growth phase.The same timing cor-relation between the lactate metabolic shift and cell growth phase transition exists in previous publications using differ-ent mammalian cell types (Burkey et al.,2006;Tsao et al.,2005;Zhou et al.,1997a).Whether there is a cause-effect relationship between lactate metabolic shift and cell growth slowdown is unknown.One possibility is that the metabolic shift results from the cell growth phase transition.High lac-

tate production is a hallmark of transformed cells,a phenom-enon established more than 70years ago (Warburg,1930).High lactate production has been attributed in the literature to the upregulation of various enzymes in the glycolytic pathway (Board et al.,1990;Dang and Semenza,1999;Kondoh et al.,2005).As cells exit the rapid proliferating phase,they may concurrently cease high lactate production.Alternatively,the lactate metabolism shift could be a result of glucose carbon limitation because of restricted ?ux through the glycolytic pathway.

Analysis of glucose consumption revealed a strong com-plementary relationship between glucose and lactate con-sumption.Cumulative glucose utilization,lactate production,and net glucose consumption (de?ned as combined glucose and lactate consumption)from the two duplicate bioreactors referred to in Figure 4a are illustrated in Figure 4b.The slope of the curves in Figure 4b represents either cell-spe-ci?c substrate consumption (glucose or net glucose)or pro-duction (lactate)rate.The glucose consumption and lactate production rates ?uctuated during the process.However,net glucose consumption rate was steady throughout the whole process.This observation inferred that (1)

cell-speci?c

Figure https://www.sodocs.net/doc/0917191069.html,parison of the CDF (solid lines)and HCF (dot-ted lines)CHO processes.

(a)Viable cell density and viability.(b)Product titer and lac-tate concentration.The results shown are the average of two duplicate

batches.

Figure 4.Cell growth and glucose and lactate metabolism of

CHO cells in the CDF process in two duplicate bioreactors.

Bioreactor 1,solid symbols;Bioreactor 2,hollow symbols.(a)Cell growth and lactate pro?le.The arrow indicates the correla-tion between shift of lactate metabolism and cell growth phase transition.(b)Weight cumulative glucose consumption,lactate production,and net glucose consumption.The net glucose con-sumption is de?ned as glucose consumption minus lactate pro-duction.The glucose and lactate metabolism can be divided into three phases:(I)high glucose consumption and net lactate production,(II)low glucose consumption and net lactate consumption,and (III)high glucose consumption and very low residual lactate concentration.

pyruvate consumption rate in TCA cycle was a constant throughout the whole process and (2)glucose and lactate consumption rate was tightly controlled so that the ?ux from glucose to pyruvate and the ?ux from lactate to pyruvate are strictly complementary to each other.

NADH homeostasis,as previously being suggested (e.g.,Zhou et al.,1997a),could be the cause of the tight control of glucose and lactate consumption.The three major path-ways related to NADH homeostasis are illustrated in Figure 5.The conversion of pyruvate to lactate recycles NADH pro-duced in glycolysis back to NAD t.When the conversion is reversed,it competes with glycolysis for NAD t.The reduced availability of NAD tcould slow down the glycoly-sis rate.NADH is also recycled back to NAD tinside mito-chondria through the aspartate–malate shuttle (Figure 5)or similar shuttle systems.We need to assume that the shuttle rate was a constant for NADH homeostasis to exert the com-plementation effect on glucose and lactate consumption.It should be noted that in Stage II and III of Figure 4b,all NADH needed to be recycled in mitochondria as no NADH was recycled through pyruvate to lactate conversion.LDH activity changes during the process

Cell-speci?c LDH activity was monitored in two bioreactor runs using the same CHO subclone and CDF process.As shown in Figure 6,cell-speci?c LDH activity oscillated in both runs.During the exponential growth phase,LDH activ-ity ?rst increased and then decreased.It further decreased and reached the lowest level during the growth phase transi-tion from exponential to stationary phase.With the decrease of cell-speci?c LDH activity,lactate metabolism shifted from net production to net consumption.After the lactate meta-bolic shift,LDH activity gradually increased and reached its maximum at the end of the process.Cell size was consistent during the fed-batch process (data not shown),so that the LDH activity variation was not due to a cell volume change.Although there seems to be a linkage between LDH activity and lactate metabolism,the results were hard to interpret.First,in the LDH assay that we used,LDH activity is quanti-?ed by the rate of lactate to pyruvate conversion,hence it is counterintuitive to ?nd that the lowest activity measured off-line correlated to the highest lactate to pyruvate conversion rate in the process.Second,although decreased LDH activity was correlated with net lactate consumption,subsequent increase in LDH activity did not reverse the metabolism back

to lactate production.More studies are needed to fully under-stand the role of LDH activity variation in lactate metabolic shift,such as the ratio between the two major LDH subunits,LDH-A and LDH-B,which favor the pyruvate–lactate con-version in opposite directions,and the ratio between the other two substrates in the pyruvate–lactate conversion reaction,NAD tand NADH,where a high NAD t/NADH ratio favors lactate to pyruvate conversion.Shift of amino acid metabolism

