Amine Kamen
McGill University, Bioengineering, Department Member
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This study aimed to establish the optimal operational conditions for hydrogen production using vermicomposting-tea and sugarcane molasses as substrate. The experiments were carried out by triplicate in 110 ml serological bottles, a... more
This study aimed to establish the optimal operational conditions for hydrogen production using vermicomposting-tea and sugarcane molasses as substrate. The experiments were carried out by triplicate in 110 ml serological bottles, a Box-Behnken design of experiments was performed in anaerobic dark conditions. The maximal hydrogen production (HP), hydrogen production rate (HPR), and hydrogen yield (HY) attained were 1021.0 mlL-1, 5.32 mlL-1h-1, and 60.3 mlLH2-1/gTCC, respectively. The statistical model showed that the optimal operational conditions for pH, molasses concentration, and temperature were 6.5; 30 % (v/v) and 25 °C. The bioreactor run showed 17.202 L of hydrogen, 0.58 Lh-1, and 77.2 mlH2gTCC-1 For HP, HPR, and HY. Chemometric analysis for the volatile fatty acids obtained at the fermentation showed that only two principal components are required to explain 90 % of the variance. The representative pathways for hydrogen production were acetic and butyric acids. This study established the operational conditions for the upstream processing amenable to pilot and industrial-scale operations. Our results add value to molasses within the circular economy for hydrogen production using a novel consortium from vermicompost.
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Research Interests: Virology and Bioprocess
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We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method... more
We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral
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In recent years, the use of adeno‐associated viruses (AAVs) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre‐clinical and clinical trials. AAV serotype 6 (AAV6) has... more
In recent years, the use of adeno‐associated viruses (AAVs) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre‐clinical and clinical trials. AAV serotype 6 (AAV6) has been found to be efficient in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG), making large‐scale production of AAV6 necessary. Suspension cell‐based platforms are currently limited to low cell density productions due to the widely reported cell density effect (CDE), which results in diminished production at high cell densities and decreased cell‐specific productivity. This limitation hinders the potential of the suspension cell‐based production process to increase yields. In this study, we investigated the improvement of the production of AAV6 at higher cell densities by transiently transfecting HEK293SF cells. The results showed that when the plasmid DNA was provided on a cell basis, the production could be carried out at medium cell density (MCD, 4 × 106 cells mL−1) resulting in titers above 1010 VG mL−1. No detrimental effects on cell‐specific virus yield or cell‐specific functional titer were observed at MCD production. Furthermore, while medium supplementation alleviated the CDE in terms of VG/cell at high cell density (HCD, 10 × 106 cells mL−1) productions, the cell‐specific functional titer was not maintained, and further studies are necessary to understand the observed limitations for AAV production in HCD processes. The MCD production method reported here lays the foundation for large‐scale process operations, potentially solving the current vector shortage in AAV manufacturing.
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Every year, millions of people are infected by the influenza virus around the world, which results in more than half a million deaths, particularly among the more vulnerable population. Since vaccination is the most efficient method of... more
Every year, millions of people are infected by the influenza virus around the world, which results in more than half a million deaths, particularly among the more vulnerable population. Since vaccination is the most efficient method of protection, millions of doses must be produced in a short period of time to supply seasonal vaccination campaigns around the globe, and billions of doses would be required to respond to a potential global influenza pandemic. The lack of flexibility of the current egg‐based production system and its long production cycles have pushed biomanufacturers to invest in more flexible alternatives for vaccine production, particularly cell culture‐based processes. While a valuable alternative, virus yields are still low, requiring extensive efforts to increase process productivity. Major efforts in the intensification of cell culture‐based viral vaccine manufacturing focus on the development of high cell density processes, which mostly involve the employment of perfusion‐based strategies. In this review, some of the advantages of cell culture‐based production of influenza vaccines will be discussed, and some of the current challenges and opportunities for the intensification of these processes will be presented. Finally, the recent advances in high cell density processes for influenza vaccine manufacturing will be reviewed.
