Ex Vivo Dermal Permeability

Ex vivo dermal studies are used to assess the feasibility of transdermal administration, to rank-order compounds in terms of permeability or accumulation, and to optimize formulations.

Such studies are performed with human or porcine skin in Franz cells. Test formulations (gels, creams, ointments, patches, etc.) are applied to the upper (external) surface, and samples are removed at pre-determined time points from the reservoir containing buffer that is in contact with the lower (serosal) surface. Reference compounds are either co-dosed (if the test compound is in solution) or run in parallel (if the test compound is in some other type of formulation) for quality control. The skin is often extracted at the end of the study to quantify accumulation of the test compound.

It is possible to determine accumulation in different layers of the skin by tape stripping or heat separation. Transepidermal water loss (TEWL) may be measured as a proxy for skin dryness. Test formulations may also be incubated with tissue homogenates to assess the metabolic stability of the test article.

Study Purpose

from formulations across synthetic
membranes in Franz cells

Evaluate the dermal permeability and/or accumulation of test articles

Type of Model

Franz Cell

Time

2-4 weeks

Test System

Human Skin

Deliverables

  • Flux or apparent permeability of the test article and reference compounds
  • Percent recovery of the test article
  • Comparison of the absorption potential of test article to that of reference compounds
  • Comparison of test and reference formulations (if applicable)
  • Accumulation in stratum corneum, epidermis, and dermis (if applicable)
  • Demographics of human donor

Model Description

  • Unidirectional permeability assessment of test article across frozen dermatomed human cadaver skin (male or female trunk region)
  • Skin mounted in Franz cells thermostatically controlled at 37°C
  • Test article may be dosed without formulation, or administered as a gel, cream, ointment, patch, or solution
  • Suitability experiments may include assessment of chemical stability and/or non-specific binding
  • Control compounds (atenolol and testosterone or caffeine) run in parallel
  • Sampling from receiver compartment at five time points up to 30 hours
  • Optional determination of test article accumulation in skin
  • Optional stability assessment in skin homogenate

Benefits

  • Screen formulations in human skin before clinical PK or bioequivalence studies
  • Rank order compounds in terms of permeability
  • Evaluate locally-acting dermal formulations and quantify dermal accumulation
  • Absorption Systems provides in-house analytical and formulation services, along with consultative assistance in study design

Analytical

Analytical Tools and Techniques

  • ELISA
    • Validate the commercial kits, assay transfer or develop the test from the scratch (LDT).
    • Can perform Assay Qualification, RUO Validation, and GxP Validation
    • Any applications related to antigen -antibodies interaction
    • Large molecules and protein PK studies
    • ADA, TAb, NAb detection
    • Types:
      • Direct
      • Indirect
      • Sandwich
      • Competitive
  • Quantitative Western Blot
    • Western blot analysis is an immunological testing method that can detect a protein of interest from a mixture of a great number of proteins. Western blotting generates information about the size of a protein and how that protein is expressed when compared directly to a control sample. Samples for analysis can include cells, tissues, and synthesized recombinant proteins. The success of the western blot method depends upon the quality and specificity of the antibodies used to detect a response against a specific protein of interest.

    • Absorption Systems can generate a method for a western blot utilized in either R&D applications or in a GLP/cGMP study. There is an additional charge for GLP and cGMP methods as compared to R&D projects. Using the Syngene System, western blots can be quantitated through the GeneSnap Software and particular methods can be validated for use in clinical trials.