In addition to lactate metabolic shift,several amino acids also demonstrated shifts of metabolism during the fed-batch process,as shown in Figure 7.The results were from a sin-gle bioreactor run.The alanine shift is of particular interest as similar to lactate,alanine can be derived from pyruvate.Figure 7showed that the pattern of alanine metabolic shift was similar to that of lactate,although the alanine production and consumption rate was much lower than that of lactate.Asparagine net consumption rate was initially very high,but decreased rapidly midway through the process.The decrease was due to asparagine supply limitation.As most asparagine is likely utilized in the TCA cycle through aspartate and then oxaloacetate,under asparagine limitation,net aspartate consumption rate would increased,as observed in Figure

7.

Figure 5.Major NAD 1and NADH conversion

pathways.

Figure 6.Variation of LDH activity in the CHO CDF process.

Results were from two similar bioreactor runs using the same clone.Bioreactor 1,solid symbols;Bioreactor 2,hollow symbols.

It should be noted that asparagine limitation was not the cause of the lactate metabolic shift.Increasing asparagine supply did not alter either cell growth or lactate metabolism (data not shown).For the other two amino acids,glycine shifted from net production to net consumption and serine shifted from high to low net consumption during the process.Glycine can be produced from 3-phosphoglycerate,a glyco-lytic pathway intermediate,through serine.But whether this link is related to the metabolic shift of glycine and serine is unknown.

Glycolytic pathway and TCA cycle metabolite pro?ling A more broad investigation of metabolites in the glyco-lytic pathway and TCA cycle was conducted as part of a metabonomics study.In this study,we monitored the change of intracellular and extracellular metabolite pools in the CDF CHO fed-batch process in one bioreactor run.One other spe-ci?c purpose of the metabonomics study was to verify whether there were any ?ux limitation steps in the glycolytic pathway that limit glucose utilization and force cells to use lactate.

The pro?ling of glycolytic pathway intermediates and the TCA cycle intermediates are shown in Figures 8and 9,respectively.It should be noted that the results shown in Fig-ures 8and 9are relative ion count results from mass spec-trometry measurements.As different metabolites have different ionization potential,data shown in Figures 8and 9cannot be used to compare relative concentration among dif-ferent metabolites.However,for a speci?c metabolite,ion counts are linearly proportional to its concentrations.Hence,the ion count data of one speci?c metabolite can be used to study its concentration change during the process and to compare its abundance between extracellular and intracellu-lar samples.Because the intracellular samples were more diluted than the medium (extracellular)samples,the intracel-lular results were adjusted based on the difference of sample dilution factors between intracellular and extracellular sam-ples,as shown in Eq.1,so that the intracellular and extracel-lular results could be directly comparable.IC indicates ion counts in Eq.1.IC Adjusted ?IC Original

Intracelluar sample dilution factor

Extracelluar sample dilution factor

(1)

Figure 8a indicates that the intracellular glucose ion count was very low,being detected only sporadically in the ?rst 5days.In contrast,the extracellular glucose ion count was much more abundant (Figure 8b).The difference between the intracellular and extracellular glucose concentration is more than two orders of magnitude,indicating a steep con-centration gradient across the plasma membrane.The intra-cellular glucose concentration is regulated by two factors,glucose transportation and its subsequent phosphorylation.Glucose transportation is accomplished through a family of 13facilitative glucose transporter (GLUT)proteins (Macheda et al.,2005).GLUT1(Harrison et al.,1991)and GLUT4(Bogan et al.,2001)are likely expressed on CHO cells.Pho-phorylation of glucose by hexokinase II (HK II)is the ?rst reaction in the glycolysis pathway.In transformed cells,HK II activity was found being upregulated by $100times (Peterson et al.,2002).This upregulation was achieved by two means:overexpression of HK II enzyme and docking of HK II to porins on the mitochondrial membrane.If glucose transportation was the rate limiting step regulating the intra-cellular glucose concentration,the immediate metabolite of glucose,glucose-6-phosphate,should be depleted because of glucose limitation.However,intracellular glucose-6-phos-phate ion count was at a relative high level during most of the process,and its extracellular concentration increased by a factor of 10during the process.These data suggest that the glycolytic pathway had an ample supply of

glucose-6-

Figure 7.Cumulative consumption of amino acids whose me-tabolism shifted in the CHO CDF process.

Other amino acids,except for glutamine,showed constant con-sumption rates.Glutamine consumption was not monitored as no glutamine was supplied in the

process.

Figure 8.Concentration of glycolytic pathway metabolites in

the CHO CDF process.

