A common framework for integrated and continuous biomanufacturing.

There is a growing application of integrated and continuous bioprocessing (ICB) for manufacturing recombinant protein therapeutics produced from mammalian cells. At first glance, the newly evolved ICB has created a vast diversity of platforms. A closer inspection reveals convergent evolution: nearly all of the major ICB methods have a common framework that could allow manufacturing across a global ecosystem of manufacturers using simple, yet effective, equipment designs. The framework is capable of supporting the manufacturing of most major biopharmaceutical ICB and legacy processes without major changes in the regulatory license. This article reviews the ICB that are being used, or are soon to be used, in a GMP manufacturing setting for recombinant protein production from mammalian cells. The adaptation of the various ICB modes to the common ICB framework will be discussed, along with the pros and cons of such adaptation. The equipment used in the common framework is generally described. This review is presented in sufficient detail to enable discussions of IBC implementation strategy in biopharmaceutical companies and contract manufacturers, and to provide a road map for vendors equipment design. An example plant built on the common framework will be discussed. The flexibility of the plant is demonstrated with batches as small as 0.5 kg or as large as 500 kg. The yearly output of the plant is as much as 8 tons.

The design basis for the integrated and continuous biomanufacturing framework.

An 8 ton per year manufacturing facility is described based on the framework for integrated and continuous bioprocessing (ICB) common to all known biopharmaceutical implementations. While the output of this plant rivals some of the largest fed-batch plants in the world, the equipment inside the plant is relatively small: the plant consists of four 2000 L single-use bioreactors and has a maximum flow rate of 13 L/min. The equipment and facility for the ICB framework is described in sufficient detail to allow biopharmaceutical companies, vendors, contract manufacturers to build or buy their own systems. The design will allow the creation of a global ICB ecosystem that will transform biopharmaceutical manufacturing. The design is fully backward compatible with legacy fed-batch processes. A clinical production scale is described that can produce smaller batch sizes with the same equipment as that used at the commercial scale. The design described allows the production of as little as 10 g to nearly 35 kg of drug substance per day.

Separation of the enantiomers of underivatized amino acids by using serially connected dual column high-performance liquid chromatography-tandem mass spectrometry.

In this article, a serially connected dual column liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous separation and enantioseparation of proteinogenic amino acids. For this purpose, different achiral and chiral stationary phases (CSP) and mobile phase compositions have been tested. As a result of the optimization studies, the best enatioseparation for amino acids were achieved with a combination of zwitterionic and crown ether stationary phases using a gradient of two mobile phases: A (water:TFA 99.5:0.5, % v/v) and B (acetonitrile:ethanol:TFA 85:15:0.5, % v/v/v). The developed method provided simultaneous enantioseparation of all proteinogenic amino acids under this study including isomeric and isobaric ones except for proline. The method was successfully applied to human lung adenocarcinoma cells (A549) and healthy human lung epithelial cells (BEAS-2B) cultivated with d-amino acid containing cocktails in order to evaluate d-amino acids transfer rate in normal and cancer lines. Thed/l amino acid ratios were different in cancer and normal cell lines cultivated as mentioned above for aspartic acid, cysteine, methionine, phenylalanine, and serine.

Best care paradigm to optimize functionality after extra-articular distal humeral fractures in the young patient.

For younger patients with extra-articular distal humerus fractures closed management is plagued with high rates of malunion, suboptimal functional outcomes, extended immobilization with loss of early motion, a delay in return to work, and a general period of lost productivity. Surgical management offers an appealing alternative. Maintaining respect for the triceps musculature and minimizing iatrogenic injury to the radial nerve are primary concerns with operative treatment. Accordingly, use of a triceps-sparing approach and single column plating may be the optimal treatment paradigm in the young patient presenting with an extra-articular distal humerus fracture.

Development of simultaneous targeted metabolite quantification and untargeted metabolomics strategy using dual-column liquid chromatography coupled with tandem mass spectrometry.

