Hollow fiber countercurrent dialysis for continuous buffer exchange of high-value biotherapeutics

Buffer exchange, desalting, and formulation of high-value biotherapeutics are currently performed using batch diafiltration (DF); however, this type of tangential flow filtration process may be difficult to implement as part of a fully continuous biomanufacturing process. The objective of this study was to explore the potential of using countercurrent dialysis for continuous protein formulation and buffer exchange. Experiments were performed using concentrated solutions of immunoglobuin G (IgG) with commercially available hollow fiber dialyzers having 1.5 and 1.8 m² membrane surface area. More than 99.9% buffer exchange was obtained over a range of conditions, as determined from the removal of a model impurity (vitamin B₁₂). The dialyzers were able to process more than 0.5 kg of IgG per day in an easily scalable low-cost process. In addition, buffer requirements were less than 0.02 L of buffer per gram IgG, which is several times less than that used in current batch DF processes. These results clearly demonstrate the potential of using low-cost hollow fiber dialyzers for buffer exchange and product formulation in continuous bioprocessing. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2763, 2019.     

Continuous production of urocanic acid by immobilized Achromobacter liquidum cells

Several microorganisms having higher L -histidine ammonia-lyase activity were immobilized into polyacrylamide gel lattice. The yield of enzyme activity by immobilization was highest in Achromobacter liquidum IAM 1667. As A. liquidum has urocanase activity, the cells were heat-treated at 70°C for 30 min to inactivate the urocanase. Enzymatic properties of the immobilized A. liquidum cells were investigated and compared with those of the intact cells. No difference was observed between the pH activity curve and optimal temperature for the intact and immobilized cells. The permeability of substrate or product through the cell wall was increased by immobilization of the cells. When an aqueous solution of 0.25M L -histidine (pH 9.0) containing 1mM Mg²⁺ was passed through a column packed with the immobilized A. liquidum cells at a flow rate of SV = 0.06 at 37°C, L -histidine was completely converted to urocanic acid. The L -histidine ammonia-lyase activity of the immobilized cell column was stable over 40 days at 37°C. From the effluent of the immobilized cell column, Urocanic acid was easily obtained in a good yield.     

Properties of invertase immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber membrane

Invertase was ionically immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2-dimethylaminoacetaldehyde dimethyl acetal. Immobilization and enzyme reaction were carried out by letting the respective solutions pass or circulate through the inside of the hollow fiber, and the activity of invertase was determined by the amount of glucose produced enzymatically from sucrose. Immobilization conditions were examined with respect to the enzyme concentration and to the time, and consequently the preferable conditions at room temperature were found to be 5 mug/mL of enzyme concentration and 4 h of immobilization time. Under those conditions the immobilization yield and the ratio of the activity of the immobilized invertase to that of the native one were 89 and 80%, respectively. For both repeating and continuous usages, the activity fell to ca. 60% of the initial activity in the early stage and after that almost kept that value. The apparent Michaelis constant K(m) (') for the immobilized invertase decreased with increasing the flow rate of the substrate solution, to be close to the value for the native one. Furthermore, the possibility of the separation of the enzymatically formed glucose from the reaction mixture through the hollow fiber membrane was preliminarily examined.     

Continuous Asymmetric Reduction Of 4-Oxoisophorone By Thermophilic Bacteria Using A Hollow Fiber Reactor

Continuous asymmetric reduction of 4-oxoisophorone by the thermophilic bacterium Thermomonospora curvata JTS321 was examined using three reactor systems: packed bed, fluidized bed and hollow fiber. T. curvata was immobilized in polyacrylamide-hydrazide gels when used in the packed and fluidized bed reactors. Of the three reactor systems, the highest productivity (964 mg.1^-1.h^-1) was observed in the fluidized bed reactor. However, many cells grew outside of the gel matrix, causing product contamination. The productivity of the hollow fiber reactor was 504 mg.1^-1.h^-1; the problem of cell contamination of the product was avoided, as the molecular cut-off of the hollow fibers (400 000) was of an appropriate size to prevent cell leakage to the product stream. We therefore consider that the hollow fiber reactor is most suitable for continuous microbial conversions.     