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Research Interests: Engineering, Biology, Virology, Quantification, Hemagglutinin, and 3 moreInfluenza, Antibody, and Dot Blot
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Research Interests: Virology and Bioprocess
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BackgroundThe Vero cell line is the most used continuous cell line for viral vaccine manufacturing with more than 30 years of accumulated experience in the vaccine industry. Nonetheless, virus production yield with Vero cells remains... more
BackgroundThe Vero cell line is the most used continuous cell line for viral vaccine manufacturing with more than 30 years of accumulated experience in the vaccine industry. Nonetheless, virus production yield with Vero cells remains limited. Therefore, given Vero cell line infection susceptibility to a wide range of viruses, alleviating limitations of viral replication and increasing production kinetics in Vero cells could significantly reduce vaccine production time and cost.Here we review the literature on the development of Vero cells as the most effective manufacturing platform for viral vaccines. Whereas various bioprocess development strategies were proposed to improve the production, this review focuses on the genomic characterization and genetic engineering aspects of Vero cells. Rational design of production cell line emerged as a state‐of‐the‐art approach to significantly enhance Vero cell viral production yield and adaptation to suspension culture which aligns with the global preparedness efforts to accelerate and intensify vaccine production capacity to better respond to pandemic situations and epidemic outbreaks.ConclusionUntil recently, the lack of a reference genome for the Vero cell line has limited the understanding of Vero cells behavior in defined culture conditions as well as host‐virus interactions underlying the affinity of the Vero cell line with emerging and re‐emerging pathogens. Importantly this limited our ability to re‐design high‐yield vaccine production processes using Vero genome editing.
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Despite their wide use in the vaccine manufacturing field for over 40 years, one of the main limitations to recent efforts to develop Vero cells as high‐throughput vaccine manufacturing platforms is the lack of understanding of virus‐host... more
Despite their wide use in the vaccine manufacturing field for over 40 years, one of the main limitations to recent efforts to develop Vero cells as high‐throughput vaccine manufacturing platforms is the lack of understanding of virus‐host interactions during infection and cell‐based virus production in Vero cells. To overcome this limitation, this manuscript uses the recently generated reference genome for the Vero cell line to identify the factors at play during influenza A virus (IAV) and recombinant vesicular stomatitis virus (rVSV) infection and replication in Vero host cells. The best antiviral gene candidate for gene editing was selected using Differential Gene Expression analysis, Gene Set Enrichment Analysis and Network Topology‐based Analysis. After selection of the ISG15 gene for targeted CRISPR genomic deletion, the ISG15 genomic sequence was isolated for CRISPR guide RNAs design and the guide RNAs with the highest knockout efficiency score were selected. The CRISPR experiment was then validated by confirmation of genomic deletion via PCR and further assessed via quantification of ISG15 protein levels by western blot. The gene deletion effect was assessed thereafter via quantification of virus production yield in the edited Vero cell line. A 70‐fold and an 87‐fold increase of total viral particles productions in ISG15−/− Vero cells was achieved for, respectively, IAV and rVSV while the ratio of infectious viral particles/total viral particles also significantly increased from 0.0316 to 0.653 for IAV and from 0.0542 to 0.679 for rVSV‐GFP.
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Abstract Transient gene expression (TGE) in animal cell cultures has been used for almost 30 years to produce milligrams and grams of recombinant proteins, virus-like particles and viral vectors, mainly for research purposes. The need to... more
Abstract Transient gene expression (TGE) in animal cell cultures has been used for almost 30 years to produce milligrams and grams of recombinant proteins, virus-like particles and viral vectors, mainly for research purposes. The need to increase the amount of product has led to a scale-up of TGE protocols. Moreover, product quality and process reproducibility are also of major importance, especially when TGE is employed for the preparation of clinical lots. This work gives an overview of the different technologies that are available for TGE and how they can be combined, depending on each application. Then, a critical assessment of the challenges of large-scale transient transfection follows, focusing on suspension cell cultures transfected with polyethylenimine (PEI), which is the most widely used methodology for transfection. Finally, emerging opportunities for transient transfection arising from gene therapy, personalized medicine and vaccine development are reviewed.
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Recent advances and discoveries in the structure and role of mRNA as well as novel lipid-based delivery modalities have enabled the advancement of mRNA therapeutics into the clinical trial space. The manufacturing of these products is... more
Recent advances and discoveries in the structure and role of mRNA as well as novel lipid-based delivery modalities have enabled the advancement of mRNA therapeutics into the clinical trial space. The manufacturing of these products is relatively simple and eliminates many of the challenges associated with cell culture production of viral delivery systems for gene and cell therapy applications, allowing rapid production of mRNA for personalized treatments, cancer therapies, protein replacement and gene editing. The success of mRNA vaccines during the COVID-19 pandemic highlighted the immense potential of this technology as a vaccination platform, but there are still particular challenges to establish mRNA as a widespread therapeutic tool. Immunostimulatory byproducts can pose a barrier for chronic treatments and different production scales may need to be considered for these applications. Moreover, long-term storage of mRNA products is notoriously difficult. This review provides a de...
