This document discusses the development of ELISAs to test Dasiprotimut-T, a therapeutic cancer vaccine produced by Biovest International. It describes qualitative and quantitative ELISAs developed in-house to test for antibody concentration, impurities from host cell proteins and bovine proteins. Automated systems using Tecan Freedom EVO liquid handlers were also implemented to increase throughput and reimbursement for quality control testing required by regulatory agencies. Quality control documentation, validation protocols, stability studies and inventory management are important aspects of ensuring the vaccine manufacturing process meets regulatory standards.
Dr. Richard Kerr - Development of an Effective Pathogen Screening Program usi...John Blue
Development of an Effective Pathogen Screening Program using Rapid, High-Throughput Molecular Detection Assays - Dr. Richard Kerr, Diagnostic R & D Lab Director, Daisy Farms, from the 2016 NIAA Annual Conference: From Farm to Table - Food System Biosecurity for Animal Agriculture, April 4-7, 2016, Kansas City, MO, USA.
More presentations at http://www.trufflemedia.com/agmedia/conference/2016_niaa_farm_table_food_system_biosecurity
Semi Automated Low-throughput Workflow for Microbial Analyses of Human StoolQIAGEN
The gut microbiota composition changes dramatically throughout aging and disease. A healthy gut microbiota is typically characterized by large bacterial taxonomic diversity and functional capacity, whereas frailty and aging are associated with loss of diversity and expansion of more pathogenic bacterial species. However, in order to accurately profile changes in microbial communities, the reproducible isolation of high-quality DNA is an important step. Automation enables reliable and reproducible isolation of DNA of superior quality, which can be used directly for downstream sequencing applications.
This webinar focuses on the development of a semi-automated workflow to profile the gut microbiota of young and old individuals and identify changes in bacterial composition and function that occur with age. This workflow will help to simplify and streamline the DNA extraction process for samples with high inhibitor content and subsequent microbial community analyses.
Bioo Scientific - Improving the Performance of SureSelectXT2 Target CaptureBioo Scientific
Agilent’s SureSelectXT2 baits are popular options for target capture because they offer offer a wide range of predesigned baits and flexible customization options which allow users to design their own capture panels. Incorporating index-specific barcode blockers during library prep allow researchers to obtain a higher percentage of on-target reads and better coverage from their SureSelectXT2 target capture experiments. The NEXTflex™ Pre- and Post- Capture Combo Kit (Agilent SureSelectXT2 Compatible) incorporates index-specific barcode blockers allowing researchers to get more useful data from their Agilent SureSelectXT2 Target Capture sequencing runs.
Dr. Richard Kerr - Development of an Effective Pathogen Screening Program usi...John Blue
Development of an Effective Pathogen Screening Program using Rapid, High-Throughput Molecular Detection Assays - Dr. Richard Kerr, Diagnostic R & D Lab Director, Daisy Farms, from the 2016 NIAA Annual Conference: From Farm to Table - Food System Biosecurity for Animal Agriculture, April 4-7, 2016, Kansas City, MO, USA.
More presentations at http://www.trufflemedia.com/agmedia/conference/2016_niaa_farm_table_food_system_biosecurity
Semi Automated Low-throughput Workflow for Microbial Analyses of Human StoolQIAGEN
The gut microbiota composition changes dramatically throughout aging and disease. A healthy gut microbiota is typically characterized by large bacterial taxonomic diversity and functional capacity, whereas frailty and aging are associated with loss of diversity and expansion of more pathogenic bacterial species. However, in order to accurately profile changes in microbial communities, the reproducible isolation of high-quality DNA is an important step. Automation enables reliable and reproducible isolation of DNA of superior quality, which can be used directly for downstream sequencing applications.
This webinar focuses on the development of a semi-automated workflow to profile the gut microbiota of young and old individuals and identify changes in bacterial composition and function that occur with age. This workflow will help to simplify and streamline the DNA extraction process for samples with high inhibitor content and subsequent microbial community analyses.
