BioMAP® Frequently Asked Questions (FAQ)

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BioMAP Platform

What is BioMAP profiling?

 

BioMAP profiling is in vitro and high throughput characterization of the clinically relevant biological effects of test agents (small molecules, biologics, etc.) on a broad range of human disease models.    

BioMAP Systems are constructed with one or more primary cell types from healthy human donors, with stimuli (such as cytokines or growth factors) added to capture defined aspects of human tissue or disease states.

BioMAP endpoints (biomarker readouts) measured in BioMAP systems are cell surface or secreted proteins selected for therapeutic and biological relevance and are detected by immuno-based techniques.

For a given test agent, the cumulative changes in biomarker readouts (above or below baseline) across a panel of BioMAP systems make up a BioMAP profile.  BioMAP Profiles are highly reproducible and can distinguish features of compounds, including target selectivity, compound liabilities and mechanisms of action (MOA).

BioMAP systems are highly validated with well-characterized drugs and pharmacological agents to ensure that BioMAP systems show responses that closely mimic what is seen in the clinic and published literature.  Over 4,000 bioactive agents have been tested at multiple concentrations and recorded using these systems in order to generate the BioMAP Reference Database, which provides the bioinformatic power behind the platform.  In house developed software is used to mathematically compare profiles of newly run test agents to those present in the database.

Comparing the BioMAP signature of a test agent to a proprietary reference database of > 4,000 BioMAP profiles of bioactive agents (e.g., drugs, biologics, chemicals, etc.) can enable drug discovery and development in a human disease-relevant manner. 

Characterization of test agents in BioMAP profiles can be used to:

  (1) correlate drug effects to in vivo biology
  (2) distinguish between multiple compounds based on MOA and target selectivity
  (3) identify potential biomarkers for in vivo studies
  (4) provide a predictive signature for in vivo toxicities
  (5) benchmark against current standards of care
  (6) test drug combinations  
 
Feature Benefit
Primary human cell-based assays Assays provide human translation for discovery programs with readouts that are clinically relevant
Standardized panels Compounds can be compared across programs over time in the same standard assay panel
Disease-relevant culture conditions Assay results are predictive of in vivo results
Large reference drug database Assay systems are validated with multiple compounds from the same target class, or for the same clinical indication, etc.  These compounds are a portion of the large BioMAP database library of agents available for analytical comparison against client compounds 
Predictive algorithms for relevant efficacy and safety related mechanisms The platform can mathematically provide identification of drug and toxicity mechanisms of action using an unbiased approach
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What is a BioMAP Platform?

 

The BioMAP platform is a cell-based compound profiling technology with 3 integrated components:

  • BioMAP Systems - primary human cell-based tissue and disease models
  • BioMAP Reference Database – BioMAP profiles of > 4,000 of bioactive agents
  • Custom Analytics - data mining and informatics-based algorithms are used in combination with a team of experts to interpret the unique profile signature of a compound (assigning mechanism class, predicting toxicities, discovering similarities or differences between other compounds, etc.).
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What are the key advantages of BioMAP in modeling human disease biology?

 

BioMAP can inform preclinical drug development in several ways:

Physiological advantages

  • Physiologically relevant to human disease – primary cells are used at early passage (passage 4 or earlier) to minimize adaptation to cell culture conditions and preserve physiological signaling responses. Cells are pooled from multiple human donors for improved reproducibility and to capture population responses.
  • Captures biological complexity – systems use either single cell types or co-culture systems and are treated with stimulatory factors designed to mimic tissue biology and signaling pathways that occur during different physiological states in the body under standardized methods.
  • Phenotypic outcome – systems are designed to determine biological impact of test agents rather than the early or intermediate events. Detection of biological impact allows these systems to be a better predictor of in vivo effects.
  • Expert analysis – the profiles of test agents are annotated with respect to therapeutic indication to facilitate preclinical interpretation and designed to predict and evaluate the in vivo safety of test agents in a clinically relevant manner.
 