(a)Intracellular concentration.(b)Extracellular concentration.Only metabolites that were detected on consecutive days are linked with lines.The sporadically detected metabolites are shown as individual data points.Missing data points were below detection limit.

phosphate.Therefore,the low intracellular glucose concen-tration is more likely due to high HK II activity,instead of low glucose transportation rate.

The extracellular lactate concentration pro?le matches that measured using a conventional chemistry analyzer (BioPro-?le of Nova Biomedical).As shown in Figure 8b,the lactate concentration increased $10-fold from Day 2to 6,after which the concentration dropped sharply by $30-fold and stayed at a low level until the end of the process.It is inter-esting to observe that on Day 9,when the extracellular lac-tate concentration decreased to a very low level,pyruvate in the broth became undetectable (Figure 8b).At the same time,the two glycolytic metabolites preceeding pyruvate for-mation,3-phosphoglycerate and phosphoenolpyruvate,had accumulated.These observations seem to suggest that the ?ow from phosphoenolpyruvate to pyruvate might be par-tially blocked after Day 9,which resulted in the buildup of metabolites in front of the blockage,and depletion of the metabolites downstream of it.However,this hypothesis of a partial glycolytic pathway blockage con?icts with the glu-cose consumption rate change illustrated in Figure 4b.Figure 4b showed that after all lactate was consumed,glucose con-sumption rate went up (stage II to stage III transition).Alter-natively,the accumulation of 3-phosphoglycerate and phosphoenolpyruvate could be due to a slow down of cell proliferation.As both 3-phosphoglycerate and phosphoenol-

pyruvate are intermediates for biomass formation,slow down of cell proliferation would reduce their consumption rates.Intracellular and extracellular TCA cycle intermediates are shown in Figures 9a,b respectively.One important metabo-lite,oxaloacetate,was not measured as its volatility makes it hard to preserve during sample preparation.All other detected intermediates were maintained at a steady concen-tration in the cells during the fed-batch process (Figure 9a).Isocitrate detection was sporadic,because of its concentra-tion being close to the detection limit.Considerably,more variation was observed with the extracellular metabolites (Figure 9b).All metabolite levels changed during the pro-cess.Citrate,isocitrate,and succinate showed a similar meta-bolic shift as lactate and alanine.Their concentration increased till Day 9or 10and then decreased.As none of these intermediates was supplemented during the process,any extracellular accumulation was due to an ef?ux of metabolites from mitochondria to the cytosol and then into the broth.

Citrate concentration was much higher in the broth.Citrate must be transported out of the cells against a steep concentra-tion gradient.Previous studies had revealed that various can-cer and transformed cells have a truncated TCA cycle where ?ux from citrate to a -ketoglutarate was downregulated (Bag-getto,1992).One proposed mechanism for the downregula-tion is a strong ef?ux of citrate out of mitochondria to the cytosol.Once in the cytosol,citrate can be used to synthesize cholesterol and fatty acids,critical building blocks for mem-brane production in rapidly growing cancer or transformed cells (Hatzivassiliou et al.,2005).In this study,it was clearly shown that citrate was not only transported out of mitochon-dria but also out of cells.The high citrate accumulation rate in the ?rst 9days suggests that the TCA cycle in CHO cells might be partially truncated in these days.After Day 9,net citrate accumulation was switched to net consumption.Because the absolute citrate concentration was not known,we cannot estimate the percentage of pyruvate carbon that was diverted out of TCA cycle as citrate.However,consider-ing the vast volume difference between broth and cells,the ef?ux of citrate out cells must be a considerable drain for the TCA cycle.The extracellular isocitrate concentration was also higher than that in the cells,indicating a transportation of isocitrate out of cells as well.It should be noted that the intracellular concentration shown in Figure 9a is neither the cytosolic concentration nor the mitochondrial concentration,but an average based on cellular volume.However,the use of intracellular average concentration does not change the citrate and isocitrate cellular ef?ux conclusion.

The extracellular malate and fumarate concentrations increased during the process.Their concentrations were much lower initially,but reached a similar level to their cor-responding intracellular concentrations at the end of the pro-cess.Malate can be a TCA cycle over?ow gate.When extra malate is produced,it is converted to pyruvate by malate enzyme (Godia and Cairo,2006).Results shown in this study indicate that the conversion of malate to pyruvate might be limited as malate concentration continued increas-ing in the medium while pyruvate became undetectable after Day 8.The concentration of a -ketoglutarate varied during the process without a clear trend.Similar metabolic shift was observed in a NS0metabonomics study where lactate,alanine,citrate,and succinate shifted from net production to net consumption (data not shown).Isocitrate was not detected in the NS0

study.

Figure 9.Concentration of TCA cycle metabolites in the CHO

CDF process.

(a)Intracellular concentration.(b)Extracellular concentration.Only metabolites that were detected on consecutive days are linked with lines.The sporadically detected metabolites are shown as individual data points.Missing data points were below detection limit.

