Production of hepatitis B core antigen in a stirred tank bioreactor: The influence of temperature and agitation

The influence of temperature and agitation on the growth ofEscherichia coli expressing hepatitis B core antigen (HBcAg) in stirred tank bioreactor were investigated. The highest specific growth rate forE. coli (0.844 h⁻¹) was achieved at a temperature of 37°C and an agitation speed of 250 rpm. The activation energy for the growth of theE. coli strain W3110IQ in the stirred tank bioreactor was estimated to be 11 kcal/mol. The highest protein yield was achieved at a temperature of 44°C and an agitation speed of 250 rpm. The relative protein concentration at 44°C is 30 and 6% higher compared to that at 30 and 37°C, respectively.     

Optimization of culture conditions and continuous production of chitosan by the fungi,Absidia coerulea

The production of chitosan from the mycelia ofAbsidia coerulea was studied to improve cell growth and chitosan productivity. Culture conditions were optimized in batch cultivation (pH 4.5 agitator speed of 250 rpm, and aeration rate of, 2 vvm) and the maximum chitosan concentration achieved was 2.3 g/L under optimized conditions. Continuous culture was carried out successfully by the formation of new growth spots under optimized conditions, with a chitosan productivity of 0.052 gL⁻¹ h⁻¹, which is the highest value to date, and was obtained at a dilution rate of 0.05 h⁻¹. Cell chitosan concentrations reached about 14% in the steady state, which is similar to that achieved in batch culture. This study shows that for the continuous culture ofAbsidia coerulea it is vital to control the medium composition.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Production of inulinase by solid-state fermentation: effect of process parameters on production and preliminary characterization of enzyme preparations

This work was aimed at producing inulinase by solid-state fermentation of sugarcane bagasse, using factorial design to identify the effect of corn steep liquor (CSL) and soybean bran concentration, particle size of bagasse and size of inoculum. Maximum inulinase activity achieved was 250 U per g of dry substrate (gds) at 20% (w/w) of CSL, 5% (w/w) of soybean bran, 1 × 10¹⁰ cells mL⁻¹ and particle size of bagasse in the range 9/32 mesh. The use of soybean bran decreased the time to reach maximum activity from 96 to 24 h and the maximum productivity achieved was 8.87 U gds⁻¹ h⁻¹. The maximum activity was obtained at pH 5.0 and 55.0°C. Within the investigated range, the enzyme extract was more thermostable at 50.0°C, showing a D-value of 123.1 h and deactivation energy of 343.9 kJ gmol⁻¹. The extract showed highest stability from pH 4.5 to 4.8. Apparent K _m and V _max are 7.1 mM and 17.79 M min⁻¹, respectively.     

Temperature management strategy for efficient gene expression in a thermally inducible <Emphasis Type="Italic">Escherichia coli</Emphasis>/bacteriophage system

In a two-phase operation, E. coli containing λSNNU1 (Q ⁻ S ⁻) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least 40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast, when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min, and a production phase at 37°C, the highest level of cloned gene expression was achieved.     

Physiological diversity within the <Emphasis Type="Italic">kluyveromyces marxianus</Emphasis> species

The Kluyveromyces marxianus strains CBS 6556, CBS 397 and CBS 712^T were cultivated on a defined medium with either glucose, lactose or sucrose as the sole carbon source, at 30 and 37°C. The aim of this work was to evaluate the diversity within this species, in terms of the macroscopic physiology. The main properties evaluated were: intensity of the Crabtree effect, specific growth rate, biomass yield on substrate, metabolite excretion and protein secretion capacity, inferred by measuring extracellular inulinase activity. The strain Kluyveromyces lactis CBS 2359 was evaluated in parallel, since it is the best described Kluyveromyces yeast and thus can be used as a control for the experimental setup. K. marxianus CBS 6556 presented the highest specific growth rate (0.70 h⁻¹) and the highest specific inulinase activity (1.65 U mg⁻¹ dry cell weight) among all strains investigated, when grown at 37°C with sucrose as the sole carbon source. The lowest metabolite formation and highest biomass yield on substrate (0.59 g dry cell weight g sucrose⁻¹) was achieved by K. marxianus CBS 712^T at 37°C. Taken together, the results show a systematic comparison of carbon and energy metabolism among three of the best known K. marxianus strains, in parallel to K. lactis CBS 2359.     