Technical advantages

  • Quantitative and reproducible – system development, data analysis and data production are done using standard operating procedures (SOP) and using standardized biomarker readouts (e.g., protein and other cellular measurements).
  • Validated platform – all systems, readouts and data analysis are verified for pharmacological relevance using known drugs and well characterized experimental compounds.
  • Captures broad disease biology50 plus validated systems that span scenarios relevant to a range of human diseases.
  • High-through put capacity – Platform is designed to handle large screening campaigns of up to 30,000 compounds, while still offering a rapid turn-around. In previous custom projects, we have run single assay/endpoint screens for small libraries of ~ 5,000 compounds.
  • Statistical prediction – With a database of over 4,000 compounds as a basis of comparison, benchmarking and comparative analysis of test agents to known compounds allows for power predictions of mechanisms of action, safety and efficacy.
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What are the readouts measured in the BioMAP platform?

 

The majority of BioMAP readouts are human protein biomarkers that are known to be predictive for disease outcomes or specific drug effects. Biomarkers are measured quantitatively by immune-based methods measuring protein (e.g., ELISA) or functional assays, such as proliferation. BioMAP readouts are diverse and include cell surface receptors, cytokines, chemokines, matrix molecules and enzymes, among others. All of the biomarker readouts (soluble and cell-based) have been selected for disease and therapeutic relevance and are analytically validated for reproducibility with well-characterized therapeutic agents. Overt adverse effects of test agents on cell proliferation and viability (cytotoxicity) are measured by sulforhodamine B (SRB) for adherent cells, or alamarBlue® staining for cells in suspension.

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How is proliferation and cytotoxicity measured in BioMAP systems?

 

End points for proliferation and cytotoxicity have been optimized for each BioMAP system and are included in each run. Cell proliferation is measured by sulforhodamine B (SRB) for adherent cell types, or alamarBlue® staining for non-adherent cells (i.e., PBMCs). SRB determines cell density by measuring the total protein content of test wells and is based on the ability of the SRB dye to bind to amino acid residues. This provides a quantifiable colorimetric end point. alamarBlue® measures cell viability based on the cell permeable compound resazurin, which is reduced into fluorescence-emitting Resorufin after entering cells. The number of living cells is proportional to the amount of fluorescence produced. Proliferation is measured in cells plated at a lower density.

In the Diversity PLUS panel (made up of 12 systems), cells are treated with the compound 48 – 96 hours prior to proliferation measurements, and 24 - 48 hours prior to cytotoxicity measurements depending on the cell type. Cytotoxicity is measured on confluent plates of cells and occurs when total protein levels decrease by more than 50% relative to vehicle controls (log10 ratio of SRB or alamarBlue levels < -0.3) and are indicated by a thin black arrow above the X-axis. A compound is considered to have broad cytotoxicity when cytotoxicity is detected in 3 or more systems. Anti-proliferative effects occur with about a 20% decrease in total protein relative to vehicle controls (log10 ratio of SRB or alamarBlue value < -0.1) from cells plated at a lower density (SUBCONFLUENT) and are indicated by grey arrows above the X-axis. The vehicle control on each plate will proliferate over the course of the assay. If the test agent has anti-proliferative (CYTOSTATIC) effects, the cells will not grow and delta between the vehicle controls will cause a decrease in the ratio. Please note a compound can be cytostatic but not cytotoxic (i.e. inhibits proliferation but does not directly kill cells) or a compound can be overtly cytotoxic but not cytostatic (i.e. kills cells but does not inhibit proliferation).

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BioMAP Services

What are the standardized BioMAP services available for profiling compounds?

 

In addition to over 50 individual BioMAP systems, we have compiled the following panels to cover broad and focused therapeutic areas.

 
Standard Panels & Services
# Systems
# Readouts
# Sample Replicates
Profile
Benchmark (optional)
Similarity  Search
Mechanism HeatMAP
Mechanism Classification
Tox Alert Analysis
Cluster (≥ 3  test agents)
Combination  Analysis
Checkerboard  Summary
T cell  Comparative  Analysis
Diversity PLUS 12 148 1 X X X X     X      
T Cell Autoimmune 4 51 3 X X         X     X
Oncology Panel – Colorectal 2 41 3 X X                
Oncology Panel – NSCLC 2 42 3 X X                
Fibrosis Panel 3 54 3 X X                
Combo ELECT Any Vary 3 X             X X  
Mechanistic Tox Alert Extra data analysis X X X   X X        
Overlay ELECT Extra data analysis   X                
Data Package Extra data analysis X                  
 

Diversity PLUS – Panel made up of 12 systems that cover a broad range of disease biology that are therapeutically relevant for agent characterization. This panel is most effectively aimed at compounds in discovery, entering preclinical testing or compounds with in vivo data from animal studies or the clinic.

