Register now to participate in the interactive, on-demand webinar: https://event.on24.com/wcc/r/3640127/2D3ACB02357328FE1A6C0F00083C5C06?partnerref=SlideShare
In this webinar, you will:
- Get an overview of the pDNA market
- Receive guidance for filter selection as a replacement for centrifugation
- Learn purification strategies using AEX chromatography resins and membranes
- Understand key considerations for sterile filtration
- Learn about a complete purification process flow for pDNA
Detailed description:
Plasmid DNA (pDNA) is an important component of mRNA, vaccine, and viral vector therapies. Scaling and optimizing downstream processes during manufacturing requires an in-depth knowledge of all unit operations. This webinar presents a design for a generic manufacturing template which overcomes the challenges associated with the purification of pDNA i.e high viscosity, large molecule size, shear sensitivity, and similarities with impurities. Key considerations for purification unit operations include harvest, lysis, clarification, tangential flow filtration, chromatography to sterilizing grade filtration. The webinar presents a comprehensive case study encompassing all downstream unit operations.
Data driven strategies and considerations for scalable purification of Plasmi...Merck Life Sciences
The document discusses strategies and considerations for the scalable purification of plasmid DNA for use in vaccine manufacturing. It covers the key upstream steps of cell harvest, lysis, and clarification. Cell harvest is typically done using centrifugation or tangential flow filtration. Lysis is usually an alkaline lysis process that must be carefully optimized to avoid damaging the plasmid DNA. Clarification can involve various pretreatment and filtration steps to reduce impurities before downstream purification. No single platform solution currently exists and processes vary between manufacturers.
This document provides an overview of DNA and RNA isolation, purification, and amplification techniques. It discusses preparing genomic DNA from bacterial cells using lysis and phenol-chloroform extraction. Plasmid DNA can be purified from bacterial cells using alkaline lysis and silica binding. The document also reviews RNA isolation, which requires rapid processing to prevent RNase degradation, and describes mRNA selection using oligo-dT cellulose. Reverse transcription PCR and quantitative PCR are introduced as methods to study gene expression.
Straight to the Point: Reaching Clinical Stage Development with a CHOZN® Cell...Merck Life Sciences
Participate in the interactive webinar: http://bit.ly/CHOZNWebinar
In this case study, we will present how we support our clients thanks to advantages provided by the CHOZN® Cell Line, and a specific strategy for clone selection where semi-automation and pool selection are leveraged, to get upstream right first time.
Explore our webinar library: www.merckmillipore.com/webinars
Straight to the Point: Reaching Clinical Stage Development with a CHOZN® Cell...MilliporeSigma
Participate in the interactive webinar: http://bit.ly/CHOZNWebinar
In this case study, we will present how we support our clients thanks to advantages provided by the CHOZN® Cell Line, and a specific strategy for clone selection where semi-automation and pool selection are leveraged, to get upstream right first time.
Explore our webinar library: www.emdmillipore.com/webinars
Principles of DNA isolation, PCR and LAMPPerez Eric
1. The document discusses principles of DNA isolation and purification as well as polymerase chain reaction (PCR). It describes how cells are broken to release DNA and the components of extraction buffers used to isolate DNA.
2. The three main steps of PCR - denaturation, annealing, and elongation - are explained. Denaturation separates DNA strands, annealing attaches primers, and elongation duplicates the DNA. Required PCR reagents and their roles are also outlined.
3. Loop-mediated isothermal amplification (LAMP) is introduced as an alternative to PCR that amplifies DNA at a constant temperature. LAMP uses multiple primers and has advantages like lower cost and faster results. The mechanism and applications of LAMP are summarized.
Fast-track solutions to address challenges with Host Cell Proteins in early d...Merck Life Sciences
Watch this webinar here: https://bit.ly/3fFRXDb
This webinar illustrates a customer case study about the challenges related to the removal of Host Cell Proteins from bioreactor harvest, the selected fast-track approach and outcome.
Our customer had one month to reduce the level of HCPs in the bioreactor harvest prior to a production run to supply drug substance for a scheduled Phase 1 clinical trial. The high level of HCPs (1,000,000 in the harvest and 700 ppm at the end of purification) unfavorably impacted the planned clarification process and subsequent downstream steps.
The goal was to reduce the level of HCPs to maximum of 300 ppm at the end of process purification and ensure clarification of the entire 2000L harvest.
In this webinar, you will learn about:
- Challenges with Host Cell Proteins
- Fast-Track Approach using caprylic acid precipitation followed by filtration using Clarisolve® filters
- The impact of Design of Experiment
Fast-track solutions to address challenges with Host Cell Proteins in early d...MilliporeSigma
This document discusses a case study where caprylic acid precipitation and depth filtration were used to address high levels of host cell proteins (HCPs) in a biotherapeutics process. A design of experiments approach was used to optimize the caprylic acid concentration and temperature for precipitation. Depth filtration with a 40MS filter was shown to improve recovery while significantly reducing HCP levels. The modified process was successfully implemented at 2000L scale within one month to meet HCP specifications for an upcoming clinical trial.
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
Data driven strategies and considerations for scalable purification of Plasmi...Merck Life Sciences
The document discusses strategies and considerations for the scalable purification of plasmid DNA for use in vaccine manufacturing. It covers the key upstream steps of cell harvest, lysis, and clarification. Cell harvest is typically done using centrifugation or tangential flow filtration. Lysis is usually an alkaline lysis process that must be carefully optimized to avoid damaging the plasmid DNA. Clarification can involve various pretreatment and filtration steps to reduce impurities before downstream purification. No single platform solution currently exists and processes vary between manufacturers.
This document provides an overview of DNA and RNA isolation, purification, and amplification techniques. It discusses preparing genomic DNA from bacterial cells using lysis and phenol-chloroform extraction. Plasmid DNA can be purified from bacterial cells using alkaline lysis and silica binding. The document also reviews RNA isolation, which requires rapid processing to prevent RNase degradation, and describes mRNA selection using oligo-dT cellulose. Reverse transcription PCR and quantitative PCR are introduced as methods to study gene expression.
Straight to the Point: Reaching Clinical Stage Development with a CHOZN® Cell...Merck Life Sciences
Participate in the interactive webinar: http://bit.ly/CHOZNWebinar
In this case study, we will present how we support our clients thanks to advantages provided by the CHOZN® Cell Line, and a specific strategy for clone selection where semi-automation and pool selection are leveraged, to get upstream right first time.
Explore our webinar library: www.merckmillipore.com/webinars
Straight to the Point: Reaching Clinical Stage Development with a CHOZN® Cell...MilliporeSigma
Participate in the interactive webinar: http://bit.ly/CHOZNWebinar
In this case study, we will present how we support our clients thanks to advantages provided by the CHOZN® Cell Line, and a specific strategy for clone selection where semi-automation and pool selection are leveraged, to get upstream right first time.
Explore our webinar library: www.emdmillipore.com/webinars
Principles of DNA isolation, PCR and LAMPPerez Eric
1. The document discusses principles of DNA isolation and purification as well as polymerase chain reaction (PCR). It describes how cells are broken to release DNA and the components of extraction buffers used to isolate DNA.
2. The three main steps of PCR - denaturation, annealing, and elongation - are explained. Denaturation separates DNA strands, annealing attaches primers, and elongation duplicates the DNA. Required PCR reagents and their roles are also outlined.
3. Loop-mediated isothermal amplification (LAMP) is introduced as an alternative to PCR that amplifies DNA at a constant temperature. LAMP uses multiple primers and has advantages like lower cost and faster results. The mechanism and applications of LAMP are summarized.
Fast-track solutions to address challenges with Host Cell Proteins in early d...Merck Life Sciences
Watch this webinar here: https://bit.ly/3fFRXDb
This webinar illustrates a customer case study about the challenges related to the removal of Host Cell Proteins from bioreactor harvest, the selected fast-track approach and outcome.
Our customer had one month to reduce the level of HCPs in the bioreactor harvest prior to a production run to supply drug substance for a scheduled Phase 1 clinical trial. The high level of HCPs (1,000,000 in the harvest and 700 ppm at the end of purification) unfavorably impacted the planned clarification process and subsequent downstream steps.
The goal was to reduce the level of HCPs to maximum of 300 ppm at the end of process purification and ensure clarification of the entire 2000L harvest.
In this webinar, you will learn about:
- Challenges with Host Cell Proteins
- Fast-Track Approach using caprylic acid precipitation followed by filtration using Clarisolve® filters
- The impact of Design of Experiment
Fast-track solutions to address challenges with Host Cell Proteins in early d...MilliporeSigma
This document discusses a case study where caprylic acid precipitation and depth filtration were used to address high levels of host cell proteins (HCPs) in a biotherapeutics process. A design of experiments approach was used to optimize the caprylic acid concentration and temperature for precipitation. Depth filtration with a 40MS filter was shown to improve recovery while significantly reducing HCP levels. The modified process was successfully implemented at 2000L scale within one month to meet HCP specifications for an upcoming clinical trial.
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
1. The document discusses different types of cloning vectors including plasmids, bacteriophages, cosmids, and phagemids that can be used to clone foreign DNA.
2. It provides details on the characteristics and components of common vectors like the pBR322 plasmid and lambda phage. Screening methods for identifying recombinant clones like blue/white selection and replica plating are also described.
3. Applications of genetic engineering using these vectors include producing recombinant proteins like insulin and hepatitis vaccines as well as studying gene function and identifying mutations associated with diseases.