Decolourization and Detoxification of Methyl Red by Aerobic Bacteria from A Wastewater Treatment Plant

Bacterial cultures from a wastewater treatment plant degraded a toxic azo dye (methyl red) by decolourization. Complete decolourization using a mixed-culture was achieved at pH 6, 30 °C within 6 h at 5 mg/l methyl red concentration, and 16 h at 20—30 mg/l. Four bacterial species were isolated that were capable of growth on methyl red as the sole carbon source, and two were identified, namely Vibrio logei and Pseudomonas nitroreducens. The Vibrio species showed the highest methyl red degradation activity at the optimum conditions of pH 6--7, and 30—35 °C. Analysis by NMR showed that previously reported degradation products 2-aminobenzoic acid and N,N-dimethyl-1,4-phenylenediamine were not observed. The decolourized dye was not toxic to a monkey kidney cell line (COS-7) at a concentration of 250 μM. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of ambient temperature and of temperature acclimation on nitrogen excretion and differential catabolism of protein and nonprotein resources in the intertidal snails,Littorina saxatilis (Olivi) and L. obtusata (L.)

Nitrogenous excretion in two snails, Littorina saxatilis (high intertidal) and L. obtusata (low intertidal) was studied in relation to temperature acclimation (at 4° and 21°C), including total N excretion rates, the fraction of urea in N excretion, corresponding O:N ratios and the partitioning of deaminated protein between catabolic and anabolic processes at 4°, 11° and 21°C. Aggregate N excretion rates in both species showed no significant compensatory adjustments following acclimation. Total weight specific N excretion rates at 21°C were higher in standard 3 mg L. saxatilis (739 ng N mg⁻¹ h⁻¹) than standard 5 mg L. obtusata (257 ng N mg⁻¹ h⁻¹) for snails acclimated to 21°C. Comparisons of Q₁₀ values of total weight specific N excretion to Q₁₀ values for weight specific oxygen consumption ({xxV}O₂) between 4° to 11 °C and 11° to 21°C indicated that, while total rates of catabolic metabolism ({xxV}O₂) and protein deamination in L. obtusata were essentially parallel, the relationship between N excretion and {xxV}O₂ in L. saxatilis revealed the partitioning of a larger share of deaminated protein carbon into anabolism at 4° and 21°C than at 11°C. Urea N accounted for a larger share of aggregate N excreted in L. saxatilis than in L. obtusata, but in both species urea N is a greater proportion of total N excreted when acclimated at 4°C (urea N: ammonia N ratio range: 1 to 2.15) than in snails acclimated to 21°C (urea N: ammonia N ratio range: 0.46 to 1.39). Molar O:N ratios indicate that the proportion of metabolism supported by protein catabolism is greater in L. saxatilis (O:N range: 2.5–8.4) than in L. obtusata (O:N range: 7.3–13.0). In both species, regardless of acclimation temperature, the O:N ratios are generally lowest (high protein catabolism) at 4°C and highest at 21°C.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Optimal conditions for chitinase production by<Emphasis Type="Italic">Serratia marcescens</Emphasis>