Lysis of Micrococcus lysodeikticus cells by lysozyme covalently immobilized on the lumen of hollow fibers

Summary A lytic enzyme reactor for microbial cell lysis is described in which lysozyme is immobilized on the lumen of hemodialyzer hollow fibers using epichlorohydrin as a coupling agent. The cell suspension flows through the lumen without any hindrance where the cells are lyzed by the immobilized lysozyme efficiently. Micrococcus lysodeikticus cells at concentrations of 0.25 g/L and 5 g/L were successfully lyzed without clogging the hollow fiber. In comparison with lysozyme immobilized on submicron particles, the activity retention was at least 8 times higher.     

Continuous two step bioconversion of 4-oxoiso-phorone (OIP) to 4-hydroxy-2,2,6-trimethylcyclo-hexanone (4-HTMCH) by thermophilic bacteria

Summary The continuous two step conversion of 4-oxoisophorone(OIP) to 4-hydroxy-2,2,6-trimethylcyclohexanone(4-HTMCH) via dihydrooxoisophorone(DOIP) was carried out using two types of thermophilic growing cells, Thermomonospora curvata and Bacillus stearothermophilus, in sequential connected continuous stirred tank reactor(CSTR) and hollow fiber reactor system. For more than 80 hours operation, 46% of OIP was converted to 4-HTMCH with productivity of 179 mg/h/l.     

1H-Nuclear magnetic resonance study on imidazole–imidazole interaction in L-histidyl-L-histidine and D-histidyl-L-histidine: Analysis including microscopic dissociation series

The ¹H-nmr titration curves of chemical shifts versus pH were observed for the protons of _D,L-histidyl-_D,L-histidine as representative of cases with two or more ionizable groups with similar pK_a values. The titration curves of L -histidyl-L -histidine and D -histidyl-L -histidine were individually analyzed according to two mathematical models: one of a macroscopic dissociation series and one of a microscopic dissociation series. Most-probable values and standard deviations were obtained for pK_a values and intrinsic chemical shifts. An analysis including the microscopic dissociation series yielded an electrostatic interaction between twoimidazole rings of about 0.3 pH units for L -histidyl-L -histidine and about 0.7 pH units for D -histidyl-L -histidine. The difference of the magnitude of imidazole-imidazole interactions between L -histidyl-L -histidyne and D -histidyl-L -histidine was interpreted in terms of the spatial arrangement of two imidazole rings in each molecule based on the solution conformation estimated from Gd(III)-induced relaxation enhancements.     

X-ray studies on crystalline complexes involving amino acids and peptides. XXIV. Different ionization states and novel aggregation patterns in the complexes of succinic acid with DL-and L-histidine

Diffusion of acetonitrile into an aqueous solution of DL -histidine and succinic acid in 1:3 molar proportions results in the crystals of DL -histidine hemisuccinate dihydrate [triclinic, P1 , a = 7.654(1), b = 8.723(1), c = 9.260(1) Å, α = 77.23(1), β = 72.37(1) and γ = 82.32 (1)°]. The replacement of DL -histidine by L -histidine in the crystallization experiment under identical conditions leads to crystals of L -histidine semisuccinate trihydrate [orthorhombic, P2₁2₁2₁, a = 7.030 (1), b = 8.773 (1), and c = 24.332 (3) Å]. The structures were solved using counter data and refined to R values of 0.056 and 0.054 for 2356 and 1778 observed reflections, respectively. Histidine molecules in both the complexes exist in open conformation I. Succinate and semisuccinate ions in them are planar, and exactly or nearly centrosymmetric. In the DL -histidine complex, the amino acid molecules form double ribbons and the succinate ions occupy voids left behind when the double ribbons aggregate, as in inclusion compounds. In the L -histidine complex, the amino acid molecules form columns; so do the semisuccinate ions and water molecules. The two columns interdigitate to form the complex crystal. There are similarities between the molecular aggregation in the complexes and that in the crystals of L - and DL -histidine. However, the presence of succinic acid has the effect of disrupting, partially or totally, head-to-tail sequences involving amino acid molecules. © 1993 John Wiley & Sons, Inc.     

