Technology outlook for real-time quality attribute and process parameter monitoring in biopharmaceutical development-A review.

Real-time monitoring of bioprocesses by the integration of analytics at critical unit operations is one of the paramount necessities for quality by design manufacturing and real-time release (RTR) of biopharmaceuticals. A well-defined process analytical technology (PAT) roadmap enables the monitoring of critical process parameters and quality attributes at appropriate unit operations to develop an analytical paradigm that is capable of providing real-time data. We believe a comprehensive PAT roadmap should entail not only integration of analytical tools into the bioprocess but also should address automated-data piping, analysis, aggregation, visualization, and smart utility of data for advanced-data analytics such as machine and deep learning for holistic process understanding. In this review, we discuss a broad spectrum of PAT technologies spanning from vibrational spectroscopy, multivariate data analysis, multiattribute chromatography, mass spectrometry, sensors, and automated-sampling technologies. We also provide insights, based on our experience in clinical and commercial manufacturing, into data automation, data visualization, and smart utility of data for advanced-analytics in PAT. This review is catered for a broad audience, including those new to the field to those well versed in applying these technologies. The article is also intended to give some insight into the strategies we have undertaken to implement PAT tools in biologics process development with the vision of realizing RTR testing in biomanufacturing and to meet regulatory expectations.

Engineering a Thermostable Keto Acid Decarboxylase Using Directed Evolution and Computationally Directed Protein Design.

Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form our best variant, LLM4. Compared to wild-type Kivd, a 13 °C increase in melting temperature and over 4-fold increase in half-life at 60 °C were observed. LLM4 will be useful for keto acid derived alcohol production in lignocellulosic thermophiles.

Fuelling the future: microbial engineering for the production of sustainable biofuels.

Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.

The Expression of Plasma Membrane Ca2 + -ATPase Isoforms 1~4 and the Splice Variants at Sites A and C in the Neonatal Rat Vestibular Organ / 听力学及言语疾病杂志

Objective To study the expression of plasma membrane Ca2 + -ATPase isoforms 1 -4 and the splice variants at sites A and C in the neonatal rat vestibular organ.Methods Ten rats at postnatal 2 days (P2 ) were decapitated and their vestibular organs (macula utriculi and macula sacculi)were isolated.The total proteins of the vestibular organs were extracted.The expression of PMCA1-4 splice variants at sites A and C was detected by RT-PCR.Results The splice variants of PMCA1-4 at sites A and C in macula utriculi and macula sacculi of neo-natal rat vestibular organs were PMCA1x/b,PMCA2w/(a,b),PMCA3z/(a,b,c)and PMCA4 (x,z)/b.Conclusion The splice variants at sites A and C among PMCA1,PMCA2,PMCA3 and PMCA4 were different in the vestibu-lar organs of neonatal rats,which could be explained that macula utriculi and macula sacculi had different require-ments of Ca2 + turning for these PMCA isoforms.

The Expression of Plasma Membrane Ca2 + -ATPase Isoforms 1 ~3in the Basi lar Membrane of Neonatal Rat Cochlea / 听力学及言语疾病杂志

Objective To study the expression of plasma membrane Ca2+-ATPase isoforms 1~3 (PMCA 1~3 )in the basilar membrane (BM)of the neonatal rat cochlea by Western blot.The PMCA2 content in single BM of the neonatal rat was also examined.Methods Four rats at postnatal 2 days (P2)and 8 days (P8)were respective-ly decapitated and their BMs were isolated.The total proteins of BMs were extracted.The 20μg total proteins were respectively loaded to the gel.The expression of PMCA1-3 was detected by Western blot.Likewise,3μg total proteins from P2 and P8 rat BM were loaded.The expression of PMCA2 was detected by Western blot.Four rats at P8 were decapitated and their BM was isolated.The 5μg,10μg and 20μg total proteins of P8 rat BM were added to the gel and 100 ng,400 ng and 800 ng bovine serum albumin (BSA)were also loaded as reference.After electro-phoresis,the gel was separated into two parts.One part was used for SYPRO staining and the other part was used for PMCA2 detection by Western blot.Results In the 20μg BM total proteins of P2 and P8 rats,the expression of PMCA1 was weak (0.126±0.024,0.131±0.012,respectively),PMCA2 was strong (4.16±0.528,4.25±0.319, respectively),and PMCA3 was barely expressed (0 ).There was a statistical difference among PMCA1 ,PMCA2 and PMCA3(P<0.05).In the 3μg BM total proteins of P2 and P8 rats,the expression of PMCA2 in P8 (4.571± 0.336)was higher than P2 (3.622±0.285).There was a statistical difference(P<0.05).The PMCA2 content in the BM of a P8 rat was about 2 .5 ng.Conclusion There was a different-level expression of PMCA1~3 in the neonatal rat BM with highest expression of PMCA2 ,which could be explained that cochlear hair cells had different requirements of Ca2+ turning for these PMCA isoforms.

Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum.

Consolidated bioprocessing (CBP) has the potential to reduce biofuel or biochemical production costs by processing cellulose hydrolysis and fermentation simultaneously without the addition of pre-manufactured cellulases. In particular, Clostridium thermocellum is a promising thermophilic CBP host because of its high cellulose decomposition rate. Here we report the engineering of C. thermocellum to produce isobutanol. Metabolic engineering for isobutanol production in C. thermocellum is hampered by enzyme toxicity during cloning, time-consuming pathway engineering procedures, and slow turnaround in production tests. In this work, we first cloned essential isobutanol pathway genes under different promoters to create various plasmid constructs in Escherichia coli. Then, these constructs were transformed and tested in C. thermocellum. Among these engineered strains, the best isobutanol producer was selected and the production conditions were optimized. We confirmed the expression of the overexpressed genes by their mRNA quantities. We also determined that both the native ketoisovalerate oxidoreductase (KOR) and the heterologous ketoisovalerate decarboxylase (KIVD) expressed were responsible for isobutanol production. We further found that the plasmid was integrated into the chromosome by single crossover. The resulting strain was stable without antibiotic selection pressure. This strain produced 5.4 g/L of isobutanol from cellulose in minimal medium at 50(o)C within 75 h, corresponding to 41% of theoretical yield.

Analysis of sites of Toxoplasma gondii SAG2 gene in blood of HIV-positive people in Lincang City,Yunnan Province / 中国血吸虫病防治杂志

Objective To preliminarily understand the genotype characteristics of Toxoplasma gondii in blood of HIV?posi?tive persons in Lincang City,Yunnan Province. Method Two segments of SAG2 gene of T. gondii from blood samples of HIV?positive persons in Lincang City were extracted and amplified by using the nested PCR method and the genotype was identified and compared with the standard strain(Type I)of Toxoplasma gondii. Results Thirty?five SAG2 genes(241 bp)and 35 SAG2 genes(221 bp)of T. gondii were amplified from 170 blood samples of the HIV?positive people,and 4 of each case were selected and digested with enzyme,then 2 aim gene fragments of each case were chosen and compared with the standard strain (Type I)of T. gondii. The digestion of SAG2 gene(241 bp)showed the genotype of the blood samples was Type I or Type II, and the digestion of SAG2 gene(221 bp)confirmed that the genotype was Type I. Conclusion It is preliminarily confirmed that the genotype of T. gondii in blood of HIV?positive persons in Lincang City,Yunnan Province is Type I.

Differences in cationic and anionic charge densities dictate zwitterionic associations and stimuli responses.

Zwitterionic materials have shown their excellent performance in many biological and chemical applications. Zwitterionic materials possess moieties that own both cationic and anionic groups. The associations among zwitterionic moieties through electrostatic interactions play an important role in properties of zwitterionic materials. However, the relationship between the molecular structures and associations of zwitterionic moieties are still not well understood. This work compared thermal- and salt-responsive behaviors of sulfobetaine and carboxybetaine polymers by examining their rheological properties as a function of temperature and their hydrodynamic sizes as a function of salt concentration. Results showed that carboxybetaine polymers do not exhibit stimuli responses as expected from the antipolyelectrolyte behavior of zwitterionic polymers as observed in sulfobetaine polymers. We studied and compared the associations among zwitterionic moieties in these two zwitterionic polymers using molecular dynamic simulations. Simulation results show that the charge-density difference between cationic and anionic groups determines the associations among zwitterionic moieties, which are responsible for different stimuli responses of carboxybetaine and sulfobetaine polymers.

Integrated antimicrobial and nonfouling zwitterionic polymers.