A chitinase-producing bacterium was isolated from seashore mud around Beobseongpo in Chunmam province through the use of a selective enrichment culture. The best chitinase producing strain was isolated and identified asSerratia marcescens KY from its characteristics. For effective production of chitinase, optimum pH, temperature, and agitation speed were investigated in flask cultures. The optimum pH usingSerratia marcescens KY was between pH 6 and 7 and the chitinase produced was 37.9 unit/mL. On the other hand, the optimal pH of theSerratia marcescens ATCC 27117 was 7.5, and the produced amount of chitinase was 35.2 unit/mL. The optimal temperature for chitinase production forSerratia marcescens KY andSerratia marcescens ATCC 27117 was 30°C. The cell growth pattern at different temperature was almost identical to the chitinase production. To investigate the optimal shaking speed under optimal culture, speeds were varied in the range of 0≈300 rpm. The maximum production of chitinase was carried at 200 rpm although the cell growth was the highest at 150 rpm. It indicates that oxygen adjustment is required for the maximum chitinase production. Using optimal conditions, batch cultures for comparingSerratia marcescens KY andSerratia marcescens ATCC 27117 were carried out in a 5 L fermentor. The oxygen consumption was increased with the increase of culture. Especially, at 120 h of cultureSerratia marcescens KY andSerratia marcescens ATCC 27117 produced 38.3 unit/mL, and 33.5 unit/mL, respectively.     

Effect of salinity and pH on growth and agar yield of Gracilaria tenuistipitata var. liui in laboratory and outdoor cultivation

Acclimation responses of the red alga Gracilaria tenuistipitata var. liui collected on the northwest coast of Philippines were determined in laboratory setups and outdoor cultivation tanks in Haifa, Israel. Growth under laboratory conditions was influenced by all three variables studied, namely, temperature (20 or 30 ^°C), salinity (20, 30 or39‰) and seawater pH (6.5, 7.0, 8.0 or ≥ 9.0). In 250 mL flasks lacking pH control growth was influenced by temperature only at 20 ‰, whereas at 39 ‰, growth rates were similar at 20 or 30 ^°C. In 500 mL cylinders in which pH was controlled, growth rates were significantly different at a pH of 6.5 and 7.0 for all salinities, with maximal rates occurring in 39 ‰. At pH 8.0, and above, growth rates between salinities were similar and reduced to approximately 50% at a pH of 9.0 compared to rates at a pH of 6.5. Photosynthesis responses generally resembled growth responses both, in 250 mL and 500 mL cultures. In 40-L outdoor tanks, weekly growth and agar yields were apparently enhanced by increasing light intensities (up to full sunlight) and nutrient concentrations (up to 0.2 mM PO₃ ^2- and 2.0 mM NH₄ ⁺), and rates averaged four times higher than rates determined in the smaller flask cultures. This study shows broad salinity tolerance of G. tenuistipitata var. liui and its ability to sustain growth rates that are among the highest measured for Gracilaria spp. in outdoor cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Pilot-scale production of carboxymethylcellulase from rice hull by Bacillus amyloliquefaciens DL-3

Optimal conditions for pilot-scale production of the carboxymethylcellulase (CMCase) by Bacillus amyloliquefaciens DL-3 were investigated. The best carbon and nitrogen sources for the production of CMCase by B. amyloliquefaciens DL-3 were found to be rice hull and peptone and their optimal concentrations were 5.0 and 0.20% (w/v), respectively. Optimal temperature and initial pH for the production of CMCase were 37°C and 6.8. Optimal agitation speed and aeration rate for the production of CMCase were 300 rpm and 1.0 vvm in a 7 L bioreactor, which were different from those for the cell growth of B. amyloliquefaciens DL-3. The highest productions of CMCase by B. amyloliquefaciens DL-3 from 5.0% (w/v) rice hull as a carbon source under optimal conditions in a 7 or 100 L bioreactor were 220 and 367 U/mL, respectively.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Optimization of Citric Acid Production from Glucose by Yarrowia lipolytica