Incorporation of granular activated carbon in an immobilized membrane bioreactor for the biodegradation of phenol by <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Granular activated carbon (GAC) was incorporated into hollow fiber membrane bioreactors for the biodegradation of 1,000 mg phenol l⁻¹ through immobilization of Pseudomonas putida. The phenol was removed within 25 h in the hybrid bioreactor, comparing with 31 h for a GAC-free bioreactor. Sorption, biodegradation, desorption, and bioregeneration were four steps for the phenol removal during batch operation.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: IV. Effects of temperature

A lipase from Aspergillus niger, immobilized by adsorption on microporous polypropylene hollow fibers, was used to effect the hydrolysis of the glycerides of melted butterfat at pH. 7.0 at 40, 50, 55, and 60 degrees C. Mcllvane buffer was pumped upward through the lumen, and melted butterfat was pumped upward through the shell side of a hollow fiber reactor. Nonlinear regression methods were employed to determine the kinetic parameters of models based on combinations of three nested rate expressions for the hydrolysis reaction with three nested rate expressions for thermal deactivation of the enzyme. A rate expression containing four lumped parameters is sufficient to model the release of free fatty acids as a function of reactor space time and time elapsed after immobilization. Nonlinear regression methods were also employed in global fits of the data to rate expressions containing an explicit dependence on temperature. For the reaction conditions used in this research, a 14-parameter rate expression is necessary to accurately model the overall release of free fatty acids as a continuous function of the absolute temperature, initial substrate concentrations, reactor space time, and time elapsed after immobilization of the lipase.     

Multi-stage high cell continuous fermentation for high productivity and titer

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.     

Demonstration of efficient trichloroethylene biodegradation in a hollow‐fiber membrane bioreactor

Rapid cometabolism of trichloroethylene (TCE) by pure cultures of Methylosinus trichosporium OB3b PP358 was demonstrated in a two‐stage hollow‐fiber membrane bioreactor over the course of 3 weeks. PP358 was grown in a continuous‐flow chemostat and circulated through the shell of a hollow‐fiber membrane module (HFMM), while TCE contaminated water (160 to 1450 μg/L) was pumped through the fiber lumen (fiber interior). In parallel‐flow HFMM biological experiments, 82% to 89% of the influent TCE was removed from the lumen (5.1‐min residence time) with 99% of the transferred TCE undergoing biodegradation. Biological experiments in a larger capacity baffled radial‐flow HFMM resulted in 66% to 99% TCE transferred and 93% to 96% TCE biodegradation at lumen residence times of between 1.5 and 3.7 min. Biodegradation was maintained throughout the experiments at pseudo‐first‐order biodegradation rate constants of 0.41 to 2.8 L/mg TSS/day. Best‐fit computer modeling of the baffled radial‐flow biological process estimated mass transfer coefficients as large as 2.7 × 10⁻² cm/min. The computer model was also shown to simulate the experimental results quite well. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 681–692, 1999.     

Raman spectra of polypeptides containing L-histidine residues and tautomerism of imidazole side chain

Raman spectra were measured for poly(L -histidine) in H₂O, poly(L -histidine-d₂ and -d₃) in D₂O, L -histidine in H₂O, L -histidine-d₃ (and d₄) in D₂O, and 4-methylimidazole in H₂O with various pH (or pD) values. The Raman scattering peaks observed for these samples were ascribed to the neutral and positively charged imidazole groups on the basis of the spectral changes due to the pH variation and to the deuterium substitution of the imino protons. The vibrational modes of these peaks were deduced from the normal coordinate analysis made on the positively charged and neutral 4-ethylimidazoles. The Raman scattering peaks from the imidazole groups in the neutral form clearly indicate that these imidazole groups exist in the equilibrium between the two tautomeric forms, the 1-N protonated from (tautomer I) and the 3-N protonated one (tautomer II). For example, the breathing vibration of the 1-N protonated form is observed at 1282 cm^?1 for L -histidine and at 1304 cm^?1 for 4-methylimidazole, while the breathing vibration of the 3-N protonated form is observed at 1260 cm^?1 for L -histidine and 4-methylimidazole. From the temperature dependence of the relative intensities of the tautomer I peak to that of the tautomer II, it was concluded that the tautomer I is energetically more stable than the tautomer II, and the ΔH value is 1.0 ± 0.3 kcal/mol for L -histidine and 0.4 ± 0.1 kcal/mol for 4-methylimidazole. Poly(L -histidine) with the neutral imidazole side chains shows the amide I peak at 1672 cm^?1, indicating that the sample assumes the antiparallel pleated-sheet structure. Poly(L -Ala⁷⁵L -His²⁵) and poly(L -Ala⁵⁰L -His⁵⁰) were found to take the α-helical and β-form conformations, respectively.     