Key Report Features:

  • Profile Analysis of 4 concentrations per test agent run in 12 systems in singlicate
  • Benchmark Analysis against any single compound in the diverse BioMAP database
  • Similarity Analysis and mathematical comparison against compounds in the database
  • Mechanism HeatMAP Analysis of each agent against 19 mechanism class profiles
  • Cluster Analysis (cluster analysis only for 3 or more client test agents)
 

T cell Autoimmune Panel – Models the adaptive immune cell microenvironment as well as T and B cell responses in a platform amenable to compound screening.

Key Report Features:

  • Profile Analysis of 4 concentrations per test agent run in 4 systems in triplicate
  • Benchmark Analysis against 6 autoimmune standards of care
  • Cluster Analysis of project agents with 6 standards of care
  • T Cell Comparative Analysis of each agent vs. 6 standards of care
 

Oncology Panels – Colorectal or NSCLC – Systems are stimulated to model tumor-immune-stromal, immune-stromal, tumor-immune-vascular or immune-vascular microenvironments and disease relevant signaling pathways that manifest during tumorigenesis.

Key Report Features:

  • Profile Analysis of 4 concentrations per test agent run in 2 systems in triplicate
  • Benchmark Analysis
 

Fibrosis Panel – Models fibrotic disease biology in two different organs (kidney, lung), under inflammatory and pro-fibrotic conditions.

Key Report Features:

  • Profile Analysis of 4 concentrations per test agent run in 3 systems in triplicate
  • Benchmark Analysis
 

For the most up-to-date list of available BioMAP systems, please click here.

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What are the BioMAP key deliverables?


Definition of BioMAP deliverables:
  • Profile analysis – annotation of key activities with respect to biological relevance
  • Benchmark analysis – Head to head comparison of the top non-cytotoxic concentration of a test compound with a specified reference benchmark.
  • Similarity search analysis – Results from an unsupervised search for biologically similar agents from the BioMAP reference database of > 4,000 agents including biologics, approved drugs and experimental chemical compounds (Diversity PLUS Only)
  • Cluster analysis – Overall comparison of compounds using Pairwise correlation analysis and clustering of most similar profiles (Diversity PLUS and T Cell Panel Only).
  • Mechanism HeatMAP Analysis – The biomarkers of each test agent are compared in a heatmap format to the BioMAP profiles of 19 consensus mechanism class profiles of well-characterized drugs (Diversity PLUS Only).
  • T Cell Comparative Analysis – Test agents and 6 standard-of-care compounds are compared across categories of biomarker readouts representing different biological processes.  Agents are assigned a score for each category that accounts for both the number of annotated biomarker readouts across categories, and the amplitude of this activity compared to the historical range (T Cell Panel Only).
  • Combination Analysis – Agents are evaluated for biomarker activity individually, and then also evaluated against the combination of these agents (Combo ELECT Only).
  • Checkerboard Analysis – Summary of the number of biomarker activities in the combination for each concentration pair that are significantly different than the corresponding concentration of individual test agents (Combo ELECT Only).
  • EC50 Analysis – Measure of the potency of a drug for an on-target effect.  Determines the concentration of agent that induces a response that is halfway between the baseline and maximum (EC50 Custom Projects Only).
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Applications – What can I do with BioMAP?

What can Diversity PLUS do for me?


 The Diversity PLUS panel is a valuable resource for:
  • Compound validation in human cells – panel allows for the verification of results of recombinant cell-based assays and animal models in a human primary cell-based system.  Identifies positive activities in human cells that are consistent with the target. 
  • Compound prioritization – program leaders often have to select one or more candidate compounds from a larger library to move forward in development.  Diversity PLUS can help prioritize compounds by identifying the positive and negative attributes between different agents. 
  • Identification of unexpected activities – unexpected off-target activities can be identified by Diversity PLUS profiling due to the breadth of biology that is covered by this panel. 
  • Investigative toxicology – When preclinical compounds induce unanticipated animal toxicities, Diversity PLUS can be used to investigate the potential mechanisms of toxicity, as well as help identify an assay (i.e., system and sentinel biomarker activity) that can be used to counter-screen back up compounds.  A Mechanistic Tox Data Analysis is available as part of an add-on option to a Diversity PLUS panel.
  • Competitor assessment – clients are often working on the same target as their competitors and would like to know how their compounds “stack-up” to their competitor molecules.
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Is cellular screening a suitable approach for chemists and their requirements for candidate lead optimization?