Citric acid (CA) is mainly produced in a biotechnological process using Aspergillus niger. In this process, large amounts of wastes have to be removed. Since the use of Yarrowia lipolytica for CA production is an environmental compatible alternative method, the CA production was optimized in regard to growth temperature and pH as well as substrate and product inhibition. The highest value of the maximum specific growth rate at pH 6.5 was found to be μ_max = 0.192 h^–1, whereas the largest amount of CA of 24.91 g/L as well as the highest selectivity of the bioprocess (89.9 % CA) and the maximum yield (0.22 g_CA/g_Glucose) were obtained at pH 6.0. During the growth phase, the temperature optimum was found to be in the range of 30–34 °C (μ_max = 0.132 h^–1). Nevertheless, the highest concentration of CA during the production phase was obtained at 30 °C (41 g/L CA, 93.1 % CA, 0.55 g_CA/g_glucose). In studying the substrate inhibition of the process, a clear tendency of decrease in the maximum specific growth rate was detected when the initial glucose concentration was increased from 50 g/L (μ_max = 0.17 h^–1) to 200 g/L (μ_max = 0.055 h^–1). The addition of 120 g/L CA to the culture broth at the start of the production phase reduced the production of CA from 32.1 g/L to 7.4 g/L.     

Effect of crab shell size on bio-demineralization with lactic acid-producing bacterium, <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">tolerans</Emphasis> KCTC-3074

Demineralization (DM) from crab shell (CS) waste was carried out using a lactic acid-producing bacterium, Lactobacillus paracasei subsp. tolerans KCTC-3074 for 7 days at 25, 30, and 35°C. DM rates were 89∼92% and slightly affected by temperature. DM was also performed for four particle-sized shell samples (0.84∼3.35, 3.35∼10, 10∼20, and 20∼35 mm) with 10% inoculum, 5% shell, and 10% glucose at 30°C and 180 rpm for 7 days. It was found out that the shell size had a slight effect on the rate of DM. Negative relationships were found between DM and residual dry weight (r² = 0.960), and between DM and pH (r² = 0.906). Conversely, positive relationships were found between DM and medium protein (r² = 0.696), and between DM and total titratable acidity (r² = 0.630).     

Fermentative Production of Piperazinium Dilactate

In order to test whether piperazinium dilactate can be produced by fermentation in its exact stoichiometric composition without losses of yield, the kinetics of cell growth and lactate production were investigated in the batch cultivation of Lactobacillus paracasei, using piperazine as a neutralizer in pH control. It was found that piperazine dilactate is formed in its stoichiometric composition at about pH 5.0, and lactic acid fermentation occurred with yields of about 90% under these conditions. Piperazine at concentrations less than or equal to 50 g/l did not affect growth and product formation. The presence or absence of piperazine did not produce any significant differences in either the maximum specific growth rate or the maximum specific lactate formation rate when piperazine was present or absent (0.65 h^—1 compared to 0.68 h^—1 and 3.86 g/g × h compared to 3.63 g/g × h, respectively). The Luedeking‐Piuret relationship between the two quantities was also not changed significantly when piperazine was added. To estimate the optimum parameters for cell growth and lactate formation in the presence of piperazine, a factorial experiment was designed and carried out under consideration of the parameter ranges 5.0 ≤ pH ≤ 7.0 and 30 °C ≤ T ≤ 36 °C. In this way, three‐dimensional models of the specific growth rate, the specific lactate formation rate and the lactate yield were obtained.     

Large‐scale culture of a megakaryocytic progenitor cell line with a single‐use bioreactor system

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single‐use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large‐scale production and feasibility of meeting clinical‐grade standards. The current work evaluated the capacity of a single‐use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k_La) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h^?1, respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7‐fold with a total cell number of 1.2 ± 0.2 × 10⁹ cells L^?1. In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single‐use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362–369, 2018     

Relationship between agitation speed and the morphological characteristics of Verticillium lecanii CS-625 during spore production

