Production of bioactive recombinant monoclonal antibody fragment in periplasm of Escherichia coli expression system

Abstract The microbial expression system (Escherichia coli) is the most widely studied host for the production of biotherapeutic products, such as antibody fragments, single chain variable fragments and nanobodies. However, recombinant biotherapeutic proteins are often expressed as insoluble proteins, thereby limiting the utility of E. coli as expression system. To overcome this limitation, various strategies have been developed, such as changes at DNA level (codon optimization), fusion with soluble tags and variations in process parameters (temperature), and inducer concentration. However, there is no “one size fits all” strategy. The most commonly used approach involves induction at low temperature, as reducing the temperature during cultivation has been reported to increase bioactive protein production in E. coli. In this study, we examine the impact of various process parameters, such as temperature and inducer concentration, as well as, high plasmid copy number vector for achieving enhanced soluble expression of TNFα inhibitor Fab. An interaction amongst these parameters has been observed and their optimization has been demonstrated to result in expression of 30 ± 3 mg/L antibody fragment using E. coli. This case study illustrates how process optimization can contribute toward making biotherapeutics affordable.


Introduction
Recombinant biotherapeutic molecules, such as singledomain antibody fragments (dAbs), antigen-binding fragments (Fabs), and single-chain variable fragments (scFvs) are produced in Escherichia coli due to its well-known physiology and genetics.Factors, such as lack of glycosylation, easier production, high expression levels, high cell densities, and low production costs compared to other available expression platforms contribute to popularity of the E. coli platform.
The last few decades have witnessed significant advancements in cloning strategies, expression, and purification, thereby resulting in improved production of useful antibody-like fragments utilizing E. coli expression system. [1,2]he production process is typically quite simple.The desired gene of interest is cloned into a suitable expression vector, which is further transformed into choice of host, cultivated and induced, and protein is extracted and purified.Post purification, the protein is further characterized for structure and function. [3]However, production of recombinant antibody fragments is typically marred by low expression level and poor refolding yield (if expressed as inclusion bodies).Researchers have reported poor growth of cells and formation of inclusion bodies as the practical obstacles associated to expression of recombinant proteins in E. coli.A wide range of cultivation techniques, such as media composition, temperature, agitation, aeration and concentration of inducers, induction time and molecular techniques are currently in use for high level production of recombinant proteins to overcome the practical problems.Synergistic effects of the physical parameters along with feeding strategies might significantly improve the expression of Fab in microbial expression system.Modified strain with oxidative environment and cell free system developments have been reported to offer enhancement in the expression of biomolecules. [1]One approach involved co-expression of chaperones with the Fab to facilitate its proper folding into a desired conformation retaining its target-binding activity.Alternatively, in vitro Fab refolding from inclusion bodies has been demonstrated.However, these strategies suffer from adverse effects, such as enhanced proteolysis, increased soluble aggregates, growth inhibition, and lack of scalability, and they are therefore difficult to employ during commercial production. [1,4,5]rotein expression in E. coli generally takes place in either cytoplasm or periplasmic space.Reductive environment of the cytoplasm tends to express the proteins into inclusion bodies due to inefficient formation of disulfide bridges, whereas periplasmic space is the preferred choice for the soluble expression due to presence of oxidizing environment for proper folding.Antibody fragments are engineered with signal sequence for directing them to periplasmic space for proper folding. [6,7]he resultant periplasmic extract is thereby already enriched for the recombinant protein of interest, with the periplasmic containing only around 4-8% of the natural E. coli cellular proteins, making purification simpler.The periplasmic extract is also of lower volume than a whole cell lysate.In addition, periplasmic protease activity is far lower than that in the cytoplasm, reducing proteolysis of the recombinant protein product. [6]arious reports have shown that increased production of recombinant antibody fragments have soluble expression below 30 C. Lowering the cultivation temperature during induction increases the refolding of protein and reduces aggregation due to the lowered translation rate.Control of the growth temperature during fermentation is a facile method that allows stable expression of heterologous proteins.A direct consequence of temperature reduction is the partial elimination of heat shock proteases that are induced under overexpression conditions. [8]A similar transcriptional effect is achieved when a moderately strong or weak promoter is used or when a strong promoter is partially induced. [3]Low induction levels have been found to result in higher amounts of soluble protein. [9]It was concluded that the low temperature was beneficial to folding and the system was suggested as a tool for expression and correct folding of recombinant proteins in the cytoplasm of E. coli.Temperature downshift to 15 or 23 C abolished degradation of the fusion protein and the toxic phenotype associated with expression at 37 C was suppressed.12][13][14][15] Most of the recent work has focused on minimization of stress encountered by bacteria generating cytoplasmic recombinant proteins by decreasing growth temperature and inducer concentration.This stress-minimizing approach results in slower recombinant protein synthesis, but permits correct folding.The result is an increase in not only the yield but also the solubility of recombinant protein.Additionally, stress minimization dramatically limits plasmid loss, increases cell viability, and improves process robustness.In this study, stress minimization [6] strategies were used to: (i) increase the yield of a Fab fragment in the periplasm of E. coli; and (ii) optimize its release using osmotic shock. [16]Through manipulation of growth temperature, inducer concentration, and the point of induction, the Fab fragment could be directed to accumulate in the periplasm, significantly simplifying purification.
While secretion of protein is preferred in eukaryotic cells, the same is not easy in prokaryotic organisms.Therefore, the periplasmic space has shown promising yields in bacteria making use of oxidizing environment.SEC and TAT pathways are the commonly preferred mode of secretion into periplasmic region. [5]n this work, we report on how a combination of high plasmid copy number dual promotor, low induction temperature, and expression in periplasmic space, without the usage of any additives and chaperones in basic strain BL 21DE3, results in favored soluble expression of a functional antibody fragment against TNFa in conventional (LB) media and expression levels of 30 ± 3 mg/L could be achieved.

