ME/CFS Patient advocate Patricia Carter was the source for finding the 2014 Nano-filter paper for vaccines information. She sent it to me and I researched and explained what the paper meant. I sent my detailed posting to researchers. Others wrote articles which came as result my e-mails sent. And after seeing the facebook postings on the our pages. Patricia Carter and I (from the ME/CFS community) are the original sources for finding and detailing this important information.
We have to ask why are they now looking to filter vaccines? We also know biologicals have not been filtered for decades.
Vaccine production using mammalian or insect cell cultures are emerging to overcome the limitations of traditional egg-based vaccines production systems. Applications in this therapeutic class includes the bioprocessing of
• Whole particle virus (animal and human)
• Virus-like Particles
https://www.repligen.com/applications/therapeutic-class/vaccines
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Original E-mail I sent:
A Size-Exclusion Nanocellulose Filter Paper for Virus Removal
"From the attached paper: Furthermore, robust and affordable virus removal filters are also demanded by the biotechnology industry as there are hundreds of products possessing the potential risk of viral infection, including cell-derived monoclonal antibodies, plasma-derived coagulation factors (e.g., factors VIII and IX) and immunoglobulins, or proteins manufactured with processes that employ viruses as biological expression systems, e.g., human or animal vaccines. [ 3] Absence of viral contamination further needs to be ensured for all types of therapeutic proteins, for example, human antithrombin III, derived from the milk of transgenic mammals. [ 4] Since cell cultures and explants are often cultivated in serum-derived media, both endogenous retroviruses and adventitious viral contaminants introduced during manufacturing also constitute a risk factor, for example, bovine viral diarrhoea virus, bovine parainfl uenza 3 virus (PI-3), parvoviruses. [ 3a, b] The threat of viral contamination of biotechnology products is manifold, including active human infection, for instance, human immunodefi ciency virus (HIV), hepatitis C (HVC), and enhanced oncogenic risk due to dormant infection, for example, avianleucosis virus. [ 3a, b] In addition, the advances in viral vectors of gene delivery to cure cancer stipulate the development of purifi cation techniques wherein the non-immunogenic virus particles can be effi ciently separated from proteins and other cell debris during production. [ 3c]"
A Size‐Exclusion Nanocellulose Filter Paper for Virus Removal
https://doi.org/10.1002/adhm.201300641
This swedish scientist is on both papers: Albert Mihranyan So the one that just came out and the ones on pdf. Here's where he's from:
Prof. A. Mihranyan
Division of Materials Science
Luleå University of Technology
971 87 Luleå, Sweden
Prof. A. Mihranyan
Nanotechnology and Functional Materials
Department of Engineering Sciences
Box 534, Uppsala University
75121 , Uppsala , Sweden
Citing Literature:
Number of times cited according to CrossRef: 56
Amir Sheikhi, Emerging Cellulose-Based Nanomaterials and Nanocomposites, Nanomaterials and Polymer Nanocomposites, 10.1016/B978-0-12-814615-6.00009-6, (307-351), (2019).
Crossref
Deepu A. Gopakumar, Shilpa Thomas, Owolabi F.A.T, Sabu Thomas, Ange Nzihou, Samsul Rizal and H.P.S. Abdul Khalil, Nanocellulose Based Aerogels for Varying Engineering Applications, Reference Module in Materials Science and Materials Engineering, 10.1016/B978-0-12-803581-8.10549-1, (2019).
Crossref
Mohammad E.T. Yazdi, Mohammad S. Amiri and Majid Darroudi, Biopolymers in the Synthesis of Different Nanostructures, Reference Module in Materials Science and Materials Engineering,10.1016/B978-0-12-803581-8.10560-0, (2019).
Crossref
Yi Li, David Stern, Lye Lin Lock, Jason Mills, Shih-Hao Ou, Marina Morrow, Xuankuo Xu, Sanchayita Ghose, Zheng Jian Li and Honggang Cui, Emerging Biomaterials for Downstream Manufacturing of Therapeutic Proteins, Acta Biomaterialia, 10.1016/j.actbio.2019.03.015, (2019).
Crossref
Andreas Mautner, Yosi Kwaw, Kathrin Weiland, Mlando Mvubu, Anton Botha, Maya Jacob John, Asanda Mtibe, Gilberto Siqueira and Alexander Bismarck, Natural fibre-nanocellulose composite filters for the removal of heavy metal ions from water, Industrial Crops and Products, 10.1016/j.indcrop.2019.03.032, 133, (325-332), (2019).
Crossref
Gui Ye, Zhiyong Yu, Yiming Li, Lei Li, Li Song, Li Gu and Xuebo Cao, Efficient treatment of brine wastewater through a flow-through technology integrating desalination and photocatalysis, Water Research, 10.1016/j.watres.2019.03.058, 157, (134-144), (2019).
Crossref
Levon Manukyan, Justine Padova and Albert Mihranyan, Virus removal filtration of chemically defined Chinese Hamster Ovary cells medium with nanocellulose-based size exclusion filter, Biologicals, 10.1016/j.biologicals.2019.03.001, (2019).
Crossref
Andrés Felipe Alzate Arbeláez, Eva Dorta Pérez, Camilo López-Alarcón, Farid B. Cortés and Benjamín A. Rojano, Immobilization of Andean berry (Vaccinium meridionale) polyphenols on nanocellulose isolated from banana residues: a natural food additive with antioxidant properties, Food Chemistry, 10.1016/j.foodchem.2019.05.085, (2019).
Crossref
K. Obi Reddy, C. Uma Maheswari, M.S. Dhlamini, B.M. Mothudi, V.P. Kommula, Jinming Zhang, Jun Zhang and A. Varada Rajulu, Extraction and characterization of cellulose single fibers from native african napier grass, Carbohydrate Polymers, 188, (85), (2018).
Crossref
Gergő P. Szekeres, Zoltán Németh, Krisztina Schrantz, Krisztián Németh, Mateusz Schabikowski, Jacqueline Traber, Wouter Pronk, Klára Hernádi and Thomas Graule, Copper-Coated Cellulose-Based Water Filters for Virus Retention, ACS Omega, 3, 1, (446), (2018).
Crossref
Olof Gustafsson, Levon Manukyan and Albert Mihranyan, High‐Performance Virus Removal Filter Paper for Drinking Water Purification, Global Challenges, 2, 7, (2018).
Wiley Online Library
Levon Manukyan, Pengfei Li, Simon Gustafsson and Albert Mihranyan, Growth Media Filtration Using Nanocellulose-based Virus Removal Filter for Upstream Biopharmaceutical Processing, Journal of Membrane Science, 10.1016/j.memsci.2018.11.002, (2018).
Crossref
Igor Rocha, Natalia Ferraz, Albert Mihranyan, Maria Strømme and Jonas Lindh, Sulfonated nanocellulose beads as potential immunosorbents, Cellulose, 10.1007/s10570-018-1661-2, 25, 3, (1899-1910), (2018).
Crossref
Guy-Alain Junter and Laurent Lebrun, Cellulose-based virus-retentive filters: a review, Reviews in Environmental Science and Bio/Technology, 10.1007/s11157-017-9434-1, 16, 3, (455-489), (2017).
Crossref
Jun Liu, Stefan Willför and Albert Mihranyan, On importance of impurities, potential leachables and extractables in algal nanocellulose for biomedical use, Carbohydrate Polymers,10.1016/j.carbpol.2017.05.002, 172, (11-19), (2017).
Crossref
Subrata Mondal, Preparation, properties and applications of nanocellulosic materials, Carbohydrate Polymers, 10.1016/j.carbpol.2016.12.050, 163, (301-316), (2017).
Crossref
Jeyabalan Sangeetha, Devarajan Thangadurai, Ravichandra Hospet, Prathima Purushotham, Kartheek Rajendra Manowade, Mohammed Abdul Mujeeb, Abhishek Channayya Mundaragi, Sudisha Jogaiah, Muniswamy David, Shivasharana Chandrabanda Thimmappa, Ram Prasad and Etigemane Ramappa Harish, Production of Bionanomaterials from Agricultural Wastes, Nanotechnology, 10.1007/978-981-10-4573-8_3, (33-58), (2017).
Crossref
Andreas Mautner, Thawanrat Kobkeatthawin and Alexander Bismarck, Efficient continuous removal of nitrates from water with cationic cellulose nanopaper membranes, Resource-Efficient Technologies, 10.1016/j.reffit.2017.01.005, 3, 1, (22-28), (2017).
Crossref
Simon Gustafsson, Levon Manukyan and Albert Mihranyan, Protein–Nanocellulose Interactions in Paper Filters for Advanced Separation Applications, Langmuir, 10.1021/acs.langmuir.7b00566, 33, 19, (4729-4736), (2017).
Crossref
Nathan Grishkewich, Nishil Mohammed, Juntao Tang and Kam Chiu Tam, Recent advances in the application of cellulose nanocrystals, Current Opinion in Colloid & Interface Science, 10.1016/j.cocis.2017.01.005, 29, (32-45), (2017).
Crossref
Ming-Guo Ma, Yan-Jun Liu and Yan-Yan Dong, Nanocellulose and Nanocellulose Composites: Synthesis, Characterization, and Potential Applications, Handbook of Composites from Renewable Materials, (109-134), (2017).
Wiley Online Library
Rudi Dungani, Abdul Khalil H.P.S., Nurjaman A. Sri Aprilia, Ihak Sumardi, Pingkan Aditiawati, Atmawi Darwis, Tati Karliati, Aminudin Sulaeman, Enih Rosamah and Medyan Riza, Bionanomaterial from agricultural waste and its application, Cellulose-Reinforced Nanofibre Composites, 10.1016/B978-0-08-100957-4.00003-6, (45-88), (2017).