BioMAP is a platform that can be used for phenotypic lead optimization when there is in an interest in a biological approach.   Importantly, BioMAP has the capacity to yield insights into potency, selectivity and toxicity of compounds.  The broad BioMAP profiling achieved in Diversity PLUS can determine selectivity and cytotoxicity, while more focused panels like the T cell Autoimmune, Oncology, Fibrosis and Combo ELECT service offerings can be used for a deep dive into a particular area.  For larger, more custom studies, a cellular EC50 in key assays can be used to measure potency.  For example, a typical “HTS project” for screening a focused library of about 8,000 compounds would involve:
  • Preliminary broad BioMAP profiling in Diversity PLUS of a subset of the library (~ 30 - 40 compounds) to first determine:
    1. Expected hit rate
    2. % of cytotoxic compounds
    3. nomination of the best BioMAP assay/readouts for HTS
  • Screen a compound library (~8,000 compounds) using a single BioMAP readout.
  • Conduct hit confirmation
  • Broad BioMAP profiling of lead compounds of interest in Diversity PLUS
    1. Statistical classification of hits into mechanistic and functional classes
    2. Ranking of compound performance based on the potency, efficacy and safety.

Recommendations of the most promising compound class(es) for lead optimization will be given based on a biological perspective that incorporates the therapeutic goals of the project.
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Is BioMAP applicable towards biomarker discovery and validation?

 

Yes, BioMAP is an excellent platform for biomarker discovery and validation. The BioMAP systems are designed to quantitatively measure the protein expression of therapeutically relevant factors in the absence and presence of a drug of interest. This platform is set up to screen, identify and characterize potential biomarkers suitable for pharmacodynamic measurements and/or patient stratification in clinical trials.

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What if gene expression of my target is not detectable in any of the BioMAP systems?

 

While gene expression may be below the level of detection, protein expression of the target may be present. For targets with undetectable gene expression and the absence of protein expression, BioMAP is not suitable for on-target biology, but is appropriate for the investigation of secondary activities and safety.

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How can BioMAP help me understand the mechanism of action of my compound?

 

Our Diversity PLUS panel has 12 systems that cover a broad range of biology. The Diversity PLUS deliverables include a mathematical comparison between the biomarker signature of a test agent against > 4,000 BioMAP profiles of bioactive agents (biologics, approved drugs, chemicals and experimental agents) present within the BioMAP reference database, as well as a direct comparison with a client-selected benchmark from our database. Benchmark comparison and the results of the unsupervised similarity search against the BioMAP database are both helpful in identifying compound targets and mechanisms of action.

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How can BioMAP help me understand the safety of my compound?

 

BioMAP can help connect drug induced biomarker changes in disease models to clinical outcomes. BioMAP panels cover a variety of different mechanisms associated with specific human toxicity outcomes including mitochondrial inhibition, proteasome inhibition, microtubule destabilization, and others. Overt cytotoxicity to human primary cell types is associated with risk of acute toxicity. Activities shared with drugs that cause organ specific toxicity by 10 particular mechanisms have been validated as potential toxicity alerts and are available as part of the Mechanistic Tox Alert service, including acute toxicity, hepatotoxicity, skin sensitization, immunosuppression, skin irritation, thrombosis related side effects and skin rash. While this analysis can benefit many different scenarios, BioMAP profiling is particularly useful when there is a specific toxicity finding identified and there is a need to understand the mechanism of this toxicity. Note: assessment of toxicity potential is not included in the standard Diversity PLUS panel analysis, but is included as an add-on service.

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How long does it take typically to complete Diversity PLUS?

 

Typically the time frame for report delivery for Diversity PLUS is 30 business days from receipt of compounds and required paperwork, and 40 business days for other panels.

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New Offerings – Tell me more

What are new service offerings I can expect in 2016?

