Genetic Variations of IL-12B,IL-12Rβ1, IL-12Rβ2 in Behcet's Disease and VKH Syndrome

Purpose

To investigate the associations of single nucleotide polymorphisms (SNPs) of three genes (IL-12B, IL-12Rβ1 and IL-12Rβ2) in Behcet''s disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population.

Methods

A total of 806 BD cases, 820 VKH patients, and 1600 healthy controls were involved in this study. The first investigation included 400 BD patients, 400 VKH cases, and 600 healthy individuals. A second confirmatory study included a separate set of 406 BD patients, 420 VKH cases and another 1000 normal controls. Genotyping was carried out by PCR-restriction fragment length polymorphism assay and results were validated by using direct sequencing. The χ2 test was performed to compare the allele and genotype frequencies between cases and healthy controls.

Results

This study comprised two phases. In the first phase study, a significantly increased frequency of the rs3212227/IL-12B genotype CC and C allele was found in BD patients as compared to controls (Bonferroni corrected p value (p_c) = 0.009, OR 1.8; p_c = 0.024, OR 1.3, respectively). Moreover, the frequency of the C allele of rs3212227/IL-12B was also significantly increased in VKH patients (p_c = 0.012, OR 1.3, 95% CI 1.1 to 1.6). No associations were found for the other seven tested SNPs either in BD or VKH disease. The second study as well as the combined data confirmed the significant association of rs3212227/IL-12B with BD (CC genotype: combined p_c = 6.3×10⁻⁷, OR = 1.8; C allele: combined p_c = 2.0×10⁻⁵, OR = 1.3, respectively) and the C allele frequency of rs3212227/IL-12B as the risk factor to VKH patients (combined p_c = 2.5×10⁻⁵, OR 1.3, 95% CI 1.2 to 1.5).

Conclusions

Our study revealed that the IL-12B gene is involved both in the susceptibility to BD as well as VKH syndrome.     

KEY WORD INDEX TO VOLUME 12, 2002

Mdo]ase A.................AaIT……...........….accines.....,..............actin .....................actin—binding protein.....AFLP….…..............alpha—fetoprotein.........ascular smooth muscle cellsastacin.......。..ATF/CREB......auxin effiux carrierB and C mRNAsB chromosome.Brassica junceaBrassica juncea 85143104207207 63151123163177247 85 63235247cancer..........................]l cardioascular diseases...............105cDNA clone……….……..199cell cycle…............…….…     

Measles Case Fatality Rate in Bihar,India, 2011–12

Background

Updated estimates of measles case fatality rates (CFR) are critical for monitoring progress towards measles elimination goals. India accounted for 36% of total measles deaths occurred globally in 2011. We conducted a retrospective cohort study to estimate measles CFR and identify the risk factors for measles death in Bihar–one of the north Indian states historically known for its low vaccination coverage.

Methods

We systematically selected 16 of the 31 laboratory-confirmed measles outbreaks occurring in Bihar during 1 October 2011 to 30 April 2012. All households of the villages/urban localities affected by these outbreaks were visited to identify measles cases and deaths. We calculated CFR and used multivariate analysis to identify risk factors for measles death.

Results

The survey found 3670 measles cases and 28 deaths (CFR: 0.78, 95% confidence interval: 0.47–1.30). CFR was higher among under-five children (1.22%) and children belonging to scheduled castes/tribes (SC/ST, 1.72%). On multivariate analysis, independent risk factors associated with measles death were age <5 years, SC/ST status and non-administration of vitamin A during illness. Outbreaks with longer interval between the occurrence of first case and notification of the outbreak also had a higher rate of deaths.

Conclusions

Measles CFR in Bihar was low. To further reduce case fatality, health authorities need to ensure that SC/ST are targeted by the immunization programme and that outbreak investigations target for vitamin A treatment of cases in high risk groups such as SC/ST and young children and ensure regular visits by health-workers in affected villages to administer vitamin A to new cases.     

Cancer Immunotherapy 2005: Mainz, Germany, 12–13 May 2005

Cancer Immunotherapy 2005 was the third international meeting organized by the Association for Immunotherapy of Cancer (AIC). About 200 participants were attracted by the excellent scientific program that consisted of overview lectures from 25 international speakers in the plenary auditorium and four guided poster sessions during both days of the meeting. The first day of the symposium mainly focused on experience with, and new perspectives in, antibody therapy. On the second day of the meeting, organized as a joint conference together with the Combined Research Grant “Mechanisms of Tumor Defense and Therapeutic Intervention” funded by the German Research Council, the participants had the chance to gain deeper insights into the principles of antigen processing and the regulation of immune responses. Further topics that were discussed mainly in the poster sessions and in the special lecture given by M. Nishimura (Chicago, USA), were “cellular therapies” and “vaccination against cancer”. The lectures selected for this report aim to provide an overview of the complete scientific program and give an impression of the lively atmosphere that could be felt from the first until the last session of CIMT 2005. C.M. Britten and C. Gouttefangeas contributed equally to this report.     

