Molecular Biology

Move your drug development program forward, faster

Pfēnex Expression Technology™ is a robust Pseudomonas fluorescens-based platform uniquely employing a high-throughput methodology for identification of optimized strains for therapeutic protein and vaccine production. Moreover, it has been used to produce tool proteins as well as biosimilars in a variety of therapeutic areas. Pfēnex Expression Technology has also been used successfully as a high throughput screen for discovery-based applications, particularly in early stages of hit-to-lead applications and novel antigen identification. In total, the technology provides a system by which proteins can be produced with the highest quality and speed, at the lowest time and cost, minimizing CMC issues in product development.

SPEED

The conventional approach to expression strain or cell line development has relied on a few tools available in several different expression systems in an attempt to arrive at a solution producing the protein of interest at sufficiently high titer and quality. This involves significant time, and internal resource and capability costs. Further, the drug developer also incurs significant direct, variable and opportunity costs due to the high rate of expression failures. The overall costs associated with maintaining these capabilities and developing expression strains are simply no longer compatible with the present day need to develop and evaluate protein drug candidates and facilitate market entry as rapidly as possible. The Pfēnex Expression Technology platform enables the identification of an expression strain that produces soluble, active protein, in vivo, within 5 weeks from the initiation of cloning. This is accomplished with a parallel high throughput strain screening process capable of evaluating hundreds of unique expression strains by high throughput analytical processes, enabling the selection of top performing strains with unprecedented speed.

QUALITY

The Pfēnex Expression Technology platform is specifically designed to produce high quality protein. Expressing soluble, active protein in vivo as opposed to protein refolding, as is often the case with proteins expressed with E. coli, is a significant quality advantage. Secretion of proteins to the periplasm of the P. fluorescens cell, while simultaneously over-expressing folding modulators, enables the proper formation of disulfide bonds. Subsequently in order to recover the protein, the use of periplasmic release methods selectively permeabilizes the outer membrane of the cell, while keeping the inner membrane intact resulting in a much purer intermediate. Periplasmic extracts obtained with Pfēnex Expression Technology are of much higher purity than standard, whole cell bacterial lysates thus simplifying downstream purification processes with increased overall yields. A large collection of protease knockout host strains helps to ensure that the expressed protein is not subject to proteolytic clipping or degradation. P. fluorescens will not glycosylate proteins, thus avoiding the costly problem of extensive heterogeneity frequently encountered in yeast expression systems.

COST OF GOODS

Using inputs from customers and CMO's, the range of production rate reduction for proteins derived from Pfēnex Expression Technology is between 41-60% with estimated annual cost savings of between $17MM and $31MM per protein project. This includes E.coli soluble, E.coli inclusion body, Pichia pastoris, and mammalian cell processes, with proteins as diverse as antibody fragments to enzymes to extracellular proteins.

DEVELOPED TO MEET REGULATORY REQUIREMENTS

Pfēnex Expression Technology is based on novel strains of Pseudomonas fluorescens, a non-pathogenic (BSL-1) Gram-negative bacterium. The platform has been developed with regulatory requirements in mind, specifically eliminating the need for antibiotics for plasmid maintenance, while also eliminating animal-derived materials through the use of a fully defined medium. By improving expression and meeting regulatory expectations while reducing costs, we can enable our clients to increase the number of products that are commercially viable and which ultimately go to market. Find out more in our Regulatory Summary

Molecular Biology Capability:

Gene cloning
Gene design
Vector construction
Directed strain improvement
Gene expression
Maintenance of recombinant microbial expression strains

Our scientists work with you to achieve your technical goals.

Cutting-Edge Technologies

We employ cutting-edge technologies on every customer project to speed strain development and process development resulting in robust manufacturing processes:

Bioinformatics

Full range of computational tools for whole genome analysis and manipulation

Genomics

The entire genome of P. fluorescens biovar 1 strain MB214 has been sequenced and annotated
Four other P. fluorescens genomes are publicly available

Genome-Scale Metabolic Modeling

Constraint-based, genome-based mathematical model for P. fluorescens

Functional Genomics

Transcriptomics
Proteomics
Metabolomics

A Deeper Look at Functional Genomics: Enabling Rapid, Directed Strain Process Improvement:

Primary application of transcriptomics

Characterizing genes which are involved with:

Carbon utilization
Protein degradation pathways
Secretion pathways
Protein folding
Disulfide bond formation

Identifying genes which are:

Growth-phase regulated
Constitutively expressed

Primary application of proteomics

Analysis of fermentation experiments

Obtain information about metabolic state of host cells
Determine if desired protein or peptide is produced and in what state
Protein yield and stability
Temporal profile of desired protein production
Signal sequence processing

Protein purification optimization:

Identify co-purifying proteins to drive purification processes
Steer direct genetic engineering to eliminate interfering proteins

Establishing a Host Strain - An Overview

We help you establish a robust host strain for the production of recombinant proteins. Below is an overview of the process:

DNA Design and Synthesis

Develop cloning strategy
Analyze existing gene
Synthesize gene; optimize codons if necessary

Strain Construction

Design and prepare primers
PCR amplify gene and secretion leaders
Subclone PCR product containing gene into expression vector
Perform DNA sequencing
Prepare glycerol stocks

Expression Analysis

Perform fermentations at 96 well scale
Develop high throughput analytical methods
Determine yield and quality of protein
Perform purification and analysis
Transfer recombinant protein or peptide to customer for evaluation

For more information about our molecular biology capabilities, contact us.