 

There are several new service offerings coming soon that are briefly described below:

  • New report format for Diversity PLUS – includes a more in depth description of the systems, biomarkers, methods and analysis.  This report format also includes a new deliverable that is a Mechanism HeatMAP Analysis where the biomarkers of each test agent are compared in a heatmap format to the BioMAP profiles of 19 consensus mechanism class profiles of well-characterized drugs.
  • Mechanistic Tox Alert Analysis – Add-on analysis service for Diversity PLUS profiling that includes computational analysis predicting mechanisms of action (over 28 pathways), as well as identification of activities (potential alerts) associated with specific human clinical outcomes (acute toxicity, hepatotoxicity, skin sensitization, immunosuppression, skin irritation, thrombosis related side effects and skin rash).
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Scalability/Reproducibility

How is the quality of the human primary cells used in BioMAP monitored?

 

BioSeek has extensive quality-control procedures to carefully monitor the quality of the primary cells used in BioMAP.  Specifically, cells are:

  • Sourced from qualified vendors
  • Limited to early passage (≤ P4)
  • Evaluated for phenotypically-appropriate marker expression
  • Assessed for viability and morphology in each experiment
  • Derived from multiple donors – cells are pooled from different donors to reduce the potential donor to donor variation.
  • All experiments include controls for expected biological responses and tested cells must fall within pre-determined assay acceptance criteria (i.e., specific requirements for CVs, performance similar to historical data, positive and negative controls, etc.).
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What is your assay acceptance criteria?

 

In standard BioMAP panels, stringent assay acceptance criteria are used in profiling projects that are based on both historical as well as assay specific performance. The BioMAP platform generates multi-parameter data sets for each sample tested and uses multiple controls within and across assays. The assays are plate based, and their performance is assessed by two positive controls (non-stimulated condition and vehicle control; stimulated condition and active test agent) and a negative control (stimulated condition and vehicle control) included on each individual plate. The data acceptance criteria for BioMAP panels requires consistent overall assays, as measured by plate performance (%CV of negative controls) and performance of positive controls compared to historical controls.

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What is the reproducibility of your platform?

 

We, as well as external collaborators, have published studies demonstrating the reproducibility of the BioMAP platform. BioSeek has been a contractor to the US Environmental Protection Agency (EPA) since 2007, and several blinded studies evaluating the reproducibility of BioMAP data have been executed and full results published in high impact peer-reviewed journals. Materials and methods for BioMAP systems and profiling have been previously described.

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Can BioMAP be used to compare 1,000’s of candidates against a single readout (HTS)?

 

Yes, the BioMAP can used to screen large numbers of candidates against a single readout. The BioMAP platform can screen libraries of up to 30,000 compounds.

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Can you use disease-specific patient cells in the BioMAP format?

 

Yes we can, however due to the complexity of the BioMAP platform we can only currently accommodate large custom projects. Individual patients are exposed to many different drugs and their responses are highly variable, requiring a large number of patients to be needed (20 – 30 donors). Due to the time and expense (> $ 100,000) involved in sourcing patient samples, these studies are performed for clinical development groups and not at the research stage.

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Biological Considerations and Applications

Why are compounds added before the stimulation of disease-related pathways?

 

Our approach calls for first exposing BioMAP systems to drug modulation (inhibition or activation), followed by stimulation with disease activating factors. We believe this maximizes the magnitude of readouts and reveals how a drug affects underlying human disease biological processes. Reversing these steps may not necessarily achieve this. For example, a small molecule kinase inhibitor binding to an already activated (e.g., phosphorylated) receptor tyrosine kinase may not yield as robust of a BioMAP response within the timeframe of the assay (typically 24-48 hours).

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How long do we pre-incubate with the test agent before stimulation?

 

Test agent pre-incubation for all systems and panels is one hour before stimulation.

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How long are cells treated with compound?

 

Treatment with compound depends on the system. Diversity PLUS systems are treated with compound for the duration of the assay with the exception of a few readouts. In most systems this duration is 24-hrs, but in the MyoF system it is 48-hrs and in the BT system it is 3 days for cytokine measurements and 6 days for the IgG measurement.

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Have you ever profiled natural compounds in BioMAP?

 

We have profiled numerous natural product compounds that were broadly active in BioMAP systems including oils, conditioned media and complex biological samples including plant, vegetable and herbal extracts.

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Is the source of serum used in BioMAP assays human or bovine?