Crossref
Kirubanandan Shanmugam, Swambabu Varanasi, Gil Garnier and Warren Batchelor, Rapid preparation of smooth nanocellulose films using spray coating, Cellulose, 10.1007/s10570-017-1328-4, 24, 7, (2669-2676), (2017).
Crossref
Simon Gustafsson and Albert Mihranyan, Strategies for Tailoring the Pore-Size Distribution of Virus Retention Filter Papers, ACS Applied Materials & Interfaces, 10.1021/acsami.6b03093, 8, 22, (13759-13767), (2016).
Crossref
Changqing Ruan, Maria Strømme and Jonas Lindh, A green and simple method for preparation of an efficient palladium adsorbent based on cysteine functionalized 2,3-dialdehyde cellulose, Cellulose, 10.1007/s10570-016-0976-0, 23, 4, (2627-2638), (2016).
Crossref
Paola Orsolini, Tommaso Marchesi D’Alvise, Cristiana Boi, Thomas Geiger, Walter R. Caseri and Tanja Zimmermann, Nanofibrillated Cellulose Templated Membranes with High Permeance, ACS Applied Materials & Interfaces, 10.1021/acsami.6b12107, 8, 49, (33943-33954), (2016).
Crossref
Simon Gustafsson, Pascal Lordat, Tobias Hanrieder, Marcel Asper, Oliver Schaefer and Albert Mihranyan, Mille-feuille paper: a novel type of filter architecture for advanced virus separation applications, Mater. Horiz., 10.1039/C6MH00090H, 3, 4, (320-327), (2016).
Crossref
Chao Liu, Bin Li, Haishun Du, Dong Lv, Yuedong Zhang, Guang Yu, Xindong Mu and Hui Peng, Properties of nanocellulose isolated from corncob residue using sulfuric acid, formic acid, oxidative and mechanical methods, Carbohydrate Polymers, 10.1016/j.carbpol.2016.06.025, 151,(716-724), (2016).
Crossref
M. Ramos, A. Valdés and M.C. Garrigós, Multifunctional Applications of Nanocellulose-Based Nanocomposites, Multifunctional Polymeric Nanocomposites Based on Cellulosic Reinforcements, 10.1016/B978-0-323-44248-0.00006-7, (177-204), (2016).
Crossref
Junji Nemoto, Toshihiko Soyama, Tsuguyuki Saito and Akira Isogai, Improvement of Air Filters by Nanocelluloses, JAPAN TAPPI JOURNAL, 10.2524/jtappij.70.1072, 70, 10, (1072-1078), (2016).
Crossref
Kai Hua, Igor Rocha, Peng Zhang, Simon Gustafsson, Yi Ning, Maria Strømme, Albert Mihranyan and Natalia Ferraz, Transition from Bioinert to Bioactive Material by Tailoring the Biological Cell Response to Carboxylated Nanocellulose, Biomacromolecules, 10.1021/acs.biomac.6b00053, 17, 3,(1224-1233), (2016).
Crossref
Zeynep Altintas, Advanced Imprinted Materials for Virus Monitoring, Advanced Molecularly Imprinting Materials, (389-411), (2016).
Wiley Online Library
Changgang Xu, Daniel O. Carlsson and Albert Mihranyan, Feasibility of using DNA-immobilized nanocellulose-based immunoadsorbent for systemic lupus erythematosus plasmapheresis,Colloids and Surfaces B: Biointerfaces, 10.1016/j.colsurfb.2016.03.014, 143, (1-6), (2016).
Crossref
, , JAPAN TAPPI JOURNAL, 10.2524/jtappij.70.1065, 70, 10, (1065-1071), (2016).
Crossref
Arne Quellmalz and Albert Mihranyan, Citric Acid Cross-Linked Nanocellulose-Based Paper for Size-Exclusion Nanofiltration, ACS Biomaterials Science & Engineering, 10.1021/ab500161x, 1, 4, (271-276), (2015).
Crossref
Hao-Cheng Yang, Yi-Fu Chen, Chen Ye, Yi-Ning Jin, Hanying Li and Zhi-Kang Xu, Polymer membrane with a mineral coating for enhanced curling resistance and surface wettability, Chem. Commun., 10.1039/C5CC03216D, 51, 64, (12779-12782), (2015).
Crossref
M. Asper, T. Hanrieder, A. Quellmalz and A. Mihranyan, Removal of xenotropic murine leukemia virus by nanocellulose based filter paper, Biologicals, 10.1016/j.biologicals.2015.08.001, 43, 6, (452-456), (2015).
Crossref
Maija Vuoriluoto, Hannes Orelma, Leena-Sisko Johansson, Baolei Zhu, Mikko Poutanen, Andreas Walther, Janne Laine and Orlando J. Rojas, Effect of Molecular Architecture of PDMAEMA–POEGMA Random and Block Copolymers on Their Adsorption on Regenerated and Anionic Nanocelluloses and Evidence of Interfacial Water Expulsion, The Journal of Physical Chemistry B, 10.1021/acs.jpcb.5b07628, 119, 49, (15275-15286), (2015).
Crossref
Kai Hua, Maria Strømme, Albert Mihranyan and Natalia Ferraz, Nanocellulose from green algae modulates the in vitro inflammatory response of monocytes/macrophages, Cellulose, 10.1007/s10570-015-0772-2, 22, 6, (3673-3688), (2015).
Crossref
Junji Nemoto, Tsuguyuki Saito and Akira Isogai, Simple Freeze-Drying Procedure for Producing Nanocellulose Aerogel-Containing, High-Performance Air Filters, ACS Applied Materials & Interfaces, 10.1021/acsami.5b05841, 7, 35, (19809-19815), (2015).
Crossref
Xuewei Wang, Yu Qin and Mark E. Meyerhoff, Paper-based plasticizer-free sodium ion-selective sensor with camera phone as a detector, Chem. Commun., 10.1039/C5CC06770G, 51, 82, (15176-15179), (2015).
Crossref
Adnan Haider, Sajjad Haider and Inn-Kyu Kang, A comprehensive review summarizing the effect of electrospinning parameters and potential applications of nanofibers in biomedical and biotechnology, Arabian Journal of Chemistry, 10.1016/j.arabjc.2015.11.015, (2015).
Crossref
Haoran Wei, Katia Rodriguez, Scott Renneckar and Peter J. Vikesland, Environmental science and engineering applications of nanocellulose-based nanocomposites, Environ. Sci.: Nano, 10.1039/C4EN00059E, 1, 4, (302-316), (2014).
Crossref
Zhaohui Wang, Petter Tammela, Peng Zhang, Jinxing Huo, Fredric Ericson, Maria Strømme and Leif Nyholm, Freestanding nanocellulose-composite fibre reinforced 3D polypyrrole electrodes for energy storage applications, Nanoscale, 10.1039/C4NR04642K, 6, 21, (13068-13075), (2014).
Crossref
D. O. Carlsson, J. Lindh, L. Nyholm, M. Strømme and A. Mihranyan, Cooxidant-free TEMPO-mediated oxidation of highly crystalline nanocellulose in water, RSC Adv., 10.1039/C4RA11182F, 4, 94, (52289-52298), (2014).
Crossref
Hugo Voisin, Lennart Bergström, Peng Liu and Aji Mathew, Nanocellulose-Based Materials for Water Purification, Nanomaterials, 10.3390/nano7030057, 7, 3, (57), (2017).
Crossref
Olof Gustafsson, Simon Gustafsson, Levon Manukyan and Albert Mihranyan, Significance of Brownian Motion for Nanoparticle and Virus Capture in Nanocellulose-Based Filter Paper,Membranes, 10.3390/membranes8040090, 8, 4, (90), (2018).
Crossref
Simon Gustafsson, Frank Westermann, Tobias Hanrieder, Laura Jung, Horst Ruppach and Albert Mihranyan, Comparative Analysis of Dry and Wet Porometry Methods for Characterization of Regular and Cross-Linked Virus Removal Filter Papers, Membranes, 10.3390/membranes9010001, 9, 1, (1), (2018).
Crossref
Nadir Yıldırım, Nanoteknoloji ve Geleceğin Çevreci Polimeri Nanoselüloz, Ormancılık Araştırma Dergisi, 10.17568/ogmoad.419758, (2018).
Crossref
Bejoy Thomas, Midhun C. Raj, Athira K. B, Rubiyah M. H, Jithin Joy, Audrey Moores, Glenna L. Drisko and Clément Sanchez, Nanocellulose, a Versatile Green Platform: From Biosources to Materials and Their Applications, Chemical Reviews, 10.1021/acs.chemrev.7b00627, (2018).
Crossref
Andreas Mautner, Thawanrat Kobkeatthawin, Florian Mayer, Christof Plessl, Selestina Gorgieva, Vanja Kokol and Alexander Bismarck, Rapid Water Softening with TEMPO-Oxidized/Phosphorylated Nanopapers, Nanomaterials, 10.3390/nano9020136, 9, 2, (136), (2019).
Crossref
Lucie Bacakova, Julia Pajorova, Marketa Bacakova, Anne Skogberg, Pasi Kallio, Katerina Kolarova and Vaclav Svorcik, Versatile Application of Nanocellulose: From Industry to Skin Tissue Engineering and Wound Healing, Nanomaterials, 10.3390/nano9020164, 9, 2, (164), (2019).
