qPCR assay Design & Validation for Human Genome


Summary

Customer
US based manufacturer of high throughput gene expression platforms for biomarker discovery & validation

Background
The customer required validated assay data to be ported on to their platform prior to a beta-launch of their equipment at a prestigious medical research institute. They required SYBRGreen qPCR primers designed (with in vitro quality checks) for all the genes in the human genome to be provided in specific gene panels based on pre-defined taxonomies & ontology

Challenge
The project required rapid design of qPCR primers (SYBR Green) for the entire human genome based on stringent platform specific parameters . The designed primers had to be classified based on the loci/ genes as per gene ontology/ KEGG databases and phylogenic / functional families.

Solution
Polyclone shortlisted 18K human genes using a clustering approach on the human genome and designed qPCR SYBRGreen primers based on stringent criteria provided by the customer and developed scripts to classify the designed primers into the specific gene panels provided.




Polyclone Case study 1 - Custom Assay Design and Validation Flow Chart



Benefits to Customer


High-throughput design
Primer design and validation for entire human genome in record time
Custom design benefits
Stringent design for custom and non-standard conditions including AA’s at 3’ end and Intron spanning primers
In vitro validation
First level of in vitro validation to support the in silico validation of primers

Design and Validation of HPV Genotyping Assay


Summary

Customer
Molecular diagnostics division of global healthcare solutions giant based in the Netherlands

Background
Customer required an HPV genotyping assay for the detection & classification of fatal strains of HPV, to be designed specifically for the Indian population and validated on patient samples

Challenge
A single qPCR assay had to be designed in such a way that it could detect 2 fatal strains of HPV(quantitatively) and indicate the presence of other common 18 Indian HPV strains. Further the assay had to be validated on patient samples.

Solution
Polyclone identified unique regions in the genomes of the 20 HPV strains and designed primers & probes to be able to detect & quantify specific strains. The assay was validated on synthetically spiked control samples followed by validation on 25 samples procured from patients using qPCR technology.







Case Study 2 - Design and validation of HPV genotyping assay - Image 2

Case Study 2 - Design and validation of HPV genotyping assay - Image 3

Case Study 2 - Design and validation of HPV genotyping assay - Image 4



Benefits to Customer


Turnkey solution
Complete solution involving in silico design & data analysis, patient sample sourcing and in vitro qPCR validation
Patient samples
Procurement of patient samples and data with ethical approvals within specified timelines
In vitro validation
In vitro validation of the assay using patient samples and data
Cost and time savings
Customer was able to obtain lab validated results with considerable cost and time savings

In silico Enzyme Engineering for Protease Activity Enhancement


Summary

Customer
Indian protein based drug discovery Company

Background
The customer required the structure of a human protease enzyme to be modeled and its active site refined to increase its activity by 2 logs

Challenge
The project required modeling of the enzyme based on sequence homology, spot catalytic triad and biologically relevant processes for in silico screening to identify suitable modifications & docking analysis using ligands like trypsinogen, fusion protein, substrates & inhibitor.

Solution
Polyclone performed in silico structure prediction, structural modeling, conformational sampling and flexible docking. Polyclone’s proprietary algorithms were used in the screening process and scoring functions were used to predict the increase in the activity of the enzyme

Case Study 1 - In silico enzyme engineering to enhance the activity of a protease enzyme - Workflow






Case Study 1 - In silico enzyme engineering to enhance the activity of a protease enzyme - Image 1

Case Study 1 - In silico enzyme engineering to enhance the activity of a protease enzyme - Image 2



Benefits to Customer


Hybridized theoretical modeling approaches
Combination of homology modeling, threading and ab initio methods to predict canonical and non canonical regions of enzyme
In house designed simulation processes
Complexity of the conformational space during an enzymatic reaction is addressed through in silico process which predicts the near-native state of enzyme-substrate complex
Customized scoring functions
Polyclone’s customized scoring functions provided conclusive results for ranking the docking studies as compared to contemporary tools
Cost Savings
Customer was able to obtain in silico validated results with an estimated cost and time savings of close to 50% !

In silico Modeling for Antibody Humanization


Summary

Customer
A German drug discovery company

Background
The customer wanted to conduct in silico modeling studies for antibody humanization on a preferred human Vh framework and epitope sequence , without affecting stability by structure prediction, which they wanted to further validate in vitro.

Challenge
The project required modeling studies to determine binding affinity of ligands & receptors using human Vh framework, to graft CDR sequences for anti-TNF- α Ab and Change amino acid residues in CDR and/or framework & check for increased binding to TNF- α

Solution
Polyclone performed modeling, energy minimization & validation of the resultant structure, local docking of the antigen sites with single domain Abs for all the Vh frameworks and epitope residues to determine binding affinity b/n specified Ag-Ab using energy values

















Case Study 4 - In silico modeling and docking analysis for antibody humanization studies - Image 1

Case Study 4 - In silico modeling and docking analysis for antibody humanization studies - Image 2



Benefits to Customer


In silico extended team
Customer was able to get in silico insights into humanizing their antibody – this was a non-core skill set that they didn’t possess, that Polyclone was able to complement
Quick turn-around time with statistical confidence
Hyrdropathy and structural superimposition ‘filters’ to keep number of docking studies to an optimal number without affecting statistical significance of the results
Reliable alternative
Provided a reliable in silico alternative to the patented in vitro approaches that were expensive and time consuming

Cell based Studies for Comparison of Transfection Reagents


Summary

Customer
Belgium based biologics company focused on solutions for basic research in genomics and proteomics

Background
Customer had developed 3 novel transfection reagents and required to benchmark them against the industry standard, Lipofectamine, to help in go-to-market decision and marketing communications

Challenge
The project required analysis to be performed by an external and independent team, with the expertise to work on challenging human cell-lines and molecular biology techniques

Solution
Polyclone performed toxicity and transfection assays to compare the customer’s reagents and lipofectamine on 4 human cell lines. The studies established that one of the customer’s reagents was superior to the industry standard

Polyclone Case study 5 - Cell based studies for comparison of transfection reagents - Image 1




Polyclone Case study 5 - Cell based studies for comparison of transfection reagents - Image 2

Polyclone Case study 5 - Cell based studies for comparison of transfection reagents - Image 3



Benefits to Customer


Independent confirmation
One of the customer’s reagents was shown to be more effective than Lipofectamine, providing the customer with an independent confirmation of their product’s efficacy.
Lesser time to market
The data helped the customer take the product to market much earlier than planned
Scientific data to back marketing
The analysis and data provided, enabled the customer to back their marketing claims of the product over Lipofectamine