  • Enzyme Activity Assays
  • ELISpot and FluoroSpot
    • Assays in 96-well plates allow rapid analysis of large numbers of samples
    • Quantification of the number of cytokine (for example IFN-Îł or TNF-α) secreting cells.
    • The ELISPOT assay can be used to determine effects of drugs or other compounds on cytokine secretion in vitro.
    • Monocytes/macrophages/dendritic cells characterization
    • Apolipoproteins analysis
    • The ELISPOT assay can be used to determine the frequency of antigen-specific cytotoxic T cells (CTL) in fresh unstimulated peripheral blood.
  • Luminex Multiplex Technology
    • Luminex technology is based on the principles of Flow Cytometry. It uses a single 5.6 micron size microspehere that are dyed with red and infrared dyes in different combinations to create 500 uniquely colored microspheres.
    • Luminex bead array assays are widely used for rapid biomarker quantification
    • Assessment of serological immunity after successful vaccination
    • Assessment of immunogenicity
    • Cytokines, chemokines and growth factors
    • Cell signaling pathways
    • Adverse effect diagnosis, for example, cytokine storm, by comparing a baseline samples to various time points after administration of vaccines
    • Ready to use kits from Vendors (Affymetrix, Millipore, Bio-Rad etc) or use in-house developed kits.
    • Fluorescent/Luminescent Assays
  • PCR, qPCR, and ddPCR
    • Transduction/Transfection efficiency
    • Retroviral vector-based gene therapy products testing
    • Analysis of patient PBMC for RCR-specific DNA sequences
    • Determination of efficacy of drugs on target genes
    • Analysis of the spread of diseases virus, bacterial, parasites
    • Viral Shedding
    • Gene Expression
    • Mutation Detection
    • Genetic Variation
  • LC/MS

Neutralizing Antibodies

How are neutralizing antibodies different than binding antibodies?

In the case of any virus/bacterial infection, antibodies are generated by B-cells as a part of the humoral immune response of our system. But not all the generated antibodies which bind the pathogenic part of the foreign particle are able to inhibit the infectivity of the pathogen or vector, it is only a fraction of them which can interfere and block their entry and infectivity and those are neutralizing antibodies (NAb).

NAb detection is important

Precise assessment of neutralizing antibody activities is important for infected patients and for animals and volunteers immunized with the experimental vaccines against the antigens for the infecting virus/bacteria. Whether a vaccine can elicit a rigorous immune response in terms of NAb or not, decides the measure of its potency and protectivity. Also, the preexisting NAbs which represent the adaptive immune response of an individual previously exposed to antigen/virus can interfere with efficacy and risk assessment of a biotherapeutics. Therefore, it is critical to design reproducible and quantitative in vitro virus neutralization assays.

Development of NAb assay

Virus neutralization is a specialized type of immunoassay, where we measure the ability of antibody to block the infectivity of the virus. Reporter cells are engineered to express reporter genes for ex firefly luciferase under the control of the desired virus LTR permitting sensitive and accurate measurements of infection. Expression of the reporter genes is induced by viral proteins soon after infection. Luciferase activity is quantified by luminescence and is directly proportional to the number of infectious virus particles present in the test sample.  If the test serum/ plasma has neutralizing antibodies, they will neutralize the virus and it will not turn on the luminescence in the cells. Antibody titer is obtained by identifying the serum dilution point at which blocking of virus activity is < 50% (Figure 1)

Considerations and challenges for NAb assay design

              Neutralization assays could be designed either in vitro or in vivo.

  • In vivo assays require embryonated eggs or animals. Although in vivo assays could be the ideal model for quantitative analyses of neutralization antibodies for evaluation of the potential vaccine or antiviral agents. But In vivo evaluation of antivirals involves monitoring the survival rate of the animals following lethal challenges with the wt viruses or with antivirals. This requires a long time and exhibits variability due to differences in immune response amongst animals and the consistency across the programs. 
  • The in vitro assays are cell-based assays. Cell-based assays are better and preferred as they provide a functional readout using relevant cell lines, which closely reflects the in vivo However, cell-based NAb assays are difficult to establish, as suitably relevant cell lines and a proper assay endpoint have to be identified and the sensitivity of the assay is difficult to establish. While completely protected cells can be easily distinguished from the damaged cells, partially protected cell populations are hard to evaluate. Therefore, it is difficult to come up with a titration curve to measure the strength of a neutralizing antibody with serially diluted testing antibodies.
  • Types of viral vector: There can be either lentiviral or AAV transduction vectors. AAV serotypes are known to have tissue tropism hence an ideal cell line that allows AAV transduction is needed.
  • NAb assay development for clinical assays/trials is challenging as it must be qualified and validated for human use.