Bioo Scientific - Improving the Performance of SureSelectXT2 Target CaptureBioo Scientific
Agilent’s SureSelectXT2 baits are popular options for target capture because they offer offer a wide range of predesigned baits and flexible customization options which allow users to design their own capture panels. Incorporating index-specific barcode blockers during library prep allow researchers to obtain a higher percentage of on-target reads and better coverage from their SureSelectXT2 target capture experiments. The NEXTflex™ Pre- and Post- Capture Combo Kit (Agilent SureSelectXT2 Compatible) incorporates index-specific barcode blockers allowing researchers to get more useful data from their Agilent SureSelectXT2 Target Capture sequencing runs.
Profile Multiple Cytokines and Chemokines Simultaneously with Very High Sensi...QIAGEN
Learn how to profile multiple cytokines and chemokines simultaneously with very high sensitivity and specificity using the standard ELISA reader. Available in different formats to suit your research needs such as single-analyte, multi-analyte or custom mix-n-match format for human, mouse and rat.
Subsequent library screening will fish out the antibody mutants that have high affinity. Two library screening strategies are available. In the first "surface-panning" strategy, decreasing concentrations of antigen is surface immobilized. In the second "solution-sorting" strategy, in which a labeled antigen in solution is used, we have two approaches, selection based on the equilibrium constant (Kd) and selection based on binding kinetics. In the first approach, sub-library phage is incubated with biotinylated antigen at controlled concentrations and bound phages are captured by immobilized NeutrAvidin. Selection based on binding kinetics is also termed off-rate (Koff) selection, in which phage population is allowed to saturate the labeled antigen before a large molar excess of unlabeled antigen is added to the mix for controlled periods of time. This allows the selection of mutant antibodies that have slower off-rates. Since a reduction in Koff usually results in a higher affinity, this selection approach singles out antibody variants with improved Kd.
Quantitative Real-Time PCR for Rapid and Accurate Titration of Recombinant Ba...Bushra Hafeez
The baculovirus expression system is widely used for the production of recombinant protein in insect cells.
Baculovirus can be amplified to very high titers in suspension culture facilitating the production of large quantities of recombinant protein.
In order to maximize and ensure reproducibility of protein production, it is important to obtain an accurate estimate of the recombinant virus titer.
An improved QPCR titration method using Applied Biosystems Taqman fluorogenic probes is used.
It is faster and give increased accuracy across a wide range of virus titers.
This technology takes advantage of the 5’exonuclease activity of Taq polymerase,
To digest a probe.
Targeted RNAseq for Gene Expression Using Unique Molecular Indexes (UMIs): In...QIAGEN
Traditional RNA sequencing (RNA-Seq) is a powerful tool for expression profiling, but is hindered by PCR amplification bias and inaccuracy at low expressing genes. QIAseq RNA is a flexible and precise tool developed for mitigating these complications, allowing digital gene expression analysis. This in-depth webinar will cover sample requirements, experimental design, NGS platform-specific challenges and workflow for gene enrichment, library prep and sequencing. The applications of QIASeq RNA Panels in cancer research, stem cell differentiation and elucidating the effects small molecules on signaling pathways will be highlighted.
Critical Steps for Real-Time PCR Analysis: Tips and Solutions to Achieve Effi...QIAGEN
In this slidedeck, we cover the following topics which are critical steps for efficient and precise gene expression studies using real-time PCR technology:
1) Effect of RNA integrity on real-time PCR results – tips to achieve a true RNA profiling suitable for real-time PCR studies
2) Improved methods for cDNA synthesis, optimized for real-time PCR
3) Real-time PCR analysis:
• Real-time PCR essentials and background information on different quantification strategies
• SYBR Green real-time PCR – factors influencing specificity
• Introduction to probe technology
• New, fast and efficient real-time PCR solutions
Snippy - Rapid bacterial variant calling - UK - tue 5 may 2015Torsten Seemann
Using Snippy to call variants in bacterial short read datasets via alignment to reference, and then using these alignments to produce core SNP alignments for phylogenomics.