6aR,12aR,12a-羟基紫穗槐苷元上调Bim诱导肝癌细胞SMMC-7721发生凋亡的机制

肝细胞癌(HCC)是一种高度恶性的肿瘤。化疗是临床上一种重要的治疗方法,但由于化疗药物的毒副作用和耐药效应,现有的化疗药物或多或少有一定的局限性。因此,寻找毒副作用小且有效的化疗药物一直是肝癌患者的迫切需求。本研究从紫穗槐种子中分离纯化出一种天然产物6aR,12aR,12a-羟基紫穗槐苷元,采用CCK-8法检测了其对人肝癌SMMC-7721细胞的杀伤作用,应用流式细胞术检测了其对肝癌细胞的凋亡诱导情况,通过免疫印迹法检测了6aR,12aR,12a-羟基紫穗槐苷元上调Bim蛋白表达进而诱导肝癌细胞凋亡的作用机制。结果表明,6aR,12aR,12a-羟基紫穗槐苷元可呈浓度依赖性和时间依赖性抑制人肝癌SMMC-7721细胞的活力,24 h、48 h和72 h的半数抑制浓度(IC50)分别为21.77μmol/L、5.65μmol/L、5.0μmol/L;同时可浓度依赖性地提高细胞凋亡率;可通过下调抑凋亡蛋白Survivin、Mcl-1、Bcl-XL、Bcl-2,上调促凋亡蛋白Bak、Bim、Bax、Bid的表达,进而促进PARP和caspase-3的活化,从而诱导细胞发生凋亡;进一步地研究发现:6aR,12aR,12a-羟基紫穗槐苷元通过延长Bim的半衰期而增加Bim的积累,且具有和MG132类似的生物学功能,能够阻断蛋白的降解途径来抑制Bim降解。上述研究表明,6aR,12aR,12a-羟基紫穗槐苷元可通过抑制蛋白降解从而上调细胞中Bim蛋白的水平,进而诱导肝癌细胞SMMC-7721发生凋亡。     

Second International Conference on Accelerating Biopharmaceutical Development: March 9–12, 2009, Coronado,CA USA

The Second International Conference on Accelerating Biopharmaceutical Development was held in Coronado, California. The meeting was organized by the Society for Biological Engineering (SBE) and the American Institute of Chemical Engineers (AIChE); SBE is a technological community of the AIChE. Bob Adamson (Wyeth) and Chuck Goochee (Centocor) were co-chairs of the event, which had the theme “Delivering cost-effective, robust processes and methods quickly and efficiently.” The first day focused on emerging disruptive technologies and cutting-edge analytical techniques. Day two featured presentations on accelerated cell culture process development, critical quality attributes, specifications and comparability, and high throughput protein formulation development. The final day was dedicated to discussion of technology options and new analysis methods provided by emerging disruptive technologies; functional interaction, integration and synergy in platform development; and rapid and economic purification process development.

• MAbs. 2009 May-Jun; 1(3): 190–209.
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• March 10, 2009 Day 1, Emerging Disruptive Technologies and Cutting-Edge Analytical Techniques

MAbs. 2009 May-Jun; 1(3): 190–209.

March 10, 2009 Day 1, Emerging Disruptive Technologies and Cutting-Edge Analytical Techniques