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Research Interests: Chemistry, Analytical Chemistry, Chromatography, Virology, Medicine, and 15 moreLinear models, Western blotting, Virus, Animals, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, High Pressure Liquid Chromatography, Reproducibility of Results, Sensitivity and Specificity, Organic Chemicals, Linear System and Response, Biochemistry and cell biology, Nucleic Acid, Spodoptera litura, Elution, and Pharmacology and pharmaceutical sciences
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ABSTRACT Gas mass spectroscopy is used in this study to monitor on-line CO 2 production and O 2 consumption rates during growth of Sf-9 insect cell batch culture and recombinant protein production using BEVS (Baculovirus Expression Vector... more
ABSTRACT Gas mass spectroscopy is used in this study to monitor on-line CO 2 production and O 2 consumption rates during growth of Sf-9 insect cell batch culture and recombinant protein production using BEVS (Baculovirus Expression Vector System). Specific production and consumption rates are then calculated for different metabolic phases based on viable cell counts. In animal cell bioreactors the dissolved oxygen (DO) is generally controlled with a strategy based on oxygen supplementation in the inlet gas. Determination of oxygen consumption is then achieved under quasi-steady state conditions without any DO control to avoid controller signal interference. A steady state method is presented here to overcome this limitation. The relationship between oxygen partial pressure in the headspace and viable cell count is linear, hence a simultaneous determination of volumetric mass transfer coefficient allows calculation of oxygen consumption rate.
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An anion exchange high-performance liquid chromatography (HPLC) method for the quantification of human Reovirus type 3 particles was validated according to the performance criteria of precision, specificity, linearity of calibration and... more
An anion exchange high-performance liquid chromatography (HPLC) method for the quantification of human Reovirus type 3 particles was validated according to the performance criteria of precision, specificity, linearity of calibration and working range, limits of detection and quantification, accuracy and recovery. Samples taken at various stages of Reovirus purification were used for the validation of the method. The method was specific for Reovirus which eluted around 9.8min without interference from any other component in the sample. Reovirus can be detected between 0.32E+12 and 2.10E12VP/mL by the proposed method that has the correlation coefficient of linearity equal to 0.9974 and the slope of linearity equal to 5.74E-07 area units/(VPmL).
Research Interests: Chemistry, Analytical Chemistry, Chromatography, Medicine, Quantitative analysis, and 15 moreHPLC, Calibration, High Performance Liquid Chromatography, Particle, Particle Size, Reproducibility of Results, Correlation coefficient, Quantitative Analysis, Sensitivity and Specificity, Linearity, Reference standards, Elution, Pharmacology and pharmaceutical sciences, HPLC Chromatography, and limit of detection
Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding... more
Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)8 to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%.
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The Vero cell line is the most used continuous cell line for viral vaccine manufacturing. Its anchorage-dependent use renders scaling up challenging and operations very labor-intensive which affects cost effectiveness. Thus, efforts to... more
The Vero cell line is the most used continuous cell line for viral vaccine manufacturing. Its anchorage-dependent use renders scaling up challenging and operations very labor-intensive which affects cost effectiveness. Thus, efforts to adapt Vero cells to suspension cultures have been invested, but hurdles such as the long doubling time and low cell viability remain to be addressed. In this study, building on the recently published Vero cell line annotated genome, a functional genomics analysis of the Vero cells adapted to suspension is performed to better understand the genetic and phenotypic switches at play during the adaptation of Vero cells from anchorage-dependent to suspension cultures. Results show downregulation of the epithelial-to-mesenchymal transition (EMT) pathway, highlighting the dissociation between the adaptation to suspension process and EMT. Surprisingly, an upregulation of cell adhesion components is observed, notably the CDH18 gene, the cytoskeleton pathway, an...
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The use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has risen considerably in recent years. Consequently, the amount of AAV vectors required during the validation and clinical trials has also increased. AAV... more
The use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has risen considerably in recent years. Consequently, the amount of AAV vectors required during the validation and clinical trials has also increased. AAV serotype 6 (AAV6) is well-documented for its efficiency in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG). Overall, this means that large-scale production of AAV6 is needed. Suspension cell-based platforms are currently limited to low-cell-density productions, hindering the potential of this production process to increase yields. Here, we investigate the improvement of the production of AAV6 at higher cell densities. The production was performed by transient transfection of HEK293SF cells. When the plasmid DNA is provided on a cell basis, the production can be ca...
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The recombinant adeno-associated virus (rAAV) is a viral vector technology for gene therapy that is considered the safest and most effective way to repair single-gene abnormalities in non-dividing cells. However, improving the viral titer... more
The recombinant adeno-associated virus (rAAV) is a viral vector technology for gene therapy that is considered the safest and most effective way to repair single-gene abnormalities in non-dividing cells. However, improving the viral titer productivity in rAAV production remains challenging. The first step to this end is to effectively monitor the process state variables (cell density, GLC, GLN, LAC, AMM, and rAAV viral titer) to improve the control performance for an enhanced productivity. However, the current approaches to monitoring are expensive, laborious, and time-consuming. This paper presents an extended Kalman filter (EKF) approach used to monitor the rAAV production using the online viable cell density measurements and estimating the other state variables measured at a low frequency. The proposed EKF uses an unstructured mechanistic kinetic model applicable in the upstream process. Three datasets were used for parameter estimation, calibration, and testing, and the data wer...
