Fluidized bed ion exchange for improving purification of lactic acid from fermentation

A strong anionic exchange resin was used to recover lactic acid directly from fermentation in an upflow fluidized bed column, resulting in 0.18 g lactic acid/g resin bound with a subsequent elution of 94%. When the culture broth was heated and adjusted pH to 8.0, 0.4 g lactic acid was bound per g resin, with a subsequent elution of 90%. L(+) and D(–) lactic acid isomers distribution was analyzed in the elution product resulting in an increase of L(+) isomer concentration. The resin did not alter its binding capacity after 23 cycles.     

Fluidized bed adsorption of cephalosporin C.

Fluidized bed adsorption can substantially simplify the recovery of products from fermentation. There are, however, several critical parameters, which have a significant influence on the performance of such systems. This paper presents experimental results on the adsorption of an antibiotic, Cephalosporin C, on macroporous adsorbents of the polystyrene type and on an ion exchanger. Internals (static mixers) were used to control bed expansion and mixing, the range of flow rates could thus be extended significantly. An integrated mathematical model was developed comprising bed expansion, residence time distribution and mixing, adsorption kinetics and equilibria.     

Fluidized bed adsorption for whole broth extraction.

Fluidized bed adsorption using a high-density synthetic resin has proven to be an invaluable technique for separating novel compounds from unfiltered fermentation broths during the very early stages of fermentation development, where product concentrations are typically in the parts per million range. Previous initial downstream processing strategies consisted of cell separation from whole broth or direct extraction with water-immiscible solvents, both of which resulted in lengthy time cycles, conflicts with existing operations requiring the use of high-cost centrifugal separators, and environmental/solvent recovery concerns. Laboratory and subsequent pilot plant process development work along with concomitant improvements in yield, quality, and time cycles are presented for one of several fluidized bed processes piloted in Merck's Natural Product Isolation facility.     

Variation of parameters affecting the start-up of methanogenic fluidized bed reactors

Summary The influence of the medium composition, the inoculum and the inoculation procedure on initial biofilm development in methanogenic fluidized bed reactors was studied on laboratory scale. Trace minerals but not vitamins were found to be essential for biofilm development. Inoculation with heterogeneous bacterial cultures of potentially sand-colonizing microorganisms and/or with pure cultures ofMethanothrix soehngenii did not accelerate biofilm development significantly as compared to inoculation with effluent from a fully operative fluidized bed reactor.     

三相流化床中固定化米根霉萃取发酵生产L-乳酸

以TRPO／磺化煤油为萃取剂,在2L三相流床反应器中进行了固定化米根霉原位萃取和异位萃取发酵生产L-乳酸的实验,结果表明,发酵液中的pH值能被控制在3．5左右．产酸速率高达每小时．每1L固定化颗粒产生11gL-乳酸。提出了一个数学模型用以描述萃取发酵中L-乳酸的积累及在各相的分配情况。模型计算曲线与实验值符合良好。     

Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines

A BHK 21 cell line expressing a recombinant antibody was grown in a fixed bed reactor (FBR) system using a porous support made of Siran glass beads. The contribution of five process variables (bead and inoculum sizes; circulation and dilution rates; glutamine concentration of the feed) to the productivity of the process (defined as production rate, effluent product concentration or yield of product on medium supplied) was investigated using a partial factorial experimental design. Individually, none of the variables tested had a significant affect upon productivity. The combination of smaller bead and inoculum sizes, higher circulation and dilution rates, plus higher feed glutamine concentration gave a markedly higher productivity than any other combination of variable levels tested. This combination of variable levels suggested that better results shold be obtained using a fluidised bed reactor system. However, comparison of the productivities of the two systems showed that the FBR gave the better results. This result can be explained in terms of the relationship of Qs_rAb to .Abbreviations C concentration - D dilution rate - FBR fixed bed bioreactor - FIBR fluidised bed bioreactor - Gln glutamine - Qs cell specific rate - Qv volumetric rate - rAb recombinant antibody - X_v viable cell density - specific growth rate     

Exploring the linkage between cell culture process parameters and downstream processing utilizing a plackett‐burman design for a model monoclonal antibody

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett‐Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow‐up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163–170, 2017     

Reverse micellar extraction for downstream processing of lipase: Effect of various parameters on extraction

Reverse micellar extraction of lipase using cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated. The effect of various process parameters on both forward and backward extraction of lipase from crude extract was studied to optimize its yield and purity. Forward extraction of lipase was found to be maximum using Tris buffer at pH 9.0 containing 0.10 M NaCl in aqueous phase and 0.20 M CTAB in organic phase consisting of isooctane, butanol and hexanol. In case of backward extraction, lipase was extracted from the organic phase to a fresh aqueous phase in 0.05 M potassium phosphate buffer (pH 7.0) containing 1.0 M KCl. The activity recovery, extraction efficiency and purification factor of lipase were found to be 82.72%, 40.27% and 4.09-fold, respectively. The studies also indicated that the organic phase recovered after back extraction could be reused for the extraction of lipase from crude extract.     