Janice M ReichertAuthor information Article notes Copyright and License information DisclaimerTufts Center for the Study of Drug Development; Boston, MA USACorresponding author.Correspondence to: Janice M. Reichert; Tufts Center for the Study of Drug Development; 75 Kneeland Street; Suite 1100; Boston, MA 02111 USA; Email: ude.stfut@trehcier.ecinajReceived 2009 Mar 20; Accepted 2009 Mar 20.Copyright © 2009 Landes BioscienceThe meeting was opened by Bob Adamson (Wyeth) who remarked that it is the responsibility of biological engineers to develop technologies that will produce drug products rapidly and cost effectively. On average, protein therapeutics cost more than small molecule drugs. However, technological advances can help to drive down the cost of these products. For example, penicillin was scarce in the 1930s and 1940s because of production issues, but this drug is easily and cheaply obtained today.Recent developments in biosimilars, a potentially disrutive group of products, were discussed by Rob Garnick (Lone Mountain Biotechnology). He first noted the various names by which biosimilars are known. While the term biosimilars is favored in Europe, the US Food and Drug Administration (FDA) uses the term ‘follow-on biologics,’ and Health Canada prefers the phrase ‘subsequent entry biologics.’ The term biogenerics is not usually used because this word implies that the products are identical to approved innovator biologics. The European Medicines Agency and Health Canada have issued regulatory guidances for approval of biosimilars, and some of these products have been approved in Europe.¹ However, the process has gotten stalled in the US for various reasons, including questions surrounding the reliability of sourcing, magnitude of price reduction, need for clinical trials done against a national comparator and comparability issues. On the other hand, the global economic problems have focused political attention on healthcare reform and unsustainable increases in the cost of healthcare.Dr. Garnick noted that in the US the general consensus is that the Drug Price Competition and Patent Term Restoration Act of 1984, also known as the Hatch-Waxman Act, has been successful in promoting generics while still providing financial incentives for research and development by innovators. In addition, Congress believes that scientific issues surrounding biosimilars are addressable and so a regulatory pathway can be established for approval of biosimilars. Due to the progress in defining a regulatory pathway, some major pharmaceutical firms, including Pfizer, AstraZeneca, Novartis and Merck, have recently indicated that they will develop these products. Biosimilars development is attractive because the success rates should be 100% if the products are developed correctly, the manufacturing processes are well-understood and can be out-sourced, and the markets are potentially large. With a global market over US $5 billion, rituximab will certainly be targeted as a biosimilar. As with the Hatch-Waxman Act, the key to success of any US biosimilars legislation will be the maintenance of incentives to innovate.There are still numerous scientific and legal problems to address,² including the exact nature of legislation, patent issues, design of clinical trials, substitutability, interchangeability, safety and post-approval surveillance. The challenge lies in the details, e.g., establishing product specifications and test methods, and defining comparability. Dr. Garnick noted that cautionary tales on comparability come from the experience of a number of innovator companies. For example, efalizumab produced by XOMA was found to have differences when compared to efalizumab produced by Genentech. The differences, which included minor changes in acidic forms, galactosylation, charge heterogeneity and an increase in C-terminal processing, were expected to be inconsequential, but translated into different clinical study results. This experience suggests that a combination of written procedures, training, analytical testing and regulatory agency inspections are needed to control the production of biological products. Quality control release tests need to be supported by rigorous product and process characterization and process control.Outside the US, the reality is that biosimilars are being marketed in Europe, India and China as well as other countries. Marketed biosimilars span a broad range of complexities and include monoclonal antibodies. Reditux, a rituximab biosimilar, was approved in India in April 2007 for non-Hodgkin lymphoma and rheumatoid arthritis. However, the clinical trials included relatively few patients and limited analytical data has been made available. In conclusion, Dr. Garnick remarked that the world is gaining experience with biosimilars, and the products will likely become a reality in the US by 2009. FDA will need input to develop effective guidance documents for assessment of biologics. Immunogenicity will be a key concern of regulators. Comparability studies will be required, product differences will need to be investigated and appropriate clinical studies must be done, but biosimilars will come to market.Global trends in antibody development by innovator companies were presented by Janice Reichert (Tufts University). Clinical development of protein therapeutics is on the rise worldwide.^3–5 Approximately 120 recombinant proteins and 240 monoclonal antibodies (mAbs) are currently in clinical studies. While recombinant proteins have historically entered the clinic at a rate of fewer than 20 candidates per year, mAbs are now approaching the 40 candidate per year mark. Recombinant proteins have somewhat higher success rates on average (approximately 30% vs. 20% for mAbs) and have been studied in a wider array of therapeutic categories compared to mAbs, but, because of their versatility as therapeutics, mAbs are clearly the focus of the biopharmaceutical industry''s attention. A total of 22 mAbs are approved in the US, and eight of these products have global markets over US$1 billion. Six additional candidates are currently undergoing regulatory review.Dr. Reichert noted that the ascendancy of mAbs is due to technologies that addressed immunogenicity, affinity, specificity, stability and production challenges. While murine versions dominated in the 1980s, the