 

Fetal bovine serum (FBS) is the source of serum used in BioMAP assays. For example, all of the BioMAP systems in Diversity PLUS contain either fetal bovine serum (FBS) or an enriched bovine protein supplement. Most of the systems contain varying levels of FBS ranging from 0.1% - 5%, with only one system having a maximum level of 10% (BT system). Other systems (BE3C and BF4T systems) do not contain any FBS, but do have an enriched bovine protein supplement.

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What disease biology is covered by BioMAP?

 

Therapeutic applications include

  • Autoimmune disease
  • Respiratory disease
  • Dermatological disease
  • Cancer
  • Cardiovascular disease (inflammation and atherosclerosis)
  • Safety profiling
  • Fibrosis
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How can BioMAP help drugs targeting neuronal cells?

 

The BioMAP platform covers the biology of multiple sclerosis (MS) and toxicity screening which are strengths of BioMAP in the CNS space. Our expertise is in inflammation, autoimmunity and tissue remodeling and BioMAP systems can be used to inform on underlying mechanisms that are universal to all cell types regardless of the organ. For example, toxicity is not necessarily organ specific and toxicity that manifests within a particular organ does not mean that is the only organ affected. Tubulin inhibitors show a neurotoxic side effect – tubulin biology is extensively covered in our systems. While we do not model/cover neuron-based BioMAP systems, we do cover related disease biology. Examples of this are that MS-related inflammation biology is covered in Diversity PLUS and the T Cell Autoimmune panel. Likewise, the lMphg system models microglial-type biology since microglia are brain macrophages. We additionally have a case study for GI side effects of neurological compounds.

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What immune cells are present in PBMCs?

 

After peripheral whole blood has been drawn for human studies, fractionation methods separate out populations of immune cells called the peripheral blood mononuclear cells (PBMC). PBMCs include lymphocytes (T cells, B cells, and NK cells) (70 – 90%), monocytes (10 – 30%), and dendritic cells (1 – 2%), with population frequencies varying across individuals. The lymphocyte population ranges from 70 – 85% CD3+ T cells (45 – 70% of PBMC; CD4+ and CD8+ T cells in a roughly 2:1 ratio), 5 – 20% B cells (up to 15% of PBMC), and 5 – 20% NK cells (up to 15% of PBMC). Erythrocytes (red blood cells) and polymorphonuclear cells (PMNs), which include neutrophils and eosinophils, are generally removed during this fractionation due to larger densities, but basophils may be present to a small degree.

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What are technical considerations of using BioMAP?

What limitations exist around the type of agents that can be profiled by BioMAP?

 

BioMAP systems model the complex biology of human disease. Factors that often limit the use of compounds in animal models may also be limited in BioMAP systems. These include:

  • Stability – compounds (including natural peptides) that are not stable in serum or in cell line cultures may also require significantly larger concentrations to see biological effects in BioMAP systems relative to in vitro experiments. 
  • Solubility – compounds that are not soluble at room temperature for prolonged periods of time may precipitate out of solution.
  • Selectivity/specificity – appropriate dose selection is ESSENTIAL to generating informative results when using BioMAP systems.  Based on our experience, many drugs will generate off-target responses in BioMAP systems at sufficiently high doses.  It is important for clients to test a range of concentrations and benchmark against compounds with known in vivo activity.  Importantly, modified compounds that may have been useful in other HTS platforms (such as biotinylated molecules) may also generate non-specific activity in BioMAP systems. 
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Are there special considerations when profiling biologics?

 

Yes, there are a few things to keep in mind when profiling biologics. BioMAP systems are perfectly suited for profiling biologics, including recombinant proteins, human/humanized antibodies, rabbit and goat antibodies. However, BioMAP systems are absolutely NOT suitable for testing mouse or rat monoclonal antibodies because many of the BioMAP readouts rely on mouse monoclonal antibody-based assays. Additionally we have seen interference with some human-mouse chimeric antibodies such as Rituxan. Of note, we are able to differentiate different forms of antibodies with variances in their FC regions.

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How much endotoxin can be present in a biological preparation?

 

Biological preparations should contain very low (less than 1 EU/mL < 0.1 ng/mL) or no endotoxin. BioMAP systems are highly sensitive to endotoxin contamination and this contamination can also induce signaling associated with TLR-activation. One EU equals approximately 0.1 to 0.2 ng endotoxin/mL of solution. The 100x stock should be 0.1 ng of endotoxin/mL with the 1x test well ideally being 1 pg/mL.

