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Preparing Biotherapeutic Extracellular Vesicles: Ultrafilters, Mesenchymal Stem Cells, and the Regulatory Horizon

by Chandreyee Das, PhD
Volume 14, Issue 2 (Summer 2015)

Extracellular vesicles (EVs) are particles of varying size, structure, and composition, which are secreted from cells and frequently mediate intercellular communication. Because they have been shown to travel through the circulatory system and also through biological barriers to deliver their molecular contents to distant target cells, there has been growing interest in using EVs, such as exosomes, as drug delivery vehicles. In the past ten years, the number of published articles linking EVs to drug delivery has increased 20-fold. EVs are being engineered to deliver protein, RNA, and small molecule cargo to target cells, tissues, and entire systems. Also, EVs derived from certain cells show inherent, therapeutically beneficial activity. For example, EVs prepared from antigen-presenting cells can be used like vaccines to elicit a desired immune response from a host. The idea of using lipid-based carriers to deliver drugs is, of course, not new. Liposomes were first introduced as delivery vehicles in the 1960s, and some liposome-formulated drugs have made it to market. However, developers of liposome-based therapies are still trying to overcome the challenges of particle clearance by the immune system, immunogenicity, and the lack of specificity in targeting these particles to particular cell types. From this perspective, EVs are an attractive alternative to synthetic liposomes. They have already demonstrated stability in the circulatory system, and because of the proteins embedded in EV membranes, they reach target cells and are taken up more efficiently...

Citation:
Das C. Preparing biotherapeutic extracellular vesicles: ultrafilters, mesenchymal stem cells, and the regulatory horizon. BioProcess J, 2015; 14(2): 50–3. http://dx.doi.org/10.12665/J142.Das.

Posted online July 10, 2015.

 
Billion-Cell Hypoxic Expansion of Human Mesenchymal Stem Cells in BioBLU® 5c Single-Use Vessels

by Khandaker Siddiquee, PhD and Ma Sha, PhD
Volume 14, Issue 2 (Summer 2015)

Stem cell-based regenerative medicine has great potential to advance the therapeutic treatment of human diseases. Among the various stem cell platforms, mesenchymal stem cells (MSCs) represent one of the most promising options. Currently, there are over 500 clinical trials based on MSCs registered at the NIH’s ClinicalTrials.gov website. Although successful expansion of MSCs in vitro has been well-established, higher-yield, billion-cell expansion of MSCs remains a bottleneck. In this study, we successfully demonstrated large-scale culture of human adipose-derived mesenchymal stem cells (AdMSCs) in an industrial, single-use vessel at 3.75 L scale. The vessel offers a precision-controlled environment for the ideal growth of stem cells under simulated hypoxic physiological conditions. Stem cells and their culture media were monitored, analyzed, and controlled, allowing production of AdMSCs in substantial quantities. At the same time, the specific properties of the stem cell were maintained, as evidenced by stem cell marker assays and differentiation assays performed at the conclusion of the run. Finally, because all steps in the platform were conducted employing single-use consumables, this study demonstrates the fact that the process can be conveniently scaled up to industrial levels of production without having to rely on stainless steel culture facilities...

Citation:
Siddiquee K, Sha M. Billion-cell hypoxic expansion of human mesenchymal stem cells in BioBLU® 5c single-use vessels. BioProcess J, 2015; 14(2): 22–31. http://dx.doi.org/10.12665/J142.Sha.

Posted online July 1, 2015.

 
Design and Optimization of a Purification Process for MY32/Ls Protein Solubilizing Inclusion Bodies for a New Vaccine Against Sea Lice

by Carlos Perez Heredia, Nemecio González Fernández, Eladio Salazar Gómez, Eulogio Pimentel Vázquez, Yamila Carpio González, and Miladys Limonta Fernández
Volume 14, Issue 1 (Spring 2015)

Sea lice are the most problematic marine pathogens the salmon industry has to deal with, significantly affecting Europe and America. The worst offenders are genera: Pseudocaligus, Caligus, and Lepeophtheirus. Over €305 million in losses are estimated. Recent results have suggested that subolesin/akirin/myosin32 are good candidate antigens for the control of arthropod infestations such as sea lice. The aim of this study was to design and optimize the purification step of MY32/Ls protein to obtain the active pharmaceutical ingredient against sea lice. Non-chromatographic purification strategies were employed, based on published works, to establish rupture, washing, solubilization, and refolding conditions...

Citation:
Heredia CP, Fernández NG, Gómez ES, Vázquez EP, González YC, Fernández ML. Design and optimization of a purification process for MY32/Ls protein solubilizing inclusion bodies for a new vaccine against sea lice. BioProcess J, 2015; 14(1): 49–59. http://dx.doi.org/10.12665/J141.Heredia.

Posted online May 1, 2015.

 
Two-Step Purification of Antibody from Tobacco Plants for Vaccine Manufacturing: Aqueous Two-Phase Extraction and Affinity Chromatography

by Williams Ferro, Tatiana Álvarez, Déborah Geada, Yenisley Medina, Yarysel Guevara, José Montero, Andrés Tamayo, Ariadna López, Daily Hernández, Mayra Wood, Tatiana González, Regla Somoza, and Rodolfo Valdés
Volume 14, Issue 1 (Spring 2015)

The purification of PHB-01 plantibody derived from tobacco leaves imposed difficulties when the plantibody solid-liquid extraction design was performed. Thus, our study focused on assessing a combination of an aqueous two-phase extraction (ATPE) procedure and affinity chromatography for solving some of the issues in plantibody purification. This was done using a complete factorial redesign, different polyethylene glycol (PEG)/K2PO4 proportions, and pH values in each partitioning variant. Out of the results of 27 variants, ten were selected for the subsequent purification step, considering an antibody recovery of ≥ 80% and a leaf-soluble protein removal capacity of ≥ 30%. Regarding this, the best ATPE combination was PEG 6000 (20%)/K2PO4 (10%), pH 5.5, showing 95.44 ± 7.89% of antibody recovery and 32.86 ± 17.02% of leaf-soluble protein removal capacity. Besides, the organoleptic properties of ATPE preparation were similar to those observed in solid-liquid extraction preparation, but a reduction in operation unit number and bioprocessing time was demonstrated. Next, the antibody extracted in the three of ten selected variants was submitted to affinity chromatography for increasing molecule recovery and purity. The highest recovery was measured in PEG 4000 (10%)/K2PO4 (15%), pH 5.5, (92.69 ± 4.81%). Conversely, a significant decrease in antibody recovery (p = 0.000) was observed when antibody was purified after ATPE with PEG 6000 (ranging from 54.81 ± 17.05% to 65.37 ± 13.47%). The antibody electrophoretic mobility was unmodified by the addition of PEG in ATPE, and antibody purity measured by SDS-PAGE did not show significant differences (> 97%). These preliminary results allowed us to predict that a combination of ATPE and affinity chromatography could be useful for solving issues in industrial-scale PHB-01 purification...

Citation:
Ferro W et al. Two-step purification of antibody from tobacco plants for vaccine manufacturing: aqueous two-phase extraction and affinity chromatography. BioProcess J, 2015; 14(1): 43–8. http://dx.doi.org/10.12665/J141.Valdes.

Posted online May 1, 2015.

 
Virotherapy Process Optimization

by Michael Artinger, PhD
Volume 14, Issue 1 (Spring 2015)

An emerging application of viruses involves engineering them to treat diseases using a number of approaches. Broadly defined under the “virotherapy” umbrella, these include viral vectors used for gene therapy, oncolytic viruses, and viral immunotherapy. Although a majority of these products are in various stages of clinical development, the diversity of the therapeutic targets and wealth of future opportunities is encouraging. A significant challenge, as it is for any virus-based technology, is gaining a clear picture of the quality of a sample at any given point—from early research and development through manufacturing and product release. Of prime concern is the quantification of viruses, which in the past, has relied on slow, labor-intensive, subjective methods such as plaque titer assays and electron microscopic imaging. However, the diversity of new viral technologies now being used as the basis for innovative drugs and vaccines requires advanced, sophisticated analytical systems. In this white paper, we discuss how the real-time enumeration of viruses made possible by the ViroCyt® Virus Counter® 3100 can significantly enhance the pace of virotherapy product development...

Citation:
Artinger M. Virotherapy process optimization. BioProcess J, 2015; 14(1): 26–9. http://dx.doi.org/10.12665/J141.Artinger.

Posted online May 1, 2015.

 
Successful High Density Escherichia coli Fermentation Using the Eppendorf BioFlo® 320 Advanced Bioprocess Control System

by Bin Li, PhD and Ma Sha, PhD
Volume 14, Issue 1 (Spring 2015)

The gram-negative bacterium, Escherichia coli, has a long history in the world of laboratory and industrial processes due to its ease of manipulation and well-understood genome. It is widely cultured under aerobic conditions. High cell density cultivation of E. coli is a powerful technique for the production of recombinant proteins. Indeed, 30% of the FDA-approved biopharmaceuticals on the market are produced in E. coli. An Escherichia coli fermentation run conducted using the Eppendorf BioFlo® 320 bioprocess control station achieved high cell density at 12 hours, as determined by a maximum optical density (OD600) measurement of 215.2. The weights of dry and wet cells were also measured...

Citation:
Li B, Sha M. Successful high density Escherichia coli fermentation using the Eppendorf BioFlo® 320 advanced bioprocess control system. BioProcess J, 2015; 14(1): 20–4. http://dx.doi.org/10.12665/J141.LiSha.

Posted online May 1, 2015.

 
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