Materials
Escherichia coli (BL21 DE3) strain was procured from Thermo Fisher Scientific and TNFa inhibitor Fab gene along with duet vector was purchased from Synbio Technologies (US).Taq Polymerase (M0272S) was obtained from New England Biolabs.Primary antibody anti-TNFa mAb was obtained from Abcam (ab1793).Deoxynucleoside triphosphates and anti-mouse IgG-HRP, developed in goat was were purchased from Real Gene (415131).3,3 0 ,5,5 0 -Tetramethylbenzidine (TMB) was obtained from SRL. Liquid Bertani (LB), Kanamycin and IPTG (Isopropyl ß-D-1-thiogalactopyranoside) was obtained from Himedia.Tris, Sucrose, Glycine and EDTA and Sodium Phosphate buffers were purchased from SRL. Tumor Necrosis Factor a was procured from Abcam.Bicinchoninic acid (BCA) was purchased from Sigma Aldrich (B6943).AKTA FPLC (Cytiva) was used for purification.MS Grade Trypsin (V5111) was procured from Promega.In-house developed IgG is used as a standard in the present study.

Construction of plasmids
Gene synthesis was done by Synbio Technologies.Codon optimized gene of TNFa inhibitor antibody fragment was cloned into a dual promotor high plasmid copy number vector pRSF-Duet vector, with outer membrane protein A (Omp A) signal sequence upstream of both light and heavy chain in MCS1 and MCS2 under the regulation of T7 promotor, respectively.Accuracy of the sequence was confirmed using DNA sequencing and PCR.One microliter of DNA was transformed into E. coli (BL21 DE3) competent cells, heat shocked at 42 C for 45 s, and plated on LB agar media.Colonies were selected based on colony PCR using T7 universal primers.Conditions used for PCR are as follows: Initial denaturation at 95 C (2 min), followed by 30 cycles of denaturation (95 C) for 60 s, annealing (55 C) for 45 s, and extension (72 C) for 60 s and final extension (72 C) for 10 min followed by termination of reaction at 4 C.

Temperature based induction expression
Escherichia coli BL21 (DE3), harboring the TNFa inhibitor Fab plasmid, was inoculated into Luria Bertani (LB) medium supplemented with 50 mg/mL kanamycin.Prior to expression studies, positive transformants were inoculated in the LB medium and grown overnight.From the overnight grown culture, 100 ml LB media in 500 mL flasks was inoculated with 1% of pre-inoculum and incubated at 37 C (180 rotation per minute) till 0.6 OD was achieved.At this point, 0.1, 0.5, and 1 mM of IPTG was added and cultivation temperature was lowered to 15, 20, 25, 30, and 37 C and were harvested at 4 C/14,000 RPM/for 15 min at eighteenth hour, post induction followed by cell lysis.