Metabolic shift of glycolytic pathway and TCA cycle intermediates

Metabolites discussed in the previous sections are sum-marized in Figure 10.The intermediates showing a transition from net accumulation to net consumption are in bold,and the intermediates that were undetectable or not monitored are labeled with an asterisk.It is interesting to note that the shifted metabolites are clustered together,downstream of py-ruvate and upstream of fumarate with citrate considered the start of the TCA cycle.Of all the intermediates monitored in this segment,a -ketoglutarate is the only one showing a dif-ferent metabolic pro?le from lactate.The similar metabolism of a group of closely related metabolites suggests that the metabolic shift was controlled by ?ux or by a group of highly coordinated enzymes.A possible ?ux controlling point is the synthesis of pyruvate from phosphoenolpyruvate.Again,the ?ux limitation hypothesis does not agree with the increased glucose consumption rate (Figure 4b from Stage II to Stage III).More studies are needed to elucidate the true mechanism of the metabolic shift.First,in this study,we monitored only a single enzyme (LDH).A more broad pro-teomics study is needed to assist interpreting the metabolo-mics results.Second,the metabolic analysis was largely based on trend analysis.A quantitative ?ux analysis could increase the quality of the analysis and might lead to new ?ndings.Third,it might be necessary to consider other path-ways,such as urea cycle and lipid synthesis pathways that

link closely to the central metabolic pathway,to understand the mechanism(s).

Conclusions

A CDF with companion medium preloading for a fed-batch NS0process was successfully developed to replace a HCF.The new feed not only allowed us to achieve a fully chemically de?ned process but also offered higher peak cell density and higher product titer.When the CDF with its companion medium preloading was tested in a CHO process,it again demonstrated multiple signi?cant bene?ts.In addition to allowing the removal of hydroly-sates,both viability and product titer were substantially improved.A lower lactate concentration is an additional bene?t of the CDF processes in both NS0and CHO cul-tures.In the CDF processes,there was a rapid shift in lac-tate metabolism that resulted in a nearly complete depletion of lactate in the second half of the process.It was also observed that the lactate metabolic shift occurred concurrently with the transition of cell growth from expo-nential to stationary phase.

Analysis of net glucose consumption by CHO cells indi-cated that the glycolytic pathway and pyruvate to lactate conversion are closely coordinated.Their ?ows are strictly complementary to each other so that the combined cell spe-ci?c glucose and lactate consumption rate is a constant.

A

Figure 10.Glycolytic pathway and TCA cycle.

Metabolites that demonstrated a shift from net accumulation to net consumption during CHO CDF process are in bold.

plausible factor that could exert this tight control is cellular maintenance of the NADt/NADH balance.

LDH activity oscillated during the process.The lowest ac-tivity measured was during the lactate metabolic shift from net accumulation to net consumption.However,the results seem to be counterintuitive so that a more in-depth study of the LDH subunits and the other pair of substrates in the lac-tate–pyruvate reaction,NADtand NADH,is needed to determine the link between the LDH activity change and the lactate metabolic shift.

Additional studies with the CHO process indicated that several amino acids demonstrated metabolic shifts as well. Alanine,another byproduct of pyruvate,showed a similar net accumulation to net consumption shift as lactate.This demonstrates that the metabolic shift is not limited to pyru-vate–lactate conversion only.

Finally,pro?ling of most glycolytic and TCA cycle inter-mediates in the CHO CDF process was conducted.In this study,both intracellular and extracellular metabolite levels were measured.A steep glucose concentration gradient was observed across the plasma membrane,where intracellular glucose concentration was more than two orders of magni-tude lower than the extracellular concentration.The low glu-cose concentration is likely due to the high phosphorylation rate of glucose in the cytosol,rather than a glucose transpor-tation limitation.

When lactate was nearly depleted,the medium pyruvate became undetectable in the CHO CDF process.Meanwhile, two glycolytic intermediates upstream of pyruvate formation,3-phosphoglycerate and phosphoenolpyruvate,accumulated in the medium.The opposite pro?le between phosphoenolpyruvate and pyruvate suggests that the?ux from the former to the latter might be partially blocked around the time at which the lactate metabolic shift occurred.However,this hypothesis is not sup-ported by the glucose consumption data,where glucose con-sumption rate increased after the lactate metabolic shift.

A considerable amount of citrate was exported out of mi-tochondria into the medium in the?rst9-day culture,after which citrate metabolism converted from net accumulation to net consumption in the CHO CDF process.The extracel-lular citrate concentration increased up to10-fold higher than the intracellular average concentration.This observation suggests that the TCA cycle in CHO cells might be partially truncated.Besides citrate,isocitrate and succinate also dem-onstrated similar metabolic shift as lactate and alanine.More interestingly,all the metabolites showing a metabolic shift are clustered together,generally downstream of pyruvate and upstream of fumarate.

