Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.     

Comparative genetic analysis of grayling (Thymallus spp. Salmonidae) across the paleohydrologically dynamic river drainages of the Altai-Sayan mountain region

Hydrobiologia - A high number of grayling (Thymallus) species have been described from the Altai-Sayan mountain region, for which little to no genetic information is available. We investigated...     

FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.     

Loss of interleukin-10 exacerbates early Type-1 diabetes-induced bone loss

Type-1 diabetes (T1D) increases systemic inflammation, bone loss, and risk for bone fractures. Levels of the anti-inflammatory cytokine interleukin-10 (IL-10) are decreased in T1D, however their role in T1D-induced osteoporosis is unknown. To address this, diabetes was induced in male IL-10 knockout (KO) and wild-type (WT) mice. Analyses of femur and vertebral trabecular bone volume fraction identified bone loss in T1D-WT mice at 4 and 12 weeks, which in T1D-IL-10-KO mice was further reduced at 4 weeks but not 12 weeks. IL-10 deficiency also increased the negative effects of T1D on cortical bone. Osteoblast marker osterix was decreased, while osteoclast markers were unchanged, suggesting that IL-10 promotes anabolic processes. MC3T3-E1 osteoblasts cultured under high glucose conditions displayed a decrease in osterix which was prevented by addition of IL-10. Taken together, our results suggest that IL-10 is important for promoting osteoblast maturation and reducing bone loss during early stages of T1D.     

Human immune response against salivary antigens of Simulium damnosum s.l.: A new epidemiological marker for exposure to blackfly bites in onchocerciasis endemic areas

BackgroundSimulium damnosum sensu lato (s.l.) blackflies transmit Onchocerca volvulus, a filarial nematode that causes human onchocerciasis. Human landing catches (HLCs) is currently the sole method used to estimate blackfly biting rates but is labour-intensive and questionable on ethical grounds. A potential alternative is to measure host antibodies to vector saliva deposited during bloodfeeding. In this study, immunoassays to quantify human antibody responses to S. damnosum s.l. saliva were developed, and the salivary proteome of S. damnosum s.l. was investigated.Methodology/Principal findingsBlood samples from people living in onchocerciasis-endemic areas in Ghana were collected during the wet season; samples from people living in Accra, a blackfly-free area, were considered negative controls and compared to samples from blackfly-free locations in Sudan. Blackflies were collected by HLCs and dissected to extract their salivary glands. An ELISA measuring anti-S. damnosum s.l. salivary IgG and IgM was optimized and used to quantify the humoral immune response of 958 individuals. Both immunoassays differentiated negative controls from endemic participants. Salivary proteins were separated by gel-electrophoresis, and antigenic proteins visualized by immunoblot. Liquid chromatography mass spectrometry (LC–MS/MS) was performed to characterize the proteome of S. damnosum s.l. salivary glands. Several antigenic proteins were recognized, with the major ones located around 15 and 40 kDa. LC–MS/MS identified the presence of antigen 5-related protein, apyrase/nucleotidase, and hyaluronidase.Conclusions/SignificanceThis study validated for the first time human immunoassays that quantify humoral immune responses as potential markers of exposure to blackfly bites. These assays have the potential to facilitate understanding patterns of exposure as well as evaluating the impact of vector control on biting rates. Future studies need to investigate seasonal fluctuations of these antibody responses, potential cross-reactions with other bloodsucking arthropods, and thoroughly identify the most immunogenic proteins.     

The effects of pesticides on bacterial nitrogen fixers in peanut-growing area

In the peanut production, the applications of herbicides and fungicides are a common practice. In this work, studies done under field conditions demonstrated that pesticides affected negatively the number and nitrogenase activity of diazotrophic populations of soil. Agrochemical effects were not transient, since these parameters were not recovered to pre-treatment levels even 1 year after pesticides application. Results obtained from greenhouse experiments revealed that the addition of herbicide or fungicides diminished the free-living diazotrophs number reaching levels found in soil amended with the pesticides and that the number of symbiotic diazotrophs was not affected by the insecticide assayed. The soil nitrogenase activity was not affected by fungicides and glyphosate. The effect of pesticides on the nitrogen-fixing bacteria diversity was evaluated both in field and greenhouse experiments. Analysis of clone libraries generated from the amplification of soil nifH gene showed a diminution in the genetic diversity of this bacterial community.     

Oxidized albumin. The long way of a protein of uncertain function

Background

Proteins are extremely reactive to oxidants and should represent a potential target of instable reactive oxygen. This may represent a problem for plasma proteins since they may be directly modified in vivo in a compartment where antioxidant enzymatic systems are scarcely represented. On the other hand, it is possible that some plasma components have evolved over time to guarantee protection, in which case they can be considered as anti-oxidants.

Scope of review

To present and discuss main studies which addressed the role of albumin in plasma antioxidant activity mainly utilizing in vitro models of oxidation. To present some advances on structural features of oxidized albumin deriving from studies carried out on in vitro models as well as albumin purified in vivo from patients affected by clinical conditions characterized by oxidative stress.

Major conclusions

There are different interaction with HOCl and chloramines. In the former case, HOCl produces an extensive alteration of ²³⁸Trp and ¹⁶²Tyr, ⁴²⁵Tyr, ⁴⁷Tyr, while thiol groups are only partially involved. Chloramines are extremely reactive with the unique free SH group of albumin (³⁴Cys) with the formation of sulfenic and sulfinic acid as intermediates and sulfonic acid as end-product. Oxidized albumin has a modified electrical charge for the addition of an acidic residue and presents α-helix and random coil reorganization with subtle changes in domain orientation.

General significance

Albumin, is the major antioxidants in plasma with a concentration (0.8 mM) higher than other antioxidants by an exponential factor. Functional and protective roles in the presence of oxidative stress must be defined. This article is part of a Special Issue entitled Serum Albumin.     

Development of a universal radioactive DNA methyltransferase inhibition test for high-throughput screening and mechanistic studies

DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying new small inhibitors of C5 DNA methyltransferases (DNMTs). Despite the development of numerous in vitro DNMT assays, there is a lack of reliable tests suitable for high-throughput screening, which can also give insights into inhibitor mechanisms of action. We developed a new test based on scintillation proximity assay meeting these requirements. After optimizing our assay on human DNMT1 and calibrating it with two known inhibitors, we carried out S-Adenosyl-l-Methionine and DNA competition studies on three inhibitors and were able to determine each mechanism of action. Finally, we showed that our test was applicable to 3 other methyltransferases sources: human DNMT3A, bacterial M.SssI and cellular extracts as well.