Periplasmic extraction, quantification, and purification
Harvested cells were re-suspended in the ice cold periplasmic extraction buffer (100 mM Tris (pH 7), 50 mM sucrose, 5 mM EDTA and 1 mM PMSF) and incubated on ice for 30 min.Post incubation lysates was centrifuged for 10 min at 6000 rpm at 4 C. Supernatant was collected in a fresh tube and saved as the periplasmic fraction.After osmotic shock, the pellet was re-suspended in 100 mM NaCl, 20 mM Tris (pH 7.5), 1 mM PMSF, 50 mM sucrose, and 1 mM EDTA, and the cells were disrupted by mild sonication for 5 min (pulse on 10 s and pulse off 15 s) at 25 kHz on ice.The sonicated lysate was centrifuged at 14,000 rpm at 4 C for 10 min.The supernatant was saved as cytoplasmic soluble fraction and the pellet was saved as insoluble fraction.Each of the fractions was further quantified using bicinchoninic acid assay (BCA) and was visualized on SDS PAGE.Protein concentration was determined by the BCA reagent using antibody fragment (Ranibizumab) as a reference standard.After quantification, equal amounts of samples were loaded onto the SDS PAGE for the analysis.
Soluble extracts were loaded onto the affinity column of 1 mL volume, which was pre-equilibrated with 10 CV of buffer A (100 mM sodium phosphate, pH 6.5).After washing the unbound proteins with 10 CV of buffer A, Fab was eluted with 5 CV of (50 mM glycine-HCl pH 3).Eluted fraction was neutralized to pH 7 and was quantified using molar extinction coefficient of 1.59 and stored for further use.

SDS PAGE and densitometry analyses
SDS PAGE was performed according to the Laemmli protocol using Biorad mini Protean gel system.Sonicated samples were analyzed on 12% SDS PAGE in reducing condition.Samples were reduced and denatured by boiling the samples for 5 min at 95 C in the SDS-PAGE sample buffer containing 200 mM Tris HCl, 20% glycerol, 10% SDS, 0.2% (w/v) bromophenol blue, and 2 mM DTT.The gels were stained using Coomassie Brilliant Blue-G250 dye.The intensity of the band corresponding to TNFa inhibitor Fab was determined by densitometry scanning using Image lab version 6.Then, based on the intensity observed, fold increment of TNFa inhibitor fab was calculated which was corroborated with the purified TNFa inhibitor Fab.Quantification of the TNFa inhibitor Fab was performed using the densitometry method based on SDS PAGE. [17]External protein of known concentration (1 mg/ml) was for creating a linear plot to enable quantification.One to 15 mg of standard protein was loaded on SDS PAGE.Image Lab (version 6) analysis by Gel Doc was made using the profiles of the lanes and calibrated to a known standard.Peak area of the each lane was used for analysis and developing a linear plot.This densitometry method was applied to quantify the periplasmic expressed TNFa inhibitor Fab (Supplementary Figure S1).

Native PAGE
Native PAGE was performed using Biorad mini Protean gel system.Ten percent NATIVE gel was casted using the composition similar to SDS PAGE except the addition of SDS.Sample buffer dye contained 20% glycerol, 0.2% (w/v) bromophenol blue and 62.5 mM Tris-HCl (pH 6.8).The gel was run at 100 V followed by development using silver nitrate.

Size exclusion chromatography for aggregate analysis
Size-exclusion high-performance liquid chromatography (SEC-HPLC) was used to determine the purity and aggregates formation of the in-house developed product.SEC-UV analysis was performed with a TSKgel V R SuperSW mAb HTP SEC column (4.6 Â 150 mm, 4 mm, Tosoh Bioscience LLC, PA) on 1290 infinity UHPLC system (Agilent Technologies) at 30 C. The separation was carried out in isocratic mode by using 100 mM sodium phosphate (pH 7) with 100 mM sodium chloride buffer as mobile phase, in HPLC-grade water, a flow rate of 0.35 mL/min, and injection volume of 10 mL, and monitored at 280 nm.

