The Increasingly Human and Profitable Monoclonal Antibody Market.

The monoclonal antibody (mAb) market has changed rapidly in the past 5 years: it has doubled in size, becoming dominated by fully human molecules, launched bispecific molecules, and faced competition from biosimilars. We summarize the market in terms of therapeutic applications, type and structure of mAbs, dominant companies, manufacturing locations, and emerging markets.

On the model-based optimization of secreting mammalian cell (GS-NS0) cultures.

The global bio-manufacturing industry requires improved process efficiency to satisfy the increasing demands for biochemicals, biofuels, and biologics. The use of model-based techniques can facilitate the reduction of unnecessary experimentation and reduce labor and operating costs by identifying the most informative experiments and providing strategies to optimize the bioprocess at hand. Herein, we investigate the potential of a research methodology that combines model development, parameter estimation, global sensitivity analysis, and selection of optimal feeding policies via dynamic optimization methods to improve the efficiency of an industrially relevant bioprocess. Data from a set of batch experiments was used to estimate values for the parameters of an unstructured model describing monoclonal antibody (mAb) production in GS-NS0 cell cultures. Global Sensitivity Analysis (GSA) highlighted parameters with a strong effect on the model output and data from a fed-batch experiment were used to refine their estimated values. Model-based optimization was used to identify a feeding regime that maximized final mAb titer. An independent fed-batch experiment was conducted to validate both the results of the optimization and the predictive capabilities of the developed model. The successful integration of wet-lab experimentation and mathematical model development, analysis, and optimization represents a unique, novel, and interdisciplinary approach that addresses the complicated research and industrial problem of model-based optimization of cell based processes.

Chemotherapy drug scheduling for the induction treatment of patients with acute myeloid leukemia.

Leukemia is an immediately life-threatening cancer wherein immature blood cells are overproduced, accumulate in the bone marrow (BM) and blood and causes immune and blood system failure. Treatment with chemotherapy can be intensive or nonintensive and can also be life-threatening since only relatively few patient-specific and leukemia-specific factors are considered in current protocols. We have already presented a mathematical model for one intensive chemotherapy cycle with intravenous (i.v.) daunorubicin (DNR), and cytarabine (Ara-C). This model is now extended to nonintensive subcutaneous (SC) Ara-C and for a standard intensive chemotherapy course (four cycles), consistent with clinical practice. Model parameters mainly consist of physiological patient data, indicators of tumor burden and characteristics of cell cycle kinetics. A sensitivity analysis problem is solved and cell cycle parameters are identified to control treatment outcome. Simulation results using published cell cycle data from two acute myeloid leukemia patients are presented for a course of standard treatment using intensive and nonintensive protocols. The aim of remission-induction therapy is to debulk the tumor and achieve normal BM function; by treatment completion, the total leukemic population should be reduced to at most 10(9) cells, at which point BM hypoplasia is achieved. The normal cell number should be higher than that of the leukemic, and a 3-log reduction is the maximum permissible level of population reduction. This optimization problem is formulated and solved for the two patient case studies. The results clearly present the benefits from the use of optimization as an advisory tool for treatment design.

Computational modeling for the optimization of a cardiogenic 3D bioprocess of encapsulated embryonic stem cells.

We present a computational fluid dynamics (CFD)-based model aimed at the identification of optimized culture conditions promoting efficient cardiogenesis of hydrogel-bead-encapsulated embryonic stem cells (ESCs) within a rotating bioreactor. The numerical approach, integrating diffusion, convection, and multiphase fluid dynamics calculations, allowed to evaluate (i) the microgravity motion of the floating beads, (ii) the O(2) delivery to the cells, also (iii) taking into account the cellular O(2) consumption, as a function of different rotation speeds of the breeding chamber. According to our results, a 25 rpm rotation (i) enhances an adequate mixing of the cell carriers, avoiding sedimentation and excessive packing, also maintaining a quite homogeneous distribution of the suspended beads and (ii) imparts a proper cellular O(2) supply, providing cells close to a normoxia condition. The bioreactor working conditions derived from the numerical analysis allowed the attainment of in vitro long-term cell viability maintenance, supporting efficient large-scale generation of ESC-derived cardiomyocytes (ESC-DCs) through a chemical-based conditioning bioprocess. In conclusion, we demonstrated the feasibility of using CFD-based tools, as a reliable and cost-effective strategy to assist the design of a 3D cardiogenic bioprocess.

The adverse skeletal effects of selective serotonin reuptake inhibitors.

Selective serotonin reuptake inhibitors (SSRIs) are a widely used group of antidepressants (ADs) with reported potential detrimental effects on bone mineral density (BMD) and increased fracture risk. Here, a comprehensive review of the in vitro, in vivo and clinical studies to date was carried out using the medical search engines MEDLINE (1950 to September 2010) and EMBASE (1980 to September 2010). Serotonin (5-HT) receptors have been identified on osteoclast, osteoblast and osteocyte cell lines. The effect of SSRIs on bone formation and resorption appears to be governed by the activation of a number of 5-HT receptors on osteoblasts and osteoclasts via endocrine, autocrine/paracrine and neuronal pathways. In vitro, in vivo and clinical collective data appears to indicate that SSRIs have a negative effect on bone at the therapeutic dose levels widely used for the treatment of depression in current clinical practice. Caution may therefore have to be employed with the use of SSRIs in patients at an increased risk of falls and osteoporosis. Further studies are needed in order to fully elicit the role of SSRIs in bone formation and their effects in the low oestrogen state.