Polymerase Chain Reaction (PCR) is a technique used to amplify a specific DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. Each cycle doubles the amount of target DNA. After 20-30 cycles, there can be over a billion copies of the original DNA sequence. PCR is used for a variety of applications including disease diagnosis, cloning genes, forensic analysis, and more. It is a powerful technique that has revolutionized molecular biology.
DNA cloning is a technique used to reproduce DNA fragments. It involves inserting a DNA fragment into a vector, which is then used to transform host cells. The key steps are digesting the DNA and vector with restriction enzymes, ligating the DNA fragment into the vector, transforming host cells with the vector, and selecting for cells containing the vector through growth on antibiotic-containing media. This allows unlimited amplification of the DNA fragment for study, manipulation, and protein expression.
DCN Diagnostics offers OEM development services for complete lateral flow diagnostic systems. They have expertise in assay development, product engineering, manufacturing process development and commercialization assistance. Their services include reagent development, assay design, reader integration, design of plastic cassettes and cartridges, and manufacturing transfer. DCN has experience across various industries like medical diagnostics, veterinary, food testing, and can develop qualitative or quantitative lateral flow assays.
Fruitbreedomics workshop wp6 dna extraction methodsfruitbreedomics
The document summarizes methods for DNA extraction that were tested for use in marker-assisted breeding of fruit trees. Four extraction methods were evaluated: 1) "quick and dirty" commercial kits, 2) "direct PCR" kits, 3) magnetic particle-based kits, and 4) a homemade CTAB method. The homemade CTAB method was found to provide high quality DNA at the lowest cost and was well-suited for marker-assisted breeding work requiring analysis of hundreds of samples. The document also provides details on optimization of the KAPA 3G Plant PCR kit for short DNA fragments and highlights CTAB and KAPA 3G PCR as good extraction methods.
Molecular Techniques For Disease DiagnosisPriyanka Gupta
Molecular techniques are used to analyze biological markers in genomes and proteomes. They provide several advantages over traditional diagnostic methods such as faster diagnosis, increased sensitivity and specificity, and ability to detect pathogens more rapidly. Common molecular techniques include PCR, real-time PCR, nucleic acid sequencing, microarrays, and nucleic acid amplification methods like NASBA. These techniques are useful for diagnosing infectious diseases and genetic conditions.
This document describes the polymerase chain reaction (PCR) technique. PCR is used to amplify a specific DNA sequence by repeating cycles of heating and cooling of the DNA sample. The key components of a PCR reaction are DNA template, primers, nucleotides, DNA polymerase, buffer, and magnesium ions. During each cycle, the DNA is denatured by heating, the primers anneal to the DNA at a lower temperature, and the DNA polymerase extends the DNA chain. This process is repeated many times, exponentially amplifying the target DNA sequence. PCR is a powerful, sensitive, specific and reliable method for detecting small amounts of DNA.
The document provides information on PCR methods and thermostable DNA polymerases. It discusses the history of PCR and how it was developed. It then explains the basic steps of PCR including denaturation, annealing and extension. It also discusses factors that influence optimal PCR such as primers, DNA polymerase, annealing temperature and melting temperature. Finally, it outlines several variations of PCR including inverse PCR, ligation-mediated PCR, and multiplex ligation-dependent probe amplification PCR along with their applications.
Back to Basics: Fundamental Concepts and Special Considerations in gDNA Isola...QIAGEN
This document provides an overview of genomic DNA (gDNA) isolation. It discusses key considerations for gDNA isolation including sample stabilization, disruption, and storage. Common isolation technologies like silica membrane and magnetic bead kits are described. The document reviews measuring gDNA concentration and quality via UV spectroscopy and gel electrophoresis. It also provides guidance on selecting appropriate QIAGEN gDNA isolation kits based on sample type.
The document provides information about polymerase chain reaction (PCR), including its history, definition, applications, and process. It summarizes that PCR was invented in 1984 by Kary Mullis to selectively reproduce portions of DNA through repeated heating and cooling cycles using DNA polymerase. PCR is now an essential tool in biology for applications like detecting genes and diagnosing infections. The process involves extracting DNA, performing PCR cycles of denaturation, primer annealing, and extension, and then analyzing the amplified DNA products through electrophoresis.
This document discusses polymerase chain reaction (PCR), its process and troubleshooting strategies. PCR is used to amplify a specific segment of DNA. It requires a DNA template, primers, DNA polymerase, nucleotides and buffer solution. The PCR process involves cycles of heating and cooling to denature and extend the DNA. Issues like contamination, reaction conditions, primer design and template quality can affect PCR results. Troubleshooting aims to optimize conditions, increase yield and specificity. It may involve adjusting magnesium concentration, annealing temperature, primer concentration, cycle number and ensuring high-quality reagents and template DNA. References on PCR optimization and troubleshooting techniques are also provided.
This presentation provides an introduction to the M Lab™ Collaboration Centers, an overview of chromatography theory, and highlights the benefits of next-generation chromatography.
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.emdmillipore.com/mlab
This presentation provides an introduction to the M Lab™ Collaboration Centers, an overview of chromatography theory, and highlights the benefits of next-generation chromatography.
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.merckmillipore.com/mlab
In Situ Polymerase Chain Reaction (In situ PCR) is a powerful method that detects minute quantities of rare or single-copy number nucleic acid sequences in frozen or paraffin-embedded cells or tissue sections for the localization of those sequences within the cells. The principle of this method involves tissue fixing (to preserve the cell morphology) and subsequent treatment with proteolytic digestion (to provide access for the PCR reagents to the target DNA). The target sequences are amplified by those reagents and then detected by standard immunocytochemical protocols. In situ PCR combines the sensitivity of PCR or RT-PCR amplification along with the ability to perform morphological analysis on the same sample, and thus it is an attractive tool in diagnostic applications. One of the most prominent applications is the detection of infectious disease agents including HIV-1, HBV, HPV, HHV-6, CMV, and EBV.
Enhanced bioseparations peptide mapping and m absOskari Aro
This document discusses Thermo Scientific products for peptide mapping and characterization of monoclonal antibodies (mAbs). It summarizes:
1) The SMART Digest kit which provides a rapid, reproducible, and hands-free workflow for protein digestion compared to standard protocols, reducing variability.
2) Chromatography columns including Acclaim C18 for peptide separation, MAbPac columns for intact protein and fragment analysis, and MAbPac SCX for charge variant analysis which provide high resolution, reproducibility, and stability.
3) Considerations for sample handling depending on properties like volume, surface interactions, and recovery using vials or plates.
4) Thermo Scientific offers an integrated solution for biomolecule separations from sample preparation
Process Development for Cell Therapy and Viral Gene TherapyMerck Life Sciences
Today’s viral vector manufacturing processes remain challenging. Process development is a critical enabler to bring safe, effective, sustainable products to market to address patient needs. When done properly, it can reduce the timeline of the project and the cost of producing the therapeutic product.
The webinar discusses our strategies for developing lentivirus and adeno associated virus (AAV) and the impact these early decisions can have on commercial readiness.
Watch the interactive webinar now: https://bit.ly/2VplwQq
Process Development for Cell Therapy and Viral Gene TherapyMilliporeSigma
Today’s viral vector manufacturing processes remain challenging. Process development is a critical enabler to bring safe, effective, sustainable products to market to address patient needs. When done properly, it can reduce the timeline of the project and the cost of producing the therapeutic product.
The webinar discusses our strategies for developing lentivirus and adeno associated virus (AAV) and the impact these early decisions can have on commercial readiness.
Watch the interactive webinar now: https://bit.ly/2VplwQq
Mycenax is a CDMO located in Taiwan that provides services for biologics development from cell line development through fill/finish. They have expertise in mammalian and microbial manufacturing up to 2000L scale using disposable technology. Mycenax has 7 biosimilars in development and is evaluating opportunities for novel biologics. They aim to be a full service CDMO partner for biologics development and manufacturing.
Preparing DNA for sequencing requires careful purification of the template DNA to ensure high quality results. Key steps include extracting DNA, quantifying the DNA concentration and quality, purifying DNA or PCR products to remove contaminants like primers, salts and proteins, and checking for purity and fragment size on a gel. Using purified template DNA free of impurities is crucial for accurate sequencing reads.
The Viscosity Reduction Platform: Viscosity-reducing excipients for improveme...MilliporeSigma
Protein viscosity is a major challenge in preparing highly concentrated protein formulations suitable for subcutaneous injection. Recently, the Viscosity Reduction Platform (VRP) was introduced and its technical key features and benefits for formulations were discussed. However, highly viscous solutions do not only pose a challenge when administering a drug to a patient, they can also impose technical limitations in the manufacturing process.
This white paper evaluates the effect of the excipients in the Viscosity Reduction Platform on ultrafiltration processes used to produce a highly concentrated formulation of a monoclonal antibody (mAb). Two filtration methods are demonstrated in this work.
Find more information about the Viscosity Reduction Platform on our website: https://www.sigmaaldrich.com/products/pharma-and-biopharma-manufacturing/formulation/viscosity-reduction-platform
More Related Content
Similar to Webinar: Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA
1. The document discusses different types of cloning vectors including plasmids, bacteriophages, cosmids, and phagemids that can be used to clone foreign DNA.
2. It provides details on the characteristics and components of common vectors like the pBR322 plasmid and lambda phage. Screening methods for identifying recombinant clones like blue/white selection and replica plating are also described.
3. Applications of genetic engineering using these vectors include producing recombinant proteins like insulin and hepatitis vaccines as well as studying gene function and identifying mutations associated with diseases.
Polymerase Chain Reaction (PCR) is a technique used to amplify a specific DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. Each cycle doubles the amount of target DNA. After 20-30 cycles, there can be over a billion copies of the original DNA sequence. PCR is used for a variety of applications including disease diagnosis, cloning genes, forensic analysis, and more. It is a powerful technique that has revolutionized molecular biology.