Although the CDF fed-batch process demonstrated signi?-cant empirical bene?ts over the previous HCF process and provides a platform for the study of cellular metabolism,the cause(s)of the metabolic shifts in the CDF CHO process is still unknown.It could be a?ux restriction,where a possible restriction point is the conversion of phosphoenolpyruvate to pyruvate or a systematic enzymatic shift during cell growth phase transition.Follow-up studies,especially a companion proteomics study,will help elucidate the mechanism(s).

Acknowledgments

The authors appreciate Dr.Kurt Droms and Dr.Amit Bane-rjee for their critical review of the manuscript and Process De-velopment Analytical group for the amino acid analyses.

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企业合理避税的分析

摘要 自改革开放以来我国经济飞速发展，经济的快速发展推动我国企业经营模式走上了另一个巅峰。越来越多的企业走上了合理避税的道路，我们不难发现企业合理避税已经成了绝大部分企业的战略部署。合理避税的方法各式各样关键取决于企业自身适合什么样的税务筹划方案。不同的企业有不一样的避税方式，但目的不外乎就是实现企业利润最大化。然而在我国这样的法制大国一旦涉及税收便是一个严肃的问题，于法律而言偷税、漏税都是触犯国家法律的。所以在这里我们提出了“合理避税”这样一个名词，就是要让企业在合情、合理、合法的范围内最大限度的减少财务成本，实现企业盈利最大化，在市场竞争中占据有利位置。但是，即使是这样企业合理避税还是存在一定程度问题的。本文首先介绍了合理避税概念、意义、影响让大家对合理避税初步认识，然后又总结了一些常见合理避税的办法，从这些办法中不难发现企业合理避税中还存在一些问题，然后针对这些问题深入了解并找出解决方案，最后对全文进行总结。 关键词：合理避税; 税务筹划; 财务成本

ABSTRACT The rapid economic development of our country since reform and opening up, the rapid development of economy to promote our country enterprise management pattern on the another peak. More and more enterprises in the path of a reasonable tax avoidance, we is not hard to find reasonable tax avoidance has become most enterprise strategy. Key depends on the reasonable tax avoidance method of all kinds enterprise itself is suitable for what kind of tax planning scheme. Different companies have different ways of tax avoidance, but nothing more than a maximize profits yet in our country the legal powers when it comes to tax is a serious problem, the law for tax evasion, tax evasion are breaking the laws of the state. So here we put forward the "fair tax" such a word, is to make our enterprise within the scope of reasonable, fair and legal maximum reduce financial cost, maximizing profits, occupy the favorable position in the market competition. But, even so, the enterprise reasonable tax avoidance or problems to a certain extent. This paper first introduces the concept of reasonable tax avoidance, meaning, influence let everybody to the reasonable tax avoidance preliminary understanding, then summarizes some common reasonable tax avoidance, from the way it is not difficult to find there are still some problems in the enterprise reasonable tax avoidance, and then to understand and find out solutions to solve these problems, finally, to summarize the full text. Key words: A reasonable tax avoidance; The tax planning;Financial cost

工程施工合同中EPC、BOT、3P解释

【工程总承包（）模式】 工程总承包( )模式，又称设计、采购、施工一体化模式。是指在项目决策阶段以后，从设计开始，经招标，委托一家工程公司对设计采购建造进行总承包。在这种模式下，按照承包合同规定的总价或可调总价方式，由工程公司负责对工程项目的进度、费用、质量、安全进行管理和控制，并按合同约定完成工程。有很多种衍生和组合，例如、、、、等。 【优点】 、业主把工程的设计、采购、施工和开工服务工作全部托付给工程总承包商负责组织实施，业主只负责整体的、原则的、目标的管理和控制，总承包商更能发挥主观能动性，能运用其先进的管理经验为业主和承包商自身创造更多的效益；提高了工作效率，减少了协调工作量； 、设计变更少，工期较短； 、由于采用的是总价合同，基本上不用再支付索赔及追加项目费用；项目的最终价格和要求的工期具有更大程度的确定性。 【缺点】

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国际工程项目BOT投资合同协议 (完整版)

编号：_____________国际ＢＯＴ投资合同 甲方：________________________________________________ 乙方：___________________________ 签订日期：_______年______月______日