Liquid chromatography-mass spectrometry for intact mass determination
Intact mass of the in-house developed TNFa inhibitor Fab was determined using LC-MS analysis.Unreduced sample was passed through Agilent AdvanceBio RP mAb C4 (4.6 Â 100 mm, 5 mm, Agilent Technologies) column operated at 45 C using Agilent 1290 Infinity II UHPLC system coupled online to Agilent 6545XT AdvanceBio LC/Q-TOF with a Dual Agilent Jet Stream source.The mobile phase used for the separation consisted of buffer A (water þ 0.1% formic acid) and buffer B (Acetonitrile þ 0.1% formic acid) at a linear gradient from 2%B to 65%B in 30 min.The mass spectrometry parameters were drying gas temperature at 325 C, drying gas flow 13 L/min, nebulizer pressure 35 psig, sheath gas temperature at 275 C, sheath gas flow at 12 L/min, nozzle voltage 5500 V, and fragmentor voltage 175 V. MS data acquisition was conducted at a rate of 1 spectrum per second in profile mode with a mass range of 400-3200 m/z using the MassHunter Workstation LC/MS Data Acquisition 10.0 software (Agilent Technologies), which was used for data analysis.

Peptide mass fingerprinting using proteomics analyses
Peptide mapping was performed for the in-house developed Fab against TNFa.50 mg of sample was denatured using 10 mM dithiothreitol (DTT) for 30 min in the dark and was further alkylated using 15 mM Iodoacetamide.Post alkylation samples were digested overnight using MS grade trypsin (1:20 w/w) at 37 C. Digestion reaction was stopped by acidification using 1% (v/v) MS grade formic acid.Generated peptides were separated using C18 column (2.1 Ã 150 mm, 2.7 mm Agilent Technologies) using 1290 infinity UHPLC system coupled to 6545XT AdvanceBio LC-QTOF system.Data acquisition was performed using Mass Hunter BioConfirm software version 10 and analyzed for MS/MS and peptide mass fingerprinting.

Bioactive characterization using ELISA
Ninety-six well plates were coated overnight with increased amount of in-house expressed TNFa inhibitor Fab (0-20 ug) in bicarbonate buffer (pH-9) at 4 C.The plates were washed thrice with PBS followed by blocking with 5% skimmed milk for 1 h at 37 C.After the blocking step, the washing step was repeated.At this point, 10 nanogram/well of TNFa was added in the each well and incubated for 1 h at 37 C, followed by washing step.After this, primary antibody (1:1000) i.e., anti-TNFa monoclonal antibody was added into each well and incubated at 37 C for 1 h.Post this, the plate was washed again with PBS followed by addition of secondary antibody conjugated with HRP with dilution (1:2000) and incubated at 37 C for 1 h.In the last step towards development, TMB was added.The reaction was arrested with 0.2 M of H 2 SO 4 stop solution.The absorbance was measured at 450 nm using spectrophotometer (Thermo Fischer).Standard fab (Ranibizumab) was used as a negative control.