Streamlining NGS data processing with GENALICE MAP - Remco Ursem (Rijk Zwaan)GENALICE
Rijk Zwaan develops vegetable varieties and sells the seeds they produce. Rijk Zwaan has over 1.000 varieties in 25 different vegetable crops. The company shares challenges of sequencing data and compares deployment of GENALICE MAP with BWA/GATK on storage footprint, analysis speed and shares first in house experiences. For more information on GENALICE MAP, please visit http://www.genalice.com/product/genalice-map/
Advancing Microbiome Research: From challenging samples to insight with Confi...QIAGEN
Microbiome research encompasses sample types as diverse as the human gut, Antarctic soil, ocean water and acidic hot spring biofilms. These samples are challenging because they are difficult to lyse, with some microbes containing a tough extracellular matrix. Incomplete lysis of a microbial community results in an inaccurate representation of the microbial content of the sample. Additionally, PCR inhibitors present in these samples, especially humic acids, polysaccharides, polyphenolics, lipids and heavy metals result in inaccurate quantification of nucleic acids that may inhibit downstream applications such as qPCR and NGS.
Comparison of Different NGS Library Construction Methods for Single-Cell Sequ...QIAGEN
Recent advances in whole genome amplification (WGA), whole transcriptome amplification (WTA) technologies and next-generation sequencing (NGS) have enabled whole genome or transcriptome sequencing at the single-cell level. Single-cell sequencing studies have yielded new insights into the heterogeneity of the genome and transcriptome in individual cells. Such heterogeneity at the single-cell level has been shown to be closely related to cellular function, differentiation, development, and diseases. A critical element of the single-cell sequencing workflow is sequencing library construction following WGA or WTA. An efficient library construction method is required to convert a high percentage of the DNA fragments to an adaptor-ligated sequencing library and to ensure high sequence complexity of the library. Furthermore, uniform representation of all genomic regions in a sequencing library is essential for retaining all important sequence information.
Here we compared 2 library construction methods following a REPLI-g MDA-mediated WGA or WTA:
• A ligation-based library construction method using a GeneRead Library Prep Kit (QIAGEN)
• A ‘tagmentation’-based method using Nextera DNA Sample Prep Kit (Illumina), which simultaneously fragments and tags DNA.
Our results demonstrated that the Nextera library construction method can be directly used with the REPLI-g-amplified DNA following MDA reaction, without the need for DNA purification. This could be beneficial if working with a high number of samples or if the complete workflow of single WGA/WTA and library construction should be automated. However, compared with the tagmentation method, the ligation-based library construction method is more flexible with regard to the input DNA amount and delivers sequencing libraries with higher complexity and less bias. This is critical for sensitive applications, such as identification of genomic variants or comprehensive profiling of transcriptomes.
Learn more about the Valitacell fluorescent polarisation based IgG quantification assay 'ValitaTITER' and about our novel ChemStress fingerprinting assay for cell line development. For more information about our products and pricing, please contact info@valitacell.com
From Screening to QC: Development Considerations for Octet MethodsKBI Biopharma
The Octet is a powerful platform that can be used for rapid binding analysis of samples throughout development, stability testing and can be implemented or release of GMP material. For potency analysis of GMP materials, methods must demonstrate precision, accuracy, specificity and linearity across the range of specifications.
Profile Multiple Cytokines and Chemokines Simultaneously with Very High Sensi...QIAGEN
Learn how to profile multiple cytokines and chemokines simultaneously with very high sensitivity and specificity using the standard ELISA reader. Available in different formats to suit your research needs such as single-analyte, multi-analyte or custom mix-n-match format for human, mouse and rat.
Subsequent library screening will fish out the antibody mutants that have high affinity. Two library screening strategies are available. In the first "surface-panning" strategy, decreasing concentrations of antigen is surface immobilized. In the second "solution-sorting" strategy, in which a labeled antigen in solution is used, we have two approaches, selection based on the equilibrium constant (Kd) and selection based on binding kinetics. In the first approach, sub-library phage is incubated with biotinylated antigen at controlled concentrations and bound phages are captured by immobilized NeutrAvidin. Selection based on binding kinetics is also termed off-rate (Koff) selection, in which phage population is allowed to saturate the labeled antigen before a large molar excess of unlabeled antigen is added to the mix for controlled periods of time. This allows the selection of mutant antibodies that have slower off-rates. Since a reduction in Koff usually results in a higher affinity, this selection approach singles out antibody variants with improved Kd.