less immunogenic humanized versions comprised 45% of the total mAbs in clinical study in the 1990s. In the 2000s, the human versions have comprised the largest contingent. Historically, mAbs have not been discontinued while in regulatory review. Assuming the six mAbs in review are approved, then cumulative FDA approval success rates for humanized and human mAbs will be nearly identical (19 and 18%, respectively). mAbs are commonly studied as either anticancer⁶ or immunological treatments. There are currently nine anticancer and ten immunological mAbs approved in the US. These products have taken approximately the same length of time for clinical development (6.5 years). The length of the FDA review period was found to vary depending on whether the product was given priority or standard review (average 6.9 or 20.4 months, respectively).Looking forward, Dr. Reichert suggested that antibody fragments and modified versions (pegylated, alternate glycosylation, Fc engineered) are likely to enter the clinical pipeline in increasing numbers. The focus is likely to remain on human IgG, but designed protein scaffolds and domain antibodies will also be included in company pipelines.The meeting then turned to discussion of potentially disruptive science and technologies. Stefan Wildt (Merck) reviewed the development of glycoengineered yeast, which he described as a versatile glycoprotein expression platform. He first emphasized the importance of glycosylation, which affects circulating half life, tissue distribution, potency and immunogenicity of therapeutic proteins. Any new bioprocesses need to be scalable, portable, and provide analytically comparable protein at all scales. The primary biomanufacturing platforms are bacterial (e.g., E. coli), fungal (e.g., Pichia pastoris), and mammalian cell culture (e.g., CHO cells). Fungal platforms are currently used for production of common industrial enzymes, but have not been used extensively for production of therapeutics because the yeast glycosylation pathway yields products that are potentially immunogenic in humans. GlycoFi Inc., a wholly owned subsidiary of Merck & Co., Inc. has developed Pichia with humanized glycosylation to circumvent this problem.In yeast, the carbohydrate processing occurs sequentially in the secretory pathway, like an assembly line. The enzymes act one after another and their actions are separated in time and space. As a consequence, humanized yeast produce proteins with human glycans that are highly uniform. In contrast, traditional mammalian cell production systems produce functional glycoproteins that are heterogeneous and contain non-human glycoforms. As reported by Hamilton et al.,⁷ GlycoFi eliminated yeast-specific glycosylation in Pichia pastoris and introduced 14 heterologous genes; this process yielded yeast capable of producing complex glycoproteins with greater than 90% terminal sialylation. Candidate protein can be produced in a bioreactor process that takes three to seven days, which is somewhat shorter than the time for production in mammalian cells.For example, Dr. Wildt discussed MK2578, which is a pegylated erythropoietin that is terminally sialylated with N-glycans. The candidate is currently in Phase 1 studies as a potential treatment for anemia. The glycosylation fidelity from the Pichia platform is retained when protein is produced at laboratory scale to up to 2,000 L. The yeast can also be used to produce antibody as IgG1. Compared to CHO cell produced IgG1, candidates produced in yeast were found to be more potent in inducing ADCC and could bind antigen as well. In preclinical studies, yeast-produced mAb glycovariants demonstrated good results in PK studies in Rhesus monkeys and C57BL mice. In conclusion, Dr. Wildt remarked that the selection process, which includes screening for titer, fermentability, glycosylation and protein quality, can result directly in production strains. Yield is approximately 1.4 grams per liter for antibody candidates, but yields up to 2 grams per liter can be achieved.Annie De Groot (EpiVax) presented information on methods to reduce protein immunogenicity by design through deimmunization and tolerance induction. She noted the parallels between vaccine use, when an immune response is desired, and immunogenic therapeutic proteins, which elicit an immune response that is not desired. In both cases, a payload coupled with a delivery vehicle and an adjuvant determine immunogenic potential. T-cell epitopes are a key contributing factor. Like proteins, antibodies are processed by antigen presenting cells. The activated T-cells in turn activate B-cells; in the absence of T-cells, no antibody formation is observed.EpiVax has developed an array of in silico tools and techniques to predict whether proteins will be immunogenic. These include EpiMatrix, in which overlapping 9-mer peptide frames are evaluated for binding potential to eight common class II HLA alleles. The ClustiMer algorithm can be used to find regions of high immunogenicity. Using these methods, an overall immunogenicity score can be estimated. These in silico results can be validated in vitro and in vivo (e.g., HLA transgenic mice).The approach has been clinically validated. Koren et al.⁸ reported on use of EpiMatrix analysis of a recombinant fusion protein that predicted promiscuous T-cell epitopes in the C-terminal region. In a phase 1 study of 76 subjects, 37% developed antibodies after one injection of the protein candidate. EpiMatrix correctly predicted the immunogenic region and the likelihood that the protein would be immunogenic in the clinic.These results suggest that the technology might be useful as part of an overall strategy for assessing antibody responses in non-clinical and clinical settings.