Verticillium lecanii is recognized as an entomopathogenic fungus, and has high potential in the biological control of pests. In this study, it was investigated that the relationship between agitation speed in a 2.5 L stirred tank reactor (STR) at 25°C and initial pH 5.5, and the morphological characteristics of V. lecanii CS-625, such as hyphal length/width, spore length/width, and the number of tips during spore production. The agitation speed affected the hyphae patterns and the number of tips. The number of spores rapidly increased at 48 to 60 h of cultivation, and the highest spore productivity (2.5 × 10¹⁰ spore/L·h at 60 h) occurred with an agitation speed of 350 rpm and an aeration rate of 1.0 vvm. The number of tips increased in proportion to the increase in spore production during the same culture time. The highest number of tips (4.8 × 10⁸ tipJ.mL) was obtained at 72 h of cultivation. The shortest mean spore length (2.8 μm) was obtained at 60 h of cultivation. Therefore, it was determined that the increased number of tips and decreased mean spore length were closely related to the production of V. lecanii spores.     

Characteristics of fed-batch cultures of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> containing human-like collagen cDNA at different specific growth rates

Fed-batch cultures of recombinant Escherichia coli BL21 for producing human-like collagen were performed at different specific growth rates (0.1~0.25 h⁻¹) before induction and at a constant value of 0.05 h⁻¹ after induction by the method of pseudo-exponential feeding. Although the final biomass (around 69 g l⁻¹) was almost the same in all fed-batch cultures, the highest product concentration (13.6 g l⁻¹) was achieved at the specific growth rate of 0.15 h⁻¹ and the lowest (9.6 g l⁻¹) at 0.25 h⁻¹. The mean productivity of human-like collagen was the highest at 0.15 h⁻¹ (0.57 g l⁻¹ h⁻¹) and the lowest at 0.1 h⁻¹ (0.35 g l⁻¹ h⁻¹). In the phase before induction, the cell yield coefficient (Y_X/S) decreased when the specific growth rate increased, while the formation of acetic acid increased upto 2.5 g l⁻¹ at 0.25 h⁻¹. The mean product yield coefficient (Y_P/S) also decreased with specific growth rate increasing. The respiration quotient (RQ) increased slightly with specific growth rate increasing before induction, and the mean value of RQ was around 72%. The optimum growth rate for human-like collagen production was 0.15~0.2 h⁻¹.     

Production and scale‐up of a monoclonal antibody against 17‐hydroxyprogesterone

The hybridoma 192 was used to produce a monoclonal antibody (MAb) against 17‐hydroxyprogesterone (17‐OHP), for possible use in screening for congenital adrenal hyperplasia (CAH). The factors influencing the MAb production were screened and optimized in a 2 L stirred bioreactor. The production was then scaled up to a 20 L bioreactor. All of the screened factors (aeration rate, stirring speed, dissolved oxygen concentration, pH, and temperature) were found to significantly affect production. Optimization using the response surface methodology identified the following optimal production conditions: 36.8°C, pH 7.4, stirring speed of 100 rpm, 30% dissolved oxygen concentration, and an aeration rate of 0.09 vvm. Under these conditions, the maximum viable cell density achieved was 1.34 ± 0.21 × 10⁶ cells mL^?1 and the specific growth rate was 0.036 ± 0.004 h^?1. The maximum MAb titer was 11.94 ± 4.81 μg mL^?1 with an average specific MAb production rate of 0.273 ± 0.135 pg cell^?1 h^?1. A constant impeller tip speed criterion was used for the scale‐up. The specific growth rate (0.040 h^?1) and the maximum viable cell density (1.89 × 10⁶ cells mL^?1) at the larger scale were better than the values achieved at the small scale, but the MAb titer in the 20 L bioreactor was 18% lower than in the smaller bioreactor. A change in the culture environment from the static conditions of a T‐flask to the stirred bioreactor culture did not affect the specificity of the MAb toward its antigen (17‐OHP) and did not compromise the structural integrity of the MAb. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013