Crossref
Ragab E. Abouzeid, Ramzi Khiari, Nahla El-Wakil and Alain Dufresne, Current State and New Trends in the Use of Cellulose Nanomaterials for Wastewater Treatment, Biomacromolecules, 10.1021/acs.biomac.8b00839, (2018).
Crossref
Dong Wang, A critical review of cellulose-based nanomaterials for water purification in industrial processes, Cellulose, 10.1007/s10570-018-2143-2, (2018).
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ChengBo Zhan, Priyanka R. Sharma, LiHong Geng, Sunil K. Sharma, RuiFu Wang, Ritika Joshi and Benjamin S. Hsiao, Structural characterization of carboxylcellulose nanofibers extracted from underutilized sources, Science China Technological Sciences, 10.1007/s11431-018-9441-1, (2019).
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Zhongde Dai, Vegar Ottesen, Jing Deng, Ragne M. Lilleby Helberg and Liyuan Deng, A Brief Review of Nanocellulose Based Hybrid Membranes for CO2 Separation, Fibers, 10.3390/fib7050040, 7, 5, (40), (2019).
Crossref
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by Youness Cherradi, Sarah Le Merdy, Li-Jun Sim, Takao Ito, Priyabrata Pattnaik, Josselyn Haas and Anissa Boumlic Thursday, April 19, 2018 5:27 pm
Figure 1: Usual clarification options for viral vaccines and vectors
Viral vaccines rely on the antigen properties of a virus or virus-like entity to trigger an immune response and induce immune protection against a forthcoming viral infection. Through development of recombinant viral vaccines, developers can reduce risks associated with the presence of live and inactivated viruses. Instead, recombinant vaccines induce immunity against a pathogen by relying on the capacity of one or more antigens delivered by means of viral vectors or the baculovirus/plasmid system (1). Viral vaccines are formulated with or without adjuvants and categorized as shown in Table 1.
Table 1: Classification of viral vaccines
Viral vaccine manufacturing processes can be templated. They follow a general scheme, starting with production in either an embryonated egg or mammalian/insect cell culture. After production, the bulk harvest material is processed to purify a vaccine of interest. Following upstream production and lysis (optional), a clarification step typically is introduced to start the purification process by either centrifugation or filtration. This is a critical unit operation because it strongly affects product recovery and subsequent downstream purification.
Considerations for Clarification of Viral Vaccines: Type of Substrate
Composition, type, and level of contaminants to be removed during clarification mainly depend on the upstream process and expression system used for production. In most cases, the virus particles must be kept integral during the clarification step.
For decades, embryonated chicken eggs have been used to produce both human and animal vaccines. However, the resulting allantoic fluid harvest (rich in virus particles and cellular debris) is a challenging feed for clarification. This fluid has a high solids content (>25%, which increases with embryo age) and high mineral and protein content, hence its highly viscous consistency. It also contains rudimentary tissue compounds from chicken embryos such as feathers, beaks, blood vessels, and/or blood cells.
Several viral vaccines have moved away from an egg-based process toward the use of live cells (e.g., plant, microbial, avian, or mammalian). The composition of these feed streams can vary significantly. Based on cell viability — and lysis method selected (e.g., chemical or mechanical), if applicable — the impurity profile in the fluid to be clarified can differ considerably. For example, low cell viability can indicate high levels of contaminants released into a feed stream from cell lysis. Typical contaminants such as host cell DNA, host cell proteins (HCPs), lipids, and bigger particles such as cell debris can be identified. The proportion of solids in the feed usually is an indicator of purifying challenges ahead (~6–8% mammalian cells or up to 40% in yeast). Compared with allantoic fluid, however, cell culture harvests are considerably cleaner in terms of solids load and soluble content.
Another expression method is the baculovirus expression vector system (BEVS) used with insect cells. This is gaining interest particularly for producing viral vectors and virus-like particles (VLPs). Other expression systems such as bacteria, yeast, and plant cells also can be used to produce viral particles.
Considerations on Key Product Quality Criteria and Control Strategies
Yield: In most cases, yield is an off-line measurement conducted at the end of a number of process steps. Depending on the success criteria for each step, yield can be the main parameter to consider when selecting one option over another for a given step. Based on the size and properties of viral particles, yield could be affected by the clarification method used. For example, whereas positive charges increase nucleic acids and HCP capture, diatomaceous earth can retain viruses by adsorption.
Some viruses are shear sensitive and can be damaged by high shear exposure in disk-stack centrifuges or by high cross-flow and multiple pump passages through TFF. Due to their large size, viruses larger than 100 nm also can be retained simply by tight filters. Thus, companies should take such factors into consideration when selecting depth filters because some depth filter devices include a 0.1-μm membrane that could cause retention-driven product loss. Other process elements such as aggregation and an excess of impurities can compromise viral particle recovery.
Final Product Purity Levels: Regulatory agencies provide recommendations and requirements regarding acceptable residual amounts of contaminants in final drug products. For reasons of patient safety and tolerance, host cell DNA in a final product must be reduced to appropriate levels. In 1998, the World Health Organization (WHO) specified the maximum residual DNA content in a vaccine to be <10 ng/dose. Since then, the European Medicines Agency (EMA) proposed more stringent conditions based on the type of cell line (tumorigenic origin) used in vaccine manufacturing. The US Food and Drug Administration (FDA) follows a case-by-case evaluation approach and recommends that manufacturers reduce both the size (~200 bp) and amount of DNA per dose.
To date, a final DNA content of <10 ng/ dose commonly is accepted for most biologics. Similarly, a recombinant monoclonal antibody (MAb) product must reach clearance of impurities to <100 ppm of HCP, ≤10 ng/dose of DNA, and <5% of immunogenic aggregates (Table 2).
Table 2: Acceptable remaining impurities in vaccine products
Feed Quality Evaluation Criteria and Processing Parameters: Several parameters can be used to assess the clarity or quality of a product, either during process development or after each manufacturing process step. Turbidity is an easy parameter to monitor and provides an immediate assessment of feed quality. For example, it enables the detection of depth filter breakthrough.
The two primary methods of ascertaining the effectiveness of the clarification step are centrifugation and filtration. Turbidity can be monitored simply by absorbance/scatter in the visible range, providing an immediate assessment of particle load within the filtrate or centrate. Turbidity also relates to filter capacity, which is the volume of feed a depth filter can process before the pressure drop breaches specifications. Capacity relating to both pressure drop and turbidity breakthrough are linked, and specifications for both should be set during process development. For some processes (particularly those with a smaller average particle size in the feed), turbidity breakthrough is the limiting factor in sizing a filtration train. Often, capacity limit is attributed to pressure drop. But because high pressures or flow rates can cause premature turbidity breakthrough, both mechanisms can be related.
Technology Options for Clarification of Virus Vaccines: Because of the extreme diversity of viral vaccines in terms of size, structure, shape, and expression system, no unified template exists for their production and purification. Those processes can be divided into four different phases: upstream/production, clarification, purification, and formulation.
To reduce burden on downstream purification steps, the main objective of a clarification process is to remove undesirable materials, including whole cells, cell debris, colloids, and large aggregates. As the first downstream process, clarification should be optimized to maximize product yield and purity. Several serial operational steps often are required to achieve a desired level of clarification. The first operation (often referred to as primary clarification) removes larger particles, and the second (often referred to as secondary clarification) removes colloids and other submicron particles (Figure 1).
In theory, all available technologies (low-speed centrifugation, microfiltration TFF, NFF) can be selected and potentially combined to clarify viruses. As with other manufacturing process steps, a clarification process should have predictable scalability, be manufacturing-friendly (e.g., be easy to use, reduce holdup volume, provide operator safety), and have a low cost of goods (CoG). However, the clarification process of viral vaccines has two unique characteristics that can require more tailored solutions:
low solids content with a high nucleic acid and colloid content, requiring higher retention capacity
high feed variability and cell culture enhancements, requiring more robustness (2).
Although centrifugation can handle a high solids load and traditionally has been used in batch and continuous modes, it requires large capital investment and high maintenance costs. More important, centrifugation scale-up can be problematic because of unreliable scale-down models with nonlinear scalability and high-shear operation for shear-sensitive vaccines. But NFF and TFF have gained interest for vaccine clarification because they are significantly easier to scale-up and implement.
NFF: Primary clarification using NFF typically involves depth filters that often contain positively charged material and filter aids (e.g., diatomaceous earth) that improve retention of cell debris, colloids, and negatively charged contaminants. NFF relies on two main mechanisms for particle retention: size exclusion and adsorption.
NFF membrane filters can be used in secondary filtration because they retain particles by size exclusion. Certain grades of depth filters have a tighter pore-size distribution (which offers greater colloidal particle retention) but do not have high holding capacity. Noncharged depth filters also can be used for clarification while offering higher cost-effectiveness for small batches (≤1,000 L). They typically use three media types:
melt-blown media fabricated in a pleated format to achieve higher flow rates and holding capacities
graded density of concentrically wrapped media to allow the filter to remove finer contaminants progressively membranes to provide higher retention efficiency.
TFF membranes with retention ratings in the range of 0.1–0.65 μm have been used to retain cells, cell debris, and other large contaminants. Most TFF devices are linearly scalable and reusable after cleaning, and hence greatly reduce consumable costs. However, certain viruses (e.g., extracellularly produced enveloped virus-like particles) can be damaged by high-shear exposure in disk-stack centrifuges or by high cross-flow and multiple pump passages in TFF processes. Open-channel TFF devices (cassette format without screen) are preferred to minimize shear.