本合同由________（下称Ａ）代表________国政府（下称Ｂ）和根据中华人民共和国法律组建的________公司（下称Ｃ）签署，Ｃ主要办公地点在中华人民共和国________。 鉴于Ａ和Ｃ于________年________月________日签订的基础设施建设备忘录对Ｂ授予Ｃ勘测和实施基础设施工程的专有权，方式为Ｂ与Ｃ共同投资。鉴于，贷款协议、担保协议、保函等为本合同不可分割的一部分，基础设施总装机容量为________，以及________等主要内容达成了一致意见。 因此双方达成协议如下 １．定义与解释： “工程”指基础设施建设的规划、可行性研究、设计与工程技术服务、建造、供货、竣工、调试、试运行和运行。 “工程造价”指第３款的费用。 “运行期”指从基础设施商业运行开始日计算的日期。 “竣工期”指Ｃ证明基础设施调试成功并可以开始运行期的日期。 “不可抗力”含义见第１６款。 “工程范围”指实施本工程时附件二规定的供货范围和服务范围。 “转让日期”指运行期最后一天的第二天。 “新公司”指由第５款规定的Ｃ和________国实体组建的公司。 “投资协定”指《中华人民共和国政府和________国政府关于相互鼓励和保护投资协定》。 “专有权”指备忘录、协议以及附属文件中授权Ｃ或新公司的特殊权力。 “日”指公历日。 ２．工程 ２．１本工程命名为________工程。 ２．２基础设施位于________国________地区。其确切位置可以根据现场条件在详细设计阶段予以调整。 ２．３本工程应在建造、运行和转让（ＢＯＴ）的基础上实施。 ２．４本工程的组成。 ２．５经Ｂ和Ｃ接受后，最终可行性研究报告和详细设计应作为本工程开发和竣工的基础。 ３．工程造价 ３．１工程造价________亿美元，建设期利息________万美元，工程总造价________万美元，见附件。 ３．２工程总造价应由下述费用组成但不限于下述费用，工程造价的细目见附件： １）可行性研究、设计和工程技术服务及其他咨询服务的费用；

中央空调维修合同范本

中央空调维修合同范本 中央空调维修合同范本 随着法律观念的深入人心，越来越多的人通过合同来调和民事关系，签订合同也是避免争端的最好方式之一。那么大家知道合法的合同书怎么写吗？下面是精心整理的中央空调维修合同范本，欢迎大家借鉴与参考，希望对大家有所帮助。 中央空调维修合同1 甲方： 乙方： 依照《中华人民共和国合同法》、《中华人民共和国建筑法》及其他有关法律、行政法规、遵循平等、自愿、公平和诚实信用的原则，双方就本建设工程施工事项协商一致，订立本合同。 第一条工程概况 工程名称：_____ 工程地点：_____ 第二条合同工期 开工日期：合同签订后，预付款到账后两天内开工 工期：管路维修20天，压缩机更换5天 合同工期总日历天数20天 第三条维修内容 ____________________。

第四条合同价款及支付方式 合同金额：壹拾万元整100000元付款方式：合同签订后付合同款的30%作为开工预付款，压缩机更换完毕三日内再付合同款的30%，工程完工合同后付合同款的’30%，余款在合同满一年付清。 第五条乙方向甲方承诺按照合同约定进行施工、竣工并在质量保修期内承担工程质量保修责任。 第六条甲方向乙方承诺按照合同约定的期限和方式支付合同价款及其他应当支付的款项。 第七条违约责任 如一方违约对方有权解除合同，违约责任由违约方承担。 第八条要本着安全第一的原则，施工中的一切安全损失由乙方负责。 第九条合同签字生效，双方各持一份。 甲方：_________乙方：_________ 法定代表人：_________法定代表人：_________ _________年____月____日_________年____月____日中央空调维修合同2 甲方： 乙方： 依据《中华人民共和国合同法》和《建筑安装工程承包条例》及有关规定，结合本工程具体情况，经甲、乙双方充分协商签订本合同，以便双方共同遵守，顺利完成本项工程。

税收筹划 企业利益最大案例分析

税收筹划企业利益最大案例分析 【最新资料，WORD文档，可编辑】

税收筹划是每个企业都必须有的一个环节，通过税收筹划可以制定合理的税收方式，最大限度的减少公司的损失，让公司利益最大化。 本文将以某研究所企业设立、经营活动中的会计核算、职工薪酬三方面管理决策中的税收筹划实例，表明通过加强对企业税收筹划在管理决策中的应用，来合理减轻企业的税收负担，保证企业价值最大化目标的实现。 一、企业设立决策的税收筹划 某研究所为使剩余资金充分发挥效能，实现收益最大化，有意向投资设立一家下属公司，希望公司设立后能使单位获得更大的收益水平，同时税负最轻，这就需要对建立子公司或分公司进行决策。 如果决定采用公司制组织形式投资设立一个新企业，将面临成立子公司或是分公司决策的选择。对企业的税收有直接的影响。从法律地位上讲，子公司必须具备独立的法人资格，这是子公司与分公司的根本区别所在。子公司和母公司之间是一种法律上财产权益关系，没有直接的隶属关系，子公司应当以其全部财产独立地承担民事责任，当然也包括独立纳税的义务。 分公司没有独立的法律地位，不具有法人资格。分公司业务活动的结果由总公司承受，总公司应以自己的全部财产对分公司所产生的债务承担责任。分公司的设立只须在当地履行简单的登记和营业手续即可，因此，分公司一般不独立履行纳税义务，而将全部的经营成果都汇总到总公司一并纳税。 一家盈利情况较好的企业，拟投资设立一新公司，预计这家新公司未来3年均会出现经营亏损，经过税收筹划，企业应做出以非法人身份投资设立分公司的管理决策。分公司不具有法人资格，由总公司汇总缴纳企业所得税，可实现总公司调节盈亏，合理减轻企业所得税的负担。 如果当无税收优惠的企业投资设立能够享受税收优惠的机构时，适合设立子公司，以使子公司在独立纳税时享受税收优惠政策。 除税收因素外，企业在决定采用何种组织形式时，也要考虑分公司与子公司在其他诸多方面存在的差异：如分公司无独立经营权与决策权，而子公司有独立经营与决策权；分公司不能独立签署合同，子公司能签署合同；分公司设立程序简单，费用低，子公司设立程序复杂，费用高等，以此进行综合的判断。 二、企业经营管理活动中会计核算的税收筹划 通过会计核算也能进行税收策划，必须“未雨绸缪”。企业会计核算中的税收筹划，实际上就是按照企业会计核算的要求和特点，设计企业的全部业务和经营活动，通过会计核算结果的变化，实现企业税收筹划的目的。会计核算是整个税收筹划方案的起点和终点。 合同文本设计是企业“分别核算”的基础环节，财务核算是企业“分别核算”的核心环节。如果两项业务分别核算会使企业税收负担最小、企业价值最大，那么企业在承接业务时，便可与客户确定两份不同业务合同。如果两项业务合并核算会使企业税收负担最小、企业价值最大，那么企业在承接业务时，便可与客户确定一份单一业务合同。