^{9, 10} In addition, rational modification of epitopes identified using the technology could effectively ‘deimmunize’ protein candidates. Dr. De Groot also discussed the discovery of ‘Tregitopes’ (highly conserved regulatory T-cell epitopes) that are promiscuous, high affinity HLA binders found in IgG. She noted that there is a correlation of antibody immunogenicity with the presence of Tregitopes. Dr. De Groot and co-workers have demonstrated that co-incubation of peripheral blood mononuclear cells (PBMCs) with the Tregitopes can lead to suppression of immune response to other antigens. This suggests that the engineering of Tregitopes into antibodies or other proteins might lead to the development of less immunogenic candidates.Modular IMmune In vitro Constructs (MIMIC), which is an in vitro biomimetic human immune system developed to accurately model the immunotoxicity and immunogenicity of drug candidates, was reviewed by William Warren (Vaxdesign). The MIMIC system is designed to serve as a ‘clinical trial in a well’ by providing predictive HTP in vitro immunology assessment of drug candidates. Primary human donor cells are used to simulate human responses to agents such as vaccines and drugs. The system consists of three modules: (1) Simulation of innate immunity with a peripheral tissue equivalent (PTE) module; (2) Simulation of adaptive immunity with the lymphoid tissue equivalent (LTE); and (3) a functional assay or disease model. The PTE module comprises one monolayer of endothelial cells grown over a 3D collagen matrix. Human PBMCs from donors are used to seed the module; monocytes extravasate through the endothelial cells and differentiate into antigen presenting cells. The PTE module can be used to assess reactogenicity and immunotoxicity responses. The LTE module functionally reproduces the environment of a human lymph node. Within the module, T-cells, B-cells, antigen-presenting cells or follicular dendritic cells interact, leading to immune stimulation that results in activation of lymphocytes, cytokine generation and antibody production. The activated lymphocytes, cytokine profiles and antibodies are then characterized using various methods.Dr. Warren discussed use of the modules to measure the magnitude and quality of T-cell response to vaccines. He noted that primary CD8 T-cell response, in vitro humoral and B-cell response, antibody titer, and microneutralization can be assessed. A correlation analysis of MIMIC response versus serum titer in hepatitis B and influenza vaccination has been performed. Results suggest that the MIMIC system can be used to predict whether a vaccine would be efficacious before going to the clinic. In addition to immunogenicity, the system acts as a biomimetic for evaluation of immunotoxicity and can be used as an inflammation model or in vitro infectious disease model. Use of the system has the potential to accelerate the entire drug development timeline, and decrease failures by providing better data for evaluation of preclinical candidates.Karyn O''Neil (Centyrex, a Johnson & Johnson Internal Venture) described alternative scaffolds that are being used as new biotherapeutic platforms by Johnson & Johnson. She started by wondering whether mAbs are always the best choice since there are reasons to develop alternatives. For example, desirable epitopes might be immunologically silent, alternatives to injection delivery are a challenge, full-size antibodies penetrate tissue and tumors poorly, and royalties might be due on numerous phases of the mAb discovery, screening, development and production process. However, requirements for a next-generation platform, which include the expansion of the range and possibility of targets, lower cost of development and manufacturing complexity, novel delivery, elimination of cold storage, clear freedom to operate and no intellectual property issues, are difficult to meet. Alternative scaffolds do meet many of the aforementioned requirements, and these molecules have favorable biophysical characteristics. Alternative scaffolds can be readily formatted into multi-specific binders with relevant biological activity. For example, Lu et al.¹¹ combined variable regions of two antagonistic antibodies to produce a human IgG-like bispecific antibody that could strongly inhibit the growth of two different human tumors in HT29 xenografts in vivo.Johnson & Johnson''s strategy toward the use of alternative scaffolds involves development of both Centyrins^™ and DARPins^™, which are viewed as complimentary molecule types. Both are small (10 to 18 kDa) single domain proteins that have high affinity (low picomolar to femtomolar range) and high selectivity for their targets. They are compatible with technologies that improve serum half-lives and seem to have low immunogenicity and low toxicity. They are also very stable and can be expressed at high levels in soluble form. DARPins^™ have flat wide surfaces that are better suited for disrupting protein-protein interactions,¹² whereas Centyrins^™ have extended loops that can interact in protein clefts, enzyme active sites and protein channels.¹³DARPins^™, which are being developed as part of a collaboration between Molecular Partners and Johnson & Johnson, are selected from in vitro display of very large (10¹²) libraries. The method uses PCR and affinity maturation, and candidates with slow off-rates can be selected. In this way, high affinity, neutralizing DARPins^™ can be selected within weeks. Melting properties can be used for selection, resulting in DARPins^™ candidates with good biophysical properties. Small scale (2 mL) expression of DARPins^™ can yield approximately 1 mg each for additional characterization.The Centyrins^™ scaffolds have loops that are analogous to the CDRs of antibodies. The molecules have excellent biophysical properties (>100mg/mL expression, >170mg/mL solubility, >82°C melting temperature, low predicted immunogenicity, stable in serum for more than one month), and can be engineered for improved stability. An in vitro display system licensed from Isogenica utilizes CIS-display technology for Centyrins^™ selection. This is proven technology for peptide display. Libraries are potentially quite large (10¹³). The CIS-display allows rapid panning and selection of binders with a PCR step that allows for in vitro evolution of binders. Rational design of library diversity can improve scaffold properties.