A real-time multi-channel monitoring system for stem cell culture process.

A novel, up to 128 channels, multi-parametric physiological measurement system suitable for monitoring hematopoietic stem cell culture processes and cell cultures in general is presented in this paper. The system aims to measure in real-time the most important physical and chemical culture parameters of hematopoietic stem cells, including physicochemical parameters, nutrients, and metabolites, in a long-term culture process. The overarching scope of this research effort is to control and optimize the whole bioprocess by means of the acquisition of real-time quantitative physiological information from the culture. The system is designed in a modular manner. Each hardware module can operate as an independent gain programmable, level shift adjustable, 16 channel data acquisition system specific to a sensor type. Up to eight such data acquisition modules can be combined and connected to the host PC to realize the whole system hardware. The control of data acquisition and the subsequent management of data is performed by the system's software which is coded in LabVIEW. Preliminary experimental results presented here show that the system not only has the ability to interface to various types of sensors allowing the monitoring of different types of culture parameters. Moreover, it can capture dynamic variations of culture parameters by means of real-time multi-channel measurements thus providing additional information on both temporal and spatial profiles of these parameters within a bioreactor. The system is by no means constrained in the hematopoietic stem cell culture field only. It is suitable for cell growth monitoring applications in general.

Evidence of species succession during chlorobenzene biodegradation.

We have previously reported the disappearance of a specific strain degrading chlorobenzene from a functionally stable bioreactor. In the present work, we investigated this species succession and isolated a new dominant strain, identified as Pandoraea pnomenusa sp. strain MCB032. A specific 16S rRNA-targeted oligonucleotide probe was designed and validated to identify strain MCB032 using fluorescence in situ hybridisation (FISH). The results confirmed the presence of strain MCB032 in samples collected over time, and showed that it was primarily located within the biofilm. Denaturing gradient gel electrophoresis (DGGE) provided evidence that the species succession occurred early in the operating period. The application of these biomolecular tools highlighted the remarkable stability of this new strain during the 15 months of reactor operation. The succession was attributed to the competitive kinetic behaviour of strain MCB032, which exhibited faster growth (micro(max) = 0.34 h(-1)) and higher substrate affinity (K(s) = 0.35 mg L(-1)) than strain JS150. Finally, this study contributed to the characterisation of the recently established Pandoraea genus, an emerging group in the biodegradation field.

Strain stability in biological systems treating recalcitrant organic compounds.

The availability of molecular probing technology in recent years has facilitated investigation of microbial community composition during bio-treatment of organic wastes. Particularly, it has allowed the study of microbial culture stability and correlation between stability and treatment performance. However, most studies to date have only addressed mixed cultures and there is limited information regarding single strain stability. Here we have investigated the microbial community dynamics in two bioreactors, each inoculated with a pure bacterial strain capable of degrading a recalcitrant substrate, namely Xanthobacter aut. GJ10 degrading 1,2-dichloroethane (DCE) and Burkholderia sp. JS150 degrading monochlorobenzene (MCB). Universal and strain specific 16S rRNA oligonucleotide probes were designed and used to follow strain stability. The bioreactor fed with DCE was functionally stable and the percentage of GJ10 cells in the community remained high (around 95% of total cells) throughout, even after introduction of foreign microorganisms. The bioreactor fed with MCB was also functionally stable, but in contrast to the DCE bioreactor, probing results revealed the disappearance of strain JS150 from the bioreactor within a week. The difference in behavior between the two systems is attributed to the specific pathway required to degrade DCE.

Modelling of Mammalian cells and cell culture processes.

Mammalian cell cultures represent the major source for a number of very high-value biopharmaceutical products, including monoclonal antibodies (MAbs), viral vaccines, and hormones. These products are produced in relatively small quantities due to the highly specialised culture conditions and their susceptibility to either reduced productivity or cell death as a result of slight deviations in the culture conditions. The use of mathematical relationships to characterise distinct parts of the physiological behaviour of mammalian cells and the systematic integration of this information into a coherent, predictive model, which can be used for simulation, optimisation, and control purposes would contribute to efforts to increase productivity and control product quality. Models can also aid in the understanding and elucidation of underlying mechanisms and highlight the lack of accuracy or descriptive ability in parts of the model where experimental and simulated data cannot be reconciled. This paper reviews developments in the modelling of mammalian cell cultures in the last decade and proposes a future direction - the incorporation of genomic, proteomic, and metabolomic data, taking advantage of recent developments in these disciplines and thus improving model fidelity. Furthermore, with mammalian cell technology dependent on experiments for information, model-based experiment design is formally introduced, which when applied can result in the acquisition of more informative data from fewer experiments. This represents only part of a broader framework for model building and validation, which consists of three distinct stages: theoretical model assessment, model discrimination, and model precision, which provides a systematic strategy from assessing the identifiability and distinguishability of a set of competing models to improving the parameter precision of a final validated model.