国际BOT投资合同

国际BOT投资合同 目录 1.定义与解释 2.工程 3.工程造价 4.工程实施责任 5.新公司 6.基础设施的建造 7.工程进度 8.调试 9.生效日和特权 10.基础设施的运行 11.基础设施的服务 12.基础设施的服务费用和收入分配 13.转让所有权 14.赔偿责任 15.文件和专利 16.不可抗力 17.保险 18.情况的变化 19.通知

20.争议解决 21.放弃主权豁免权 22.法律和语言 23.仲裁 本合同由________________（下称a）代表________________国政府（下称b）和根据中华人民共和国法律组建的_____________公司（下称c）签署，c主要办公地点在中华人民共和国________________. 鉴于a和c于__________年______月______日签订的基础设施建设备忘录对b授予c勘测和实施基础设施工程的专有权，方式为b与c共同投资。鉴于，贷款协议、担保协议、保函等为本合同不可分割的一部分，基础设施总装机容量为____，以及_____________等主要内容达成了一致意见。 因此双方达成协议如下： 1.定义与解释： “工程”指基础设施建设的规划、可行性研究、设计与工程技术服务、建造、供货、竣工、调试、试运行和运行。 “工程造价”指第3款的费用。 “运行期”指从基础设施商业运行开始日计算的日期。 “竣工期”指c证明基础设施调试成功并可以开始运行期的日期。 “不可抗力”含义见第16款。 “工程范围”指实施本工程时附件二规定的供货范围和服务范围。 “转让日期”指运行期最后一天的第二天。 “新公司”指由第5款规定的c和______国实体组建的公司。 “投资协定”指《中华人民共和国政府和________________国政府关于相互鼓励和保护投资协定》。

热水工程太阳能+热泵

太阳能加空气能中央热水工程 设 计 预 算 书 东莞市发辉热水器设备厂

一、设计方案说明 1、工程名称：广西酒店热水工程 1）日用热水总量：17吨（190间客房，每间客房85升水设计） 2）用热水方式：花洒 3）热水供给方式：不定时供应热水 4）冷水计算温度：15℃； 5）热水计算温度：55℃； 6）太阳能产水量：8 L/日/ 管 9）FH-SKR100型10P循环机组技术参数 17吨热水空气能主机选择2台10匹主机，2台10匹机制热量为76KW。