Zwitterionic polymers are generally viewed as a new class of nonfouling materials. Unlike their poly(ethylene glycol) (PEG) counterparts, zwitterionic polymers have a broader chemical diversity and greater freedom for molecular design. In this Minireview, we highlight recent microbiological applications of zwitterionic polymers and their derivatives, with an emphasis on several unique molecular strategies to integrate antimicrobial and nonfouling properties. We will also discuss our insights into the bacterial nonfouling performance of zwitterionic polymers and one example of engineering zwitterionic polymer derivatives for antimicrobial wound-dressing applications.

Comparation of Toxoplasma gondii separated from HIV-positive people and RH strain GRA6 gene / 中国血吸虫病防治杂志

Objective To comparatively analyze Toxoplasma gondii separated from HIV-positive people and RH strain GRA6 gene. Methods By using the nested PCR the amplification of Dali HIV-positive blood samples and RH strains of Toxo-plasma GRA6 genome was performed. The GRA6 gene amplification positive product was selected and the electrophoresis imag-ing was performed by being digested with the Mse I endonuclease and the gene sequences were measured and analyzed. Re-sults The GRA6 gene fragment 800 bp was successfully amplified and about 600 bp and 200 bp bands were got by Mse I. The sequencing results showed that T. gondii GRA6 gene positive samples had 2 nucleotide variation compared with T. gondii strain RH namely 447 base pair at C becoming G and 623 base pair at G becoming T. At 146 bp and 690 bp the Mse I restric-tion sites TTAA were found. Conclusion The preliminary judgment shows that the Dali HIV-positive T. gondii genotype is consistent with RH strain belonging to genotype I.

Study and application of surface antigen in tachyzoites of Toxoplasma gondii / 中国血吸虫病防治杂志

Toxoplasma gondii is an intracellular protozoan parasite that infects all warm?blooded animals. The surface anti?gens of T. gondii with the potential for application as antigens of diagnosis and vaccines have been studied extensively in recent years especially for P43 P35 P30 P23 and P22. The studies on the surface antigen in tachyzoites of T. gondii are reviewed in this paper.

Synchronizing nonfouling and antimicrobial properties in a zwitterionic hydrogel.

In this work, we report a new approach to integrate antimicrobial and nonfouling properties into a single platform without compromising each other. To achieve this, a zwitterionic hydrogel is conjugated with an antimicrobial agent as a leaving group in a way that maintains the zwitterionic form of the hydrogel before, during and after drug release, preventing bacteria surface adhesion and bulk proliferation simultaneously. The antibacterial salicylate anion contributes the negative charge to the initial zwitterionic state and is released through the ester linkage hydrolysis. The hydrogel then switches to its final zwitterionic state with the carboxylate as its new negatively charged group. We prove that this hydrogel can reach one-salicylate-per-monomer drug loading while still retaining the nonfouling property at protein and bacteria levels. It was also shown that its drug release profile was dictated by the hydrolysis rate of the monomer, making it possible to control and tailor the release rate of small hydrophilic drugs from the highly hydrated nonfouling polymer matrix.

Interactions of alginate-producing and -deficient Pseudomonas aeruginosa with zwitterionic polymers.

In this work, we report a study of the interactions between bacteria and zwitterionic polymers using a long-range surface plasmon resonance (LR-SPR) sensor. The LR-SPR with an extended probing field allows one to perform in-situ monitoring of bacterial adhesion and to investigate bacteria-surface interactions under various conditions. Alginate-producing and -deficient Pseudomonas aeruginosa were used in order to study the role of capsular exopolysaccharide in bacterial adhesion onto a surface. Polycarboxybetaine polymers with one and two carbons in spaces between two oppositely charged moieties on polymer side chains (pCBAA-1 and pCBAA-2) were prepared in order to unveil the effects of material properties on surface resistance to bacterial adhesion. In addition, environmental factors (e.g., divalent cations) were investigated. This work demonstrates the role of polysaccharides beyond proteins in bacteria adhesion and the effective resistance of pCBAA-1 against bacterial adhesion.

Reversibly switching the function of a surface between attacking and defending against bacteria.

Attack or defend! A smart polymer surface has two reversibly switchable equilibrium states, a cationic N,N-dimethyl-2-morpholinone (CB-Ring) and a zwitterionic carboxy betaine (CB-OH). CB-Ring will kill bacteria upon contact under dry conditions, whereas CB-OH will release the previously attached and dead bacteria and further resist adhesion of bacteria under wet conditions.

Divalent cation-mediated polysaccharide interactions with zwitterionic surfaces.