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Chun Fang Shen, National Research Council of Canada chunfang.shen@cnrc-nrc.gc.ca Claire Guilbault, National Research Council of Canada Xiuling Li, China National Biotech Group, China Mehdy Elahi, National Research Council of Canada Sven... more
Chun Fang Shen, National Research Council of Canada chunfang.shen@cnrc-nrc.gc.ca Claire Guilbault, National Research Council of Canada Xiuling Li, China National Biotech Group, China Mehdy Elahi, National Research Council of Canada Sven Ansorge, National Research Council of Canada Amine Kamen, National Research Council of Canada & McGill University Lakshmi Krishnan, National Research Council of Canada Rénald Gilbert, National Research Council of Canada
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All influenza vaccines currently sold in Canada require one fertilized chicken egg to produce roughly one dose of vaccine. During pandemic influenza outbreaks, the limited availability of eggs stresses the ability of this method to... more
All influenza vaccines currently sold in Canada require one fertilized chicken egg to produce roughly one dose of vaccine. During pandemic influenza outbreaks, the limited availability of eggs stresses the ability of this method to deliver vaccine in a timely manner (1). Unlike eggs, cell lines grow exponentially, resulting in virtually limitless substrate for cultivating influenza vaccines. This ability to rapidly scale production during periods of increased demand is ideal for effectively responding to pandemic influenza outbreaks. While promising, cell-based influenza vaccine production suffers from low volumetric yield (~10-fold lower) compared to egg-based methods (2).
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Training Deep Learning (DL) models with missing labels is a challenge in diverse engineering applications. Missing value imputation methods have been proposed to try to address this problem, but their performance is affected with Massive... more
Training Deep Learning (DL) models with missing labels is a challenge in diverse engineering applications. Missing value imputation methods have been proposed to try to address this problem, but their performance is affected with Massive Proportion of Missing Labels (MPML). This paper presents a approach for handling MPML in Multivariate Long-Term Time Series Forecasting. It is an two-step process where interpolation (using Gaussian Processes Regression (GPR) and domain knowledge from experts) and prediction model are separated to enable the integration of prior domain knowledge. First, a set of samples of the possible interpolation of the missing outputs are generated by the GPR based on the domain knowledge. Second, the observed input sensor data and interpolated labels from GPR are used to train the prediction model. We evaluated our approach with the development of a soft-sensor with one real datasets to forecast the biomass during recombinant adeno-associated virus (rAAV) produ...
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Catherine Cleuziat, Boehringer-Ingelheim, France catherine.cleuziat@boehringer-ingelheim.com Sophie Biard, Boehringer-Ingelheim, France Géraldine Popovic, Boehringer-Ingelheim, France Sylvain Lagresle, Boehringer-Ingelheim, France Chloé... more
Catherine Cleuziat, Boehringer-Ingelheim, France catherine.cleuziat@boehringer-ingelheim.com Sophie Biard, Boehringer-Ingelheim, France Géraldine Popovic, Boehringer-Ingelheim, France Sylvain Lagresle, Boehringer-Ingelheim, France Chloé Damiany, Boehringer-Ingelheim, France Christine Coupier, Boehringer-Ingelheim, France Chun Fang Shen, NRC, USA Amine Kamen, MacGill University, Canada Hervé Poulet, Boehringer-Ingelheim, France Noël Detraz, Boehringer-Ingelheim, France Zahia Hannas, Boehringer-Ingelheim, France
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The Vero cell line is the most used continuous cell line for viral vaccine manufacturing. Its anchorage-dependent use renders scaling-up challenging and operations very labor intensive which affects cost effectiveness. Thus, efforts to... more
The Vero cell line is the most used continuous cell line for viral vaccine manufacturing. Its anchorage-dependent use renders scaling-up challenging and operations very labor intensive which affects cost effectiveness. Thus, efforts to adapt Vero cells to suspension cultures have been invested but hurdles such as the long doubling time and low cell viability remain to be addressed. In this study, building on the recently published Vero cell line annotated genome, a functional genomics analysis of the Vero cells adapted to suspension is performed to better understand the genetic and phenotypic switches at play during the adaptation of Vero cells from anchorage-dependent to suspension cultures. Results show a downregulation of the epithelial to mesenchymal transition (EMT) pathway, highlighting the dissociation between the adaptation to suspension process and EMT. Surprisingly, an upregulation of cell adhesion components is observed, notably the CDH18 gene, the cytoskeleton pathway, a...
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We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific to the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood... more
We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific to the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS).