Leveraging single‐pass tangential flow filtration to enable decoupling of upstream and downstream monoclonal antibody processing

Decoupling upstream and downstream operations in biopharmaceutical production could enable more flexible manufacturing operations and could allow companies to leverage strategic or financial benefits that would be otherwise unattainable. A decoupling process was developed and scaled up utilizing single‐pass tangential flow filtration for volume reduction, followed by bulk freezing in single‐use bags prior to purification. Single‐pass tangential flow filtration can be used to continuously concentrate harvested cell culture fluid, reducing the volume by 15‐25× with a step yield of >96%. These concentration factors were reproduced with a second product, indicating that the process could be amenable to platform processes. Experimental data indicate that the product tested was stable for at least one year at ?40 or ?70°C. The concentration of the harvested cell culture fluid—either with or without a subsequent period of frozen storage—had no impact on the product quality attributes that were tested. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:405–411, 2018     

A low volume 3D-printed temperature-controllable cuvette for UV visible spectroscopy

We report the fabrication of a 3D-printed water-heated cuvette that fits into a standard UV visible spectrophotometer. Full 3D-printable designs are provided and 3D-printing conditions have been optimised to provide options to print the cuvette in either acrylonitrile butadiene styrene or polylactic acid polymers, extending the range of solvents that are compatible with the design. We demonstrate the efficacy of the cuvette by determining the critical micelle concentration of sodium dodecyl sulphate at 40 °C, the molar extinction coefficients of cobalt nitrate and dsDNA and by reproducing the thermochromic UV visible spectrum of a mixture of cobalt chloride, water and propan-2-ol.     

青少年运动员途中跑足底连续三维力的研究

目的测量青少年运动员途中跑阶段足底连续三维力,以揭示不同水平青少年运动员途中跑阶段三维力特征。方法选取10名身体健康高中男性运动员作为受试者,应用作者研发的新型足底三维测力跑鞋,测试与记录每位受试者100m短跑途中跑阶段的足底连续三维力,并加以分析。结果青少年运动员在冲击时相时Fx分量有明显的峰值,其中优秀组运动员的Fx第一负波峰值均值小于对照组;Fy分量是客观存在的,优秀组运动员Fy的标准差较对照组要小,并且优秀组运动员的Fy分量的连续波动更加平稳;Fy分量与Fz分量具有相同的变化趋势。结论结果表明,前撑阶段Fx第一负波峰值对运动员向前有阻力的作用;Fy分量是客观存在的且与Fz分量具有相同的变化趋势;Fy分量的大幅波动影响运动员的身体平稳性,不利于运动员大腿充分发力。     

Single pass tangential flow filtration to debottleneck downstream processing for therapeutic antibody production

As the therapeutic monoclonal antibody (mAb) market continues to grow, optimizing production processes is becoming more critical in improving efficiencies and reducing cost-of-goods in large-scale production. With the recent trends of increasing cell culture titers from upstream process improvements, downstream capacity has become the bottleneck in many existing manufacturing facilities. Single Pass Tangential Flow Filtration (SPTFF) is an emerging technology, which is potentially useful in debottlenecking downstream capacity, especially when the pool tank size is a limiting factor. It can be integrated as part of an existing purification process, after a column chromatography step or a filtration step, without introducing a new unit operation. In this study, SPTFF technology was systematically evaluated for reducing process intermediate volumes from 2× to 10× with multiple mAbs and the impact of SPTFF on product quality, and process yield was analyzed. Finally, the potential fit into the typical 3-column industry platform antibody purification process and its implementation in a commercial scale manufacturing facility were also evaluated. Our data indicate that using SPTFF to concentrate protein pools is a simple, flexible, and robust operation, which can be implemented at various scales to improve antibody purification process capacity.     

Continuous, high level production and excretion of a plasmid-encoded protein by Escherichia coli in a two-stage chemostat

The stable continuous overproduction of a plasmidencoded protein, beta-lactamase, for at least 50 days by Escherichia coli K-12, RB791(pKN), with release into the culture medium has been demonstrated in two-stage chemostats. The second-stage culture was continuously induced with 0.1 mM IPTG. Continuous expression of beta-lactamase could not be sustained with this strain in a single-stage chemostat because of cell death and selection for lac(-1) cells. beta-Lactamase production in the second stage was sensitive to the second-stage dilution rate and the distribution of the limiting substrate (i.e., glucose) between the first and second stages. The fraction of viable, excreting cells and the average copy number in the induced culture was measurably higher under those conditions of dilution rate and substrate distribution which yielded high beta-lactamase levels. The best operating conditions found at 20 degrees C were a first-stage dilution rate of 0.12 h(-1), a second-stage dilution rate of 0.03 h(-1), and equal glucose feed supplied to each stage. Enzymatically active beta-lactamase was produced at a level of 25% of total cellular protein with 90% excretion yielding 300 mg beta-lactamase/L that was 50% pure at an OD(600) < 6. (c) 1993 Wiley & Sons, Inc.     