Case Studies
Egg-Based Vaccines: In influenza vaccine processes, a typical allantoic fluid harvest is rich in proteins (e.g., ovalbumin, lysozyme, ovomucin) and contains 45 μg hemagglutinin antigen per egg (~3–4 μg HA and 108–109 infectious units/mL of allantoic fluid). The typical gravity-settled turbidity of a virus-containing allantoic fluid (VCAF) generally is 46–132 NTU. Low-speed zonal continuous centrifugation around 4,000–5,000g often is the preferred option to remove large particles and thus gets used for primary clarification, typically providing a recovery yield of 70% (3, 4). Many vaccine manufacturers use sucrose gradient zonal centrifugation to purify and concentrate viruses. However, polypropylene and cellulose-based depth filters also can be implemented for filtration. Fair capacities of 150–210 L/m² and up to 3× reduction of feed stream turbidity can be achieved with those allantoic fluid harvests. That option is appropriate for influenza vaccines, which are prone to adsorption loss during clarification on charged filters.
NFF also can be used for secondary clarification. Combinations of polypropylene, cellulose, and glass-fiber materials generally demonstrate good efficiency (5). Using a salt solution can reduce association between a virus vaccine and solid debris, resulting in a yield increase of about twofold without compromising viral particle integrity.
One study demonstrated that higher ionic strength on allantoic debris increased influenza virus yields (6). In that case, 1.5 M NaCl was applied to pooled allantoic fluids of various influenza strains, and the sample was centrifuged for different durations to understand the amount of virus partitioned in the supernatant and pellet. Control samples were kept at 0.15M NaCl. This test scope was expanded to include other influenza strains — A/New Caledonia (H1N1), A/Texas (H3N2), B/Jiangsu and B/Hong Kong — to demonstrate an average twofold yield increase. Further work verified the integrity of the purified virus in control and higher ionic strengths. Data showed that higher ionic strength did not adversely affect the live titer of the tested influenza virus strains (2). Specifically, studies have reported use of a 1.2-μm cellulose nitrate (CN) filter polypropylene media followed by 0.45-μm filter polyvinylidene fluoride (PVDF) membrane for clarification of cell-based influenza with a loading of 111 L/m² and 105 L/m², respectively (7).
Another option is use of TFF with a 0.65-μm or 0.45-μm microfiltration membrane device operated with permeate flux control (8). Using a “two-pump process” with a permeate pump in addition to the standard TFF feed pump allows for permeate flux control to manage/reduce polarization and fouling. That provides better characterization of the “critical” flux — the limiting flux above which a process becomes unstable (9).
Moreover, researchers conducted primary clarification of allantoic fluid using a 40-μm bag filter followed by an open-channel microfiltration device (Prostak 0.65 μm; data not shown). Using a crossflow of 3 LPM/channel, with transmembrane pressure (TMP) at 0.2 bar and dP at 0.4 bar, a capacity of 33 L/m² was achieved. The MF process was designed for 10× concentration and 5× diafiltration, and the Prostak allowed for 60× reuse.
Viruses in Adherent Cells on Microcarriers: Microcarriers can be used as a support matrix for the growth of adherent cells such as Vero cells. Primary clarification of these cells grown on microcarriers can be performed using a 75-μm stainless steel sieve to remove microcarriers from harvest. A Millistak+ C0HC depth filter medium then can be used for secondary clarification and directly followed by a sterilizing-grade filter (10). This study was reported for a cell density of 0.78 x 106 cells/mL. Results showed varying filter capacity based on cell density or cell viability of harvest.
Viral Vectors: Trial results on lentiviruses from the supernatant of HEK293T cells show positive performance (data not shown). The negative charge of lentiviruses is known to be responsible for poor recoveries with positively charged depth filters. Therefore, despite the fact that no sign of plugging was observed using a Millistak+ C0HC depth filter, very low viral vector recovery was recorded. However, use of a 1.0/0.5 μm Polysep II filter led to both a high capacity and high 84% recovery. The Polygard CN filter, on the other hand, behaved quite well in terms of recovery (75%) but showed more signs of plugging. An extra 10–20% lower virus recovery should be considered for the following 0.45 μm or 0.22μm (sterile) filtration step.
Two other studies on lentivirus feed streams confirm the positive recovery performance using the Polygard CN filters (CN25, CN12, CN10, and CN06 with more than 80% recovery). Other studies report an interesting result with recoveries reaching up to 90% for the Millistak+ CE50 filter that does not contain inorganic filter aid (e.g., diatomaceous earth; data not shown).
Adenoviruses also can be prone to adsorption, but divergent results have been reported. In some cases, good adenovirus recovery is observed even when a positively charged depth filter medium containing diatomaceous earth such as the Millistak+ HC medium is used (11). If adenovirus is lost, Polygard and Clarigard filters can be used instead, but a secondary clarification might be needed to reduce turbidity (12).
Clarification Options
Different approaches are used to produce viral vaccines, making designing a typical template for their clarification difficult. Indeed, these products can be produced by different expression systems and possess a range of physicochemical properties. The clarification method selected should take those factors into account to ensure that yield and contaminants removal are sufficient.
NFF and microfiltration TFF technologies are increasingly preferred to centrifugation because of their more predictable scalability and robustness. Low CoG also can be achieved by tailoring the choice of media chemistry and porosity to gain high recovery and productivity with satisfactory impurity removal. Continuing innovation in this field by suppliers of purification and filtration systems will help vaccine manufacturers meet their current and future challenges on the road to developing novel and efficient therapies.
References
1 Nascimento IP, Leite LCC. Recombinant Vaccines and the Development of New Vaccine Strategies. Braz. J. Med. Biol. Res. 45(12) 2012: 1102–1111.
2 Besnard L, et al. Clarification of Vaccines: An Overview of Filter-Based Technology Trends and Best Practices. Biotechnol. Advances 34(1) 2016: 1–13.
3 Hendriks J, et al. An International Technology Platform for Influenza Vaccines. Vaccine 29 Suppl 1, 2011: A8–11.
4 Eichhorn U. Influenza Vaccine Composition. US Patent US7316813 B2, 2008.
5 Lampson GP, Machlowitz RA. Process for Producing Purified Concentrated Influenza Virus.US Patents US3547779 A, 1970.
6 Hughes K, et al. Yield Increases in Intact Influenza Vaccine Virus from Chicken Allantoic Fluid through isolation from Insoluble Allantoic Debris. Vaccine 25(22) 2007: 4456–4463.
7 Thompson M, Wee J, Nagpal A. Methods for Purification of Viruses. European Patent EP2334328 A4, 2012.
8 Lau SY, et al. Impact of Process Loading on Optimization and Scale-Up of TFF Microfiltration. BioProcess J. 13(2) 2014: 46–55; doi:10.12665/J132.Pattna.
9 Raghunath B, et al. Best Practices for Optimization and Scale-Up of Microfiltration TFF Processes. Bioprocess. J. 11(1) 2012: 30–40.
10 Thomassen YE, et al. Scale-Down of the Inactivated Polio Vaccine Production Process. Biotechnol. Bioeng. 110(5) 2013: 1354–1365.
11 Namatovu HH, et al. Evaluation of Filtration Products in the Production of Adenovirus Candidates Used in Vaccine Production: Overview and Case Study. BioProcess J. 5(3) 2006: 67–74.
12 Weggeman M, van Corven EJJM. Virus Purification Methods. US Patent: US8124106 B2, 2012.
Corresponding author Anissa Boumlic (anissa.boumlic@merckgroup.com) is associate director of the vaccine segment at Millipore S.A.S.
Wed 9/9/2015 12:21 PM
It looks like they knew about this nano paper and they have been looking at it since 2014. It's published here: Adv. Healthcare Mater. 2014. It was the Swedish scientists who have been looking at it:
It looks like they knew about this nano paper and they have been looking at it since 2014. It's published here: Adv. Healthcare Mater. 2014. It was the Swedish scientists who have been looking at it:
A Size-Exclusion Nanocellulose Filter Paper for Virus Removal
"From the attached paper: Furthermore, robust and affordable virus removal filters are also demanded by the biotechnology industry as there are hundreds of products possessing the potential risk of viral infection, including cell-derived monoclonal antibodies, plasma-derived coagulation factors (e.g., factors VIII and IX) and immunoglobulins, or proteins manufactured with processes that employ viruses as biological expression systems, e.g., human or animal vaccines. [ 3] Absence of viral contamination further needs to be ensured for all types of therapeutic proteins, for example, human antithrombin III, derived from the milk of transgenic mammals. [ 4] Since cell cultures and explants are often cultivated in serum-derived media, both endogenous retroviruses and adventitious viral contaminants introduced during manufacturing also constitute a risk factor, for example, bovine viral diarrhoea virus, bovine parainfl uenza 3 virus (PI-3), parvoviruses. [ 3a, b] The threat of viral contamination of biotechnology products is manifold, including active human infection, for instance, human immunodefi ciency virus (HIV), hepatitis C (HVC), and enhanced oncogenic risk due to dormant infection, for example, avianleucosis virus. [ 3a, b] In addition, the advances in viral vectors of gene delivery to cure cancer stipulate the development of purifi cation techniques wherein the non-immunogenic virus particles can be effi ciently separated from proteins and other cell debris during production. [ 3c]"
A Size‐Exclusion Nanocellulose Filter Paper for Virus Removal
https://doi.org/10.1002/adhm.201300641
This swedish scientist is on both papers: Albert Mihranyan So the one that just came out and the ones on pdf. Here's where he's from:
Prof. A. Mihranyan
Division of Materials Science
Luleå University of Technology
971 87 Luleå, Sweden
Prof. A. Mihranyan
Nanotechnology and Functional Materials
Department of Engineering Sciences
Box 534, Uppsala University
75121 , Uppsala , Sweden
Citing Literature:
Number of times cited according to CrossRef: 56
Amir Sheikhi, Emerging Cellulose-Based Nanomaterials and Nanocomposites, Nanomaterials and Polymer Nanocomposites, 10.1016/B978-0-12-814615-6.00009-6, (307-351), (2019).
