Development of a scale down cell culture model using multivariate analysis as a qualification tool

In characterizing a cell culture process to support regulatory activities such as process validation and Quality by Design, developing a representative scale down model for design space definition is of great importance. The manufacturing bioreactor should ideally reproduce bench scale performance with respect to all measurable parameters. However, due to intrinsic geometric differences between scales, process performance at manufacturing scale often varies from bench scale performance, typically exhibiting differences in parameters such as cell growth, protein productivity, and/or dissolved carbon dioxide concentration. Here, we describe a case study in which a bench scale cell culture process model is developed to mimic historical manufacturing scale performance for a late stage CHO‐based monoclonal antibody program. Using multivariate analysis (MVA) as primary data analysis tool in addition to traditional univariate analysis techniques to identify gaps between scales, process adjustments were implemented at bench scale resulting in an improved scale down cell culture process model. Finally we propose an approach for small scale model qualification including three main aspects: MVA, comparison of key physiological rates, and comparison of product quality attributes. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:152–160, 2014     

Performance of a novel Viresolve NFR virus filter

Mammalian cell-expressed therapeutic proteins are particularly vulnerable to contamination by endogenous retrovirus-like particles (RVLPs). The Viresolve NFR filter was designed to meet the critical requirement of manufacturing a safe and virus-free therapeutic by retaining RVLPs by a minimum of six log reduction value (LRV). The NFR designation refers to retrovirus removal in a normal flow format. To qualify the product, we tested two model viruses: the 78 nm diameter phi6 bacteriophage and the 80-110 nm diameter Xenotropic Murine Leukemia Virus (X-MuLV). Robust retention was demonstrated over a wide range of process parameters. Viresolve NFR filters also retain other model adventitious viruses including 70-85 nm diameter Reovirus 3 (Reo3), 70-90 nm diameter Adenovirus 2 (Ad2), and 53 nm diameter PR772 by >6 LRV. In addition to these model viruses, the filter retains >7 LRV of both the mycoplasma Acholeplasma laidlawii and the bacterium Brevundimonas diminuta. Protein passage is shown to be consistently high (95-100%) for a variety of therapeutic protein products, including monoclonal antibodies. Characterization of the filter in specific applications is made simple by availability of ultralow surface area (5 cm(2)) disks, which are shown to scale linearly to the manufacturing scale pleated-filters. Viresolve NFR filters provide consistent water permeability performance (34-37 LMH/psi) and show very little plugging for all feedstocks evaluated. The Viresolve NFR filter incorporates Retropore, a unique asymmetric polyethersulfone membrane, the surface of which has been modified to minimize protein binding.     

Qualification of a novel inline spiking method for virus filter validation

Virus filters are widely used in bioprocessing to reduce the risk of virus contamination in therapeutics. The small pores required to retain viruses are sensitive to plugging by trace contaminants and frequently require inline adsorptive prefiltration. Virus spiking studies are required to demonstrate virus removal capabilities of the virus filter using scale down filters. If prefiltration removes viruses and interferes with the measurement of virus filter LRV, the standard approach is to batch prefilter the protein solution, spike with virus, and then virus filter. For a number of proteins, batch prefiltration leads to increased plugging and significantly lower throughputs than inline prefiltration. A novel inline spiking method was developed to overcome this problem. This method allows the use of inline prefiltration with direct measurement of virus filter removal capabilities. The equipment and its operation are described. The method was tested with three different protein feeds, two different parvovirus filters, two virus injection rates; a salt spike, a bacteriophage spike, and two mammalian virus spikes: MMV and xMuLV. The novel inline method can reliably measure LRV at throughputs representative of the manufacturing process. It is recommended for applications where prefiltration is needed to improve throughput, prefiltration significantly reduces virus titer, and virus filter throughput is significantly reduced using batch vs. inline prefiltration. It can even help for the case where the virus preparation causes premature plugging.     

Field applications of a bio-trickling filter for the removal of nitrogen oxides from flue gas

A bio-trickling filter (BTF) packed with polyhedral spheres was used to remove nitrogen oxides (NOx) from the flue gas of a coal-fired power plant. The BTF system consistently removed 64–95% of the NOx after start-up and acclimation under dynamic conditions (e.g., 120–240 m³/h flue gas flow rate and inlet 300–900 mg NOx/m³). Scanning electron microscopy of the biofilms that were formed showed a shift in the predominating bacteria. Analyses by PCR-denaturing gradient gel electrophoresis showed that the naturally-selected mixed cultures in the biofilm under a flue gas environment were mainly Klebsiella sp. and Pseudomonas sp.     

Development of a novel polycationic adsorbent for cryogel removal.