DNA cloning is a technique used to reproduce DNA fragments. It involves inserting a DNA fragment into a vector, which is then used to transform host cells. The key steps are digesting the DNA and vector with restriction enzymes, ligating the DNA fragment into the vector, transforming host cells with the vector, and selecting for cells containing the vector through growth on antibiotic-containing media. This allows unlimited amplification of the DNA fragment for study, manipulation, and protein expression.
DCN Diagnostics offers OEM development services for complete lateral flow diagnostic systems. They have expertise in assay development, product engineering, manufacturing process development and commercialization assistance. Their services include reagent development, assay design, reader integration, design of plastic cassettes and cartridges, and manufacturing transfer. DCN has experience across various industries like medical diagnostics, veterinary, food testing, and can develop qualitative or quantitative lateral flow assays.
Fruitbreedomics workshop wp6 dna extraction methodsfruitbreedomics
The document summarizes methods for DNA extraction that were tested for use in marker-assisted breeding of fruit trees. Four extraction methods were evaluated: 1) "quick and dirty" commercial kits, 2) "direct PCR" kits, 3) magnetic particle-based kits, and 4) a homemade CTAB method. The homemade CTAB method was found to provide high quality DNA at the lowest cost and was well-suited for marker-assisted breeding work requiring analysis of hundreds of samples. The document also provides details on optimization of the KAPA 3G Plant PCR kit for short DNA fragments and highlights CTAB and KAPA 3G PCR as good extraction methods.
Molecular Techniques For Disease DiagnosisPriyanka Gupta
Molecular techniques are used to analyze biological markers in genomes and proteomes. They provide several advantages over traditional diagnostic methods such as faster diagnosis, increased sensitivity and specificity, and ability to detect pathogens more rapidly. Common molecular techniques include PCR, real-time PCR, nucleic acid sequencing, microarrays, and nucleic acid amplification methods like NASBA. These techniques are useful for diagnosing infectious diseases and genetic conditions.
This document describes the polymerase chain reaction (PCR) technique. PCR is used to amplify a specific DNA sequence by repeating cycles of heating and cooling of the DNA sample. The key components of a PCR reaction are DNA template, primers, nucleotides, DNA polymerase, buffer, and magnesium ions. During each cycle, the DNA is denatured by heating, the primers anneal to the DNA at a lower temperature, and the DNA polymerase extends the DNA chain. This process is repeated many times, exponentially amplifying the target DNA sequence. PCR is a powerful, sensitive, specific and reliable method for detecting small amounts of DNA.
The document provides information on PCR methods and thermostable DNA polymerases. It discusses the history of PCR and how it was developed. It then explains the basic steps of PCR including denaturation, annealing and extension. It also discusses factors that influence optimal PCR such as primers, DNA polymerase, annealing temperature and melting temperature. Finally, it outlines several variations of PCR including inverse PCR, ligation-mediated PCR, and multiplex ligation-dependent probe amplification PCR along with their applications.
Back to Basics: Fundamental Concepts and Special Considerations in gDNA Isola...QIAGEN
This document provides an overview of genomic DNA (gDNA) isolation. It discusses key considerations for gDNA isolation including sample stabilization, disruption, and storage. Common isolation technologies like silica membrane and magnetic bead kits are described. The document reviews measuring gDNA concentration and quality via UV spectroscopy and gel electrophoresis. It also provides guidance on selecting appropriate QIAGEN gDNA isolation kits based on sample type.
The document provides information about polymerase chain reaction (PCR), including its history, definition, applications, and process. It summarizes that PCR was invented in 1984 by Kary Mullis to selectively reproduce portions of DNA through repeated heating and cooling cycles using DNA polymerase. PCR is now an essential tool in biology for applications like detecting genes and diagnosing infections. The process involves extracting DNA, performing PCR cycles of denaturation, primer annealing, and extension, and then analyzing the amplified DNA products through electrophoresis.
This document discusses polymerase chain reaction (PCR), its process and troubleshooting strategies. PCR is used to amplify a specific segment of DNA. It requires a DNA template, primers, DNA polymerase, nucleotides and buffer solution. The PCR process involves cycles of heating and cooling to denature and extend the DNA. Issues like contamination, reaction conditions, primer design and template quality can affect PCR results. Troubleshooting aims to optimize conditions, increase yield and specificity. It may involve adjusting magnesium concentration, annealing temperature, primer concentration, cycle number and ensuring high-quality reagents and template DNA. References on PCR optimization and troubleshooting techniques are also provided.
This presentation provides an introduction to the M Lab™ Collaboration Centers, an overview of chromatography theory, and highlights the benefits of next-generation chromatography.
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.emdmillipore.com/mlab
This presentation provides an introduction to the M Lab™ Collaboration Centers, an overview of chromatography theory, and highlights the benefits of next-generation chromatography.
To learn more about this topic or collaborate with our technical experts, schedule an in-person or remote visit at our M Lab™ Collaboration Centers: www.merckmillipore.com/mlab
In Situ Polymerase Chain Reaction (In situ PCR) is a powerful method that detects minute quantities of rare or single-copy number nucleic acid sequences in frozen or paraffin-embedded cells or tissue sections for the localization of those sequences within the cells. The principle of this method involves tissue fixing (to preserve the cell morphology) and subsequent treatment with proteolytic digestion (to provide access for the PCR reagents to the target DNA). The target sequences are amplified by those reagents and then detected by standard immunocytochemical protocols. In situ PCR combines the sensitivity of PCR or RT-PCR amplification along with the ability to perform morphological analysis on the same sample, and thus it is an attractive tool in diagnostic applications. One of the most prominent applications is the detection of infectious disease agents including HIV-1, HBV, HPV, HHV-6, CMV, and EBV.
Enhanced bioseparations peptide mapping and m absOskari Aro
This document discusses Thermo Scientific products for peptide mapping and characterization of monoclonal antibodies (mAbs). It summarizes:
1) The SMART Digest kit which provides a rapid, reproducible, and hands-free workflow for protein digestion compared to standard protocols, reducing variability.
2) Chromatography columns including Acclaim C18 for peptide separation, MAbPac columns for intact protein and fragment analysis, and MAbPac SCX for charge variant analysis which provide high resolution, reproducibility, and stability.
3) Considerations for sample handling depending on properties like volume, surface interactions, and recovery using vials or plates.
4) Thermo Scientific offers an integrated solution for biomolecule separations from sample preparation
Process Development for Cell Therapy and Viral Gene TherapyMerck Life Sciences
Today’s viral vector manufacturing processes remain challenging. Process development is a critical enabler to bring safe, effective, sustainable products to market to address patient needs. When done properly, it can reduce the timeline of the project and the cost of producing the therapeutic product.
The webinar discusses our strategies for developing lentivirus and adeno associated virus (AAV) and the impact these early decisions can have on commercial readiness.
Watch the interactive webinar now: https://bit.ly/2VplwQq
Process Development for Cell Therapy and Viral Gene TherapyMilliporeSigma
Today’s viral vector manufacturing processes remain challenging. Process development is a critical enabler to bring safe, effective, sustainable products to market to address patient needs. When done properly, it can reduce the timeline of the project and the cost of producing the therapeutic product.
The webinar discusses our strategies for developing lentivirus and adeno associated virus (AAV) and the impact these early decisions can have on commercial readiness.
Watch the interactive webinar now: https://bit.ly/2VplwQq
Mycenax is a CDMO located in Taiwan that provides services for biologics development from cell line development through fill/finish. They have expertise in mammalian and microbial manufacturing up to 2000L scale using disposable technology. Mycenax has 7 biosimilars in development and is evaluating opportunities for novel biologics. They aim to be a full service CDMO partner for biologics development and manufacturing.
Preparing DNA for sequencing requires careful purification of the template DNA to ensure high quality results. Key steps include extracting DNA, quantifying the DNA concentration and quality, purifying DNA or PCR products to remove contaminants like primers, salts and proteins, and checking for purity and fragment size on a gel. Using purified template DNA free of impurities is crucial for accurate sequencing reads.
Similar to Webinar: Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA (20)
The Viscosity Reduction Platform: Viscosity-reducing excipients for improveme...MilliporeSigma
Protein viscosity is a major challenge in preparing highly concentrated protein formulations suitable for subcutaneous injection. Recently, the Viscosity Reduction Platform (VRP) was introduced and its technical key features and benefits for formulations were discussed. However, highly viscous solutions do not only pose a challenge when administering a drug to a patient, they can also impose technical limitations in the manufacturing process.
This white paper evaluates the effect of the excipients in the Viscosity Reduction Platform on ultrafiltration processes used to produce a highly concentrated formulation of a monoclonal antibody (mAb). Two filtration methods are demonstrated in this work.
Find more information about the Viscosity Reduction Platform on our website: https://www.sigmaaldrich.com/products/pharma-and-biopharma-manufacturing/formulation/viscosity-reduction-platform
Use of Excipients in Downstream Processing to Improve Protein PurificationMilliporeSigma
Excipients are used to improve the stability of protein-based therapeutics by protecting the protein against a range of stress conditions such as temperature changes, pH changes, or agitation. Similar stresses are applied to proteins during downstream purification. Shifts in pH during Protein A chromatography, subsequent incubations at low pH for virus inactivation, and changes in conductivity in ion exchange chromatography can lead to aggregation, fragmentation, or other chemical modifications of the therapeutic protein. Given the potential impact on the protein’s structural integrity, there is a need for approaches to reduce the risk presented by the conditions during downstream processing. For example, integration of a solution to prevent aggregation of proteins would be a more efficient strategy than implementing steps to remove multimeric forms.