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Deepu A. Gopakumar, Shilpa Thomas, Owolabi F.A.T, Sabu Thomas, Ange Nzihou, Samsul Rizal and H.P.S. Abdul Khalil, Nanocellulose Based Aerogels for Varying Engineering Applications, Reference Module in Materials Science and Materials Engineering, 10.1016/B978-0-12-803581-8.10549-1, (2019).
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Mohammad E.T. Yazdi, Mohammad S. Amiri and Majid Darroudi, Biopolymers in the Synthesis of Different Nanostructures, Reference Module in Materials Science and Materials Engineering,10.1016/B978-0-12-803581-8.10560-0, (2019).
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Yi Li, David Stern, Lye Lin Lock, Jason Mills, Shih-Hao Ou, Marina Morrow, Xuankuo Xu, Sanchayita Ghose, Zheng Jian Li and Honggang Cui, Emerging Biomaterials for Downstream Manufacturing of Therapeutic Proteins, Acta Biomaterialia, 10.1016/j.actbio.2019.03.015, (2019).
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Andreas Mautner, Yosi Kwaw, Kathrin Weiland, Mlando Mvubu, Anton Botha, Maya Jacob John, Asanda Mtibe, Gilberto Siqueira and Alexander Bismarck, Natural fibre-nanocellulose composite filters for the removal of heavy metal ions from water, Industrial Crops and Products, 10.1016/j.indcrop.2019.03.032, 133, (325-332), (2019).
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Gui Ye, Zhiyong Yu, Yiming Li, Lei Li, Li Song, Li Gu and Xuebo Cao, Efficient treatment of brine wastewater through a flow-through technology integrating desalination and photocatalysis, Water Research, 10.1016/j.watres.2019.03.058, 157, (134-144), (2019).
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Levon Manukyan, Justine Padova and Albert Mihranyan, Virus removal filtration of chemically defined Chinese Hamster Ovary cells medium with nanocellulose-based size exclusion filter, Biologicals, 10.1016/j.biologicals.2019.03.001, (2019).
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Andrés Felipe Alzate Arbeláez, Eva Dorta Pérez, Camilo López-Alarcón, Farid B. Cortés and Benjamín A. Rojano, Immobilization of Andean berry (Vaccinium meridionale) polyphenols on nanocellulose isolated from banana residues: a natural food additive with antioxidant properties, Food Chemistry, 10.1016/j.foodchem.2019.05.085, (2019).
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K. Obi Reddy, C. Uma Maheswari, M.S. Dhlamini, B.M. Mothudi, V.P. Kommula, Jinming Zhang, Jun Zhang and A. Varada Rajulu, Extraction and characterization of cellulose single fibers from native african napier grass, Carbohydrate Polymers, 188, (85), (2018).
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Gergő P. Szekeres, Zoltán Németh, Krisztina Schrantz, Krisztián Németh, Mateusz Schabikowski, Jacqueline Traber, Wouter Pronk, Klára Hernádi and Thomas Graule, Copper-Coated Cellulose-Based Water Filters for Virus Retention, ACS Omega, 3, 1, (446), (2018).
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Olof Gustafsson, Levon Manukyan and Albert Mihranyan, High‐Performance Virus Removal Filter Paper for Drinking Water Purification, Global Challenges, 2, 7, (2018).
Wiley Online Library
Levon Manukyan, Pengfei Li, Simon Gustafsson and Albert Mihranyan, Growth Media Filtration Using Nanocellulose-based Virus Removal Filter for Upstream Biopharmaceutical Processing, Journal of Membrane Science, 10.1016/j.memsci.2018.11.002, (2018).
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Igor Rocha, Natalia Ferraz, Albert Mihranyan, Maria Strømme and Jonas Lindh, Sulfonated nanocellulose beads as potential immunosorbents, Cellulose, 10.1007/s10570-018-1661-2, 25, 3, (1899-1910), (2018).
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Guy-Alain Junter and Laurent Lebrun, Cellulose-based virus-retentive filters: a review, Reviews in Environmental Science and Bio/Technology, 10.1007/s11157-017-9434-1, 16, 3, (455-489), (2017).
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Jun Liu, Stefan Willför and Albert Mihranyan, On importance of impurities, potential leachables and extractables in algal nanocellulose for biomedical use, Carbohydrate Polymers,10.1016/j.carbpol.2017.05.002, 172, (11-19), (2017).
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Subrata Mondal, Preparation, properties and applications of nanocellulosic materials, Carbohydrate Polymers, 10.1016/j.carbpol.2016.12.050, 163, (301-316), (2017).
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Jeyabalan Sangeetha, Devarajan Thangadurai, Ravichandra Hospet, Prathima Purushotham, Kartheek Rajendra Manowade, Mohammed Abdul Mujeeb, Abhishek Channayya Mundaragi, Sudisha Jogaiah, Muniswamy David, Shivasharana Chandrabanda Thimmappa, Ram Prasad and Etigemane Ramappa Harish, Production of Bionanomaterials from Agricultural Wastes, Nanotechnology, 10.1007/978-981-10-4573-8_3, (33-58), (2017).
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Andreas Mautner, Thawanrat Kobkeatthawin and Alexander Bismarck, Efficient continuous removal of nitrates from water with cationic cellulose nanopaper membranes, Resource-Efficient Technologies, 10.1016/j.reffit.2017.01.005, 3, 1, (22-28), (2017).
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Simon Gustafsson, Levon Manukyan and Albert Mihranyan, Protein–Nanocellulose Interactions in Paper Filters for Advanced Separation Applications, Langmuir, 10.1021/acs.langmuir.7b00566, 33, 19, (4729-4736), (2017).
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Nathan Grishkewich, Nishil Mohammed, Juntao Tang and Kam Chiu Tam, Recent advances in the application of cellulose nanocrystals, Current Opinion in Colloid & Interface Science, 10.1016/j.cocis.2017.01.005, 29, (32-45), (2017).
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Ming-Guo Ma, Yan-Jun Liu and Yan-Yan Dong, Nanocellulose and Nanocellulose Composites: Synthesis, Characterization, and Potential Applications, Handbook of Composites from Renewable Materials, (109-134), (2017).
Wiley Online Library
Rudi Dungani, Abdul Khalil H.P.S., Nurjaman A. Sri Aprilia, Ihak Sumardi, Pingkan Aditiawati, Atmawi Darwis, Tati Karliati, Aminudin Sulaeman, Enih Rosamah and Medyan Riza, Bionanomaterial from agricultural waste and its application, Cellulose-Reinforced Nanofibre Composites, 10.1016/B978-0-08-100957-4.00003-6, (45-88), (2017).
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Kirubanandan Shanmugam, Swambabu Varanasi, Gil Garnier and Warren Batchelor, Rapid preparation of smooth nanocellulose films using spray coating, Cellulose, 10.1007/s10570-017-1328-4, 24, 7, (2669-2676), (2017).
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Simon Gustafsson and Albert Mihranyan, Strategies for Tailoring the Pore-Size Distribution of Virus Retention Filter Papers, ACS Applied Materials & Interfaces, 10.1021/acsami.6b03093, 8, 22, (13759-13767), (2016).
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Changqing Ruan, Maria Strømme and Jonas Lindh, A green and simple method for preparation of an efficient palladium adsorbent based on cysteine functionalized 2,3-dialdehyde cellulose, Cellulose, 10.1007/s10570-016-0976-0, 23, 4, (2627-2638), (2016).
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Paola Orsolini, Tommaso Marchesi D’Alvise, Cristiana Boi, Thomas Geiger, Walter R. Caseri and Tanja Zimmermann, Nanofibrillated Cellulose Templated Membranes with High Permeance, ACS Applied Materials & Interfaces, 10.1021/acsami.6b12107, 8, 49, (33943-33954), (2016).
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Simon Gustafsson, Pascal Lordat, Tobias Hanrieder, Marcel Asper, Oliver Schaefer and Albert Mihranyan, Mille-feuille paper: a novel type of filter architecture for advanced virus separation applications, Mater. Horiz., 10.1039/C6MH00090H, 3, 4, (320-327), (2016).
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Chao Liu, Bin Li, Haishun Du, Dong Lv, Yuedong Zhang, Guang Yu, Xindong Mu and Hui Peng, Properties of nanocellulose isolated from corncob residue using sulfuric acid, formic acid, oxidative and mechanical methods, Carbohydrate Polymers, 10.1016/j.carbpol.2016.06.025, 151,(716-724), (2016).
Crossref
M. Ramos, A. Valdés and M.C. Garrigós, Multifunctional Applications of Nanocellulose-Based Nanocomposites, Multifunctional Polymeric Nanocomposites Based on Cellulosic Reinforcements, 10.1016/B978-0-323-44248-0.00006-7, (177-204), (2016).