Targeted quantification and untargeted global profiling are the two mainstream approaches, a merging of which could provide enhanced analytical potential in metabolomics research. Here, a simultaneous targeted quantification and untargeted metabolomics (STQUM) strategy was developed for more efficient, accurate and comprehensive metabolomics research by using ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UPLC-HRMS/MS). First, we selected 110 cancer-related metabolites as targets and established a dual LC sequential separation method for simultaneous analysis of strong and weak polar metabolites. In order to achieve efficient acquisition for synchronous qualitative and quantitative analysis, high-resolution, data-dependent parallel reaction monitoring (PRM) method and data-independent all ion fragmentation (AIF) method were established. Their performance in targeted confirmation and quantification, and untargeted analysis were systematically investigated and assessed. In total, 78 metabolites were confidently confirmed in positive ion mode in both PRM and AIF assays, in which 73 metabolites can be accurately quantified. In addition, simultaneously untargeted profiling of 4651 features of high reliability and validity were achieved. Both AIF and PRM methods revealed high confidence, sensitivity and accuracy. In the STQUM approach, another 15 metabolites could be accurately quantified in negative ion mode. The method offers a new perspective for merging the hypothesis-based targeted quantitative validation and untargeted biomarkers discovery in one run for improved analysis efficiency and integrity.

Development of a linear dual column HPLC-MS/MS method and clinical genetic evaluation for tramadol and its phase I and II metabolites in oral fluid.

Tramadol is a centrally acting synthetic opioid analgesic and has received special attention due to its abuse potential and unexpected responses induced by CYP2D6 polymorphism. Oral fluid is an advantageous biofluid for drug analysis due to non-invasive sampling and high correlation of drug concentrations with plasma. However, few studies have been performed on distribution of tramadol and its metabolites in oral fluid. In the present study, a linear dual column HPLC-MS/MS method was developed and fully validated for the simultaneous determination of tramadol and its phase I [O-desmethyltramadol (ODMT), N-desmethyltramadol (NDMT) and N,O-didesmethyltramadol (NODMT)] and II metabolites in oral fluid. Furthermore, the distribution of tramadol and its metabolites, in relation to CYP2D6 genetic variations, in oral fluid was investigated following a clinical study including 23 subjects with CYP2D6*wt/*wt, CYP2D6*10/*10 or CYP2D6*5/*5. The validation results of selectivity, matrix effect, linearity, precision and accuracy were satisfactory. Pharmacokinetic parameters, such as Css,max and AUC0-τ of tramadol, NDMT and NODMT, in the CYP2D6*10/*10 group were significantly higher than those in the CYP2D6*wt/*wt group. Moreover, the ratios of ODMT/tramadol, NDMT/tramadol and NODMT/NDMT correlated well with the CYP2D6 genotypes. We demonstrated that oral fluid is a promising biofluid for pharmacokinetic evaluation in relation to genetic variations.

Method development for comprehensive extraction and analysis of marine toxins: Liquid-liquid extraction and tandem liquid chromatography separations coupled to electrospray tandem mass spectrometry.

A variety of toxins are produced by marine and freshwater microorganisms that present a threat to human health. These toxins have diverse chemical properties and specifically, a range of hydrophobicity. Methods for extraction and identification of these toxins are often geared toward specific classes of toxin depending on the sample type. There is a need for a general method of toxin extraction and identification for screening samples where the likely toxin content is not known a priori. We have applied a general method for metabolite extraction to toxin containing samples. This method was coupled with a simple dual liquid chromatography approach for separating a broad range of toxins. This liquid chromatography approach was coupled to triple quadrupole and quadrupole time-of-flight MS/MS platforms. The method was testing on a fish matrix for recovery of palytoxin as well as marine corals for detection of natural mixtures of palytoxin analogues. The recovery of palytoxin was found to produce a linear response (R2 of 0.95) when spiked into the fish matrix with a limit of quantitation of 2.5 ng/µL and recovery efficiency of 73% + /- 9%. The screening of corals revealed varying amount of palytoxin, and in one case, different palytoxin structural analogues. This demonstration illustrates the potential utility of this method for toxin extraction and detection.

Interference of anesthetics in blood alcohol analysis by HS-GC-FID: A case report.

One of the techniques most widely used in ethanol analysis in forensic laboratories is undoubtedly the headspace gas chromatography with flame-ionization detection (HS-GC-FID) since the determination of this substance is carried out directly, without the need for additional purification procedures, which leads to increased productivity. This is a very important factor due to the high number of alcohol analysis requested to these laboratories. The presence of other volatile substances can cause a problem given the fact that they can be interferents in ethanol analysis by HS-GC-FID, which can have legal consequences related with driving under the influence of alcohol. The authors report a case of a routine analysis by HS-GC-FID for the determination of ethanol of a driver who has suffered an accident in which the use of two chromatographic columns with different polarities was essential to obtain an unequivocally identification of this substance in presence of an interfering volatile anesthetic administered in the hospital. The method was validated according to international recommendations before being introduced into routine laboratory in terms of selectivity, limits of detection (LOD), limits of quantification (LOQ), linearity, repeatability, intermediate precision, accuracy, robustness and carryover.