Quantitative Real-Time PCR for Rapid and Accurate Titration of Recombinant Ba...Bushra Hafeez
The baculovirus expression system is widely used for the production of recombinant protein in insect cells.
Baculovirus can be amplified to very high titers in suspension culture facilitating the production of large quantities of recombinant protein.
In order to maximize and ensure reproducibility of protein production, it is important to obtain an accurate estimate of the recombinant virus titer.
An improved QPCR titration method using Applied Biosystems Taqman fluorogenic probes is used.
It is faster and give increased accuracy across a wide range of virus titers.
This technology takes advantage of the 5’exonuclease activity of Taq polymerase,
To digest a probe.
Targeted RNAseq for Gene Expression Using Unique Molecular Indexes (UMIs): In...QIAGEN
Traditional RNA sequencing (RNA-Seq) is a powerful tool for expression profiling, but is hindered by PCR amplification bias and inaccuracy at low expressing genes. QIAseq RNA is a flexible and precise tool developed for mitigating these complications, allowing digital gene expression analysis. This in-depth webinar will cover sample requirements, experimental design, NGS platform-specific challenges and workflow for gene enrichment, library prep and sequencing. The applications of QIASeq RNA Panels in cancer research, stem cell differentiation and elucidating the effects small molecules on signaling pathways will be highlighted.
Critical Steps for Real-Time PCR Analysis: Tips and Solutions to Achieve Effi...QIAGEN
In this slidedeck, we cover the following topics which are critical steps for efficient and precise gene expression studies using real-time PCR technology:
1) Effect of RNA integrity on real-time PCR results – tips to achieve a true RNA profiling suitable for real-time PCR studies
2) Improved methods for cDNA synthesis, optimized for real-time PCR
3) Real-time PCR analysis:
• Real-time PCR essentials and background information on different quantification strategies
• SYBR Green real-time PCR – factors influencing specificity
• Introduction to probe technology
• New, fast and efficient real-time PCR solutions
Snippy - Rapid bacterial variant calling - UK - tue 5 may 2015Torsten Seemann
Using Snippy to call variants in bacterial short read datasets via alignment to reference, and then using these alignments to produce core SNP alignments for phylogenomics.
Streamlining NGS data processing with GENALICE MAP - Remco Ursem (Rijk Zwaan)GENALICE
Rijk Zwaan develops vegetable varieties and sells the seeds they produce. Rijk Zwaan has over 1.000 varieties in 25 different vegetable crops. The company shares challenges of sequencing data and compares deployment of GENALICE MAP with BWA/GATK on storage footprint, analysis speed and shares first in house experiences. For more information on GENALICE MAP, please visit http://www.genalice.com/product/genalice-map/
Advancing Microbiome Research: From challenging samples to insight with Confi...QIAGEN
Microbiome research encompasses sample types as diverse as the human gut, Antarctic soil, ocean water and acidic hot spring biofilms. These samples are challenging because they are difficult to lyse, with some microbes containing a tough extracellular matrix. Incomplete lysis of a microbial community results in an inaccurate representation of the microbial content of the sample. Additionally, PCR inhibitors present in these samples, especially humic acids, polysaccharides, polyphenolics, lipids and heavy metals result in inaccurate quantification of nucleic acids that may inhibit downstream applications such as qPCR and NGS.
Comparison of Different NGS Library Construction Methods for Single-Cell Sequ...QIAGEN
Recent advances in whole genome amplification (WGA), whole transcriptome amplification (WTA) technologies and next-generation sequencing (NGS) have enabled whole genome or transcriptome sequencing at the single-cell level. Single-cell sequencing studies have yielded new insights into the heterogeneity of the genome and transcriptome in individual cells. Such heterogeneity at the single-cell level has been shown to be closely related to cellular function, differentiation, development, and diseases. A critical element of the single-cell sequencing workflow is sequencing library construction following WGA or WTA. An efficient library construction method is required to convert a high percentage of the DNA fragments to an adaptor-ligated sequencing library and to ensure high sequence complexity of the library. Furthermore, uniform representation of all genomic regions in a sequencing library is essential for retaining all important sequence information.