¹⁴ A green fluorescent protein solubility/folding reporter assay¹⁵ is used to assess library quality. The unique properties of alternative scaffolds can be exploited in numerous areas, such as bispecific molecules, medical device, encapsulation, novel formulation and delivery, drug/toxin or radionuclide conjugation, imaging, biosensor, purification technologies and intracellular expression.Cutting-edge analytical technologies became the focus of the meeting in the afternoon session. Steve Cohen (Waters Corporation) discussed chromatographic analysis for biopharmaceuticals with an emphasis on current trends and future prospects. He first discussed ultra performance liquid chromatography (UPLC) utilizing sub-2 micron particle packing (1.7 µmeter with either 130 angstrom or 300 angstrom pores). Compared to HPLC with 3.5 µmeter particle packing, UPLC gives sharper peaks, and can improve resolution of samples in the same run time or achieve comparable resolution and selectivity in a reduced run time (80 versus 120 minutes). Dr. Cohen then presented results of LC/ultraviolet (UV) analysis of a reduced monoclonal antibody, and a murine monoclonal IgG reduced and alkylated standard run at elevated temperature. He noted that the high temperature (80–90°C) is absolutely required for reasonable chromatography. Analytical methods for monitoring glycosylation of mAbs are important because bioprocess conditions can cause variation in high mannose type, truncated forms, reduction of tetra-antennary and increase in tri- and biantennary structures, less sialyated glycans and less glycosylation.Dr. Cohen also reviewed new separations technologies such as monolithic materials and chip based nanoscale separations. He presented an example of the use of a ceramic microfluidic UPLC system and the software tool BiopharmaLynx 1.2 to perform humanized peptide mapping. Three dimentional structure analysis using amide hydrogen exchange was also discussed. In a continuous labeling experiment, labeling occurs at 25°C, pH 7, and aliquots are removed and quenched at 0°C, pH 2.5. The protein sample can be subjected to HPLC/UPCL directly, or subjected to online digestion and then HPLC/UPCL. Electrospray mass spectrometry then provides information about isotope pattern and deuterium content that can be used to determine exchange rates. Use of UPLC will provide sharper peaks and improved spectral quality.¹⁶ The technique can be used for quality control or comparability of samples, e.g., differentiation of correctly folded protein from incorrectly folded protein.Tom Laue (University of New Hampshire) discussed advances in analytical ultracentrifugation (AUC) and analytical electrophoresis (AE). Dr. Laue remarked that AUC provides a framework for thinking about concentrated solutions and proximity energies. AUC can be used to characterize proteins in high concentration formulations. Proximity energies at high concentrations may be positive or negative, and are dependent on such factors as distance, orientation, solvent and time. Potential energy is dependent on forces such as charge-charge, charge-dipole, dipole-dipole, hydrogen bonding, dispersion, dipole induced dipole, charge induced dipole and van der Waals interactions. AUC with fluorescence detection can be used to characterize labeled proteins in concentrated samples such as plasma. For example, mAb interactions in plasma can be observed using fluorescence detected sedimentation. Weak electrostatic interactions will dominate molecular behavior in concentrated solutions.Dr. Laue then discussed AE as a technique to determine accurate values of protein charge. Interestingly, monoclonal IgGs have an actual charge that is aberrantly low compared to the calculated value (e.g., 2 versus 24). The low charge leads to problems with poor solubility and high viscosity. Dr Laue speculated that the mAb charge suppression may have some housekeeping function such as weakening charge interactions with anionic plasma proteins, or altering co-operativity for Fc FcR binding or other functions. The low charge may be due to a combination of pKa shifts, anion binding (territorial or site), and carbohydrate involvement. Many of the viscosity and solubility problems encountered during processing may be traced to the low charge on IgGs. He urged attendees to measure the charge and not to rely on calculated charge estimates (e.g., from isoelectric point measurements).Kermit Murray (Louisiana State University) discussed coupling microfluidic chips to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The chips can speed proteomics by serving as a single platform for automated cell culturing, digestion, separation and sample deposition. The system is based on synthetic polymer microfluidic devices, with chip components fabricated on a poly(methyl) methacrylate plate using the hot embossing method and off-line MALDI analysis. A key component of the system is a trypsin microreactor. Assembled chips are processed using a pressure-driven or electrokinetic flow; the system utilizes a Dionex LC and a Probot MALDI plate spotter for the former. Digested peptides are coaxially mixed with a MALDI matrix solution and deposited on a MALDI target. Chips are stable for about one month.Dr. Murray presented experimental results from analyses of cytochrome c under various flow rates. A flow rate of 1 µL/min, with a residence time of approximately 24 seconds within the reaction bed, provided 67% sequence coverage, which increased to 72% when residence time was increased to 48 seconds. Use of the 1 µL/min flow rate resulted in sequence coverages of 35%, 58%, and 47% for 10 µM samples of bovine serum albumin, myoglobin and phosphorylase b, respectively. The digestion efficiency was improved using an electrokinetically driven microreactor using a micro-post structured chip. Using the micro-post system, sequence coverage of 10 µM cytochrome c was 89%; sequence coverage decreased when protein concentration of the sample was lower. Whole bacterial cells can be analyzed using the system. Digestion and deposition of E. coli resulted in identification of the aminoglycoside 3′-phosphotransferase type 1, with 57% sequence coverage.A two-chamber chip developed by Dr. Murray and colleagues can also be used to provide MALDI MS results for bacteria. It has applications for analysis of sepsis, pneumonia, tuberculosis, blood supply QA/QC, and environmental pathogen samples. The cell culture chamber has sample and media inlet, as well as outlet, channels; the culture chamber itself has a 3 mm diameter and 300 µm depth. The system uses a PMMA chip and PDMS cover. The channel surfaces are sterilized with UV. After assembly, the chamber is filled with nutrient broth, approximately 4,000 E. coli cells are added and the reservoirs are closed. The bacteria are cultured for 24 hours at 37°C. One µL of E. coli is then