State-of-the-art in downstream processing of monoclonal antibodies: Process trends in design and validation

Monoclonal antibodies (mAbs) are the most important family of biopharmaceutical compounds in terms of market share. At present, 30 mAbs have been approved and are now commercialized for therapeutic purposes. mAbs are typically produced by mammalian cell culture in bioreactors that range in scale of 1–20 m³. Regardless of scale, from laboratory to commercial settings, the recovery and purification of mAbs present important challenges. Depending on the scale, the particular product, and the type of production process (bioreactor operation, process time, complexity of the culture media, cell density, etc.), many possible downstream configurations are possible and have been used. In this contribution, we review each type of unit operation that forms a downstream train for mAb production. We provide information regarding typical operation settings and critical variables for centrifugation, ultrafiltration, affinity chromatography, ion exchange chromatography, and viral removal operations. In addition, we discuss some important considerations required for the formulation of drugs based on mAbs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 899–916, 2012     

A novel high-pressure liquid-liquid extraction process for downstream processing in biotechnology: extraction of cardiac glycosides

This investigation examines phase equilibrium phenomena that can be used to create two water-like solvents for liquid-liquid extraction in downstream processing in biotechnology: a completely miscible, binary liquid mixture of water and a hydrophilic organic solvent (e. g., an alcohol) reveals a liquid phase split, when it is pressurized with a "near-critical" gas (i.e., a substance which at ambient conditions is a gas, near its critical temperature). This phase split results in two hydrophilic liquid phases. Making use of this phenomenon in process development first requires research on the phase split phenomenon and, second, research on the feasibility of biomolecule extraction and separation. In this study, basic fluid phase equilibrium phenomena are briefly described. Then, experimental results are reported for the partitioning of small amounts of cardiac glycosides (digitoxin and digoxin) on coexisting liquid phases in the high-pressure, three-phase, vapor-liquid-liquid equilibrium of the ternary system of "near critical" CO(2) + water + 1-propanol, at 313 K and 333 K. Finally, a process for extraction and separation of the aforementioned glycosides by means of the high-pressure phase equilibrium phenomenon is discussed.     

CE-UV/VIS and CE-MS for monitoring organic impurities during the downstream processing of fermentative-produced lactic acid from second-generation renewable feedstocks

Background

During the downstream process of bio-based bulk chemicals, organic impurities, mostly residues from the fermentation process, must be separated to obtain a pure and ready-to-market chemical. In this study, capillary electrophoresis was investigated for the non-targeting downstream process monitoring of organic impurities and simultaneous quantitative detection of lactic acid during the purification process of fermentatively produced lactic acid. The downstream process incorporated 11 separation units, ranging from filtration, adsorption and ion exchange to electrodialysis and distillation, and 15 different second-generation renewable feedstocks were processed into lactic acid. The identification of organic impurities was established through spiking and the utilization of an advanced capillary electrophoresis mass spectrometry system.

Results

A total of 53 % of the organic impurities were efficiently removed via bipolar electrodialysis; however, one impurity, pyroglutamic acid, was recalcitrant to separation. It was demonstrated that the presence of pyroglutamic acid disrupts the polymerization of lactic acid into poly lactic acid. Pyroglutamic acid was present in all lactic acid solutions, independent of the type of renewable resource or the bacterium applied. Pyroglutamic acid, also known as 5-oxoproline, is a metabolite in the glutathione cycle, which is present in all living microorganisms. pyroglutamic acid is found in many proteins, and during intracellular protein metabolism, N-terminal glutamic acid and glutamine residues can spontaneously cyclize to become pyroglutamic acid. Hence, the concentration of pyroglutamic acid in the lactic acid solution can only be limited to a certain amount.

Conclusions

The present study proved the capillary electrophoresis system to be an important tool for downstream process monitoring. The high product concentration encountered in biological production processes did not hinder the capillary electrophoresis from separating and detecting organic impurities, even at minor concentrations. The coupling of the capillary electrophoresis with a mass spectrometry system allowed for the straightforward identification of the remaining critical impurity, pyroglutamic acid. Although 11 separation units were applied during the downstream process, the pyroglutamic acid concentration remained at 12,900 ppm, which was comparatively high. All organic impurities found were tracked by the capillary electrophoresis, allowing for further separation optimization.

     

Enzyme stability in downstream processing. Part 2: quantification of inactivation

In biotechnological recovery processes the instability of the product can lead to large losses in the sequence of recovery processes needed to purify the product. As the cost of the final active product is strongly dependent on the recovery yield, this will lead to an increase in product cost. Therefore knowledge of factors that influence stability is important. This Part 2 provides the basic principles for design and operation of processes in which inactivation takes place. Simple kinetics and reactor modelling are discussed. These are applied to a number of unit operations: cell disruption, membrane filtration, drying and reversed micellar extraction. It is thus shown that the basic tools for modeling of biochemical processes provide us with the data needed for optimal process design and operation.     

Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences.

In a previous report, we described the presence, in pituitary tissue, of an alternatively processed species of bovine growth hormone mRNA from which the last intron (intron D) has not been removed by splicing (R. K. Hampson and F. M. Rottman, Proc. Natl. Acad. Sci. USA 84:2673-2677, 1987). Using transient expression of the bovine growth hormone gene in Cos I cells, we observed that splicing of intron D was affected by sequences within the downstream exon (exon 5). Deletion of a 115-base-pair FspI-PvuII restriction fragment in exon 5 beginning 73 base pairs downstream of the intron 4-exon 5 junction resulted in cytoplasmic bovine growth hormone mRNA, more than 95% of which retained intron D. This contrasted with less than 5% of the growth hormone mRNA retaining intron D observed with expression of the unaltered gene. Insertion of a 10-base-pair inverted repeat sequence, CTTCCGGAAG, which was located in the middle of this deleted segment, partially reversed this pattern, resulting in cytosolic mRNA from which intron D was predominantly removed. More detailed deletion analysis of this region indicated that multiple sequence elements within the exon 5, in addition to the 10-base-pair inverted repeat sequence, are capable of influencing splicing of intron D. The effect of these exon sequences on splicing of bovine growth hormone precursor mRNA appeared to be specific for the growth hormone intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. The results of this study suggest a unique interaction between sequences located near the center of exon 5 and splicing of the adjacent intron D.     

Optimization of production and downstream processing of the almond beta-glucosidase-mediated glucosylation of glycerol.

This article describes the synthesis of glyceryl glucoside from glycerol and glucose with almond beta-glucosidase as the catalyst. A yield of 54% (0.45 mmol/g) was obtained. The influence of the enzyme stability, the water concentration, and the water activity on the glucoside yield were determined. A molar fraction-based equilibrium constant of 2.4 +/- 0.6 was found, with which the glucoside yield could be calculated for all possible combinations of initial substrate and water fractions in the reaction mixture. A model was used to optimize the glucoside yield while minimizing one of the substrate concentrations at equilibrium. This straightforward model gives a good prediction of the measured glucoside yield, according to a parity plot.     

Modeling retrovirus production for gene therapy. 2. Integrated optimization of bioreaction and downstream processing

In this work a model envisaging the integrated optimization of bioreaction and downstream processing is presented. This model extends the work presented in part 1 of this pair of papers by adding ultrafiltration to process optimization. The new operational parameters include ultrafiltration time, pressure, and stirring rate. For global optimization, the model uses as constraints the final product titer and quality to be achieved after downstream processing. This extended model was validated with the same system used in part 1, i.e., PA317 cells producing a recombinant retrovirus containing the LacZ gene as a marker in stirred tanks using porous supports. Optimization of the extended model led to the conclusion that bioreaction should have two steps, batch and perfusion, similar to what was found in part 1. Ultrafiltration in a stirred cell should be performed at low pressures and stirring rates to reduce the losses of infective retroviruses. Sensitivity analysis performed on the results of the integrated optimization showed that under optimal conditions the productivity is less sensitive to the parameters related to ultrafiltration than to those associated with bioreaction. These results were interpreted as reflecting the high yield of ultrafiltration (90%). The relevance of the model extension to perform integrated optimization was also demonstrated since a restriction in the specific ultrafiltration area in downstream processing conditioned perfusion duration and perfusion rate in bioreaction. This clearly indicates that overall process optimization cannot be achieved without integrated optimization.     

A novel design of simulated moving bed (SMB) chromatography for separation of ketoprofen enantiomer

A simulated moving bed (SMB) chromatography system is a powerful tool for preparative scale separation, which can be applied to the separation of chiral compound. We have designed our own lab-scale SMB chromatography using 5 HPLC pumps, 6 stainless steel columns and 4 multi-position valves, to separate a racemic mixture of ketoprofen in to its enantiomers. Our design has the characteristics of the low cost for assembly for the SMB chromatography and easy repair of the unit, which differs from the designs suggested by other investigators. It is possible for the flow path through each column to be independently changed by computer control, using 4 multi-position rotary valves and 5 HPLC solvent delivery pumps. In order to prove the operability of our SMB system, attempts were made to separate the (S)-ketoprofen enantiomer from a ketoprofen racemic mixture. The operating parameters of the SMB chromatography were calculated for ketoprofen separation from a batch chromatography experiment as well as by the triangle theory. With a feed concentration of 1 mg/mL, (S)-ketoprofen was obtained with a purity of 96% under the calculated operating conditions.