A study of polynucleotide phosphorylase production by <Emphasis Type="Italic">Escherichia coli</Emphasis> in a hollow fibre reactor

A 30-l hollow fibre reactor with continuous fermentation for cell recycling of Escherichia coli AS 1.183 was used to remove the inhibitory effects on cell growth and extend the fast growth phase to increase the yield of polynucleotide phosphorylase (PNPase) in E. coli cells. When the dilution rate was 1.5 h⁻¹, the cell concentration of E. coli reached 235 g/l (wet wt, 70% moisture content), with PNPase activity above 90 u/g (wet wt). With the dilution rate is 1.0 h⁻¹, the fermentor volumetric productivity of PNPase in a hollow fiber reactor can reach 974 (u/h * l) compared to 20 (u/h * l) in a conventional batch culture.     

Recovery of insect cells using hollow fiber microfiltration

An efficient method was developed for media separation and cell collection for eukaryotic cells growing in suspension. The method is based on tangential flow microfiltration using an open channel arrangement in a hollow fiber configuration. Best results (highest processing flux rate) for polysulfone hollow fibers were obtained using fibers with internal diameter of 0.75 mm, 0.45 mum pore size, and a cell suspension flow at a shear rate of 14000 s(-1) (0.032 L/min per fiber). A flux rate of 500 L/m(2) h can be obtained by maintaining the surface area/cell ratio at 0.05 m(2)/10 L of cells at a concentration of 2.5 x 10(6) cells/mL. Forty liters of infected insect cells can be concentrated 10 times in 20 min without affecting cell viability. (c) 1995 John Wiley & Sons, Inc.     

Immobilization of Lactococcus lactis to cellulosic material by cellulose‐binding domain of Cellvibrio japonicus

Aims: Immobilization of whole cells can be used to accumulate cells in a bioreactor and thus increase the cell density and potentially productivity, also. Cellulose is an excellent matrix for immobilization purposes because it does not require chemical modifications and is commercially available in many different forms at low price. The aim of this study was to construct a Lactococcus lactis strain capable of immobilizing to a cellulosic matrix. Methods and Results: In this study, the Usp45 signal sequence fused with the cellulose‐binding domain (CBD) (112 amino acids) of XylA enzyme from Cellvibrio japonicus was fused with PrtP or AcmA anchors derived from L. lactis. A successful surface display of L. lactis cells expressing these fusion proteins under the P45 promoter was achieved and detected by whole‐cell ELISA. A rapid filter paper assay was developed to study the cellulose‐binding capability of these recombinant strains. As a result, an efficient immobilization to filter paper was demonstrated for the L. lactis cells expressing the CBD‐fusion protein. The highest immobilization (92%) was measured for the strain expressing the CBD in fusion with the 344 amino acid PrtP anchor. Conclusions: The result from the binding tests indicated that a new phenotype for L. lactis with cellulose‐binding capability was achieved with both PrtP (LPXTG type anchor) and AcmA (LysM type anchor) fusions with CBD. Significance and Impact of the Study: We demonstrated that an efficient immobilization of recombinant L. lactis cells to cellulosic matrix is possible. This is a step forward in developing efficient immobilization systems for lactococcal strains for industrial‐scale fermentations.     