This white paper highlights the results from a recent paper by Stange et. al., in which protein stabilizing excipients such as polyols, sugars, and polyethylene glycol (PEG4000) were used as buffer system additives. Effect of the excipients on elution patterns, stabilization of the monomer antibody, host-cell protein removal, virus inactivation rates and binding capacity of cation exchange chromatography were explored.
Exploring the protein stabilizing capability of surfactants against agitation...MilliporeSigma
Agitation of therapeutic protein solutions during manufacturing, shipping and handling is one of the major initiators for protein aggregation and particle formation during the life history of a protein drug. Adsorption of protein molecules to liquid-air interfaces leads to the formation of highly concentrated protein surface films. The rupture of these protein films due to various mechanical processes can then result in the appearance of protein aggregates and particles in the bulk solution phase.
One technique to stabilize proteins against stress induced by liquid-air interfaces is the use of non-ionic surfactants. About 91% of antibody formulations commercially available in 2021 contained a surfactant. Polysorbate 20 and 80, composed of a hydrophilic polyoxyethylene sorbitan and hydrophobic fatty acid esters, made up the largest part being employed in 87% of said formulations.
Despite their frequent use in parenteral drug products, concerns have been raised for decades about the application of polysorbates as surfactants in biopharmaceutical formulations. Autoxidation of polysorbate, caused by residual peroxides in polysorbates, can damage the proteins and can further drive the oxidative degradation of polysorbate. Chemical and enzymatic hydrolysis of polysorbate may lead to the formation of free fatty acid particles, which may become visible; and both mechanisms eventually lead to the reduction in polysorbate concentration. Therefore, the purpose of the current study was to compare various molecules for their capabilities to reduced agitation-induced protein aggregation and particle formation; and furthermore, investigate their underlying protein stabilizing mechanisms.
The Viscosity Reduction Platform: Viscosity Reducing Excipients for Protein F...MilliporeSigma
Protein viscosity is one of the major obstacles in preparing highly concentrated protein formulations suitable for subcutaneous injection.
This whitepaper examines how combining an amino acid with a second viscosity-reducing excipient circumvents adverse effects on protein stability and improves viscosity-reducing capacity.
To find more information about the Viscosity Reduction Platform, please visit our website: https://sigmaaldrich.com/products/pharma-and-biopharma-manufacturing/formulation/viscosity-reduction-platform
Characterization of monoclonal antibodies and Antibody drug conjugates by Sur...MilliporeSigma
The document discusses characterization of antibodies and antibody-drug conjugates (ADCs) using surface plasmon resonance (SPR). It provides details on:
1. Using SPR to characterize binding kinetics of ADCs and determine effects of different linker types and drug-antibody ratios on antigen binding. SPR shows reduced but detectable binding for ADCs versus the unconjugated antibody.
2. An application of SPR to study binding interactions of SARS-CoV-2 spike protein and mutants with the ACE2 receptor and anti-spike antibodies. This can aid understanding of viral mutations and inform vaccine and drug development.
3. SPR is proposed as a method to screen binding kinetics of spike protein mutants to evaluate effects
The Role of BioPhorum Extractables Data in the Effective Adoption of Single-U...MilliporeSigma
Regulatory expectation does require patient safety evaluations with supporting data for manufacturing components that directly come into contact with drug manufacturing process streams. Readily available extractables data can help manufacturers using singleuse technology to accelerate product qualifications, risk assessments and process optimization
This white paper guides you on how to save time and resources with supplier-provided single-use system extractables data and gives you an overview about the overall strategy for Extractables & Leachables. At the end you will find a case study.
Find more information about filters and single-use components on our website: https://www.sigmaaldrich.com/DE/en/services/product-services/emprove-program/emprove-filter-and-single-use-component-portfolio
The Future of Pharma- and Biopharmaceutical AuditsMilliporeSigma
The document discusses how COVID-19 has changed pharma and biopharmaceutical audits, with remote audits now accepted by regulatory authorities. It explores the benefits of different audit approaches like remote, virtual, and hybrid audits and the features that enable each. Regulatory bodies are adopting new guidance for remote inspections and assessments. Looking ahead, risk-based planning and digital solutions will allow the industry to conduct more flexible and agile audits, though on-site audits will still be necessary at times. Hybrid audits combining various approaches are positioned as the future of auditing.
Moving your Gene Therapy from R&D to IND: How to navigate the Regulatory Land...MilliporeSigma
Watch the recording of this presentation here: https://bit.ly/3SqOsoP
Novel therapies, including cell and gene therapies, continue to be central to innovation in healthcare and represent the fastest growing area of therapeutic medicine. As a consequence, the number of gene therapies undergoing clinical trials has increased significantly in the last five years.
Manufacturing processes for these novel therapeutics are very complex with a high risk of contamination. Regulatory agencies world-wide have responded by issuing guidance to outline their expectations for development and manufacture of cell and gene therapies. Currently, regulatory guidance is not harmonized globally and can often lead to confusion within industry and increased risk of non-compliance.
In this webinar, we'll answer:
• Which regulatory guidelines do you need to comply for your INDs?
• When do you start implementing GMPs and validated assays?
• How do you get your QC testing strategy ‘right the first time’?
• How do you ensure testing is not your rate limiting step for the IND submission?
Presented by:
Manjula Aysola, Senior Regulatory Consultant
Dr. Alison Armstrong, Sr. Director, Technical and Scientific Solutions
Identity testing by NGS as a means of risk mitigation for viral gene therapiesMilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3RijkHC
Detailed description:
Imagine you’ve just completed a manufacturing run for your viral vector. Identity testing is performed to confirm the vector sequence. But when the results come back the data reveals unexpected sequence variants! With an appropriate risk mitigation testing strategy, this situation can be prevented.
The situation described above is not hypothetical, and happens more that you think, costing valuable time and resources.
Investigatory testing has shown that sequence variants present in starting materials (e.g. plasmids) are likely to make their way to the final product. Adequate identification of low-level variants with an appropriately sensitive method is critical in ensuring the quality of the final product. A risk-based testing strategy, in the context of identity, for viral vector manufacturing will be presented, focusing on key testing points. NGS assays for identity and variant detection will be highlighted due to their extremely sensitive nature compared to traditional approaches.
In this webinar, we'll explore:
• Regulatory requirements for identity testing
• NGS applications for identity testing as compared to traditional methods
• A case study on the impact of not establishing a proper risk-based testing strategy
Presented by: Bradley Hasson, Director of Lab Operations for NGS Services
Latest advancements of melt based 3D printing technologies for oral drug deli...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3A2WcH4
The application of polymer excipients in 3D printing manufacturing is usually limited due to the concerns of filament strength, high processing temperature and large scale manufacturing.
Latest technology developments are targeting a direct melt deposition to simplify the process and enable a constant and efficient process. Two different processing approaches will be presented:
The advanced melt drop deposition, where individual three dimensional geometries can be created by depostition of polymer droplets and the MED® 3D printing technology which allows by precise layer-by-layer deposition to produce objects with well-designed geometric structures.
In this webinar, you will learn:
• Latest advancements of melt based 3D printing approaches
• Application examples for the individual technologies
• Deep dive in the MED® 3D printing technology to design dedicated drug release profiles
Presented by:
Dr. Thomas Kipping, Head of Drug Carriers
Dr. Xianghao Zuo, Deputy Director of R&D, Triastek
CAR-T Manufacturing Innovations that Work - Automating Low Volume Processes a...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3NDNIKe
Automated, fit-for-purpose tools are essential in CAR-T processing to support sustainable manufacturing of clinical and market-approved cell therapy products. This webinar will discuss how the ekko™ Acoustic Cell Processing System uses acoustic technology as a touchless approach to manipulate cells, enabling a modular tool across the CAR-T manufacturing workflow. Typical performance of templated ekko™ System processes for DMSO washout of leukapheresis material, low volume and high cell concentrate for electroporation preparation, and harvest of expanded T cells will be reviewed.
This webinar will also give an early glimpse at the ekko™ Select System for unmatched T cell selection.
In this webinar, you will:
• Uncover how the ekko™ System supports the broad industrialization of cell therapy, with particular focus on how to achieve low volume, high concentrate cell product for critical transduction and transfection steps
• Discover how ekko™ System for wash and concentrate processes throughout the cell therapy workflow achieve high cell recovery, viability, and effective residual removal
• Preview to ekko™ Select, our cell therapy selection platform, to achieve unmatched ease-of-use with direct processing from leukopaks reducing the need for preparation steps
Presented by:
Benjamin Ross-Johnsrud, Acoustic Technology Expert
Robert Scott, Mechanical Engineer III
How does the ICH Q5A revision impact viral safety strategies for biologics?MilliporeSigma
The document discusses the expected revision of ICH Q5A guidance on viral safety of biotechnology products. Key points include:
1) ICH Q5A is being revised to address new biologic modalities like viral vectors and advances in manufacturing and detection methods.
2) The revision will provide more flexibility in viral clearance validation strategies and acceptance of alternative detection methods like PCR and HTS.
3) Challenges for viral safety of advanced manufacturing will also be discussed, as the original guidance does not address emerging approaches.
Improve Operational Efficiency by Over 30% with Product, Process, & Systems A...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3adaxWh
When implementing new automation systems, organizations must consider things like deployment time, user adoption, and costs.
They must also consider the cost of doing nothing – that is, what competitive advantage is lost in standing still? What time and quality is lost in repetitive, manual tasks rather than an automated, digital workflow? What operational efficiencies are lost?