Figure 1.

References:

  1. Nelson EK, Piehler B, Eckels J, Rauch A, Bellew M, Hussey P, Ramsay S, Nathe C, Lum K, Krouse K et al: LabKey Server: an open source platform for scientific data integration, analysis and collaboration. BMC Bioinformatics 2011, 12:71.
  2. Pavlov IY, Carper J, Lazar-Molnar E, Delgado JC: Clinical laboratory application of a reporter-gene assay for measurement of functional activity and neutralizing antibody response to infliximab. Clin Chim Acta 2016, 453:147-153.
  3. Piehler B, Nelson EK, Eckels J, Ramsay S, Lum K, Wood B, Greene KM, Gao H, Seaman MS, Montefiori DC et al: LabKey Server NAb: a tool for analyzing, visualizing and sharing results from neutralizing antibody assays. BMC Immunol 2011, 12:33.
  4. Sprangers MC, Lakhai W, Koudstaal W, Verhoeven M, Koel BF, Vogels R, Goudsmit J, Havenga MJ, Kostense S: Quantifying adenovirus-neutralizing antibodies by luciferase transgene detection: addressing preexisting immunity to vaccine and gene therapy vectors. J Clin Microbiol 2003, 41(11):5046-5052.
  5. Tian Y, Zhao H, Liu Q, Zhang C, Nie J, Huang W, Li C, Li X, Wang Y: Development of in vitro and in vivo neutralization assays based on the pseudotyped H7N9 virus. Sci Rep 2018, 8(1):8484.
  6. Montefiori DC: Measuring HIV neutralization in a luciferase reporter gene assay. Methods Mol Biol 2009, 485:395-405.

 

Dose Ranging Studies

While a true dose-ranging study is a clinical trial, Absorption Systems offers in vivo dose ranging studies where titerable doses of a test article or compound are tested against each other to establish which dose is safely tolerated.  A typical study format would include a placebo group of animals, along with several groups that receive higher and higher doses of the test article.

A routine format would include 4-6 test groups that provides a range from low to highest concentrations of the test article along with a group that receives the buffer that the test articles have been re-constituted in. The highest dose tolerated is defined as the maximum tolerable dose (MTD), which then is used to
determine further studies.

Other endpoints that can be generated from this type of testing can be chemistries, hematology panels, blood gases as well as a variety of biomarker measurements and necropsy and histology testing.  Absorption Systems has AAALAC and USDA accredited facilities.

Potency Assay Development

The preferred method for determining potency is a single, quantitative biological assay. However, due to the complex nature of CGT products, this approach may not always be sufficient. In this case, an alternative approach of using a non-biological assay and/or a combination of biological and non-biological assays (i.e. an assay matrix) may be used. At least one assay used to measure potency should be quantitative, and there should be scientifically sound data to correlate assay results to relevant product-specific biological activity.

Roadmap of In Vitro Potency Assays for CGT Products: Tools & Techniques 

 
Potency Assay Validation

Potency assay development should start as early as possible in the product’s life cycle to allow time for evaluating multiple assays, generating data on product stability and consistency, and collecting data to support correlation studies if needed. As the product advances through clinical phases, so should the potency assay with regards to demonstrating biological relevance and establishing acceptance criteria. This phase-appropriate approach allows continuous improvement and optimization of the potency assay, which will enable consistent lot release during clinical trials and streamlined regulatory submission.

Immunoassay Validation

Absorption Systems provides a complete immunoassay validation package that is extensive and in compliance with FDA review. All work is performed under good laboratory procedure conditions (21 CFR Parts 58) and with CFR 320.29 and follows the basic parameters set forth in the “Guidance for Industry: Bioanalytical Methods Validation for Human Studies:
FDA CDER: May 2001”.

Whether the system is a simple screening assay or a quantitative assay used in stability testing, performance characteristics can be defined and assay parameters implemented for day-to-day operations.