Transport ofl-histidine by human diploid fibroblasts in culture

Summary The transport ofl-histidine has been characterized in skin derived diploid human fibroblasts, cultured under strictly controlled conditions. The transport measurements were made on cells grown to subconfluency after 60 to 90 min timed preincubation. The data, at substrate concentrations ranging from 0.050 to 10 mmol/l, were analyzed by a computer program. A saturable transport system (K _m =0.25 mmol/l, V _max =17 nmol/mg protein per min) and a nonsaturable component of influx (K _d =1.6±0.4 nmol/mg protein/min per mmol) were found.l-Histidine displayed no Na⁺ requirement at either low or high concentrations. Inhibition analysis demonstrated thatl-histidine uptake at low concentration was poorly inhibited by amino acids known to be effective inhibitors of system A. The largest fraction ofl-histidine uptake was inhibited by 2-amino-bicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), leucine, and tryptophan. These results indicated thatl-histidine is transported in human fibroblasts, mainly by the Na⁺ independent system L. The differences between this cell type and others studied previously are discussed. This work was supported in part by Grant 773 from UER de Médecine, Université Paris XI (France).     

Immobilization conditions of ketoreductase on enantioselective reduction in a gas-solid bioreactor

The immobilization conditions of commercial ketoreductase for continuous enantioselective reduction in the gas-phase reaction were investigated with respect to the immobilization efficiency (residual activity and protein loading) and the gas-phase reaction efficiency (initial reaction rate, half-life, and enantioselectivity). For the analyses, ketoreductase was first immobilized by physical deposition on glass supports and the reduction of 2-butanone to (S)-2-butanol with the concomitant regeneration of NADH by 2-propanol was used as a model reaction. The optimal conditions of enzyme immobilization were obtained using an absolute pressure of 100 hPa for drying, a pH between 6.5 and 7.0, and a buffer concentration of 50 mM. The buffer concentration in particular had a strong effect on both the enzyme activity and enantioselectivity. Under optimal immobilization conditions, the thermostability of ketoreductase in the gas-phase system was enhanced compared to the aqueous-phase system, while the enantioselectivity was successfully maintained at a level identical to that of the native enzyme. These results indicate that the gas-phase reaction has a great potential for industrial production of chiral compounds, but requires careful optimization of immobilization conditions for the reaction to progress effectively.     

Inert supports for lactic acid fermentation —a technological assessment

Production of lactic acid using Lactobacillus delbrueckii NRRL B445 recently renamed L. rhamnosus was studied in continuously recycled packed reactors at pH 6.3 and 42° C. Four inert adsorbent supports were used for immobilization: Raschig rings of sintered glass (Schott, FRG), beads of sintered glass (Schott), beads of porous glass (Poraver; Dennert, FRG) and irregular ceramic particles (Otto Feuerfest, FRG). The best support was found to be the beads of sintered glass, yielding the highest volumetric lactic acid productivity. Zeta potentials of L. rhamnosus showed the cells to be negatively charged at all pHs studied, the charge becoming less negative with increasing ionic strength. The surface charge did not control adhesion. A comparison between the immobilization carried out in batch and continuous tests with the different supports demonstrated that extrapolation from batch adsorption curves to continuous operation can introduce large errors. The effect of dilution rate was also stidied: a saturation concentration of adsorbed cells was achieved at all dilution rates, i.e., the immobilized cell component was almost invariant. Different diameters of Poraver beads were tested; clear evidence for mass transfer limitation was shown. Finally, the effects of pH and substrate concentration under immobilization were evaluated. The results indicate that pHs above or below the optimum for suspended cell systems can be used in the immobilized reactor while maintaining lactic acid productivity. To simplify downstream processing by keeping the glucose concentration close to zero in the effluent, the glucose concentration in the feed has to be chosen in conjunction with the dilution rate. Correspondence to: M. J. T. Carrondo     

耦合中空纤维膜超滤分离游离细胞催化合成ATP

对耦合中空纤维膜超滤分离进行游离细胞催化合成ATP过程进行了实验研究,考察了细胞的催化效率和膜组件的操作稳定性。结果显示,中空纤维超滤膜的耦合分离能有效地截留反应液中的游离酶,其中乙醇脱氢酶(ADH)和已糖激酶(HK)的稳态截留效率在95%以上。耦合膜分离的酵母细胞催化ATP合成反应可重复使用2.5～3.0次,酶的利用率比普通分离的细胞提高2.0～2.5倍。中空纤维超滤膜于0.1Mpa工作压力下连续11批耦合分离操作,膜的渗透性无明显下降,过滤速率保持在初速率的95%以上。在稀释速率0.25h-1下,反应体系保持了连续5h的ATP高转化率合成与分离耦合的拟稳态操作。