In this webinar we examine how a product, process, and system agnostic automation platform can be deployed faster than traditional system specific software while bringing greater operational efficiencies (in many cases over 30% improvement).
To remain competitive in the market, biopharma manufacturers must adopt automation and digital technologies, but most plants still have island of automation consisting of independently functioning, standalone unit operations. This results in operational inefficiency, regulatory concerns, and a poor understanding of the process and product life cycle.
Taking the first, right step must include considering risks, costs, timelines, and technology alternatives. Traditional automation approaches tied to specific systems, processes, and products are, by their nature, limited; while an agnostic platform will address current biomanufacturing business challenges and ensure future readiness. With the right platform, a phased automation implementation can yield operational efficiency gains of up to 30% and improved product quality and regulatory compliance.
In this webinar, let's explore:
• Challenges of automation and digital technology adoption
• What a product, process, and system agnostic platform entails
• Applications and benefits of a process orchestration platform
• Ensuring future readiness with process orchestration
Presented by:
Braj Nandan Thakur, Global Product Manager - Automation
Insights from a Global Collaboration Accelerating Vaccine Development with an...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3Nbb5ug
Get insights and best practices from a multinational team establishing a platform for vaccine production. See how a long-term collaboration on a bench-scale process used to produce a Virus Like Particle (VLP) vaccine for SARS-CoV-2 was successfully converted to a robust GMP-compatible, scalable process.
The COVID-19 pandemic further emphasized the need for collaboration in the development of urgently needed vaccines and therapeutics. In this webinar, we take you behind the scenes of our collaboration with Technovax and Innovative Biotech in which a scalable VLP vaccine platform was optimized for use in a production facility in Nigeria in response to the need for local production of SARS-CoV-2 vaccines. The flexibility and robustness of the platform will enable its rapid deployment to support the West African pandemic readiness program. Initial development of the VLP process began in late 2019 and by March 2020, was already adapted for production of a SARS-CoV-2 vaccine.
In this webinar, you will learn:
• About building a priceless collaborative network with integrated solutions
• Virus-Like Particle Vaccines
• Process Development Overview and Challenges
• Pre-clinical Results and Next Steps
Presented by:
Jose M. Galarza, PhD,
President and Founder of TechnoVax
Naomi Baer,
Business development consultant, Emerging Biotech, BioProcess division
Youssef Gaabouri, Eng. ,
Associate Director, Head of Sales Middle East & Africa, BioProcess division
Risk-Based Qualification of X-Ray Sterilization for Single-Use SystemsMilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3vQf0qv
In the single-use bioprocess industry, X-ray irradiation warrants consideration as an alternate sterilization technology. Using a risk-based qualification testing strategy is important when evaluating and implementing equivalent ionizing irradiation sterilization methods.
The urgent need for life-saving therapies as a result of the global pandemic has reinforced the criticality of flexibility in pharmaceutical manufacturing, including sterilization. The single-use bioprocess industry traditionally has employed gamma irradiation sterilization. X-ray irradiation is being considered as an additional sterilization technology for business and supply continuity. We will share a risk-based qualification testing strategy including Extractables and data generated to support comparability of gamma irradiation and X-ray irradiation as equivalent ionizing irradiation sterilization methods.
In this webinar, you will learn about:
• The comparison of gamma and X-ray irradiation sterilization
• A risk-based qualification test strategy
• Data evaluation of gamma versus X-ray sterilized single-use components
Presented by:
Monica Cardona,
Global Senior Program Manager
Paul Killian, Ph.D.,
R&D Director, Analytical Technologies
Rapid Replication Competent Adenovirus (rRCA) Detection: Accelerate your Lot ...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3MJ4u9V
Testing for presence of replication competent adenovirus (RCA) is a key component to ensure patient safety and a requirement for all biologicals manufactured using adenoviral vectors. For many adenoviral-based products, the RCA assay is a rate-limiting assay for lot release.
Join this webinar to learn about a rapid RCA detection assay currently in development, which combines a 7-day culture assay with a highly sensitive molecular endpoint specific for RCA. The method can detect presence of as little as 1 RCA in adenoviral vector material at an approximate concentration of 5x107 - 2x108 vector particles (VP)/mL, making it a suitable method to meet regulatory requirements while accelerating your lot release timelines.
In this webinar, you will learn about:
• Regulatory framework for adenoviral vector products
• Considerations for lot release testing of adenoviral-based therapies
• Advantages of a rapid method for RCA testing on production lot material
Presented by:
Axel Fun, Ph.D.,
Principal Scientist
Alberto Santana, MBA,
Product Manager, Biologics Biosafety Testing
The High Intensity Sweeteners Neotame and Sucralose: 2 Ways to ace the Patien...MilliporeSigma
Watch the presentation of this webinar here: https://bit.ly/3vQyN7K
Bitter medicines are an important issue, especially for pediatric applications. As several APIs have bitter tasting components, high intensity sweeteners for taste optimization are of great interest. Join our webinar to discover our new sweetener toolbox enabling safe and stable formulations.
Mask bitter aftertaste for a sweeter pill to swallow! Patients’ compliance and the therapeutic benefit are supported by a pleasant taste of pharmaceutical formulations. With the high intensity sweeteners Neotame and Sucralose, you have efficient tools at hand which are superior to other sweeteners in many aspects:
• excellent sugar-like taste profile
• outstanding sweetness factors
• use effectiveness
• enhanced stability
We will present our new toolbox of two high performance sweeteners and focus on aspects of stability, safety, the application in various dosage forms, and market perception.
In this webinar, you will learn:
• How to optimize the patients' taste experience of your pharmaceuticals
• How sweeteners can be differentiated by their sensory profiles and features
• How our new product offering Neotame can be effectively used in your targeted formulations
Presented by:
Almut von der Brelie,
Senior Manager Strategic Marketing, Excipients for Solid Applications
The Developability Classification System (DCS): Enabling an Optimized Approac...MilliporeSigma
This whitepaper by Dr. Daniel Joseph Price outlines how poorly soluble drug formulations can be designed using the developability classification system (DCS).
The DCS identifies the root cause of low solubility and enables lean, cost-effective and effective formulations to be developed.
#solubility #pharmaceuticalmanufacturing #oralsoliddosage #drugdevelopment
How to Accelerate and Enhance ADC TherapiesMilliporeSigma
In this webinar, you will learn about:
The advantages of using advanced intermediates to develop ADC therapies
How to increase ADC solubility and efficiency
Fast, small-scale ADC library generation
Seamless supply chain with reduced complexity and regulatory support
The ADCore product line offers versatile intermediates that simplify the synthesis of common ADC payloads (dolastatins, maytansinoids, and PBDs) by greatly reducing the number of synthetic steps. This translates to savings in development and manufacturing costs and shorter timelines to the clinic. To address the poor solubility of many ADC payloads, ChetoSensar™ was developed to significantly increase the hydrophilicity of the drug linker, which has been shown to also substantially increase the efficacy of ADCs and broaden the therapeutic window.
Lastly, the ADC Express™ service leverages conjugation chemistry and analytical expertise to help design and quickly synthesize sets of potential ADC therapies suitable for screening to simplify candidate selection and get ADC therapies to market faster.
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
• Evidence-based strategies to address health misinformation effectively
• Building trust with communities online and offline
• Equipping health professionals to address questions, concerns and health misinformation
• Assessing risk and mitigating harm from adverse health narratives in communities, health workforce and health system
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Integrating Ayurveda into Parkinson’s Management: A Holistic ApproachAyurveda ForAll
Explore the benefits of combining Ayurveda with conventional Parkinson's treatments. Learn how a holistic approach can manage symptoms, enhance well-being, and balance body energies. Discover the steps to safely integrate Ayurvedic practices into your Parkinson’s care plan, including expert guidance on diet, herbal remedies, and lifestyle modifications.
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
Local Advanced Lung Cancer: Artificial Intelligence, Synergetics, Complex Sys...Oleg Kshivets
Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
We’re talking about Vedic Meditation, a form of meditation that has been around for at least 5,000 years. Back then, the people who lived in the Indus Valley, now known as India and Pakistan, practised meditation as a fundamental part of daily life. This knowledge that has given us yoga and Ayurveda, was known as Veda, hence the name Vedic. And though there are some written records, the practice has been passed down verbally from generation to generation.
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptx
Webinar: Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA
1. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Effective and Efficient Design
of a Downstream Purification
Process for Plasmid DNA
March 24, 2022
Nargisse El Hajjami, Ph.D. Eng.
Senior Consultant, Novel Modalities Bioprocessing Strategy Operationalization
Global Lead for mRNA Technology
Laurens Vergauwen, Eng.
EMEA Process Development Scientist,
Global Vaccine and Viral Therapies PD Focal Point Lead
Thomas Elich, Eng.
Manager MSAT Americas Purification Process Engineering
2. MilliporeSigma is the U.S. and
Canada Life Science business
of Merck KGaA, Darmstadt,
Germany.
3. Agenda
1
2
3
Introduction to plasmid DNA - Nargisse
El Hajjami, Ph.D. Eng.
pDNA purification challenges &
considerations - Laurens Vergauwen
Case study - Thomas Elich
4 Summary
5. Circular double helix DNA molecules, naturally found in bacteria, intracellular replicated
Introduction
What are Plasmids?