deposited on the MALDI target plate. In an experiment using ATCC#9637, #11303, or #11775, some cellular protein peaks were found. The results suggest that such on-chip culturing could be used for fingerprint analysis. Finally, Dr. Murray discussed preliminary work on a temperature regulated chip with heating and cooling elements.The topic of mass spectrometry (MS)-based strategies to study protein architecture, dynamics and binding was reviewed by Igor Kaltashov (University of Massachusetts, Amherst). He first noted that biopharmaceuticals have higher order structure and conformational heterogeneity, and various perturbations or changes in the production process can result in alterations of the primary or higher order structure that can have deleterious effects on the efficacy, immunogenicity or stability of the protein. MS has been applied to the structural characterization of recombinant protein pharmaceuticals¹⁷ and specifically therapeutic antibodies.¹⁸ The tertiary and quaternary protein structure can be characterized directly in solution by electrospray ionization (ESI) MS.As an example of the use of MS in structure characterization, Dr. Kaltashov discussed studies done on alkylated interferon β-1a (IFN-β-1a). Alkylation at Cys-17 of the protein results in 50–90% reduction of the antiviral activity. He remarked that ‘classical’ biophysical techniques such as size exclusion chromatography, fluorescence, far-UV circular dichroism (CD) and near-UV CD were not very informative regarding conformational changes between the alkylated and unmodified forms. Two complementary MS-based techniques, analysis of ionic charge state distribution and hydrogen/deuterium exchange (HDX), were used to monitor conformational changes.¹⁹ The analysis of the ionic charge distributions indicated a decrease in conformational stability in the alkylated form; the partial unfolding was revealed by the presence of protein ions in the ESI mass spectra with significantly higher charge density compared to the unmodified version. In HDX MS, measurements can be carried out under conditions closely mimicking the formulation buffer, and thermodynamic information is derived from biophysical measurements. Global HDX MS revealed higher flexibility of alkylated IFN. Backbone flexibility was observed to be distributed unevenly across the polypeptide sequence. Structural studies suggest that the loss of antiviral activity of the alkylated form is due to destabilization of a region of IFN that binds with its low affinity receptor (IFNAR1), and disruption of ternary complex formation.Dr. Kaltashov concluded by suggesting that ESI MS can be used to characterize highly heterogeneous systems and presented findings from a study of heparin, which is very heterogeneous, and difficult to characterize by MS. With colleagues, he has developing a mass spectrometry-based strategy for characterization of anti-thrombin interaction with low molecular weight heparin and heparin oligomers.²⁰Genentech''s use of high throughput (HTP) methods in bioprocess development was discussed by Judy Chou (Genentech). She described analytical methods as the ears and eyes of the production process. Use of a high throughput platform is directly related to the need for rapid analysis of bioprocess samples. The need for speedy analysis, which enables new products to get to patients in a timely fashion, has to be balanced with the need for extensive sample characterization that might be time-consuming. The aim is to leverage new technology, especially in HTP purification and automation to increase the number of experiments while reducing resources required and shortening timelines. The HTP approach involves scaling down cell culture, use of protein A well-plate purification (PAWP), purification in plate (PiP), HTP impurity analysis and an at-line reverse phase high performance liquid chromatography (RP-HPLC) method.In the PAWP method, 24- or 96-well plates with protein A are used. The plates can be subjected to centrifugation or vacuum procedures, then samples are directly analyzed by HPLC, liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CE), or image capillary isoelectric focusing (icIEF) techniques. The method allows product quality tests to be expedited, and allows the company to address product quality issues early on and reduce resource and time cost later. Use of the PAWP method was directly compared with use of a standard purification procedure (protein A column). Samples subjected to both methods gave similar results in an array of tests (SEC IEC, CZE, icIEF analysis, CE-Glycan assay, peptide mapping).RP-HPLC rapid monitoring can be used as a fast method to monitor mAb fragments in both the purified samples and the cell culture fluids without any sample preparation procedure. It provides a powerful tool to look into antibody reduction issues and helps to monitor as well as to develop bioprocess to mitigate the risk of losing product quantity and quality. Furthermore, the on-line RP-HPLC-Mass Spectroscopy (MS) enabled the understanding of the new peaks identified in the cell culture fluids and increased the process knowledge during the development and operation phases.HTP impurity assays were also developed as a tool to quickly narrow down purification conditions. A CHO protein Meso Scale Discovery (MDS) impurity assay that utilizes electrochemiluminescence was incorporated in an HTP process. Plates can be prepared and stored for up to nine months. The MSD assay was compared to ELISA at various purification steps and the difference was found to be less than 15%, whereas only 10% of resources are used to perform assay and the total assay time was only 2.5 hours for ∼400 samples. In addition, a leached Protein A HTP assay based on MSD technology was also developed. In order to prevent the signal masking introduced by the products, a novel approach of acidification combined with effective blockers was implemented. The new method is generic to all the molecules tested so far and only takes three hours for ∼400 samples with limited amount of resources needed.A novel TEACAN system that puts all the relevant analytical assays as well as the PiP and High throughput Formulation development in an assembly line is currently being used. Dr. Chou mentioned that the details of the methods she described will be published soon.     