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Can viruses or bacteria be profiled in BioMAP systems?

 

We do not allow testing of replication competent viruses or bacteria in our facility to avoid potential cross-contamination by infectious particles. Testing of infectious viruses or bacteria must be certified as replication INCOMPETENT (heat killed or inactivated) in all cells in BioMAP systems and not only in select cell types such as proliferating cancer or immune cells.

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Does the grade of my compound matter?

 

Yes, BioMAP is able to discern differences between compounds purchased from a commercial vendor and those that are clinical grade. We recommend testing the grade and formulation that is most appropriate for your studies.

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How do I select an appropriate concentration of my agent to test?

 

The selection of an appropriate concentration range for BioMAP systems is more related to an animal study than an in vitro biochemical study.  For a standard profiling run in singlicate in the Diversity PLUS panel, we test an agent at 4 concentrations at a 3-fold dilution.  This regime has been optimized to provide informative changes in biomarker activity from singlicate data.  BioMAP systems are stimulated (conditions where target levels or activations states are amplified) and often require higher concentrations than cell line-based assays to impact the target-related biology.

  • Observed Cmax concentrations in animal preclinical models or in clinical testing can be used to guide the selection of the top concentration to test. 
  • For early stage drug leads in which no animal Cmax data exists, we suggest a top concentration of 100 to 1000 fold OVER the biochemical IC50.  For example, kinase inhibitors with in vitro IC50’s in the low nanomolar range would be tested at a top dose in the low micromolar range. 
  • For large biologics such as antibodies, we suggest 100x above the ligand concentration (most are in the pM – nM range).  Typically, starting concentrations for biologics are around 10 -30 µg/mL.
  • For peptides, we suggest testing at a top concentration of 10 µM.  This is because peptides are more susceptible to proteolytic cleavage in biological systems.
  • For safety applications, compounds should be tested at 10 – 100 fold above the expected maximal exposure level (Cmax) to assess the compound well above the concentration that might be achieved in vivo.  This is because exposures in humans are highly variable ranging 5 - 10 fold between individual patients.
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How do I select my Benchmark?

 

Benchmark selection is most often based on a competitor agent or the target of the test agent. However, a variety of factors may influence the final identification of the proper benchmark. There are lists of curated and recommended benchmarks for our BioMAP panels which include:

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How do I prepare my samples to be submitted for a BioMAP panel?

 

Information for compound preparation can be found here

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What is the buffer compatibility in a BioMAP system?

 

Considerations depending on the compound for standard BioMAP profiling are as follows:

  • Small molecules – DMSO is the recommended buffer
  • Biologics – PBS is the recommended buffer
  • Compounds in other buffers – this should be discussed with our technical support.  Typically we recommend the buffer be run as a separate sample to test if the buffer has activity.  This is especially important for recombinant proteins that are purified by column elution, etc.
  • A few buffer components are known to interfere with BioMAP systems:
    1. Buffers should NOT contain any methanol or Tween
    2. Buffers should contain NO MORE than
      1. 0.9% Ethanol
      2. 0.1 % DMSO
      3. 10 nM EDTA
      4. 0.1% glycerol
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Can I run my compound in a buffer that is not listed as compatible in BioMAP systems?

 

For buffers not listed as compatible, we have limited experience and cannot recommend an amount to be used in BioMAP systems and recommend running a side-by-side control.

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Can we accommodate gelatin-like material?

 

The BioMAP platform can accommodate a gelatin-like material if it can be aspirated and dispensed through a 100 µl pipette tip. If the test concentration of the gelatin-like material is to be 1x during the assay we recommend sending a 100x stock, however an 8x stock can be accommodated if the 100x is too concentrated.

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Can I run a mixture of [test agent A + test agent B] and only serially dilute test agent A and keep the concentration of test agent B the same?

 

For all BioMAP panels each sample is run at 4 concentrations using a standardized format that is performed in fixed serial dilutions. Due to this standardization, the top concentration of agent A and agent B in the mixture is serially diluted in parallel.

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Is there a reference I can cite for BioMAP methods?

 

The supplemental methods of the paper below can be used as a reference for Diversity PLUS:

A selective inhibitor reveals PI3Kγ dependence of T(H)17 cell differentiation.

