Scaling-up vaccine production: implementation aspects of a biomass growth observer and controller

This study considers two aspects of the implementation of a biomass growth observer and specific growth rate controller in scale-up from small- to pilot-scale bioreactors towards a feasible bulk production process for whole-cell vaccine against whooping cough. The first is the calculation of the oxygen uptake rate, the starting point for online monitoring and control of biomass growth, taking into account the dynamics in the gas-phase. Mixing effects and delays are caused by amongst others the headspace and tubing to the analyzer. These gas phase dynamics are modelled using knowledge of the system in order to reconstruct oxygen consumption. The second aspect is to evaluate performance of the monitoring and control system with the required modifications of the oxygen consumption calculation on pilot-scale. In pilot-scale fed-batch cultivation good monitoring and control performance is obtained enabling a doubled concentration of bulk vaccine compared to standard batch production.     

Maternal adverse childhood experiences,child mental health,and the mediating effect of maternal depression: A cross-sectional,population-based study in rural,southwestern Uganda

Objectives

This study aimed to examine the intergenerational effects of maternal adverse childhood experiences (ACEs) and child mental health outcomes in rural Uganda, as well as the potentially mediating role of maternal depression in this pathway. Additionally, we sought to test the extent to which maternal social group membership attenuated the mediating effect of maternal depression on child mental health.

Methods

Data come from a population-based cohort of families living in the Nyakabare Parish, a rural district in southwestern Uganda. Between 2016 and 2018, mothers completed surveys about childhood adversity, depressive symptoms, social group membership, and their children's mental health. Survey data were analyzed using causal mediation and moderated-mediation analysis.

Results

Among 218 mother–child pairs, 61 mothers (28%) and 47 children (22%) showed symptoms meeting cutoffs for clinically significant psychological distress. In multivariable linear regression models, maternal ACEs had a statistically significant association with severity of child conduct problems, peer problems, and total child difficulty scores. Maternal depression mediated the relationship between maternal ACEs and conduct problems, peer problems, and total difficulty, but this mediating effect was not moderated by maternal group membership.

Conclusions

Maternal depression may act as a potential mechanism linking maternal childhood adversity with poor child mental health in the next generation. Within a context of elevated rates of psychiatric morbidity, high prevalence of childhood adversity, and limited healthcare and economic infrastructures across Uganda, these results emphasize the prioritization of social services and mental health resources for rural Ugandan families.     

Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris GS115

A recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype E (BoNT/E) is proposed as a vaccine against the serotype E neurotoxin. This fragment, rBoNTE(Hc), was produced intracellular in Pichia pastoris GS115 by a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The rBoNTE(Hc) protein was purified from the soluble fraction of cell lysates using three ion-exchange chromatography steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography step (MEP HyperCel). Method development at the bench scale was achieved using 7-380 mL columns and the process was performed at the pilot scale using 0.5-3.1 L columns in preparation for technology transfer to cGMP manufacturing. The purification process resulted in greater than 98% pure rBoNTE(Hc) based on HPLC and yielded up to 1.01g of rBoNTE(Hc)/kg cells at the bench scale and 580mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the purified rBoNTE(Hc) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD50's of BoNT/E.     

Scale-up fermentation of recombinant <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using the GAP promoter

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml⁻¹ and a cell wet weight of ca. 500 g l⁻¹ at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.     

Investigation of the effect of different extracellular factors on the lipase production by <Emphasis Type="Italic">Yarrowia lipolityca</Emphasis> on the basis of a scale-down approach

The influence of three extracellular factors (namely, the methyl oleate dispersion in the broth, the dissolved oxygen variations, and the pH fluctuation) on the lipase production by Y. lipolytica in batch bioreactor has been investigated in different scale-down apparatus. These systems allow to reproduce the hydrodynamic phenomena encountered in large-scale equipments for the three specified factors. The effects of the extracellular factors have been observed at three distinct levels: the microbial growth, the extracellular lipase production, and the induction of the gene LIP2 encoding for the main lipase of Y. lipolytica. Among the set of environmental factors investigated, the dissolved oxygen fluctuations generated in a controlled scale-down reactor (C-SDR) have led to the more pronounced physiological effect by decreasing the LIP2 gene expression level. The other environmental factors observed in a partitioned scale-down reactor, i.e., the methyl oleate dispersion and the pH fluctuations, have led to a less severe stress traduced only by a decrease of the microbial yield and thus of the extracellular lipase specific production rate.     

Purification of a synthetic oligonucleotide by anion exchange chromatography: method optimisation and scale-up

A single-step chromatographic method for purification of a synthetic 20-mer oligonucleotide is described. Method optimisation was conducted at laboratory scale where 30 mg crude sample was purified per run with a yield of 17 mg pure oligonucleotide. The protocol was scaled-up in steps to achieve 5-, 58- and a final 230-fold scale-up. At the final scale, 7.0 g of crude material was purified with a yield of 4.1 g product. The purity of the oligonucleotide was in all scales higher than 97%. The cycle time was 110 min, which corresponds to a purification capacity of about 90 g crude oligonucleotide material per 24 h.     