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Junji Nemoto, Toshihiko Soyama, Tsuguyuki Saito and Akira Isogai, Improvement of Air Filters by Nanocelluloses, JAPAN TAPPI JOURNAL, 10.2524/jtappij.70.1072, 70, 10, (1072-1078), (2016).
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Kai Hua, Igor Rocha, Peng Zhang, Simon Gustafsson, Yi Ning, Maria Strømme, Albert Mihranyan and Natalia Ferraz, Transition from Bioinert to Bioactive Material by Tailoring the Biological Cell Response to Carboxylated Nanocellulose, Biomacromolecules, 10.1021/acs.biomac.6b00053, 17, 3,(1224-1233), (2016).
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Zeynep Altintas, Advanced Imprinted Materials for Virus Monitoring, Advanced Molecularly Imprinting Materials, (389-411), (2016).
Wiley Online Library
Changgang Xu, Daniel O. Carlsson and Albert Mihranyan, Feasibility of using DNA-immobilized nanocellulose-based immunoadsorbent for systemic lupus erythematosus plasmapheresis,Colloids and Surfaces B: Biointerfaces, 10.1016/j.colsurfb.2016.03.014, 143, (1-6), (2016).
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, , JAPAN TAPPI JOURNAL, 10.2524/jtappij.70.1065, 70, 10, (1065-1071), (2016).
Crossref
Arne Quellmalz and Albert Mihranyan, Citric Acid Cross-Linked Nanocellulose-Based Paper for Size-Exclusion Nanofiltration, ACS Biomaterials Science & Engineering, 10.1021/ab500161x, 1, 4, (271-276), (2015).
Crossref
Hao-Cheng Yang, Yi-Fu Chen, Chen Ye, Yi-Ning Jin, Hanying Li and Zhi-Kang Xu, Polymer membrane with a mineral coating for enhanced curling resistance and surface wettability, Chem. Commun., 10.1039/C5CC03216D, 51, 64, (12779-12782), (2015).
Crossref
M. Asper, T. Hanrieder, A. Quellmalz and A. Mihranyan, Removal of xenotropic murine leukemia virus by nanocellulose based filter paper, Biologicals, 10.1016/j.biologicals.2015.08.001, 43, 6, (452-456), (2015).
Crossref
Maija Vuoriluoto, Hannes Orelma, Leena-Sisko Johansson, Baolei Zhu, Mikko Poutanen, Andreas Walther, Janne Laine and Orlando J. Rojas, Effect of Molecular Architecture of PDMAEMA–POEGMA Random and Block Copolymers on Their Adsorption on Regenerated and Anionic Nanocelluloses and Evidence of Interfacial Water Expulsion, The Journal of Physical Chemistry B, 10.1021/acs.jpcb.5b07628, 119, 49, (15275-15286), (2015).
Crossref
Kai Hua, Maria Strømme, Albert Mihranyan and Natalia Ferraz, Nanocellulose from green algae modulates the in vitro inflammatory response of monocytes/macrophages, Cellulose, 10.1007/s10570-015-0772-2, 22, 6, (3673-3688), (2015).
Crossref
Junji Nemoto, Tsuguyuki Saito and Akira Isogai, Simple Freeze-Drying Procedure for Producing Nanocellulose Aerogel-Containing, High-Performance Air Filters, ACS Applied Materials & Interfaces, 10.1021/acsami.5b05841, 7, 35, (19809-19815), (2015).
Crossref
Xuewei Wang, Yu Qin and Mark E. Meyerhoff, Paper-based plasticizer-free sodium ion-selective sensor with camera phone as a detector, Chem. Commun., 10.1039/C5CC06770G, 51, 82, (15176-15179), (2015).
Crossref
Adnan Haider, Sajjad Haider and Inn-Kyu Kang, A comprehensive review summarizing the effect of electrospinning parameters and potential applications of nanofibers in biomedical and biotechnology, Arabian Journal of Chemistry, 10.1016/j.arabjc.2015.11.015, (2015).
Crossref
Haoran Wei, Katia Rodriguez, Scott Renneckar and Peter J. Vikesland, Environmental science and engineering applications of nanocellulose-based nanocomposites, Environ. Sci.: Nano, 10.1039/C4EN00059E, 1, 4, (302-316), (2014).
Crossref
Zhaohui Wang, Petter Tammela, Peng Zhang, Jinxing Huo, Fredric Ericson, Maria Strømme and Leif Nyholm, Freestanding nanocellulose-composite fibre reinforced 3D polypyrrole electrodes for energy storage applications, Nanoscale, 10.1039/C4NR04642K, 6, 21, (13068-13075), (2014).
Crossref
D. O. Carlsson, J. Lindh, L. Nyholm, M. Strømme and A. Mihranyan, Cooxidant-free TEMPO-mediated oxidation of highly crystalline nanocellulose in water, RSC Adv., 10.1039/C4RA11182F, 4, 94, (52289-52298), (2014).
Crossref
Hugo Voisin, Lennart Bergström, Peng Liu and Aji Mathew, Nanocellulose-Based Materials for Water Purification, Nanomaterials, 10.3390/nano7030057, 7, 3, (57), (2017).
Crossref
Olof Gustafsson, Simon Gustafsson, Levon Manukyan and Albert Mihranyan, Significance of Brownian Motion for Nanoparticle and Virus Capture in Nanocellulose-Based Filter Paper,Membranes, 10.3390/membranes8040090, 8, 4, (90), (2018).
Crossref
Simon Gustafsson, Frank Westermann, Tobias Hanrieder, Laura Jung, Horst Ruppach and Albert Mihranyan, Comparative Analysis of Dry and Wet Porometry Methods for Characterization of Regular and Cross-Linked Virus Removal Filter Papers, Membranes, 10.3390/membranes9010001, 9, 1, (1), (2018).
Crossref
Nadir Yıldırım, Nanoteknoloji ve Geleceğin Çevreci Polimeri Nanoselüloz, Ormancılık Araştırma Dergisi, 10.17568/ogmoad.419758, (2018).
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Bejoy Thomas, Midhun C. Raj, Athira K. B, Rubiyah M. H, Jithin Joy, Audrey Moores, Glenna L. Drisko and Clément Sanchez, Nanocellulose, a Versatile Green Platform: From Biosources to Materials and Their Applications, Chemical Reviews, 10.1021/acs.chemrev.7b00627, (2018).
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Andreas Mautner, Thawanrat Kobkeatthawin, Florian Mayer, Christof Plessl, Selestina Gorgieva, Vanja Kokol and Alexander Bismarck, Rapid Water Softening with TEMPO-Oxidized/Phosphorylated Nanopapers, Nanomaterials, 10.3390/nano9020136, 9, 2, (136), (2019).
Crossref
Lucie Bacakova, Julia Pajorova, Marketa Bacakova, Anne Skogberg, Pasi Kallio, Katerina Kolarova and Vaclav Svorcik, Versatile Application of Nanocellulose: From Industry to Skin Tissue Engineering and Wound Healing, Nanomaterials, 10.3390/nano9020164, 9, 2, (164), (2019).
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Ragab E. Abouzeid, Ramzi Khiari, Nahla El-Wakil and Alain Dufresne, Current State and New Trends in the Use of Cellulose Nanomaterials for Wastewater Treatment, Biomacromolecules, 10.1021/acs.biomac.8b00839, (2018).
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Dong Wang, A critical review of cellulose-based nanomaterials for water purification in industrial processes, Cellulose, 10.1007/s10570-018-2143-2, (2018).
Crossref
ChengBo Zhan, Priyanka R. Sharma, LiHong Geng, Sunil K. Sharma, RuiFu Wang, Ritika Joshi and Benjamin S. Hsiao, Structural characterization of carboxylcellulose nanofibers extracted from underutilized sources, Science China Technological Sciences, 10.1007/s11431-018-9441-1, (2019).
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Zhongde Dai, Vegar Ottesen, Jing Deng, Ragne M. Lilleby Helberg and Liyuan Deng, A Brief Review of Nanocellulose Based Hybrid Membranes for CO2 Separation, Fibers, 10.3390/fib7050040, 7, 5, (40), (2019).
Crossref
-------------------------------
Here was their response: Mon 9/7/2015 7:05 PM
Wow!! I Dont know any of this !!
Here's the link to that new paper but it's behind a paywall and it's German and Swedish scientists who did it.
Removal of xenotropic murine leukemia virus by nanocellulose based filter paper
M. Aspera,
T. Hanriedera,
A. Quellmalzb,
A. Mihranyanb
Abstract
The removal of xenotrpic murine leukemia virus (xMuLV) by size-exclusion filter paper composed of100% naturally derived cellulose was validated. The filter paper was produced using cellulose nanofibers derived from Cladophora sp. algae. The filter paper was characterized using atomic force microscopy, scanning electron microscopy, helium pycnometry, and model tracer (100 nm latex beads and 50 nm gold nanoparticles) retention tests. Following the filtration of xMuLV spiked solutions, LRV ≥5.25 log10TCID50 was observed, as limited by the virus titre in the feed solution and sensitivity of the tissue infectivity test. The results of the validation study suggest that the nanocellulose filter paper is useful for removal of endogenous rodent retroviruses and retrovirus-like particles during the production of recombinant proteins.
Graphical abstract
Filter paper composed of 100% naturally derived cellulose nanofibers was shown useful to remove xenotropic murine leukemia virus (MuLV) spiked solutions with LRV ≥ 5.25 as limited by titre of the feed solution and sensitivity of tissue infectivity method.
Keywords
Viral contamination;
Virus retentive filtration;
Cladophora cellulose;
Paper making;
Recombinant proteins and monoclonal antibodies
Corresponding author.
Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Note to users: Corrected proofs are Articles in Press that contain the authors' corrections. Final citation details, e.g., volume and/or issue number, publication year and page numbers, still need to be added and the text might change before final publication.
Although corrected proofs do not have all bibliographic details available yet, they can already be cited using the year of online publication and the DOI , as follows: author(s), article title, Publication (year), DOI. Please consult the journal's reference style for the exact appearance of these elements, abbreviation of journal names and use of punctuation.
When the final article is assigned to volumes/issues of the Publication, the Article in Press version will be removed and the final version will appear in the associated published volumes/issues of the Publication. The date the article was first made available online will be carried over.
https://www.sciencedirect.com/science/article/pii/S1045105615000925?via%3Dihub
Wed 10/14/2015 5:50 PM
-------------------------------
Filter-Based Clarification of Viral Vaccines and Vectors
View PDF
Original E-mail I sent:
On Sep 7, 2015 6:23 PM
Here's the link to that new paper but it's behind a paywall and it's German and Swedish scientists who did it.
Removal of xenotropic murine leukemia virus by nanocellulose based filter paper
M. Aspera,
T. Hanriedera,
A. Quellmalzb,
A. Mihranyanb
Abstract
The removal of xenotrpic murine leukemia virus (xMuLV) by size-exclusion filter paper composed of100% naturally derived cellulose was validated. The filter paper was produced using cellulose nanofibers derived from Cladophora sp. algae. The filter paper was characterized using atomic force microscopy, scanning electron microscopy, helium pycnometry, and model tracer (100 nm latex beads and 50 nm gold nanoparticles) retention tests. Following the filtration of xMuLV spiked solutions, LRV ≥5.25 log10TCID50 was observed, as limited by the virus titre in the feed solution and sensitivity of the tissue infectivity test. The results of the validation study suggest that the nanocellulose filter paper is useful for removal of endogenous rodent retroviruses and retrovirus-like particles during the production of recombinant proteins.
Graphical abstract
Filter paper composed of 100% naturally derived cellulose nanofibers was shown useful to remove xenotropic murine leukemia virus (MuLV) spiked solutions with LRV ≥ 5.25 as limited by titre of the feed solution and sensitivity of tissue infectivity method.
Keywords
Viral contamination;
Virus retentive filtration;
Cladophora cellulose;
Paper making;
Recombinant proteins and monoclonal antibodies
Corresponding author.
Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Note to users: Corrected proofs are Articles in Press that contain the authors' corrections. Final citation details, e.g., volume and/or issue number, publication year and page numbers, still need to be added and the text might change before final publication.
Although corrected proofs do not have all bibliographic details available yet, they can already be cited using the year of online publication and the DOI , as follows: author(s), article title, Publication (year), DOI. Please consult the journal's reference style for the exact appearance of these elements, abbreviation of journal names and use of punctuation.
When the final article is assigned to volumes/issues of the Publication, the Article in Press version will be removed and the final version will appear in the associated published volumes/issues of the Publication. The date the article was first made available online will be carried over.
https://www.sciencedirect.com/science/article/pii/S1045105615000925?via%3Dihub
Wed 10/14/2015 5:50 PM
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Filter-Based Clarification of Viral Vaccines and Vectors
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by Youness Cherradi, Sarah Le Merdy, Li-Jun Sim, Takao Ito, Priyabrata Pattnaik, Josselyn Haas and Anissa Boumlic Thursday, April 19, 2018 5:27 pm
Figure 1: Usual clarification options for viral vaccines and vectors
Viral vaccines rely on the antigen properties of a virus or virus-like entity to trigger an immune response and induce immune protection against a forthcoming viral infection. Through development of recombinant viral vaccines, developers can reduce risks associated with the presence of live and inactivated viruses. Instead, recombinant vaccines induce immunity against a pathogen by relying on the capacity of one or more antigens delivered by means of viral vectors or the baculovirus/plasmid system (1). Viral vaccines are formulated with or without adjuvants and categorized as shown in Table 1.
Table 1: Classification of viral vaccines
Viral vaccine manufacturing processes can be templated. They follow a general scheme, starting with production in either an embryonated egg or mammalian/insect cell culture. After production, the bulk harvest material is processed to purify a vaccine of interest. Following upstream production and lysis (optional), a clarification step typically is introduced to start the purification process by either centrifugation or filtration. This is a critical unit operation because it strongly affects product recovery and subsequent downstream purification.
Considerations for Clarification of Viral Vaccines: Type of Substrate
Composition, type, and level of contaminants to be removed during clarification mainly depend on the upstream process and expression system used for production. In most cases, the virus particles must be kept integral during the clarification step.
For decades, embryonated chicken eggs have been used to produce both human and animal vaccines. However, the resulting allantoic fluid harvest (rich in virus particles and cellular debris) is a challenging feed for clarification. This fluid has a high solids content (>25%, which increases with embryo age) and high mineral and protein content, hence its highly viscous consistency. It also contains rudimentary tissue compounds from chicken embryos such as feathers, beaks, blood vessels, and/or blood cells.
Several viral vaccines have moved away from an egg-based process toward the use of live cells (e.g., plant, microbial, avian, or mammalian). The composition of these feed streams can vary significantly. Based on cell viability — and lysis method selected (e.g., chemical or mechanical), if applicable — the impurity profile in the fluid to be clarified can differ considerably. For example, low cell viability can indicate high levels of contaminants released into a feed stream from cell lysis. Typical contaminants such as host cell DNA, host cell proteins (HCPs), lipids, and bigger particles such as cell debris can be identified. The proportion of solids in the feed usually is an indicator of purifying challenges ahead (~6–8% mammalian cells or up to 40% in yeast). Compared with allantoic fluid, however, cell culture harvests are considerably cleaner in terms of solids load and soluble content.
Another expression method is the baculovirus expression vector system (BEVS) used with insect cells. This is gaining interest particularly for producing viral vectors and virus-like particles (VLPs). Other expression systems such as bacteria, yeast, and plant cells also can be used to produce viral particles.
Considerations on Key Product Quality Criteria and Control Strategies
Yield: In most cases, yield is an off-line measurement conducted at the end of a number of process steps. Depending on the success criteria for each step, yield can be the main parameter to consider when selecting one option over another for a given step. Based on the size and properties of viral particles, yield could be affected by the clarification method used. For example, whereas positive charges increase nucleic acids and HCP capture, diatomaceous earth can retain viruses by adsorption.
Some viruses are shear sensitive and can be damaged by high shear exposure in disk-stack centrifuges or by high cross-flow and multiple pump passages through TFF. Due to their large size, viruses larger than 100 nm also can be retained simply by tight filters. Thus, companies should take such factors into consideration when selecting depth filters because some depth filter devices include a 0.1-μm membrane that could cause retention-driven product loss. Other process elements such as aggregation and an excess of impurities can compromise viral particle recovery.
Final Product Purity Levels: Regulatory agencies provide recommendations and requirements regarding acceptable residual amounts of contaminants in final drug products. For reasons of patient safety and tolerance, host cell DNA in a final product must be reduced to appropriate levels. In 1998, the World Health Organization (WHO) specified the maximum residual DNA content in a vaccine to be <10 ng/dose. Since then, the European Medicines Agency (EMA) proposed more stringent conditions based on the type of cell line (tumorigenic origin) used in vaccine manufacturing. The US Food and Drug Administration (FDA) follows a case-by-case evaluation approach and recommends that manufacturers reduce both the size (~200 bp) and amount of DNA per dose.
To date, a final DNA content of <10 ng/ dose commonly is accepted for most biologics. Similarly, a recombinant monoclonal antibody (MAb) product must reach clearance of impurities to <100 ppm of HCP, ≤10 ng/dose of DNA, and <5% of immunogenic aggregates (Table 2).
Table 2: Acceptable remaining impurities in vaccine products
Feed Quality Evaluation Criteria and Processing Parameters: Several parameters can be used to assess the clarity or quality of a product, either during process development or after each manufacturing process step. Turbidity is an easy parameter to monitor and provides an immediate assessment of feed quality. For example, it enables the detection of depth filter breakthrough.
The two primary methods of ascertaining the effectiveness of the clarification step are centrifugation and filtration. Turbidity can be monitored simply by absorbance/scatter in the visible range, providing an immediate assessment of particle load within the filtrate or centrate. Turbidity also relates to filter capacity, which is the volume of feed a depth filter can process before the pressure drop breaches specifications. Capacity relating to both pressure drop and turbidity breakthrough are linked, and specifications for both should be set during process development. For some processes (particularly those with a smaller average particle size in the feed), turbidity breakthrough is the limiting factor in sizing a filtration train. Often, capacity limit is attributed to pressure drop. But because high pressures or flow rates can cause premature turbidity breakthrough, both mechanisms can be related.
Technology Options for Clarification of Virus Vaccines: Because of the extreme diversity of viral vaccines in terms of size, structure, shape, and expression system, no unified template exists for their production and purification. Those processes can be divided into four different phases: upstream/production, clarification, purification, and formulation.
To reduce burden on downstream purification steps, the main objective of a clarification process is to remove undesirable materials, including whole cells, cell debris, colloids, and large aggregates. As the first downstream process, clarification should be optimized to maximize product yield and purity. Several serial operational steps often are required to achieve a desired level of clarification. The first operation (often referred to as primary clarification) removes larger particles, and the second (often referred to as secondary clarification) removes colloids and other submicron particles (Figure 1).