Cryogel, prevalent in the plasma of rheumatism patients, is a plasma fibronectin (pFN)-extra domain A containing FN (EDA(+)FN)-fibrinogen (Fbg) complex formed by adding heparin (HP) at a low temperature (4 degrees C). Although EDA(+)FN does not usually exist in normal plasma, its prevalence in rheumatic patients causes cryogelation in plasma. Removal of cryogel is thus a promising and novel approach to treating rheumatism. As HP-EDA(+)FN aggregate, which is induced by the main component of cryogel, is considered to be an anion, cationic materials capable of eliminating this anionic conjugate were innovated in this study. We found that an amino group density of 100-130 micromol/g (dry weight) of adsorbents prompted selective adsorption of the EDA(+)FN-HP complex. Elimination of EDA(+)FN as high as 80% accompanied by removal of the components of total FN (pFN) (10%) and Fbg (10%) in the model patient plasma was established.     

Effect of antibody solution conditions on filter performance for virus removal filter Planova™ 20N

We investigated the effect of antibody solution conditions (ionic strength, pH, IgG concentration, buffer composition, and aggregate level (dimer content)) on filter performance for a virus removal filtration process using the Planova? 20N, a virus removal filter. Ionic strength and pH affected the filter flux. A consistent high flux was maintained at an ionic strength greater than 10 mM and at pH 4–8 under a typical buffer composition (sodium chloride, citrate, acetate, and phosphate). Optimum IgG concentration was 10–20 mg/mL allowing for high throughput (kg/m² of IgG). Dimer content negligibly affected the flux level. Under high throughput conditions, virus spiking did not affect flux whereas a parvovirus logarithmic reduction value greater than 5 was maintained. From the results of zeta potential analyses for IgG and the membrane, we considered that electrostatic interactions between antibodies and the membrane affect filter performance (flux level and throughput). These results indicate that the Planova? 20N filter is applicable for a wide range of solution conditions typically used in antibody processing. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Albumin and mammalian cell culture: implications for biotechnology applications

Albumin has a long historical involvement in design of media for the successful culture of mammalian cells, in both the research and commercial fields. The potential application of albumins, bovine or human serum albumin, for cell culture is a by-product of the physico-chemical, biochemical and cell-specific properties of the molecule. In this review an analysis of these features of albumin leads to a consideration of the extracellular and intracellular actions of the molecule, and importantly the role of its interactions with numerous ligands or bioactive factors that influence the growth of cells in culture: these include hormones, growth factors, lipids, amino acids, metal ions, reactive oxygen and nitrogen species to name a few. The interaction of albumin with the cell in relation to these co-factors has a potential impact on metabolic and biosynthetic activity, cell proliferation and survival. Application of this knowledge to improve the performance in manufacturing biotechnology and in the emerging uses of cell culture for tissue engineering and stem cell derived therapies is an important prospect.     

Silicon substrate as a novel cell culture device for myoblast cells

Background

Tissue and organ regeneration via transplantation of cell bodies in-situ has become an interesting strategy in regenerative medicine. Developments of cell carriers to systematically deliver cell bodies in the damage site have fall shorten on effectively meet this purpose due to inappropriate release control. Thus, there is still need of novel substrate to achieve targeted cell delivery with appropriate vehicles. In the present study, silicon based photovoltaic (PV) devices are used as a cell culturing substrate for the expansion of myoblast mouse cell (C2C12 cells) that offers an atmosphere for regular cell growth in vitro. The adherence, viability and proliferation of the cells on the silicon surface were examined by direct cell counting and fluorescence microscopy.

Results

It was found that on the silicon surface, cells proliferated over 7 days showing normal morphology, and expressed their biological activities. Cell culture on silicon substrate reveals their attachment and proliferation over the surface of the PV device. After first day of culture, cell viability was 88% and cell survival remained above 86% as compared to the seeding day after the seventh day. Furthermore, the DAPI staining revealed that the initially scattered cells were able to eventually build a cellular monolayer on top of the silicon substrate.

Conclusions

This study explored the biological applications of silicon based PV devices, demonstrating its biocompatibility properties and found useful for culture of cells on porous 2-D surface. The incorporation of silicon substrate has been efficaciously revealed as a potential cell carrier or vehicle in cell growth technology, allowing for their use in cell based gene therapy, tissue engineering, and therapeutic angiogenesis.     