Economic improvement of continuous pharmaceutical production via the optimal control of a multifeed bioreactor

Projections on the profitability of the pharmaceutical industry predict a large amount of growth in the coming years. Stagnation over the last 20 years in product development has led to the search for new processing methods to improve profitability by reducing operating costs or improving process productivity. This work proposes a novel multifeed bioreactor system composed of independently controlled feeds for substrate(s) and media used that allows for the free manipulation of the bioreactor supply rate and substrate concentrations to maximize bioreactor productivity and substrate utilization while reducing operating costs. The optimal operation of the multiple feeds is determined a priori as the solution of a dynamic optimization problem using the kinetic models describing the time‐variant bioreactor concentrations as constraints. This new bioreactor paradigm is exemplified through the intracellular production of beta‐carotene using a three feed bioreactor consisting of separate glucose, ethanol and media feeds. The performance of a traditional bioreator with a single substrate feed is compared to that of a bioreactor with multiple feeds using glucose and/or ethanol as substrate options. Results show up to a 30% reduction in the productivity with the addition of multiple feeds, though all three systems show an improvement in productivity when compared to batch production. Additionally, the breakeven selling price of beta‐carotene is shown to decrease by at least 30% for the multifeed bioreactor when compared to the single feed counterpart, demonstrating the ability of the multifeed reactor to reduce operating costs in bioreactor systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:902–912, 2017     

拟茎点霉B3与有机肥配施对连作草莓生长的影响

通过盆栽试验探讨了内生拟茎点霉B3与有机肥配施对连作草莓土壤的改善及对草莓生长的影响。试验共设5个处理,分别为对照(CK)、施有机肥与灭菌固体培养基(A)、施有机肥与内生拟茎点霉B3固体菌种(B)、施有机肥与绿色木霉、黑曲霉、枯草芽孢杆菌固体菌种(C)、施加有机肥与绿色木霉、黑曲霉、枯草芽孢杆菌和内生拟茎点霉B3固体菌种(D)。结果表明:A、B、C和D处理平均单果鲜重分别为对照(CK)的1.1、1.4、0.9和1.1倍。B处理比对照增产19.7%,A处理增产8.2%,C和D处理产量均比CK低。B处理草莓生长最好,植株株高及叶面积均值比其它4个处理大。发病率及病情指数结果表明B处理抗病效果最明显,推断内生拟茎点霉B3可以用作生防菌剂。进一步的研究表明土壤真菌和细菌数量在整个生育期先上升后下降,在花期达到最大。成熟期A、B、C、D处理的土壤放线菌数量分别比CK增加7.2%、160.3%、124.5%及82.6%。在花期,B处理及D处理蔗糖酶酶活达到最大,其中A、B、C及D处理的蔗糖酶酶活分别比CK高11.1%、69.4%、50.3%及77.2%。B处理整个生育期都保持较高的土壤蔗糖酶活性。花期是草莓生长的关键期,需氮量较高。A、B、C及D处理脲酶酶活分别比CK处理高250.0%、700.0%、250.0%及175.0%,B处理花期土壤脲酶酶活性显著高于其它4个处理,促进了有机氮向速效氮的转化。花期A、C处理磷酸酶酶活比对照低67.0%、46.7%,B、D处理比对照高122.5%,227.5%。B处理在整个生育期都有较高的土壤磷酸酶酶活, D处理组在花期土壤磷酸酶酶活较高。可见含内生拟茎点霉B3菌的B及D处理组能增加土壤磷酸酶酶活。B处理在苗期和花期土壤纤维素酶活较低,而结果期和成熟期较高。说明内生拟茎点霉B3菌剂与有机肥配施可以改善连作草莓土壤微生物区系,提高土壤酶活性,增强草莓抗病能力,增加草莓产量,是一种有效缓解草莓连作障碍的方法。     

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration

Aims: The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla_TEM‐52‐carrying plasmid (lactose‐negative mutant of B1‐54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. Methods and Results: Fresh faeces from a healthy volunteer, negative for cephalosporin‐resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed‐field gel electrophoresis profiles (PFGE). The avian extended‐spectrum β‐lactamase‐positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1‐54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla_TEM‐52‐carrying plasmid. Conclusions: These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. Significance and Impact of the Study: The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.     

A direct continuous spectrophotometric assay for glycosidases with 3-nitro-2-pyridyl glycosides by tautomerization of 2-hydroxy-3-nitropyridine

Two kinds of 3-nitro-2-pyridyl glycosides were synthesized and evaluated as substrates for continuous spectrophotometric assay for glycosidases. The liberated aglycon, 2-hydroxy-3-nitropyridine, immediately tautomerized to 3-nitro-2(1H)-pyridone, causing an absorption shift of ca. 60 nm even under acidic conditions (pH 3-6). Consequently, the enzymatic hydrolysis of these glycosides was monitored continuously in the acidic to neutral pH range (pH 4-7), the optimum pH for most glycosidases. The absorbance of liberated aglycon increased linearly at 390 nm until 10% consumption of the substrate to enable the initial rate to be determined at once without terminating the reaction. The kinetic parameters for the hydrolysis of 3-nitro-2-pyridyl glycosides were obtained from the slopes of the progress curves and were compared with those obtained from the conventional discontinuous assay using p- and o-nitrophenyl glycosides as substrates. The kinetic parameters indicated that 3-nitro-2-pyridyl glycosides were more activated and specific substrates, but with less affinity to the enzymes than the corresponding nitrophenyl glycosides. Moreover, the absorbance shift by tautomerization should promise further applications to continuous spectrophotometric assays for other enzymes acting under acidic conditions, such as acid proteases and acid phosphatases.     

Growth kinetics of a bacteriophage in continuous culture

Lytic coliphage Qbeta was grown in continuously cultured host bacteria using a cascade of stirred flow reactors. The apparatus was constructed so that the steady stream of exponentially growing bacterial cells passing through the stirred flow reactors served to prevent coevolution brought about by host-parasite interactions. Wall growth was the primary cause for deviation from ideal continuous culture conditions and is largely dependent on the surface structure of the host bacteria. Using an Escherichia coli strain deficient in adhesive type I pili expression, the desynchronization of single burst events could easily be followed over the course of four infection latency periods. Computer simulations based on a two-stage model for the Qbeta infection cycle were in perfect agreement with the experimental data. Applications of the optimized system to strategies of molecular evolution are discussed. (c) 1996 John Wiley & Sons, Inc.