A newborn child with karyotype 47,XX,+der(12) (12pter→12q12::8q24→8qter),t(8;12) (q24;q12) pat

Summary A case of Meckel or Gruber syndrome is reported, together with a survey of the relevant literature of recent years (1971–1977), in reference to a probably autosomal recessive inheritance of this malformation.     

Margaret C. Etter (Wilmington,DE: September 12, 1943 – Minneapolis,MN: June 10, 1992)

In Memoriam

Margaret C. Etter (Wilmington, DE: September 12, 1943 – Minneapolis, MN: June 10, 1992)     

Proceedings of the XVIII Congress of the International Primatological Society Adelaide,Australia, January 7–12, 2001

International Journal of Primatology -     

IS HEALTH,MEASURED BY WORK ABILITY INDEX,AFFECTED BY 12-HOUR ROTATING SHIFT SCHEDULES?

Two forms of continuously forward rotating 12-h shift schedules exist at BASF's Ludwigshafen site. These shift schedules were compared with a daytime working system to investigate potential differential effects on employee's health status assessed with the Work Ability Index (WAI). In the 3?×?12 system, a 12-h day shift is followed 24?h later by a 12-h night shift, and after a day off the employee returns to the day shift. The 4?×?12 schedule follows the same pattern except that there are 2 days off between the night and next day shift. A total of 924 participants (278 3?×?12 and 321 4?×?12 shiftworkers and 325 day workers) were recruited. A self-administered questionnaire was used to obtain information about shiftwork schedule, demographic characteristics, and lifestyle and social factors, and the WAI was applied. The outcomes of interest were the WAI sum score and its seven dimensions. In examining the relationship with the WAI categories, a Proportional Odds Model (POM) was used to identify the potential determinants. Logistic regression models were used to estimate the impact of age on single dimensions of WAI after adjustment for potential confounding factors. Increasing age and obesity (BMI?≥?30) were the only significant determinants of poorer WAI. Although a positive association was found linking the second WAI dimension (work ability in relation to job demands) with age, an inverse association was demonstrated consistently between age and the third and fourth WAI dimensions, i.e., number of diagnosed diseases and estimated work impairment due to disease, after adjustment for potential confounders. The age-dependency was moderate overall, but seemed to be stronger among shift- than day workers, although this difference did not reach statistical significance. There was no significant differential impact of the working time systems on the WAI sum score or on the individual WAI dimensions. Thus, there is no indication of an excessive adverse health impact of these shift schedules compared to day work, to the extent that health can be measured by the WAI. (Author correspondence: mei.yong@basf.com)