Combined sulfite method for the measurement of the oxygen transfer coefficient k(L)a in bio-reactors

The combined sulfite method is proposed for the measurement of oxygen transfer coefficients, k_La, in bioreactors. The method consists of a steady-state and a dynamic measurement which are carried out under the same experimental conditions and thus yield data for both methods during one experiment. The applied experimental conditions are shown to avoid chemical enhancement during the steady-state measurement. Moreover, no parallel sulfite oxidation occurs during the oxygen saturation phase of the dynamic measurement. Under the applied experimental conditions, no information about the sulfite oxidation kinetics is required and possible metal ion impurities in sulfite salts do not influence the measurement. The characterization of a laboratory-scale bioreactor aerated with pure oxygen yields k_La values during the steady-state and the dynamic measurements that are in good agreement with the dynamic pressure method, the correctness of which is generally accepted. When air is used for absorption, the steady-state measurement yields k_La values that correlate to the correct variant of the standard dynamic method. The dynamic measurement with air absorption yields a k_La value which considers the influence of the non-uniform bubble size distribution present in bubble-aerated bioreactors.     

Design of static mixers for inclined tubular photobioreactors

Static mixers, which improve gas-liquid mass transfer inside tubular photobioreactors and move the cells between the upper and lower parts of the tubes were designed. Each static mixer was equipped with an opening (v-cut slit) at the top (for gas dispersion) and an orifice at the lower part (for liquid circulation). When the static mixers were installed in the riser tube of an inclined tubular photobioreactor, vertical movement of the liquid was induced so that cells were moved between the surface and bottom part of the photobioreactor. The mass transfer rates in the tubular photobioreactor without static mixers decreased sharply when they were scaled up by increasing the tube diameters. However, by installation of static mixers, the mass transfer rates in 12.5-cm diameter tubes were almost as high as those of 3.8-cm tubes without static mixers. The effectiveness of the static mixers in improving the mass transfer characteristics of the tubular photobioreactors was higher in large than small diameter tubes. The ratio of the diameters of the static mixers to the diameter of the tube, the areas of the v-cut slits and the orifice were the important design parameters that affected the mass transfer characteristics. The gas hold up and and k_La were higher when the areas of the slits (v-cut) and the orifice were reduced.     

国家公园遴选标准的国际经验及对我国的启示

建立以国家公园为主体的自然保护地体系,是我国生态文明思想的重大举措。国家公园作为国际普遍认可的自然保护手段,受到世界各国的欢迎。美国、加拿大、德国、南非、日本等国的国家公园建设较早、发展较好,通过分析其国家公园建设经验得出：（1）国家公园是以自然保护为首要目标,并为公众提供享受大自然的场所;（2）其评价对象与标准主要包括,自然景观/自然遗迹、生态系统、生物多样性/重要栖息地、面积/范围、自然区域/自然环境、文化景观等;（3）根据资源条件和目标定位的差异,国外国家公园的选建模式主要有基于生态区划的国家公园建设模式、基于社区共建的国家公园建设模式和基于资源主导的国家公园建设模式。在国际经验研究和分析的基础上,结合我国自然资源状况及国家公园建设需求,提出我国国家公园发展建议：（1）明确国家公园的功能定位和发展目标;（2）制定国家公园评价准则和方法,建立以国家代表性、完整性、原真性为主,包括生态区位重要性、历史文化价值等因素的国家公园评价指标体系;（3）划定面向国家公园规划布局的生态地理分区,在区域内建立国家公园,并与生态系统优先区、生物多样性保护关键区,或代表性自然景观格局相协调。     

Optimisation and scale-up of α-glucuronidase production by recombinant Saccharomyces cerevisiae in aerobic fed-batch culture with constant growth rate

α-Glucuronidase (EC 3.2.1.139) of family GH 115 from Scheffersomyces stipitis is a valuable enzyme for the modification of water-soluble xylan into insoluble biopolymers, due to its unique ability to act on polymeric xylans. The influence of growth rate on the production of α-glucuronidase by recombinant Saccharomyces cerevisiae MH1000pbk10D-glu in glucose-limited fed-batch culture was studied at 14 and 100 L scale. At and below the critical specific growth rate (μ_crit) of 0.12 h⁻¹ at 14 L scale, the biomass yield coefficient (Y_x/s) remained constant at 0.4 g g⁻¹ with no ethanol production, whereas ethanol yields relative to biomass (k_eth/x) of up to 0.54 g g⁻¹ and a steady decrease in Y_x/s were observed at μ > 0.12 h⁻¹. Production of α-glucuronidase was growth associated at a product yield (k_α-glu/x) of 0.45 mg g⁻¹, with the highest biomass (37.35 g/L) and α-glucuronidase (14.03 mg/L) concentrations, were recorded during fed-batch culture at or near to μ_crit. Scale-up with constant k_La from 14 to 100 L resulted in ethanol concentrations of up to 2.5 g/L at μ = 0.12 h⁻¹. At this scale, α-glucuronidase yield could be maximised at growth rates below μ_crit, to prevent localised high glucose concentration pockets at the feed entry zone that would induce oxido-reductive metabolism. This is the first report where recombinant production of α-glucuronidase (EC 3.2.1.139) by S. cerevisiae was optimised for application at pilot scale.