MW Size
Plasmid DNA 4 MD* 200 nm**
mAb 0.2 MD* 10 nm**
* Mega Dalton (6 kb Plasmid)
** Dynamic range
Supercoiled plasmid is recognized by regulatory authorities as the most therapeutically effective
Carsten Voß, 2007
CCC form I: covalently closed circles,
supercoiled, fully intact,
wound around itself
▪ Large size 1.5 – 150 kb
▪ Poly anion, highly negatively charged
▪ Sensitive to mechanical stress
▪ Various topological forms
OC form: one strand nicked/broken,
less compact, totally relaxed
Linear form: both strands broken,
free ends
Characteristics
5 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
6. The importance of plasmid DNA
The key-role of pDNA in various applications
Viral Vectors Non Viral Vector
In vitro
Transcription
Linear DNA
Plasmid
DNA
mRNA
plasmid
DNA
MRNA Vx & Tx
mRNA in vitro
transcription
- Delivery:
- - Liposomal
- - Nanoparticles
- - Electroporation
Plasmid DNA
Vx & Tx
The 1st DNA-based Vx
approved for Covid19
6 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
7. What is your application for plasmid DNA? (Select all that apply)
1. Viral vectors production
2. Template for mRNA in vitro transcription
3. Plasmid DNA-based vaccines / therapeutics
4. Raw material for other applications
5. Others
Poll Question #1
7 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
8. ✓ Certificate of analysis
✓ Shake-flask/high density
fermenter
✓ Alkaline lysis
✓ Chromatographic purification
✓ Master/working cell bank E. coli
✓ Purity (A260/280 1.8-2.0)
✓ >85% supercoiled (HPLC)
✓ Host protein <1% (ELISA)
✓ Host gDNA <1% (PCR)
✓ Endotoxins <10EU/mg (LAL or rabbit
pyrogenicity test)
✓ Full traceability of materials
✓ Stability studies
✓ ISO 5 validated clean rooms in
GMP suites
✓ Complete QA & QC oversight
✓ >90% supercoiled
✓ Host protein & gDNA <<0.1%
✓ BSE/TSE risk analysis (if animal
products used)
✓ Environmental monitoring
Plasmid DNA manufacturing
GMP-LIKE
RESEARCH USE ONLY
GMP
Highly documented
+ use of clean rooms
+ EM
Least documentation, no
endotoxin measurement
Different grades of plasmid DNA are produced
8 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
9. Advantages
Simple design
Easy to manipulate/optimize
Rapid production & formulation
Reproducible, large-scale
production
Requires only BL1 safety level
Temperature stable, long shelf
life
Different applications
Attractive market
General considerations
Plasmid DNA potential & limitations
Disadvantages as Tx or Vx
High production cost
Poor gene transfer efficiency
Estimated that for every 1000
plasmid molecules only one
reaches the cell and is
expressed.
Low immunogenic potential of
bacterial DNA
Possible insertional mutagenesis
Challenges
Lack of adequate infrastructure
Production scale-up efficiency
Removal of impurities and non
desired pDNA forms need
careful process design
High concentration needed (mg
level)
Delivery and protection of DNA
from degradation
Complex process
Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA
9
10. Plasmid DNA Manufacturing
Key Process Considerations
Process
Considerations
• Batch Vs Fed-batch
• Plamsid DNA sequence design
• Final cell density
• Cell harvest- Centrifugation Vs TFF
• Pressure/shear
• Cell lysis – physical Vs chemical
treatment
• Volume
• Surface Area
• Pore Size
• Media
• Flow Rate
• Pressure/shear
• Clarification – Depth filtration Vs
centrifugation
• Size exclusion Vs absorption
mechanisms
• Mixing speed
• Pre-treatment steps (Rnase
treatment
• Volume
• Media
• Purification mode
• Nature of contaminants
• Pressure/Shear
• Viscosity
• Final Filtration
• Mixing speed
• Duration of alkaline lysis
10 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
12. Typical pDNA process flow
pDNA purification challenges & considerations
Thaw Cells Fermentation Cell Harvest Cell Lysis
Clarification
Concentration
& Diafiltration
(UF/DF)
Concentration
& Diafiltration
(UF/DF)
Purification with
Chromatography
(1-2 steps)
Storage
Sterile
Filtration
12 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
13. Unique challenges of pDNA purification
pDNA purification challenges & considerations
Similarity of product and contaminants (genomic DNA (gDNA), endotoxin, RNA, plasmid isoforms)
leads to low resolution separation.
Feed often highly viscous, complicating downstream processing.
Shear sensitivity
Lack of platform process and integrated solutions
13 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
14. Cell harvest - Centrifugation Vs Filtration
pDNA purification challenges & considerations
Cost effective at batch volumes
<5 L and >500 L
Special attention is needed for high
shear generated in large scale
centrifugation
More process development required
for scale up
Centrifugation Microfiltration Tangential Flow
Filtration (MF-TFF)
Open-channel, flat-sheet TFF
devices (1000 kD, 0.1 μm, 0.2 μm)
work well in this application.
Lower capital cost
Linear scalability
Pellicon® 2 ProstakTM
14 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
15. Bacterial cells
suspended in
starting buffer
Alkaline solution
with detergent is
added to lyse
bacterial cells
Neutralization with
potassium acetate
Cell Lysis - Alkaline lysis is commonly used
pDNA purification challenges & considerations
Principle
• Alkaline condition plus detergent solubilizes the cell walls and the
alkaline environment denatures gDNA
• Sodium Dodecyl Sulfate (SDS) commonly used as the detergent
• Reaction neutralized with acid after short incubation
Challenges
• Both alkaline and shear from mixing can damage supercoiled
plasmid DNA
• Sudden increase of viscosity will impact mixing efficiency
Solutions
• Optimize mix speed, incubation time, and chemical
concentrations
• Mobius® single-use mixers
15 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
16. Clarification - The complex nature of lysed cells
pDNA purification challenges & considerations
Lysis output can be challenging for clarification
− Complex mixture of cell debris, pDNA, genomic DNA, RNA and HCP
− High solid load
− High viscosity
− Alkaline lysis leads to two-phase separation
− Froth phase (top) – cell debris and genomic DNA
− Lysate phase (bottom) – pDNA, RNA, HCP
Pretreatment can be performed to enhance further clarification,
options include:
− Gravity separation of top and bottom phase (commonly used)
− Centrifugation
− RNase digestion
− Ca2+ precipitation of RNA
Note: When using gravity separation, yield losses up to 20% were reported
Froth phase
Lysate phase
16 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
17. Clarification - Centrifugation Vs Filtration
pDNA purification challenges & considerations
Capable of handling high solid load
May require secondary clarification
More process development required for
scale up
Attention: High shear can damage
supercoiled pDNA
Centrifugation Normal flow filtration
Depth filtration based on size exclusion
and adsorption
Robust process
Linear scalable
Attention: + charged filtration aid may
interact with pDNA
17 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
18. Clarification - Depth filter selection
pDNA purification challenges & considerations
Lysate is highly plugging and viscous in nature due to cell debris
− Depth filters with pore structure >0.5 µm rating work well
− Apply low flow rates 100-150 LMH
• Media with lower adsorptive properties are preferred (dependent on media type and density)
Clarisolve®
Depth Filters
18 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
19. TFF - Tangential Flow Filtration
pDNA purification challenges & considerations
First TFF step (optional): After clarification and before chromatography
− Concentrate plasmid to reduce loading time and complete buffer exchange for chromatography steps
− Removal of impurities: RNA, small size gDNA fragments and protein
Second TFF step: After chromatography and before sterile filtration
− Achieve final concentration and exchange into formulation buffer
19 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
20. TFF - Devices and watch outs
pDNA purification challenges & considerations
Typical MWCO include 30, 100, 300 kD
− Pellicon® 2 with Biomax® or Ultracel® C screen
− Pellicon® Capsule available in 30 kD Ultracel® membrane
− Single-use format
− Gamma sterilized
− Eliminated carry-over and cleaning validation
Watch outs
− High viscosity → D or V screen may be needed to reach high concentration
− Shear stress
− Flat sheet devices can be operated at lower flux compared to hollow fiber
Pellicon® Capsule
Pellicon® 2
20 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
21. Chromatography - Common approaches
pDNA purification challenges & considerations
Goal: Separate supercoiled (ccc) plasmid from oc-/linear isoforms and residual impurities (HCP, nucleic
acid, endotoxin) by charge, size or hydrophobicity
Combination of Anion exchange and Hydrophobic interaction
Anion Exchange Chromatography (AEX)
• Applicable for capture, intermediate and polishing
• Weak AEX resins give highest recovery and selective impurity removal
• Separate plasmid from proteins, RNA and gDNA and removing endotoxin
• Separation of plasmid isoforms difficult
Hydrophobic Interaction Chromatography (HIC)
• Works by salt promoted binding (≈ 2.5 M NH4SO4)
• Separate isoforms: Supercoiled pDNA is less hydrophobic than RNA, oc- and linear- plasmid forms
and denatured gDNA
1
2
21 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
22. Membrane Chromatography
• Improved hydraulic performance due to large
convective pores and low bed height
• Large accessible surface for Plasmids enabling for
improved binding capacity (5 - 10 mg/mL)
at very short residence times < 0.2 min
• High productivity due to short cycling time
• Single use format, principally scalable
Chromatography - Two technologies to consider
pDNA purification challenges & considerations
Resin Chromatography
• Low binding capacity due to large Plasmid size
limiting diffusion into beads restricting binding to the
resin surface only (< 3 mg/mL)
• High pressure drops due to elevated viscosity feed
stream resulting in flow limitations and long
processing times (2-8 min RT typical)
• Re-use needed for economic feasibility
• Flexible installation – pack as much as needed
• Generally better selectivity than membranes
Natrix® Q membrane
Fractogel®
DEAE/DMAE
(wAEX)
22 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
23. pDNA sterile filtration optimization parameters
pDNA purification challenges & considerations
Sterile filtration can be challenging due to the large size of pDNA, shear sensitivity, and viscosity.