	Pfēnex - Molecular Biology
Capabilities Molecular Biology Fermentation Process Development Purification Process Development Analytical Development Pilot Manufacturing Process Technology and Documentation Project Lifecycle Timeline Literature and Resources News /pfenex/news/20070515a.htm /pfenex/news/20070501a.htm /pfenex/news/20070430a.htm /pfenex/news/20070227a.htm /pfenex/news/20070222a.htm /pfenex/news/20060410a.htm /pfenex/news/20060407a.htm /pfenex/news/20060228a.htm /pfenex/news/20060214a.htm Events Calendar Contact Us	Move your drug development program forward, faster Pfēnex Expression Technology™ is a robust Pseudomonas fluorescens-based platform uniquely employing a high-throughput methodology for identification of optimized strains for therapeutic protein and vaccine production. Moreover, it has been used to produce tool proteins as well as biosimilars in a variety of therapeutic areas. Pfēnex Expression Technology has also been used successfully as a high throughput screen for discovery-based applications, particularly in early stages of hit-to-lead applications and novel antigen identification. In total, the technology provides a system by which proteins can be produced with the highest quality and speed, at the lowest time and cost, minimizing CMC issues in product development. SPEED The conventional approach to expression strain or cell line development has relied on a few tools available in several different expression systems in an attempt to arrive at a solution producing the protein of interest at sufficiently high titer and quality. This involves significant time, and internal resource and capability costs. Further, the drug developer also incurs significant direct, variable and opportunity costs due to the high rate of expression failures. The overall costs associated with maintaining these capabilities and developing expression strains are simply no longer compatible with the present day need to develop and evaluate protein drug candidates and facilitate market entry as rapidly as possible. The Pfēnex Expression Technology platform enables the identification of an expression strain that produces soluble, active protein, in vivo, within 5 weeks from the initiation of cloning. This is accomplished with a parallel high throughput strain screening process capable of evaluating hundreds of unique expression strains by high throughput analytical processes, enabling the selection of top performing strains with unprecedented speed. QUALITY The Pfēnex Expression Technology platform is specifically designed to produce high quality protein. Expressing soluble, active protein in vivo as opposed to protein refolding, as is often the case with proteins expressed with E. coli, is a significant quality advantage. Secretion of proteins to the periplasm of the P. fluorescens cell, while simultaneously over-expressing folding modulators, enables the proper formation of disulfide bonds. Subsequently in order to recover the protein, the use of periplasmic release methods selectively permeabilizes the outer membrane of the cell, while keeping the inner membrane intact resulting in a much purer intermediate. Periplasmic extracts obtained with Pfēnex Expression Technology are of much higher purity than standard, whole cell bacterial lysates thus simplifying downstream purification processes with increased overall yields. A large collection of protease knockout host strains helps to ensure that the expressed protein is not subject to proteolytic clipping or degradation. P. fluorescens will not glycosylate proteins, thus avoiding the costly problem of extensive heterogeneity frequently encountered in yeast expression systems. COST OF GOODS Using inputs from customers and CMO's, the range of production rate reduction for proteins derived from Pfēnex Expression Technology is between 41-60% with estimated annual cost savings of between $17MM and $31MM per protein project. This includes E.coli soluble, E.coli inclusion body, Pichia pastoris, and mammalian cell processes, with proteins as diverse as antibody fragments to enzymes to extracellular proteins. DEVELOPED TO MEET REGULATORY REQUIREMENTS Pfēnex Expression Technology is based on novel strains of Pseudomonas fluorescens, a non-pathogenic (BSL-1) Gram-negative bacterium. The platform has been developed with regulatory requirements in mind, specifically eliminating the need for antibiotics for plasmid maintenance, while also eliminating animal-derived materials through the use of a fully defined medium. By improving expression and meeting regulatory expectations while reducing costs, we can enable our clients to increase the number of products that are commercially viable and which ultimately go to market. Find out more in our Regulatory Summary Molecular Biology Capability: Gene cloning Gene design Vector construction Directed strain improvement Gene expression Maintenance of recombinant microbial expression strains Our scientists work with you to achieve your technical goals. Cutting-Edge Technologies We employ cutting-edge technologies on every customer project to speed strain development and process development resulting in robust manufacturing processes: Bioinformatics Full range of computational tools for whole genome analysis and manipulation Genomics The entire genome of P. fluorescens biovar 1 strain MB214 has been sequenced and annotated Four other P. fluorescens genomes are publicly available Genome-Scale Metabolic Modeling Constraint-based, genome-based mathematical model for P. fluorescens Functional Genomics Transcriptomics Proteomics Metabolomics A Deeper Look at Functional Genomics: Enabling Rapid, Directed Strain Process Improvement: Primary application of transcriptomics Characterizing genes which are involved with: Carbon utilization Protein degradation pathways Secretion pathways Protein folding Disulfide bond formation Identifying genes which are: Growth-phase regulated Constitutively expressed Primary application of proteomics Analysis of fermentation experiments Obtain information about metabolic state of host cells Determine if desired protein or peptide is produced and in what state Protein yield and stability Temporal profile of desired protein production Signal sequence processing Protein purification optimization: Identify co-purifying proteins to drive purification processes Steer direct genetic engineering to eliminate interfering proteins Establishing a Host Strain - An Overview We help you establish a robust host strain for the production of recombinant proteins. Below is an overview of the process: DNA Design and Synthesis Develop cloning strategy Analyze existing gene Synthesize gene; optimize codons if necessary Strain Construction Design and prepare primers PCR amplify gene and secretion leaders Subclone PCR product containing gene into expression vector Perform DNA sequencing Prepare glycerol stocks Expression Analysis Perform fermentations at 96 well scale Develop high throughput analytical methods Determine yield and quality of protein Perform purification and analysis Transfer recombinant protein or peptide to customer for evaluation For more information about our molecular biology capabilities, contact us.
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Pfēnex - Molecular Biology