A cDNA from human bone marrow encoding a protein exhibiting homology to the ATP1gamma1/PLM/MAT8 family of transmembrane proteins.

A cDNA clone, IWU-1, was cloned from human bone marrow. Its putative open reading frame encoded a protein of 115 amino acids with a calculated molecular mass of 12.9 kDa. The deduced amino acid sequence exhibited high homology (>68%) to members of the ATP1gamma1/PLM/MAT8 family of single transmembrane proteins, primarily in the region containing the putative transmembrane domain. The sequence at the amino-terminal side exhibited high homology (>61%) to the cytoplasmic region of the angiotensin II type 1 receptors.

Localization of androgen receptor expression in human bone marrow.

Androgens have been shown to modulate the haematopoietic and immune systems and have been used clinically for stimulating haematopoiesis in bone marrow failure conditions. To identify the bone marrow cell types as potential targets of androgens, an androgen receptor (AR)-specific antibody was used to localize the AR in normal human bone marrow biopsies. The results show that AR was ubiquitously expressed in the bone marrow of both males and females. Furthermore, the AR expression pattern did not change with age. Stromal cells, macrophages, endothelial cells, myeloblasts, myelocytes, neutrophils, and megakaryocytes expressed AR. In contrast, AR was not detected in the lymphoid and erythroid cells, or in eosinophils. These results indicate that androgens may exert direct modulating effects on a wide spectrum of bone marrow cell types via AR-mediated responses.

Membrane instability in late-stage erythropoiesis.

During maturation of the red blood cell (RBC) from the nucleated normoblast stage to the mature biconcave discocyte, both the structure and mechanical properties of the cell undergo radical changes. The development of the mechanical stability of the membrane reflects underlying changes in the organization of membrane-associated cytoskeletal proteins, and so provides an assessment of the time course of the development of membrane structural organization. Membrane stability in maturing erythrocytes was assessed by measuring forces required to form thin, tubular, lipid strands (tethers) from the surfaces of mononuclear cells obtained from fresh human marrow samples, marrow reticulocytes, circulating reticulocytes, and mature erythrocytes. Cells were biotinylated and manipulated with a micropipette to form an adhesive contact with a glass microcantilever, which gave a measure of the tethering force. The cell was withdrawn at controlled velocity and aspiration pressure to form a tether from the cell surface. The mean force required to form tethers from marrow reticulocytes and normoblasts was 27 +/- 9 pN, compared to 54 +/- 14 pN for mature cells. The energy of dissociation of the bilayer from the underlying skeleton increases 4-fold between the marrow reticulocyte stage and the mature cell, demonstrating that the mechanical stability of the membrane is not completely established until the very last stages of RBC maturation.

Expression of mPer1 and mPer2, two mammalian clock genes, in murine bone marrow.

Although circadian variations in hematopoiesis have been well documented, the molecular mechanism of the circadian rhythms remains elusive. To determine if a clock system exists in bone marrow to mediate the circadian rhythms, we analyzed the expression of mPer1 and mPer2, both mouse homologues of the Drosophila period gene and known regulators of the clock system, in murine bone marrow by relative quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We demonstrated that both genes were expressed in bone marrow. Furthermore, the expression patterns of mPer1 and mPer2 in total bone marrow cells exhibited two peaks over a 24-h period. In contrast, the expression patterns of these two genes in the Gr-1-positive cells isolated from bone marrow mainly contributed to one of the two peaks. These results indicate that a clock system exists in bone marrow and suggest that the circadian rhythms in bone marrow are lineage- and/or differentiation stage-dependent.

Engineering a human bone marrow model: a case study on ex vivo erythropoiesis.

Bone marrow, with its intricate, three-dimensional tissue structure facilitating cell-cell interactions, provides a microenvironment supporting the production of hundreds of billions of multilineal blood cells everyday. We have developed a three-dimensional bone marrow culture system in which marrow cells are cultured in a reactor packed with porous microspheres. The culture supports a three-dimensional growth configuration and multilineal hemopoiesis mimicking the bone marrow in vivo. We studied ex vivo human erythropoiesis using the three-dimensional culture system. The system sustained extensive erythropoiesis at low erythropoietin concentrations (0.2 U/mL), plus stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor, and insulin-like growth factor-I. Erythroid cell production lasted for more than 5 weeks, and the percentage of erythroid cells in the nonadherent cell population was approximately 60%. Flow cytometric analysis using cell surface markers specific for erythroid cells (CD71 and glycophorin-A) indicated that the culture produced early, intermediate, and late erythroid cells. As the culture progressed, the erythroid cell population shifted gradually toward mature cell types. When compared to the three-dimensional culture, the traditional flask cultures failed to support extensive erythropoiesis under the same conditions. This indicates that the three-dimensional bone marrow culture system provides a microenvironment conducive to erythropoiesis under more physiological conditions and is a better bone marrow model.