Internal data shows that the following parameters are important:
Optimization Parameter Yield Capacity
Product
integrity
Salt concentration X X
Supercoiled pDNA content (purity) X X
Filtration endpoint X
Membrane type – PVDF or PES X – PES X - PES
pDNA concentration X X
Feed flux or pressure X
Higher concentrations → lower
radius of gyration
Higher purity gives better performance
Membrane fouling leads to lower yield
Can affect Plasmid integrity (shear)
Millipore Express® SHC best performer
Needs further investigation
23 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
24. Integrated solutions for pDNA purification
pDNA purification challenges & considerations
24 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
25. What is your involvement with plasmid DNA manufacturing?
1. CMO that manufactures & sells pDNA.
2. Company that purchases pDNA from CMO for my process.
3. Company that currently purchases pDNA from CMO, but considering to bring in-house.
4. Company that produces pDNA in-house.
5. Just here to learn!
Poll Question #2
25 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
26. Case study
Downstream process development for high
productivity purification of plasmid DNA
Tom Elich1, Jaime De Souza1,2, Herb Lutz3, John Cyganowski4
1. Technical and Scientific Solutions, MilliporeSigma, Burlington, MA
2. Formerly with MilliporeSigma
3. Ambassador to the Future, Center of Excellence MilliporeSigma, Burlington, MA
4. Downstream Customer Applications, MilliporeSigma, Burlington, MA
27. TFF cell harvest Lysis
Chromatography
Formulation
Final Filter
Storage
Plasmid DNA Case Study
Process Overview
Fermenter Clarification
Salt Addition
• 20 hr fed-batch E.Coli
• ~100 OD600 endpoint
• Dry cell wt: 20- 40 g/L
• Wet cell wt: 100-120 g/L
• 2x conc. to ~200 g/L wet cell wt.
• 2x DF cell wash
• Freeze
• Release plasmid w/NaOH & SDS
• Quench w/K-Acetate
• Phase separate floating debris
• Depth filter bottom layer
• NaCl
• Bind/elute AEX
• Clear RNA, HCP, Endotoxin
• 5x DF buffer exchange
27 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
28. TFF cell harvest Lysis
Chromatography
Formulation
Final Filter
Storage
Fermenter Clarification
Salt Addition
• 20 hr fed-batch
• ~100 OD600 endpoint
• Dry cell wt: 20- 40 g/L
• Wet cell wt: 100-120 g/L
• 2x conc. to ~200 g/L wet cell wt.
• 2x DF cell wash
• Freeze
• Release plasmid w/NaOH & SDS
• Quench w/K-Acetate
• Phase separate floating debris
• Depth filter bottom layer
• NaCl
• Bind/elute AEX
• Clear RNA, HCP, Endotoxin
• 5x DF buffer exchange
Discussed in this presentation – Feasibility at 3L fermenter scale
Plasmid DNA Case Study
Process Overview
28 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
29. Objective:
Retain, concentrate, and wash E. coli cells (2 x 0.5µm); permeate water
& impurities
Materials:
1000KD Biomax® polyethersulfone membrane retains 0.018 mm spheres,
V screen for high viscosity & particles
Schematic of a 2-pump TFF system
Methods:
Membrane preparation:
• Water flush: 20L/m2
• Clean-in-place recirculation: 0.2N NaOH, 20L/m2 single pass
• Buffer flush: 20L/m2 10mM Tris, 1mM EDTA, pH 8 (TE)
Cell concentration:
• Tank volume reduction with permeate to waste; cells are retained.
Cell wash
• Constant volume diafiltration adds TE buffer at same rate as permeate removal
Cell recovery
• Drain tank and flush retentate line and flush tank
Cell Harvest by Tangential Flow Filtration (TFF)
• Tangential flow operation provides high capacity
• Permeate pump has higher capacity than TMP control
Plasmid DNA Case Study
29 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
30. 0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
1.0 1.4 1.8 2.2
Flux
(LMH)
TMP
(psi)
Concentration Factor
TMP Flux
Critical flux testing1
• 0.1 m2 scale down cassette
• Ramp up pump 2 until TMP is unstable
• Critical flux was identified as 20 LMH
• 80% of the critical flux (~16 LMH) for operation
Cell concentration
• TMP increased from 1.5 psi to 4 psi due to
viscosity; flux was reduced to 11 LMH.
• 2.1x concentration factor (volume
reduction) achieved
0
0.5
1
1.5
2
2.5
3
3.5
0 5 10 15 20 25
TMP
(psi)
Time (min)
10LMH
20LMH
15LMH
0
2
4
6
8
10
12
14
16
18
20
0
5
10
15
20
25
0.0 0.5 1.0 1.5 2.0 2.5
Flux
(LMH)
TMP
(psi)
Diavolume
TMP
Flux
Cell wash
• 2 diavolumes (tank volumes)
exchanged with TE buffer
• Wash out impurities (media
components, HCP, nucleic acids, etc.)
Cell Harvest by Tangential Flow Filtration (TFF)
• 2.1x VCF achieved at 13 LMH average flux
• 2 diavolume wash at average flux of 12 LMH
1 Field 1995
Plasmid DNA Case Study
30 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
31. Plasmid DNA Case Study
Lysis Buffer Screening
1) Lyse: Add lysis buffer (NaOH/detergent) to
suspension in 1:1 volume ratio and incubate
2) Quench: Add neutralization buffer (3M
potassium acetate, pH 5.5) in 1:1 volume
ratio
3) Recover: Centrifuged at 12,000 x g for 30
minutes and 0.45 mm filtered
4) Assay: PAGE or Quant-iT dsDNA assay
Test Matrix
Parameters: [NaOH] added, [detergent added], lysis time
Meacle 2004
NaOH
Concentration
Detergent
Concentration
Incubation Time
0.05 M
0.075 M
0.1 M
0.125 M
0.15 M
1% SDS 0 (negative control)
1 min
5 min
10 min
60 min
Cell Alkaline Lysis- Test Procedures
31 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
32. Cell Alkaline Lysis - Assay Results
Plasmid DNA Case study
5-minute lysis, 1% SDS, variable NaOH
Plasmid
RNA
Operating setpoints for lysis:
• 5-10 min
• 0.1-0.15 M NaOH
• 1% SDS
1. Resuspension
2. 0.15M NaOH, 1% SDS – 5 min
3. 0.125M NaOH, 1% SDS – 5 min
4. 0.1M NaOH, 1% SDS – 5 min
5. 0.075M NaOH, 1% SDS – 5 min
6. 0.05M NaOH, 1% SDS -5 min
1 2 3 4 5
1. 0.1N NaOH, 1% SDS, 0 min
2. 0.1N NaOH, 1% SDS, 10 min
3. 0.1M NaOH, 1% SDS, 60 min
4. Unfiltered control, 0.15M NaOH,
1% SDS, 5 min
5. Ladder
Plasmid
RNA
0.1M NaOH, 1% SDS, variable time
0
10
20
30
40
50
60
70
80
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
ng/uL
dSDNA
in
solution
Sample #
QuantiT dsDNA [ng/uL] – dsDNA
specific
1. 0.150 M NaOH, 1% SDS – 5 min
2. 0.125 M NaOH, 1% SDS – 5 min
3. 0.100 M NaOH, 1% SDS – 5 min
4. 0.075 M NaOH, 1% SDS – 5 min
5. 0.050 M NaOH, 1% SDS – 5 min
6. 0.150 M NaOH, 1% SDS – 1 min
7. 0.125 M NaOH, 1% SDS – 1 min
8. 0.100 M NaOH, 1% SDS – 1 min
9. 0.075 M NaOH, 1% SDS – 1 min
10. 0.050 M NaOH, 1% SDS – 1 min
5 min 1 min
Confirmation by Quant-iT dsDNA assay
32 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
33. • Multi-layer depth filters
• Clarisolve® 60HX: 60 mm nominal, PP material
• Millistak+® C0HC: 2 mm nominal, DE material
• Scalable, single-use “Pod” format
• Use in series with sterilizing filters
• Batch CSTR reactor
• Single-use
• Neutralize & allow
impurities to
flocculate, ~15 min
after lysis & quench
• Pump out bottom
layer without
disturbing
• Millipore Express® SHC
• Cast PES membrane
• Sterilizing grade
• Single-use
33
1. NaOH induced 2-phase
flocculation
2. Depth filtration of bottom phase 3. Sterile filtration
Lysate Clarification
Plasmid DNA Case study
33 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
34. • E. coli lysate, 1% SDS 0.1N NaOH
• Open Clarisolve® 60HX offers low pressure
drop ; turbidity breakthrough at 300 L/m2
• Tighter Millistak+® C0HC filtrate turbidity
<2NTU protects subsequent sterile filter
Filter Train 1
Clarisolve®
60HX
Millipore
Express® SHC
Filter Train 2
Clarisolve®
60HX
Millistak+®
C0HC
Millipore
Express® SHC
0
500
1000
1500
2000
2500
3000
3500
0 100 200 300 400
J
Throughput (L/m²)
(LMH)
Filter Train 1 Filter Train 2
0
1
2
3
4
5
6
7
8
0.0
0.5
1.0
1.5
2.0
2.5
0 100 200 300 400
Turbidity
(NTU)
Pressure
Throughput (L/m2)
(psig)
Filter Train 2
pressure
turbidity
Filter Train 1
pressure
turbidity
Depth Filter Sizing
Constant flow test (Pmax™, Tmax)
Monitor pressure rise & turbidity
Sterile Filter Sizing
Constant pressure test (Vmax™)
Watch flux decay
Lysate Clarification
Filter Train 1: 98% yield, ~2x more sterile filter area
Filter Train 2: 82% yield due to charged C0HC filter
Plasmid DNA Case study
34 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
35. Natrix® Q Chromatography Membrane
Anion Exchange Capture Chromatography
➢ Porous hydrogel chromatography membrane
• A high surface area containing quaternary amine ligand.