Here we compared 2 library construction methods following a REPLI-g MDA-mediated WGA or WTA:
• A ligation-based library construction method using a GeneRead Library Prep Kit (QIAGEN)
• A ‘tagmentation’-based method using Nextera DNA Sample Prep Kit (Illumina), which simultaneously fragments and tags DNA.
Our results demonstrated that the Nextera library construction method can be directly used with the REPLI-g-amplified DNA following MDA reaction, without the need for DNA purification. This could be beneficial if working with a high number of samples or if the complete workflow of single WGA/WTA and library construction should be automated. However, compared with the tagmentation method, the ligation-based library construction method is more flexible with regard to the input DNA amount and delivers sequencing libraries with higher complexity and less bias. This is critical for sensitive applications, such as identification of genomic variants or comprehensive profiling of transcriptomes.
Learn more about the Valitacell fluorescent polarisation based IgG quantification assay 'ValitaTITER' and about our novel ChemStress fingerprinting assay for cell line development. For more information about our products and pricing, please contact info@valitacell.com
From Screening to QC: Development Considerations for Octet MethodsKBI Biopharma
The Octet is a powerful platform that can be used for rapid binding analysis of samples throughout development, stability testing and can be implemented or release of GMP material. For potency analysis of GMP materials, methods must demonstrate precision, accuracy, specificity and linearity across the range of specifications.
A workshop is intended for those who are interested in and are in the planning stages of conducting an RNA-Seq experiment. Topics to be discussed will include:
* Experimental Design of RNA-Seq experiment
* Sample preparation, best practices
* High throughput sequencing basics and choices
* Cost estimation
* Differential Gene Expression Analysis
* Data cleanup and quality assurance
* Mapping your data
* Assigning reads to genes and counting
* Analysis of differentially expressed genes
* Downstream analysis/visualizations and tables
Demonstrating Process Scalability with Robust and Turnkey PlatformsMerck Life Sciences
Upstream bioreactor process development and scale-up is a time-consuming step in recombinant protein production. Variability in the recombinant cell, cell culture media and bioreactor vessel contributes to the number of studies required to obtain a stable, productive, and scalable process. In our laboratory, we set out to develop a robust, turnkey platform that includes DNA vectors, modified cell lines, chemically defined cell culture media and single-use bioreactors. Here we demonstrate process development and scale-up of a recombinant CHOZN® GS clone in EX-CELL® Advanced™ cell culture media from small-scale flasks through bench-scale bioreactors and up to 50 L pilot scale bioreactor systems. While challenges typical of process scale-up were present, we consistently achieved the desired level of process performance across the different scales with minimal process optimization due to the robustness of the complete solution.
In this webinar, you will learn about:
- Demonstrating the process development and scale-up of a recombinant CHOZN® GS clone in EX-CELL® Advanced™ cell culture media from small-scale up to 50 L pilot scale.
- Achieving the desired level of process performance across the different scales.
Demonstrating Process Scalability with Robust and Turnkey PlatformsMilliporeSigma
Upstream bioreactor process development and scale-up is a time-consuming step in recombinant protein production. Variability in the recombinant cell, cell culture media and bioreactor vessel contributes to the number of studies required to obtain a stable, productive, and scalable process. In our laboratory, we set out to develop a robust, turnkey platform that includes DNA vectors, modified cell lines, chemically defined cell culture media and single-use bioreactors. Here we demonstrate process development and scale-up of a recombinant CHOZN® GS clone in EX-CELL® Advanced™ cell culture media from small-scale flasks through bench-scale bioreactors and up to 50 L pilot scale bioreactor systems. While challenges typical of process scale-up were present, we consistently achieved the desired level of process performance across the different scales with minimal process optimization due to the robustness of the complete solution.