A paradigm shift in the surgical reconstruction of extra-articular distal humeral fractures: single-column plating.

OBJECTIVES: The study aimed (1) to examine if there are equivalent results in terms of union, alignment and elbow functionally comparing single- to dual-column plating of AO/OTA 13A2 and A3 distal humeral fractures and (2) if there are more implant-related complications in patients managed with bicolumnar plating compared to single-column plate fixation. DESIGN: This was a multi-centred retrospective comparative study. SETTING: The study was conducted at two academic level 1 trauma centres. PATIENTS/PARTICIPANTS: A total of 105 patients were identified to have surgical management of extra-articular distal humeral fractures Arbeitsgemeinschaft für Osteosynthesefragen/Orthopaedic Trauma Association (AO/OTA) 13A2 and AO/OTA 13A3). INTERVENTION: Patients were treated with traditional dual-column plating or a single-column posterolateral small-fragment pre-contoured locking plate used as a neutralisation device with at least five screws in the short distal segment. MAIN OUTCOME MEASUREMENTS: The patients' elbow functionality was assessed in terms of range of motion, union and alignment. In addition, the rate of complications between the groups including radial nerve palsy, implant-related complications (painful prominence and/or ulnar nerve neuritis) and elbow stiffness were compared. RESULTS: Patients treated with single-column plating had similar union rates and alignment. However, single-column plating resulted in a significantly better range of motion with less complications. CONCLUSIONS: The current study suggests that exposure/instrumentation of only the lateral column is a reliable and preferred technique. This technique allows for comparable union rates and alignment with increased elbow functionality and decreased number of complications.

Study of determination method of alcohol in blood using headspace gas chromatography / 国际检验医学杂志

Objective To establish a kind of simple,rapid,accurate and reliable method to analyze the concentration of alcohol in blood by headspace gas chromatography (HS-GC) with dual-column and dual-detector.Methods The samples were pre-treated by headspace sampler,which was the basis on the extraction principle of the gas extracting volatile substances.Next,these samples were analyzed by HS-GC that the tertiary butyl alcohol was acted as the internal standard substance.The HS-GC was equipped with two chromatographic column (the DB-ALC2 chromatographic column of 001 channel;the DB-ALC1 chromatographic column of 002 channel).At the same time,the HS-GC was also equipped with two hydrogen flame ionization detector (FID1 detector;FID2 detector).The retention time of the peak was finally performed as qualitative parameter and the standard curves method of internal standard were acted as quantitative basis.Results The liner range of the method was 0.2-2.0 mg/mL.The linear regression equation of 001 channel was Y=1.057 7X+0.048 2 and the correlation coefficient was R2=0.999 05.Besides,the linear regression equation of 002 channel was Y=1.039 5X+0.046 5 and the correlation coefficient was R2=0.999 25.In short,the average recovery rate of the method was 99.70%.Relative standard deviation(RSD) was less than 4% between the analysis results of 001 channel and 002 channel for the determination of the plan sample.Conclusion The method shown satisfactorily that it could not only be applied to determine the alcohol of blood of forensic toxicological analysis,but also be applied to determine the plan sample of ability test and verify of laboratory ability accreditation.

Solid phase extraction-gas chromatography with dual tower and dual column for the simultaneous determination of 53 organophosphorus pesticides in traditional Chinese herbal medicines / 中成药

AIM: The multi-residues method was used to determine organophosphorus pesticides in traditional Chinese herbal medicines (TCHMs).METHODS: Fifty three pesticides were extracted by high-speed homogenization,and then cleaned sequentially by C_(18) and Carb/NH2 solid phase extraction (SPE) cartridges.The residues were simultaneously identified and quantified by GC-FPD equipped with dual tower,dual column and two FPD detectors.RESULTS: The analytical performance was demonstrated by the analysis of 6 TCHMs samples extracts,spiked at three concentration levels for each pesticide.In general,the recoveries ranging from 70% to 120%,with relative standard deviations (RSDs) less than 15%,were obtained.The limit of detection (LOD) for most of the targeted pesticides tested was below 0.01 mg/kg.CONCLUSION: The method has good extraction efficiency,purification effect and good reproducibility,which can be applied to the determination of organophosphorus pesticide residues in the routine analysis of TCHMs.