One popular postulation in the design of a nonfouling surface is that a surface capable of resisting nonspecific protein adsorption should also resist bacterial adhesion and subsequent biofilm formation. Such a hypothesis, though valid in certain cases, oversimplifies complex biological systems, since they contain not only proteins but also other biomacromolecules, such as polysaccharides. This work aims to re-examine this postulation by testing the biofouling of polysaccharides onto protein-resisting zwitterionic surfaces in the presence of a multivalent cation. Our results show that Mg(2+) plays an important role in mediating alginate adsorption onto zwitterionic surfaces through ion-bridged interactions from surface plasmon resonance (SPR) experiments. Three zwitterionic polymers tested in this work have clearly different responses to changes in Mg(2+) concentration, indicating that such ion-bridged adsorption is strongly dependent on cation-zwitterionic polymer binding affinities and is dictated by the specific chemical structure of the polymer betaine side chain. This work underlines the necessity to go beyond current nonfouling criteria at the protein level and to take into account polysaccharides when it comes to complex environments.

Multifunctional magnetic-plasmonic nanoparticles for fast concentration and sensitive detection of bacteria using SERS.

Multifunctional magnetic-plasmonic Fe(3)O(4)-Au core-shell nanoparticles (Au-MNPs) were prepared for simultaneous fast concentration of bacterial cells by applying an external point magnetic field, and sensitive detection and identification of bacteria using surface-enhanced Raman spectroscopy (SERS). We demonstrated that a spread of a 10 µL drop of a mixture of 10(5) cfu/mL bacteria and 3 µg/mL Au-MNPs on a silicon surface can be effectively condensed into a highly compact dot within 5 min by applying an external point magnetic field, resulting in 60 times more concentrated bacteria in the dot area than on the spread area without concentration. Surrounded by dense uniformly packed Au-MNPs, bacteria can be sensitively and reproducibly detected directly using SERS. The principle component analysis (PCA) showed that three different Gram-negative bacterial strains can be clearly differentiated. We also demonstrated that the condensed multifunctional Au-MNPs dot can be used as a highly sensitive SERS-active substrate and a limit of detection better than 0.1 ppb was obtained in detection of small molecules such as 4-mercaptopyrine. This novel platform significantly simplifies the concentration and detection process, which holds great promise for applications in food safety, environmental monitoring, medical diagnoses, and chemical and biological threat detections.

Functionalizable and ultrastable zwitterionic nanogels.

Multifunctional biomimetic nanogels based on zwitterionic poly(carboxybetaine methacrylate) (pCBMA) were synthesized by inverse microemulsion free-radical polymerization. pCBMA nanogels exhibited excellent stability in 100% fetal bovine serum. Dextran labeled with fluorescein isothiocyanate (FITC-dextran) was encapsulated in nanogels as a model drug. Encapsulated FITC-dextran exhibited controlled release from the pCBMA nanogel. Additionally, pCBMA nanogels carry abundant carboxylate groups as functional groups used to conjugate ligands to the nanogels for targeted drug delivery. Flow cytometry results obtained showed that pCBMA nanogels conjugated with cyclo[Arg-Gly-Asp-D-Tyr-Lys] ligands dramatically improved the nanogel uptake by primary human umbilical vein endothelium cells. Functionalizable zwitterionic pCBMA nanogels hold great potential as targeted drug-delivery vectors for biomedical applications.

pH responsive properties of non-fouling mixed-charge polymer brushes based on quaternary amine and carboxylic acid monomers.

In this work, we report a tunable mixed-charge copolymer surface containing positively charged quaternary amine monomers ([2-(acryloyloxy)ethyl] trimethyl ammonium chloride, TMA) and negatively-charged carboxylic acid monomers (2-carboxy ethyl acrylate, CAA). The non-fouling properties of this copolymer coating depend on environmental pH. The surface has charge neutrality under neutral and basic conditions, and is positively charged under acidic conditions due to the protonation of the carboxylic acid group. This transition in surface charge with respect to pH allows the surface to be switched from bacteria-adhesive to bacteria-resistant. We demonstrate that the bacteria adhered to the surface under acidic conditions can be easily released as bulk pH increases. This tunable surface can be used to collect a contaminant and then be externally stimulated to release the contaminant, to allow for analysis of its composition. Its bacteria attraction and release property makes it very promising for decontamination and biomedical applications.