In this webinar, you will learn about:
- Demonstrating the process development and scale-up of a recombinant CHOZN® GS clone in EX-CELL® Advanced™ cell culture media from small-scale up to 50 L pilot scale.
- Achieving the desired level of process performance across the different scales.
Presentation of John Mountzouris in 1st International Antibody Validation For...St John's Laboratory Ltd
John Mountzouris completed his Ph.D. in Medicinal Chemistry at the University of Texas, with a focus on structural biology, followed by a post-doctorate at the Scripps Research Institute. His industrial career begin with a stint as a bench scientist Pharmingen (later acquired by Becton Dickinson). He then turned to the business side, with work in sales and marketing, business development and communication at Pfizer, Abgent, and Acris Antibodies. Most recently, he has returned as Site Leader at Abgent, following its acquisition by Wuxi, a global CRO company.
For more details about 1st international antibody validation forum please check on http://www.stjohnslabs.com/ac_cms/blog
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Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
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I Introduction
II Subalternation and Theology
III Theology and Dogmatic Declarations
IV The Mixed Principles of Theology
V Virtual Revelation: The Unity of Theology
VI Theology as a Natural Science
VII Theology’s Certitude
VIII Conclusion
Notes
Bibliography
All the contents are fully attributable to the author, Doctor Victor Salas. Should you wish to get this text republished, get in touch with the author or the editorial committee of the Studia Poinsotiana. Insofar as possible, we will be happy to broker your contact.
2. • Dasiprotimut-T
• Therapeutic cancer vaccine
• Treatment for non-
Hodgkins lymphoma
• Trains immune system
against BCR
• Located in Coon Rapids,
MN
Biovest International
3. ELISAs for Dasiprotimut-T
1. Qualitative
a) Fusion Screening
2. Quantitative
a) Antibody concentration
• IgM/IgG
b) Impurities
• Host cell proteins
• Bovine proteins
6. Fusion Screening ELISAs
• Qualitative so development of assay
was faster
• Fewer requirements for validation
• Developed in house so cheaper
reagents and you can buy them in bulk
• Needed to be completely developed
• Find vendors for appropriate antibodies
and buffers
• Performed qualification study to determine
concentrations
• Precious samples, only had enough each
time for one ELISA
• Harder to be prepared for, need at least 20
ELISA plates coated at anytime
• Does not have a standard curve, harder to
monitor assay performance over time
9. Quantitative ELISAs
• Qualifications were completed
• Developed in house so cheaper
reagents and you can buy them in bulk
• Quantitative ELISA so additional
requirements are needed for validation
(accuracy, repeatability)
• Concentration of sample is unknown
• Analysis may need to be repeated until
the sample quantitates on the standard
curve
11. Impurity ELISA - HCP
TMB
TMB
HRP
Anti-K6H6/B5
Host cell
proteins
Biotinylated anti-K6H6/B5
B
B
B TMB
TMB
HRP
TMB
TMB
HRP
Streptavidin HRP
12. Impurity ELISA - HCP
• Assay antibodies were well
characterized
• Very low background
• Tailor-made for our product
• Very limited quantity without another
rabbit study
• Variability with biotinylated antibody
• Depending on # biotins per antibody
the working dilution changed per batch
• Analysis may need to be repeated until
the sample quantitates on the standard
curve
13. Impurity ELISAs –
Bovine proteins
Anti-bovine protein
Bovine
protein
Anti-bovine protein HRP
TMB
TMB
HRP
14. Impurity ELISAs -
Bovine proteins
• Plates are pre-coated and pre-blocked
• Plates don’t need to be coated ahead
of time
• Analysis time is shorter
• Each kit has been tested as a lot
• Plate strips can be separated
• Very expensive ($500-750)
• Large lot to lot variability
• Very sensitive to timing on an
automated system
17. Tecan Automated Systems
• Can process samples overnight
• Process more plates at one time
• Automation results in a higher
reimbursement from EMA
• Need fewer technicians to run ELISAs
• Timing of sample addition
• Need a programmer for plate maps
• May freeze
21. Quality Control
• Documentation
• Veeva Vault
• Writing of validation protocols
• ICH Q2(R1), FDA
• Preparation of final reports for EMA submission and protocol issuance
• Stability studies
• Lot to lot comparison
• Reagent inventory and budget
22. Tecan Automated Systems
1 2 3 4 5 6 7 8 9 10 11 12
A PTT
Blank
Control
RMQ013 Lambda
Vial 1
RMQ013 Kappa
Vial 1
B RMQ013 Lambda
Vial 21
RMQ013 Kappa
Vial 21
C Media
Blank
Control
RMQ013 Lambda
Vial 41
RMQ013 Kappa
Vial 41
D RMQ013 Lambda
Vial 61
RMQ013 Kappa
Vial 61
E IgM
Lambd
a
Control
RMQ013 Lambda
Vial 81
RMQ013 Kappa
Vial 81
F RMQ013 Lambda
Vial 100
RMQ013 Kappa
Vial 100
G IgM
Kappa
ControlH
Good morning. This morning I’m going to talk to you about the most recent project I was involved in.