Nature Chemical Biology. 8, 576-82 (2012). (PMID: 22544264)
Bergamini G, Bell K, Shimamura S, Werner T, Cansfield A, Müller K, Perrin J, Rau C, Ellard K, Hopf C, Doce C, Leggate D, Mangano R, Mathieson T, O'Mahony A, Plavec I, Rharbaoui F, Reinhard F, Savitski MM, Ramsden N, Hirsch E, Drewes G, Rausch O, Bantscheff M, Neubauer G.

Additional BioMAP references can be found here

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What are analytical considerations of using BioMAP?

What does the Y-axis of a BioMAP profile represent?

 

Biomarker measurements in a drug-treated sample are divided by the average of the vehicle control samples to generate a ratio that is then log10 transformed. This is what is plotted on the Y-axis. We do not further normalize our data.

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What types of agents are profiled in the BioMAP database? How many are there?

 

Over 4,000 agents have been profiled in our BioMAP reference database, including approved drugs, failed drugs, biologics, chemicals, nanomaterials, extracts, natural products and experimental agents. Please note that these agents have all not been run in all of our systems. The majority of test agents in the BioMAP reference data base are profiled in our Diversity PLUS panel. BioSeek continues to expand this database to include current agents of interest to the clinical community and our clients.

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Why is a 1:3 dilution recommended for Diversity PLUS?

 

Diversity PLUS is a panel designed to screen test agents across 12 systems covering broad biology. Each test agent is run in singlicate at 4 different concentrations. Our experience and previous data have demonstrated that fixed 1:3 serial dilutions informs both concentration dependent drug effects as well as provides a set of internal replicates. Additionally, annotation of notable biomarker activities in Diversity PLUS depends on how the biomarker activity is impacted by 2 or more concentrations.

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Can I have the raw data from my BioMAP project to generate standard curves for analysis?

 

BioMAP data is presented as a log transformed ratio precisely to enable cross experiment comparison in a robust and reproducible manner suitable for screening projects. Since the goal of the BioMAP system is to determine agent-related activities only, we do not calculate levels in unit/vol. BioMAP utilizes the log transformed ratio of the raw OD biomarker readout levels in the presence of a test agent relative to the vehicle control on the same plate. This ratio allows the change in biomarker activity to be attributed to the agent at a particular concentration, and allows various biomarkers with markedly different ranges of values to be aligned on the same axis as is seen in our profile plots. We cannot release the raw OD values for standard profiling projects (something that may not apply to larger collaborative projects) since biomarker readouts are measured on different technology platforms and it’s not always possible to cross compare raw values.

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I have an Excel data table of log/ratio values of triplicate data. I want to linearize the data and I am not sure what to do with the error bars. Is the standard deviation also on a log10 scale? How do I calculate the standard deviation for a linear scale?

 

To learn more about how we calculate our standard deviation and how to linearize this value for our panels run in triplicate, please see the link below.
BioMAP error bar calculations

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Is it possible to compare an experiment looking at [agent A + agent B] in combination to previous runs of [agent A] alone and [agent B] alone run at different times? Diversity PLUS is leveraged as a highly reproducible system where one can compare a compound to the existing data of a compound present in the database…why can’t I do the same thing?

 

Combinations are mathematically evaluated for statistically significant differences between a combination vs. individual agents. Many differences between a combination and individual agents are subtle, requiring a format that allows rigourous statistical evaluation. This means these samples must be run head-to head in the same experiment in triplicate in order to calculate the statistical difference of the triplicate samples between the individual agents and the combination. Moreover, this comparison needs to be done on the exact same pools of cells, the same stimulation, the same vehicle control (which serves as the denominator in the ratio) and same measurement of readouts that were on the same plate to minimize technical variability.

BioMAP Diversity PLUS profiling is a broad phenotypic screen that can be used to identify mechanistic signatures (i.e., mathematical “shape” of the data) across 148 readouts in 12 systems. We compare this overall shape to database benchmarks using a combinatorial approach that filters and ranks the Pearson correlation scores to mathematically determine which profile shape is the most similar to the test agent in question. Additionally, benchmark profiles can be consensus profiles that are the average of multiple runs to give a “stereotypical” BioMAP profile for that compound. This averaging of profiles makes benchmarks unsuitable for mathematical comparisons to profiles from a specific run. Combo ELECT was developed to enable the mathematically rigorous comparisons of how the combination differs from its individual components and not the overall mechanistic shape.

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