In theory, all available technologies (low-speed centrifugation, microfiltration TFF, NFF) can be selected and potentially combined to clarify viruses. As with other manufacturing process steps, a clarification process should have predictable scalability, be manufacturing-friendly (e.g., be easy to use, reduce holdup volume, provide operator safety), and have a low cost of goods (CoG). However, the clarification process of viral vaccines has two unique characteristics that can require more tailored solutions:
low solids content with a high nucleic acid and colloid content, requiring higher retention capacity
high feed variability and cell culture enhancements, requiring more robustness (2).
Although centrifugation can handle a high solids load and traditionally has been used in batch and continuous modes, it requires large capital investment and high maintenance costs. More important, centrifugation scale-up can be problematic because of unreliable scale-down models with nonlinear scalability and high-shear operation for shear-sensitive vaccines. But NFF and TFF have gained interest for vaccine clarification because they are significantly easier to scale-up and implement.
NFF: Primary clarification using NFF typically involves depth filters that often contain positively charged material and filter aids (e.g., diatomaceous earth) that improve retention of cell debris, colloids, and negatively charged contaminants. NFF relies on two main mechanisms for particle retention: size exclusion and adsorption.
NFF membrane filters can be used in secondary filtration because they retain particles by size exclusion. Certain grades of depth filters have a tighter pore-size distribution (which offers greater colloidal particle retention) but do not have high holding capacity. Noncharged depth filters also can be used for clarification while offering higher cost-effectiveness for small batches (≤1,000 L). They typically use three media types:
melt-blown media fabricated in a pleated format to achieve higher flow rates and holding capacities
graded density of concentrically wrapped media to allow the filter to remove finer contaminants progressively membranes to provide higher retention efficiency.
TFF membranes with retention ratings in the range of 0.1–0.65 μm have been used to retain cells, cell debris, and other large contaminants. Most TFF devices are linearly scalable and reusable after cleaning, and hence greatly reduce consumable costs. However, certain viruses (e.g., extracellularly produced enveloped virus-like particles) can be damaged by high-shear exposure in disk-stack centrifuges or by high cross-flow and multiple pump passages in TFF processes. Open-channel TFF devices (cassette format without screen) are preferred to minimize shear.
Case Studies
Egg-Based Vaccines: In influenza vaccine processes, a typical allantoic fluid harvest is rich in proteins (e.g., ovalbumin, lysozyme, ovomucin) and contains 45 μg hemagglutinin antigen per egg (~3–4 μg HA and 108–109 infectious units/mL of allantoic fluid). The typical gravity-settled turbidity of a virus-containing allantoic fluid (VCAF) generally is 46–132 NTU. Low-speed zonal continuous centrifugation around 4,000–5,000g often is the preferred option to remove large particles and thus gets used for primary clarification, typically providing a recovery yield of 70% (3, 4). Many vaccine manufacturers use sucrose gradient zonal centrifugation to purify and concentrate viruses. However, polypropylene and cellulose-based depth filters also can be implemented for filtration. Fair capacities of 150–210 L/m² and up to 3× reduction of feed stream turbidity can be achieved with those allantoic fluid harvests. That option is appropriate for influenza vaccines, which are prone to adsorption loss during clarification on charged filters.
NFF also can be used for secondary clarification. Combinations of polypropylene, cellulose, and glass-fiber materials generally demonstrate good efficiency (5). Using a salt solution can reduce association between a virus vaccine and solid debris, resulting in a yield increase of about twofold without compromising viral particle integrity.
One study demonstrated that higher ionic strength on allantoic debris increased influenza virus yields (6). In that case, 1.5 M NaCl was applied to pooled allantoic fluids of various influenza strains, and the sample was centrifuged for different durations to understand the amount of virus partitioned in the supernatant and pellet. Control samples were kept at 0.15M NaCl. This test scope was expanded to include other influenza strains — A/New Caledonia (H1N1), A/Texas (H3N2), B/Jiangsu and B/Hong Kong — to demonstrate an average twofold yield increase. Further work verified the integrity of the purified virus in control and higher ionic strengths. Data showed that higher ionic strength did not adversely affect the live titer of the tested influenza virus strains (2). Specifically, studies have reported use of a 1.2-μm cellulose nitrate (CN) filter polypropylene media followed by 0.45-μm filter polyvinylidene fluoride (PVDF) membrane for clarification of cell-based influenza with a loading of 111 L/m² and 105 L/m², respectively (7).
Another option is use of TFF with a 0.65-μm or 0.45-μm microfiltration membrane device operated with permeate flux control (8). Using a “two-pump process” with a permeate pump in addition to the standard TFF feed pump allows for permeate flux control to manage/reduce polarization and fouling. That provides better characterization of the “critical” flux — the limiting flux above which a process becomes unstable (9).
Moreover, researchers conducted primary clarification of allantoic fluid using a 40-μm bag filter followed by an open-channel microfiltration device (Prostak 0.65 μm; data not shown). Using a crossflow of 3 LPM/channel, with transmembrane pressure (TMP) at 0.2 bar and dP at 0.4 bar, a capacity of 33 L/m² was achieved. The MF process was designed for 10× concentration and 5× diafiltration, and the Prostak allowed for 60× reuse.
Viruses in Adherent Cells on Microcarriers: Microcarriers can be used as a support matrix for the growth of adherent cells such as Vero cells. Primary clarification of these cells grown on microcarriers can be performed using a 75-μm stainless steel sieve to remove microcarriers from harvest. A Millistak+ C0HC depth filter medium then can be used for secondary clarification and directly followed by a sterilizing-grade filter (10). This study was reported for a cell density of 0.78 x 106 cells/mL. Results showed varying filter capacity based on cell density or cell viability of harvest.
Viral Vectors: Trial results on lentiviruses from the supernatant of HEK293T cells show positive performance (data not shown). The negative charge of lentiviruses is known to be responsible for poor recoveries with positively charged depth filters. Therefore, despite the fact that no sign of plugging was observed using a Millistak+ C0HC depth filter, very low viral vector recovery was recorded. However, use of a 1.0/0.5 μm Polysep II filter led to both a high capacity and high 84% recovery. The Polygard CN filter, on the other hand, behaved quite well in terms of recovery (75%) but showed more signs of plugging. An extra 10–20% lower virus recovery should be considered for the following 0.45 μm or 0.22μm (sterile) filtration step.
Two other studies on lentivirus feed streams confirm the positive recovery performance using the Polygard CN filters (CN25, CN12, CN10, and CN06 with more than 80% recovery). Other studies report an interesting result with recoveries reaching up to 90% for the Millistak+ CE50 filter that does not contain inorganic filter aid (e.g., diatomaceous earth; data not shown).
Adenoviruses also can be prone to adsorption, but divergent results have been reported. In some cases, good adenovirus recovery is observed even when a positively charged depth filter medium containing diatomaceous earth such as the Millistak+ HC medium is used (11). If adenovirus is lost, Polygard and Clarigard filters can be used instead, but a secondary clarification might be needed to reduce turbidity (12).
Clarification Options
Different approaches are used to produce viral vaccines, making designing a typical template for their clarification difficult. Indeed, these products can be produced by different expression systems and possess a range of physicochemical properties. The clarification method selected should take those factors into account to ensure that yield and contaminants removal are sufficient.
NFF and microfiltration TFF technologies are increasingly preferred to centrifugation because of their more predictable scalability and robustness. Low CoG also can be achieved by tailoring the choice of media chemistry and porosity to gain high recovery and productivity with satisfactory impurity removal. Continuing innovation in this field by suppliers of purification and filtration systems will help vaccine manufacturers meet their current and future challenges on the road to developing novel and efficient therapies.
References
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2 Besnard L, et al. Clarification of Vaccines: An Overview of Filter-Based Technology Trends and Best Practices. Biotechnol. Advances 34(1) 2016: 1–13.
3 Hendriks J, et al. An International Technology Platform for Influenza Vaccines. Vaccine 29 Suppl 1, 2011: A8–11.
4 Eichhorn U. Influenza Vaccine Composition. US Patent US7316813 B2, 2008.
5 Lampson GP, Machlowitz RA. Process for Producing Purified Concentrated Influenza Virus.US Patents US3547779 A, 1970.
6 Hughes K, et al. Yield Increases in Intact Influenza Vaccine Virus from Chicken Allantoic Fluid through isolation from Insoluble Allantoic Debris. Vaccine 25(22) 2007: 4456–4463.
7 Thompson M, Wee J, Nagpal A. Methods for Purification of Viruses. European Patent EP2334328 A4, 2012.
8 Lau SY, et al. Impact of Process Loading on Optimization and Scale-Up of TFF Microfiltration. BioProcess J. 13(2) 2014: 46–55; doi:10.12665/J132.Pattna.
9 Raghunath B, et al. Best Practices for Optimization and Scale-Up of Microfiltration TFF Processes. Bioprocess. J. 11(1) 2012: 30–40.
10 Thomassen YE, et al. Scale-Down of the Inactivated Polio Vaccine Production Process. Biotechnol. Bioeng. 110(5) 2013: 1354–1365.
11 Namatovu HH, et al. Evaluation of Filtration Products in the Production of Adenovirus Candidates Used in Vaccine Production: Overview and Case Study. BioProcess J. 5(3) 2006: 67–74.
12 Weggeman M, van Corven EJJM. Virus Purification Methods. US Patent: US8124106 B2, 2012.
Corresponding author Anissa Boumlic (anissa.boumlic@merckgroup.com) is associate director of the vaccine segment at Millipore S.A.S.
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