I was part of a team to bring Dasiprotimut-T to market approval in the European Union. Dasiprotimut-T is a therapeutic vaccine for patients with non-Hodgkins lymphoma that works by eliciting a cytotoxic T-cell response against the patient’s B-cell receptor (BCR) on the surface of the lymphoma cells. The treatment was originally developed at the National Cancer Institute in collaboration with Biovest using Biovest’s AutovaxID hollow-fiber biorectors, pictured here.
During the vaccine manufacturing process there are different checkpoints that are monitored with the help of 8 different ELISAs. While I was at Biovest, a colleague and myself optimized and validated all 8 of these for use in Biovest’s facility. I’ve divided them into 2 major categories: qualitative and quantitative with the quantitative ELISAs being divided further into testing for antibody concentration and the presence of protein impurities. These ELISA assays needed to be validated for in-house use but also to support the in-process validation for vaccine manufacturing. Some of the ELISAs needed to be completely developed, some were qualified and some were kits that needed to be optimized for use at our facility.
Biovest receives a lymph node biopsy. The biopsy is macerated and a single cell suspension is generated. The single cell suspension is cryopreserved and an aliquot of cells is analyzed to determine the antibody isotype (IgM or IgG) utilizing flow cytometry.
K6H6/B5 fusion partner cell line is grown to mid-log phase (this is a non-secreting myeloma cell line). Patient tumor cells are thawed, washed and counted. Patient tumor cells are combined with K6H6/B5 cells in a polyethylene glycol fusion reagent. Cells are resuspened in culture medium and aliquoted into 96-well plates at various densities. Selection media is added, cells are grown and monitored for an average of 5 weeks. Fusion screening ELISAs are performed to identify candidate hybridoma clones.
The two qualitative ELISAs test for successful fusion of a patient’s biopsy cells with K6H6/B5. The plate is coated with either anti-IgM or IgG whichever was predetermined by flow cytometry. Since K6H6/B5 cells are non-secreting, successful fusions will express an antibody. In the beginning there are 5-15, 96-well plates per patient which is a ton of clones so we needed to develop a way to easily pick more successful clones.
So we developed a macro to process the qualitative data for the fusion screening ELISAs. A ratio is calculated using the positive control OD and sample OD. Anything over a ratio of 1, shows up red on the map, between 0.5 and 1 is orange and anything lower than 0.5 is in yellow. This is a clear visual for the technician of which fusion clones are currently secreting antibody the best for that patient. In the beginning many clones are picked per patient but as they are expanded, fewer are allowed to continue growing. After sequencing confirmation, up to 10 clones are chosen per patient.
When talking about it needing to be completely developed mention that it was also the first assay that had to be validated to support in process validation (vaccine manufacturing)
3. Fusions that are positive for secretion of the correct heavy and light chains are expanded. The fusion screening ELISA is continually performed to monitor for clones that continue to be positive for immunoglobulin secretion. In the end up to 10 positive clones are selected for further analysis/expansion and aliquots are cryopreserved. Immunoglobulin production of each cell line is measured by HPLC or quantitative ELISA. The IgH variable region of the candidate production banks are amplified by RT-PCR and sequenced. Production banks are chosen for scale-up based on immunoglobulin secretion, growth rate and variable region identity matching to the patient’s biopsy.