• Convective flow path: high capacity at 6 second residence time.
• Open pore structure suitable for large molecule purification.
➢ Disposable device format
• 0.2 mL, 15 mL, 115 mL, 460 mL devices.
35 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
36. AEX Capture Chromatography - Test Overview and Methods
Test 1
No NaCl
added
Test 2
35mM
NaCl
Addition
Test 3
75mM
NaCl
Addition
Step Mobile Phase
Membrane
Volumes
Flowrate
Equil
1M K-Acetate + 150 mM NaCl, pH 5.0
(75 mS/cm)
50 MV 10 MV/min
Load Clarified, sterile filtered lysate pH 5.2
11 mg pDNA/mL
membrane
10 MV/min
Wash
1M K-Acetate + 150 mM NaCl, pH 5.0
(75 mS/cm)
20 MV 10 MV/min
Elute 100 mM Tris, pH 9 + 1M NaCl 50 MV 5 MV/min
CIP 1M NaOH + 2 M NaCl 20 MV 10 MV/min
Impact of NaCl supplementation
on RNA clearance
Capture pDNA while impurities (RNA) flowthrough
Clarified lysate conditions:
• 6.5 kbp pDNA, 24 µg/mL titer. 1.5M K-acetate buffer, pH 5.3, 86.9 ms/cm
• Nucleic acid content: 3.8% pDNA, 96.2% RNA. Endotoxin content: 380,000 EU/mg pDNA
Analytics:
• DNA and RNA content assessed by HPLC (Tosoh DNA-NPR method)1
• Endotoxin content assessed by Charles River Endosafe assay
Plasmid DNA Case Study
1 Urthaler 2005
36 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
37. AEX Capture Chromatography - Results
35 mM NaCl supplementation offers best
balance of capacity, purity, recovery:
• Capacity = 8 mg pDNA/mL membrane
• Nucleic acid purity = 77% pDNA
• pDNA recovery = 88%
• Endotoxin content = 3,100 EU/mg pDNA
Can RNA and endotoxin purity be further
improved with an alternative wash strategy?
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 35 75
NaCl Supplementation (mM)
pDNA Binding Capacity (mg pDNA/mL membrane)
pDNA purity (% of total nucleic acids)
pDNA Recovery
11
8
2
46%
100%
77%
88%
36%
61%
Plasmid DNA Case Study
37
Impact of Salt Supplementation on Capacity, Purity, Recovery
37 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
38. AEX Capture Chromatography- Wash Strategy
Step Mobile Phase
Equilibration 1M K-Acetate + 150 mM NaCl, pH 5.0 (75 mS/cm)
Load Clarified, sterile filtered lysate pH 5.2 + 35mM NaCl
Wash 1M K-Acetate + 150 mM NaCl, pH 5.0 (75 mS/cm)
Elute 100 mM Tris, pH 9 + 1M NaCl
CIP 1M NaOH + 2 M NaCl
Step Mobile Phase
Equilibration 1M K-Acetate + 150 mM NaCl, pH 5.0 (75 mS/cm)
Load Clarified, sterile filtered lysate pH 5.2 + 35mM NaCl
Wash 1M K-Acetate + 150 mM NaCl, pH 5.0 (75 mS/cm)
Detergent
Wash
0.1M Tris, 10mM NaCl, + 0.5% detergent, pH 7.5
EDTA Wash 0.1M Tris, 10mM NaCl, + 2mM EDTA, pH 7.5
Elute w/EDTA 100 mM Tris, 1M NaCl + 2mM EDTA, pH 9
CIP 1M NaOH + 2 M NaCl
Nucleic Acid Content Endotoxin Content Cycle Time
Feed Conditions 4% DNA, 96% RNA 380,000 EU/mg N/A
Elution w/ Control wash
(measured from eluate pool) 77% DNA, 23% RNA 3,100 EU/mg 55 min
Elution w/ Detergent wash
(measured from eluate pool) 95% DNA, 5% RNA 500 EU/mg 65 min
Control Wash Detergent Wash
Results
38
Plasmid DNA Case Study
38 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
39. Membrane Chromatography Resin Chromatography
AEX Capture Chromatography- Membrane Vs Resin
100 L batch of clarified lysate, 3.6 g pDNA
Binding Capacity 8 g/L
Membrane Volume 0.46 L
Flow Rate 4.6 LPM
Step Time 1.1 hr
Cycles 1 cycle
Productivity 7.3 g pDNA/L/hr
Plasmid DNA Case Study
Binding Capacity 3 g/L
Resin Volume 1.18 L
Flow Rate 0.3 LPM
Step Time 9.9 hr
Cycles 1 cycle
Productivity 0.3 g pDNA/L/hr
Natrix® Q membrane is 24x
more productive than resin
39 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
40. What is your level of interest in membrane chromatography for pDNA?
1. Not interested – resin chromatography works well for me.
2. Interested to evaluate for my process.
3. Currently using in my process.
4. Not enough knowledge to comment.
Poll Question #3
40 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
41. 0
20
40
60
80
100
120
140
160
180
5 LMM 6 LMM 7 LMM 8 LMM
Permeate
Flux
(LMH)
Critical Flux
0
5
10
15
20
25
30
35
40
45
50
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0.0 1.0 2.0 3.0 4.0 5.0
Flux
(LMH)
TMP
(psi)
Diavolume (X)
Diafiltration
TMP Flux
• Pellicon® 2 cassette retains plasmids; passes water,
ionic and low MW impurities
• 300KD Ultracel® Membranes (regenerated cellulose membrane)
retains ~10KD DNA, C screen for moderate viscosities
• Tangential flow w/permeate pump for high capacity
• Constant volume DF at 4 LMM feed flow with
buffer (10mM Tris, 1mM EDTA, 10mM NaCl, pH 8.0)
• Declining TMP due to reduced viscosity w/ionic strength over
5DV
• 96% yield
• Critical flux: high 110-170 LMH depending on feed flow
for 0.1 m2 test device, pick 75% (80-125 LMH)
• Dilute formulation: no concentration
Diafiltration Buffer Exchange using TFF
Plasmid DNA Case Study
41 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
42. Cell harvest
4.0m2 Pellicon® 2
Biomax® MF-TFF 1000 kDa
V-screen
Lysis
0.15N NaOH, 1% SDS
5 min incubation
200L Mobius® mixer
Chromatography
460mL Natrix® Q membrane
1 cycle
Formulation
3.0m2 Pellicon® 2 cassette
Ultracel® membrane UF-TFF
300kDa C-screen
Final Filter
Storage
Plasmid DNA Case Study
Proposed Scaled Up Process Flowsheet
Fermenter
100L
Clarify
1.65m2 Clarisolve® 60HX
2x30 in. Millipore Express® SHC
Capsule
Salt Addition
+35mM NaCl
83% yield
3.2 g plasmid
42 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
43. 1
pDNA is a rapidly growing
therapeutic modality for
use with viral vectors,
mRNA, gene therapy.
Summary
2
Feasibility of high productivity
pDNA purification was
demonstrated at lab scale:
MF-TFF harvest, alkaline
lysis, depth filter clarification,
chromatography membrane,
and UF formulation. 3
Implementation at larger
scales requires verification
studies for lysis mixing &
flocculation, membrane
module manifolding, etc.
4
Additional purification
steps may be investigated
to enhance performance,
i.e. CaCl2 precipitation,
isoform separation, final
product concentration,
etc.
Acknowledgements
The authors thank Greenlight BioSciences for their assistance in providing valuable discussions,
feedstocks, and assays.
IP Disclaimer/ At the current time, MilliporeSigma is not aware of any potentially relevant third-party patents, however, a full freedom-to-operate analysis has not
been completed and is the responsibility of the party interested to use pDNA for plasmid purification.
43 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022
44. References
1. JD Watson, et. al., Recombinant DNA, Scientific American books, 1992
2. M Gagnon, Purification of nucleic acids, BIA separations, Slovenia, 2020
3. DR Latulippe, Biotechnol. Bioeng. 107(2010), 134
4. S Levy, et. al., “Biochemical engineering approaches to the challenges of producing pure plasmid DNA”, TIBTECH, July 2000, Vol 18
5. RW Field, et. al., “Critical flux concept for microfiltration fouling”, J Member Sci, 100(1995)259-72
6. H Lutz, Ultrafiltration for bioprocessing, Woodhead Publishing, Cambridge, UK 2015
7. FJ Meacle, et. al., “Impact of Engineering Flow Conditions on Plasmid DNA Yield and Purity in Chemical Lysis Conditions”, Biotechnol.
Bioeng., 87(2004)
8. MilliporeSigma, Millistak+® Pod Disposable Depth Filter Performance Guide, Lit No. PF1119EN00 Rev. B 2013
9. MilliporeSigma, Depth Filters at a Glance, Lit. No. PB1900EN00, Ver. 3.0, 2017
10. MilliporeSigma, Filter Sizing Methods, Lit. No. AN1512EN00, Rev 3, 2000
11. MilliporeSigma, Plasmid DNA Downstream Process, Lit. No. MS WP7523EN, Ver 0.1, 2021
12. Urthaler et. al., “Improved downstream process for the production of plasmid DNA for gene therapy”, Acta Biochimica Polonica, 2005.
44 Effective and Efficient Design of a Downstream Purification Process for Plasmid DNA - March 2022