Results
Light chain and heavy chain of the TNFa inhibitor was cloned into duet vector as depicted in the construct map in Figure 1.DNA sequencing and PCR confirmed the accuracy and presence of gene of TNFa inhibitor Fab and its orientation (Data shown in Supplementary Figure S2).Gene of the TNFa inhibitor fragment was transformed into E. coli BL21 (DE3), followed by analysis of recombinantly expressed TNFa inhibitor Fab using standard culturing conditions (37 C and 0.1, 0.5, and 1 mM IPTG).As seen in Figure 2, the expression host and plasmid without the gene of interest were taken as negative controls for the partial confirmation of expression of TNFa inhibitor antibody fragment on SDS PAGE.Presence of band in the test clone and absence of the same in the negative controls partially confirmed that the expressed protein is TNFa inhibitor Fab.Based on the analysis of expression on SDS PAGE along with the reduced IgG as marker, it was confirmed that the 0.1 mM of IPTG  was optimal for expression of TNFa inhibitor Fab as marked in the red box in Figure 2.
For soluble expression of TNFa inhibitor Fab in E. coli, induction was performed at lower temperature than the cultivating temperature i.e., induction with 0.1 mM IPTG was performed at 15, 20, 25, 30, and 37 C, and periplasmic fractions were analyzed with the SDS PAGE (Supplementary Figures S3 and S4).The band corresponding to 25 kDa (equivalent to light chain of IgG) is present in the periplasmic fraction, thus confirming the soluble expression of the same as seen in Supplementary Figure S4.Densitometry analysis confirmed soluble expression of TNFa inhibitor Fab at 30 ± 2 mg/L in the periplasmic extract at 20 C whereas expression at 37 C is 9 ± 1 mg/L (Figure 3).Expression of soluble TNFa inhibitor increased as the cultivation temperature was reduced and the expression of insoluble protein increased as the temperature increased as seen in Table 1.SDS PAGE analysis partially confirmed the soluble expression of recombinant TNFa inhibitor Fab in E. coli.Purification and quantitative analysis of recombinant TNFa inhibitor Fab was checked using single step Protein L chromatography yielding 30 ± 3 mg/L in periplasmic fraction.Quality of the purified recombinant TNFa inhibitor Fab was characterized using SEC-HPLC for purity and intact molecular mass was determined using LC-MS.According to the   SEC results (Figure 4), there was a single peak observed with other minor contaminants, thus confirming the purity to be above 95% which corroborated with the NATIVE PAGE (Figure 4B).Affinity purified samples were analyzed on SDS PAGE stained with silver nitrate based on the densitometry; the purity noted was 97% as can be seen in Figure 5.Standard IgG (trastuzumab) was run as standard in Native PAGE.
Based on the intact mass analysis (Figure 6), the affinity purified TNFa inhibitor Fab showed the molecular weight of 47,879 Da which is 119 Da above theoretical mass of 47,753.49Da, which could be related the adduct formation due to s-cysteinylation [18,19] (equivalent to mass of 119 Da) with ppm error below 5. Intact mass confirmation and SEC based purity assessment confirmed the expression of recombinant TNFa Fab.
Analysis of amino acids sequence was performed using peptide mass fingerprinting via MS/MS.Digested peptides were matched with the theoretically predicted mass-bycharge ratio by comparing against their theoretical sequences.Sequence coverage of 81.04% was obtained, confirming the expressed protein to be TNFa inhibitor Fab at primary structure level (Supplementary Figure S6).
Purified TNFa inhibitor Fab was analyzed for specific activity against TNFa via sandwich ELISA.Increased concentration (0-20 mg) of in-house expressed recombinant TNFa inhibitor Fab was coated in each well.The neutralization effect of TNFa inhibitor Fab confirmed the activity of the soluble protein.Absorbance at 450 nm directly correlated with the increased amount of TNFa inhibitor Fab and confirmed the antigen binding specificity against TNFa (Figure 7).