4. One of the five vials of the production bank are thawed, washed and counted. Cells are grown and expanded four to six times over two to three weeks depending on doubling time. Then the cells are transferred to a bioreactor and the hybridoma clones grows for an average of 4-6 weeks. During this time antibody concentration is monitored.
The quantitative ELISA for the antibody concentration is very similar to the fusion screening ELISAs. However they utilize a standard curve so the amount of antibody can be determined.
5. Once the cells begin dying, the contents of the bioreactor is harvested and purification begins. Over 5 different steps, the patient idiotype is purified from the cell culture supernatant. These steps vary depending on whether the isotype was IgM or IgG. Using different salt buffers, the harvest undergoes a round of affinity chromatography, then anion exchange, another round of chromatography followed by virus reduction and finally 0.2 micron filtration. During each stage of purification certain threshold need to be met for protein impurities and that is monitored using ELISA.
The first protein impurity I’m going to talk about is the host cell protein quantification ELISA. K6H6/B5 cells were weaned from serum, pelleted and sheered to make a pool of host cell proteins. These proteins were used as the reference standard in the ELISA and also used to immunize rabbits to produce anti-host cell protein antibodies. This was probably the most important ELISA because it’s unique to the product and you have to make sure the host cell proteins are removed during purification so you’re not injecting foreign proteins into the patient. The purification process effectively eliminated these proteins so streptavidin was needed to amplify the signal.
The remaining three assays share two things in common: they are all bovine proteins and they are all related to bovine proteins found in the serum added to the tissue culture during the cell expansion process. These assays detected bovine serum albumin, bovine IgG and bovine plasma proteins. Bethyl Laboratories manufactures the BSA kit and Cygnus Technologies produced the BSA and bovine IgG.
The automated systems we used were made by Tecan. The EVO 100 made dilutions and transferred samples to the ELISA plate. There are slots on the bay for sample diluent, 1 mL, deep well plates for making dilutions, 96-well ELISA plates and conductive tips.
Once the samples were loaded on the ELISA plate, the plate is transferred to the EVO 150 to complete the ELISA assay. In addition to having the same reagent pipetting capabilities of the EVO 100, the 150 also has an integrated 96-head plate washer. “Hotels” which hold the ELISA plates protecting them from light during incubation samples, are capable of shaking the plate at a specific speed or holding the plate at a certain temperature. And finally there is an integrated plate reader to measure the sample OD when the assay is finished.
So once all of the ELISAs are completed and the patient idiotype was successfully purified, conjugation of the product can begin. Vials for the vaccine product and KLH are sterilized in a glove box. Purified idiotype, KLH and buffer are mixed. Glutaraldehyde is added and vaccine is mixed for 2-4 hours. Glycine is added to stop the reaction and final product is dialyzed against saline. Vials are then filled with patient vaccine to await release to clinic.
The Committee for Medicinal Products for Human Use adopted a negative opinion for dasiprotimut T for the treatment of Follicular non-Hodgkin’s lymphoma in European Union and requested additional clinical trials. Since we were privately funded, the project was discontinued at the end of March of this year.
The instrument division of Biovest does still exist. And for comparison this is how the bioreactors perform compared to other cell culture methods so you can generate large amount of antibody in a relatively short time in a cost-effective manner compared to other methods.
Once you receive samples you needed to submit a plate map, similar to this one. Indicating where on the 96-well ELISA plate blank, standard curve, controls and samples are located. You also needed to indicated whether any dilutions are necessary. Which can be very difficult especially in actual practice because at best you only have a ballpark for the concentration of the samples. So during the in-process validation where we made the vaccine from beginning to end for 3 different patients, we tracked concentrations and were able to determine set dilutions for the different stages on the vaccine product so the samples would quantitate on the standard curve.