Development of a novel compact sonicator for cell disruption

Ultrasound microbial cell disrupters operating at around 20 kHz are often physically large and, due to significant heating, can be unsuitable for small sample volumes where biochemical integrity of the extracted product is required. Development of a compact device based on a 63.5-mm diameter, 6.5-mm thick tubular transducer for rapid cell disruption in small-volume samples in a high-intensity acoustic cavitation field with minimal temperature rises is described here. Suspensions of Saccharomyces cerevisiae were exposed to cavitation for various times in the compact device and a 20-kHz probe sonicator. Cell disruption was assessed by protein release and by staining. Yeast cell disruption was greater in the novel 267-kHz sonicator than in the 20-kHz probe sonicator for the same exposure time. A 1-dimensional (1-D) transfer matrix model analysis for piezoelectric resonators was applied to an axial cross-section of the tubular sonicator to predict frequencies of mechanical resonance in the sample volume associated with maximum acoustic pressure. Admittance measurements identified frequencies of electrical resonance. Ultrasonic cavitation noise peaks were detected by a hydrophone at both the mechanical and electrical resonances. Cell breakage efficiency was twice as great in terms of protein released per dissipated watt at the mechanical resonance predicted by the model, compared to those at the electrical resonance frequencies. The results form a basis for rational design of an ultrasound cell disruption technique for small-volume samples.     

Optimization of an acoustic cell filter with a novel air-backflush system

Increasing worldwide demand for mammalian cell production capacity will likely be partially satisfied by a greater use of higher volumetric productivity perfusion processes. An important additional component of any perfusion system is the cell retention device that can be based on filtration, sedimentation, and/or acoustic technologies. A common concern with these systems is that pumping and transient exposure to suboptimal medium conditions may damage the cells or influence the product quality. A novel air-backflush mode of operating an acoustic cell separator was developed in which an injection of bioreactor air downstream of the separator periodically returned the captured cells to the reactor, allowing separation to resume within 20 s. This mode of operation eliminated the need to pump the cells and allows the selection of a residence time in the separator depending on the sensitivity of the cell line. The air-backflush mode of operating a 10L acoustic separator was systematically tested at 10(7) cells/mL to define reliable ranges of operation. Consistent separation performance was obtained for wide ranges of cooling airflow rates from 0 to 15 L/min and for backflush frequencies between 10 and 40 h(-1). The separator performance was optimized at a perfusion rate of 10 L/day to obtain a maximum separation efficiency of 92 +/- 0.3%. This was achieved by increasing the power setting to 8 W and using duty cycle stop and run times of 4.5 and 45 s, respectively. Acoustic cell separation with air backflush was successfully applied over a 110 day CHO cell perfusion culture at 10(7) cells/mL and 95% viability.     

Packed-bed bioreactors for mammalian cell culture: bioprocess and biomedical applications

This article describes the development history of packed-bed bioreactors (PBRs) used for the culture of mammalian cells. It further reviews the current applications of PBRs and discusses the steps forward in the development of these systems for bioprocess and biomedical applications. The latest generation of PBRs used in bioprocess applications achieve very high cell densities (>10(8) cells ml(-1)) leading to outstandingly high volumetric productivity. However, a major bottleneck of such PBRs is their relatively small volume. The current maximal volume appears to be in the range of 10 to 30 l. A scale-up of more than 10-fold would be necessary for these PBRs to be used in production processes. In biomedical applications, PBRs have proved themselves as compact bioartificial organs, but their metabolic activity declines frequently within 1 to 2 weeks of operation. A main challenge in this field is to develop cell lines that grow consistently to high cell density in vitro and maintain a stable phenotype for a minimum of 1 to 2 months. Achieving this will greatly enhance the usefulness of PBR technology in clinical practice.     

Preparation of 3D fibrin scaffolds for stem cell culture applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue. A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside. This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach.     

戊型肝炎病毒细胞培养模型的研究进展

戊型肝炎病毒（ HEV）体外细胞培养模型的建立有助于阐明HEV的感染、复制及其致病机理,有利于新型抗病毒药物和疫苗的研发与评估。近年来国内外对HEV细胞培养模型的报道很多,但所用细胞系、感染方法及病毒的复制效果等不尽相同。因此,对近年来HEV细胞培养模型的研究进展进行了综述。     

Selective removal of ammonia from animal cell culture broth

Serum-free perfusion cultures of hybridoma TO-405 cells were carried out in spinner flasks coupled with zeolite A-3 packed beads. Ammonia was selectively removed from the culture broth by passing cell free permeate from ceramic cross flow filtration, through the zeolite packed bed. Ammonia concentration in the culture broth was effectively maintained between 1 to 4 mmol/l which was below the inhibitory concentration for cell growth. Maximum cell density levels of 10⁷ cells/ml as well as improved percentage cell viability higher than in serum-supplemented cultures were feasible in this system.The possible effects of shear stress, generated by variation of the flow rates of the broth through the ceramic filter module, on the growth of the hybridoma cells were investigated. Backwashing, by reversing the direction of the permeate, was found necessary to prolong the life of the filter. Variation of the flow rates of the broth through the ceramic module between 0.29 m/s to 0.59 m/s did not cause immediate cell damage but growth was repressed at the higher flow rate.This study also showed that glutamine appears to be one of the factors limiting the growth of the hybridoma cells.