Discussion
Amongst the broadly used expression system, E. coli is the most attractive choice of host for the heterologous proteins.However, due to high expression of recombinant proteins, many form insoluble aggregates within the cytoplasm, thereby resulting in formation of inclusion bodies.The protein in this form is devoid of any functional activity and hence, developing an efficient technique that can express the bioactive protein without compromising the expression level would lessen in the burden on downstream processing and also offer higher process yield.Although there is no universal approach that would be effective for all cases, numerous strategies have been attempted.Single promoter vectors are generally preferred for the expression of recombinant Fabs, with affinity tags for the ease of purification whereas recently upgraded vectors with dual promoters are slowly replacing the former ones due to expression of multidomain heterologous proteins, such as antibody fragments, rendering a cost effective approach and simultaneously reducing the time of expression. [2,7]Plasmids are categorized based on the copy number as high copy number plasmids (>100) and low copy plasmids (10-12). [2,20]igh copy number plasmids are related to increased protein production in bacteria. [3,21]However, these plasmids also overburden the cell and take a toll on cell metabolism and may even lead to cell death eventually, thereby resulting in low product yield. [9]Therefore, achieving appropriate cell growth and bioactive product yield while minimizing the stress on the cells would be desirable.In this paper, we report on soluble expression of antibody fragment against TNFa cultivating at low temperature using dual promotor vector without affinity tag and chaperones in E. coli (BL21 DE3).Combination of high copy plasmid number and low induction temperature yielded soluble TNFa inhibitor Fab up to 30 ± 3 mg/L in periplasmic region.We observed that soluble expression of recombinant TNFa inhibitor Fab was higher at low temperature (20 C) when compared to 37 C (Figure 5, Supplementary Figure S4, and Table 1).The mechanistic reason for this observation is that the level of ribosomes working on the peptide elongation of the proteins during translation is dependent on the growth rate which in turn is dependent on the cultivating temperature [22] .This may be plausibly attributed to decrease in the formation of in vivo aggregation due to slowing down of cellular synthetic machinery leading to reduced transcription and translation.Due to the reduced growth rate, nutrient uptake is reduced, thus curtailing the formation of toxic by-products.
Cellular proteolysis activity decreases as the proteases are less active at low temperature due to number of ribosomes per unit of cell mass, corresponding to excess of protein synthetic machinery indicating increased protein degradation at high temperature, [23][24][25] thus partially explaining the plausible correct folding at low temperature.Generally, E. coli is grown up to log phase at 37 C and then induced at low temperature (15-30 C).At such low temperatures, as the recombinant protein expression is progressive, cultivation time is prolonged generally up to 16-18 h. [26]In our case, we observed that the expression of recombinant TNFa inhibitor Fab was seen at the sixth hour post induction, particularly at temperature lower than 25 C, whereas at 30 C and above, protein expression was observed post 1 h of induction (Supplementary Figure S3).Protein translation occurs at a rate of 10-15 amino acids/second at optimal (37 C) temperature when compared to 3-5 amino acids/ second at low temperature in E. coli. [27]Expression of soluble TNFa inhibitor Fab at 15 and 25 C was similar.As seen in Supplementary Figure S5, reduction in optical density was noted which indicated reduction in growth.E. coli grows inadequately at temperature below 15 C and almost ceases at or below 5 C, as at such extremely low temperature population of non-translating ribosomes is predominant contributing little to cell growth, supporting poor protein initiation. [8,24,25,28]Strocchi et al have reported failure of chaperons in refolding of housekeeping proteins associate toward the cellular machinery maintenance at low temperature. [8]Based on the findings, 22 housekeeping proteins are involved in the cell growth mechanism which at low temperature resulted in impaired growth. [27,29]This might be the probable reason for reduced growth along with low titer at 15 C when compared to 20 C, which is in line with earlier reports in the case of recombinant enzymes. [28,30]esearchers have also reported expression levels of 7.25 mg/L [31] and 10 mg/L [32] using single promoter expression vector with affinity Tag in E. coli.Above 25 C, another group of researchers [33] have reported 30 mg/L of recombinant anti MM14 Fab at 30 C in E. coli with the help of coexpression of DsbA/C chaperons with His-Tag, similar to what we have achieved by lowering the temperature using a dual vector.Few other reports have showed expression levels of 70 mg/L [34] of recombinant his-tagged Fab in E. coli at 30 C, using PASylation and increasing the molecular weight of the protein by 150 kDa which may be useful for the expression of non-biopharmaceuticals molecules.
For production of biosimilars in the microbial expression system, a tagless (affinity or fusion) expression strategy would be desirable, such that it can be achieved without compromising on the yield of the final product.The proposed tagless strategy in this paper utilizes a dual promoter at low induction temperature in E. coli basic strain and delivers 30 mg/L of recombinant Fab against TNFa.Thus, TNFa inhibitor Fab was obtained with purity up to 95% using single step chromatography, assessed by SE-HPLC in corroboration with native and SDS-PAGE.Intact molecular mass was confirmed by LC-MS and the primary sequence of expressed TNFa inhibitor Fab was confirmed using LC-MS/MS analysis up to 81% of sequence coverage.Specificity against antigen TNFa showed that the expressed Fab has retained the binding domain when expressed at low temperature.

Conclusions
Combinatorial optimization of high plasmid copy dual promotor vector and; low temperature induction can produce soluble and functional Fab, equivalent to that produced by using chaperons in periplasmic space of E. coli.Synergistic effect of low temperature and dual promoter vector in microbial system may be advantageous for the commercial production of bioactive recombinant biologics.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Construct map for TNFa inhibitor Fab showing the cloning strategy.

Figure 3 .
Figure 3. Quantitative analysis of recombinant TNFa inhibitor Fab using densitometry based on SDS PAGE.

Figure 6 .
Figure 6.LC-MS analysis of intact mass determination of recombinant TNFa Fab.

Figure 7 .
Figure 7. Antigen-binding activity of TNFa inhibitor Fab performed using ELISA.

Table 1 .
Densitometry based quantitative analysis of expressed recombinant TNFa fab at various cultivation conditions.