此表中的额定制热量和产热水量为环境温度10℃、湿球温度10℃、冷水温度15℃、热水温度55℃时的测试数据。 2、设计方案 2.1设计方案：根据用户的要求，结合贵方用热水特点，从热泵、太阳能集热器合理布局及优化设计等技术经济角度考虑，结合太阳能、空气能热水系统特点及我方现场实际勘察情况，为了实现节能和有效降低日常运行费用，同时保证可靠供应热水并延长系统设备的使用寿命，我方高级技术设计人员特此为本项目提供了太阳能-热泵中央热水系统优化设计方案，具体说明如下： 本优化设计对太阳能按春、夏、秋三季晴天情况下能基本满足正常热水供应来配置集热器数量（阴雨天或日照不足情况下通过热泵机组来进行辅助加热），为保证系统在冬季最不利情况下仍能满足正常热水供应，系统配备足够的高效空气源热泵机组进行辅助加热。太阳能系统按照温差循环相结合方式工作，由一级太阳能储热水箱以定量、定时方式为二级热泵辅助加热储热水箱补水。系统以太阳能为主热泵为辅，不仅太阳能、热泵皆可在有效时段高效率独立工作，又能相互弥补。 本优化设计，充分利用太阳能集热器在日照有效时间来加热冷水，因为热泵机组不同于锅炉加热设备，同等投资条件下，热泵机组单位时间的产热量远低于锅炉类加热设备的出热量，所以，太阳能热水系统用热泵作辅助时，要想初期投资不致过大就须尽可能延长热泵机组工作时间，对此，可以按照近似相等原则，把系统总用水量按照太阳能集热器每日8～10小时有效加热时间，分批（定量）先由太阳能系统进行预加热（一次加热），一级太阳能储热水箱的水经过太阳能系统预加热后，通过过渡水泵将一级储热水箱中的水分批定量补给二级储热水箱，按照补入水温情况，再由热泵热水机组以定温方式作定温加热（二次加热）；这样热泵与太阳能系统均可在日照有效时间或环境温度较高的时段同时工作，既达到了最大限度利用太阳能，又能使热泵机组工作时间相对延长，合理的解决热泵机组单位时间产水量较小的问题，能够控制初期投资在一定范围内，实现环保节能的目的。所以本优化设计综合了太阳能集热器及热泵工作特点，

家用中央空调安装合同范本

编号：_____________ 家用中央空调安装合同 甲方：___________________________ 乙方：___________________________ 签订日期：_______年______月______日

委托方(甲方)： 承接方(乙方)： 根据《中华人民共和国合同法》以及有关法律、法规的规定，结合家用中央空调系统安装施工的特点，经双方友好协商，就甲方委托乙方承担施工家用中央空调系统事宜，达成如下协议(包括本合同附件和所有协议)，以资共同遵守。 一、工程概况 1．工程名称： 2．工程地址： 3．甲方联系人或委托联系人：电话： 4．甲方所提供机组 室外机： ┌────┬──────┬─────┬─────┬──────┐ │序号│品牌│品名│型号│数量(台) │ ├────┼──────┼─────┼─────┼──────┤ │ 1 │││││ ├────┼──────┼─────┼─────┼──────┤ │ 2 │││││ ├────┼──────┼─────┼─────┼──────┤ │ 3 │││││ ├────┼──────┼─────┼─────┼──────┤ │ 4 │││││ ├────┼──────┼─────┼─────┼──────┤

│ 5 │││││ └────┴──────┴─────┴─────┴──────┘室内机： ┌────┬──────┬──────┬──────┬──────┐│序号│品牌│品名│型号│数量(台) │├────┼──────┼──────┼──────┼──────┤│ 1 │││││├────┼──────┼──────┼──────┼──────┤│ 2 │││││├────┼──────┼──────┼──────┼──────┤│ 3 │││││├────┼──────┼──────┼──────┼──────┤│ 4 │││││├────┼──────┼──────┼──────┼──────┤│ 5 │││││├────┼──────┼──────┼──────┼──────┤│ 6 │││││├────┼──────┼──────┼──────┼──────┤│7 │││││├────┼──────┼──────┼──────┼──────┤│8 │││││└────┴──────┴──────┴──────┴──────┘

某公司职工薪酬的个人所得税筹划方案分析

某公司职工薪酬的个人所得税筹划方案分析 一、基本情况介绍 某公司按照绩效考核制度，实行每季考核办法，对员工实行四档工资制度：第一档，每月12000元；第二档，每月8000元；第三档，每月5000元；第四档，每月3500元。加上每季考核奖和年终考核奖并扣除五险一金的成本因素，第一档，第二档，第三档，第四档员工的年度总收入大概分别为：60万、23万、18万、14万。由于四挡员工年总收入的大部分是通过发放年终奖的办法，在每年的1月份按照年终奖的计算方法进行申报纳税。从调研来看，以上第一档，第二档，第三档，第四档员工年终奖在申报个人所得税时，分别适用了30%、25%、25%、20%的税率，致使公司为员工承担一笔较高的个人所得税。请分析如何降低公司的个人所得税？ 二、法律依据 根据《关于调整个人取得全年一次性奖金等计算征收个人所得税方法问题的通知》（国税发[2005]9号）的规定，全年一次性奖金是指行政机关、企事业单位等扣缴义务人根据其全年经济效益和对雇员全年工作业绩的综合考核情况，向雇员发放的一次性奖金。纳税人取得全年一次性奖金，单独作为一个月工资、薪金所定适用税率和速算扣除数，然后将雇员个人当月内取得的全年一次性奖金，按相得计算纳税。 计算方法是先将雇员当月内取得的全年一次性奖金，除以12个月，按其商数确应的适用税率和速算扣除数计算征税。如果在发放年终一次性奖金的当月，雇员当月工资、薪金所得低于税法规定的费用扣除额，应按全年一次性奖金减除“雇员当月工资、薪金所得与费用扣除额的差额”后的余额，确定适用的税率和速算

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