Validations can be applied to bioassays, RIA’s, EIA’s, and any other similar type of detection system. A typical validation package would include the following parameters:

  • Performance Characteristics – Parameters such as reproducibility, operator-to-operator variation, sample parallelism, sensitivity, specificity, beginning to end variation, assay ruggedness, spike and recovery, freeze-thaw effects and cutoff analysis can be performed to evaluate the performance of the particular assay.
  • Assay Acceptability Criteria – Acceptability parameters are established from the validation, which then are used for the determination of assay acceptability. Control charts are utilized to track assay performance and trends that may occur over time. By using this method, overall performance can be monitored assay-to-assay, operator-to-operator, and day-to-day.
  • Supporting Documentation – Depending upon the client’s requirements, Standard Operating Procedures (SOP’s), worksheets, and reagent logs are generated and incorporated into the TGA BioServices QA documentation system, resulting in full traceability of all analysis.
  • Database Formation and Handling – Specific macros are tailored to each project to ensure that all raw data is electronically transferred during database generation and statistical analysis, thereby eliminating manual input errors.

Immunoassay Development

Absorption Systems offers our clients a complete package of development, optimization and quantification systems utilized in pre-clinical through Phase IV studies, all. in compliance with GLP and cGMP requirements when requested by the client. There is an additional charge for GLP and cGMP methods as compared to RD projects.

We have a strong track record of expertise in the development of complex and infrequently used methods to assist our clients in their research. We offer weekly progress reports and/or teleconferences to ensure the client is up to date on all aspects of their projects.

Capabilities include:

  • Measurement of small molecules, peptides and large proteins for RD or product release (cGMP)
  • Immunoassays for the monitoring of antibody or protein levels throughout a clinical study in complete compliance with GLP guidelines
  • Development of an Immunoassay-based screening protocol against your lead compound
  • Assay design and detection systems for antibodies with unique specifications; Quantitative detection systems for antibodies with unique specifications
  • Quantitative detection systems for the determination of contaminants in highly purified products
  • Immunoassays utilized for the detection of cytokines or cell surface markers
  • Use of commercial kits in compliance with cGMP or GLP guidelines
  • Monoclonal antibody screening (Western blot or ELISA)

Immunoassay formats include:

  • Bridging Assay
  • Competitive Technique
  • Direct Binding Assay Study
  • HACA
  • HAMA
  • Immunogenicity Assays
  • Immunoglobulin Screening
  • Isotyping
  • Client Specific Assay Formats

Immunoassays

Absorption Systems (FDA/EMA registered and inspected) provides GLP, GCLP, and cGMP regulatory-compliant services for RUO, pre-clinical toxicity studies, clinical trials, and batch release testing. We offer dedicated QA/QC personnel to insure compliance to required guidelines and the highest quality of the
data.

Our team has many years of experience in the development and validation of immunoassays for compound screening, ADA, Nab, PK, biomarkers testing using variety of methodologies. We provide the white gloves service covering all aspect of your bioanalytical needs from preparing the sample collection kits and training the clinical sites for proper specimen’s management to creating a FDA-ready Validation and/or Sample Analysis Reports.

Immunological Testing (R&D, cGMP, GLP)

  • Anti-Drug Antibody (ADA) Testing 
  • Bioequivalence Studies
  • Cytokine and Biomarker Testing
  • ELISA (Bridging, Indirect, Direct, Inhibition)
  • ELISpot/FluoroSpot testing
  • Enzymatic Activity Assays
  • Flow Cytometry Analysis
  • Fluorescent and Luminescent Assays
  • Kinetic Assays
  • LAL Endotoxin Testing (RUO)
  • Luminex Suspension Array Technology
  • Metabolic Markers Testing
  • Neutralizing Antibodies (NAb)
  • PCR/ddPCR/qPCR
  • Protein Analysis
  • Quantitative Western Blot

Core Expertise and Technology:

  • ELISA
    • Validate the commercial kits, assay transfer or develop the test from the scratch (LDT).
    • Can perform Assay Qualification, RUO Validation, and GxP Validation
    • Any applications related to antigen -antibodies interaction
    • Large molecules and protein PK studies
    • ADA, TAb, NAb detection
    • Types:
      • Direct
      • Indirect
      • Sandwich
      • Competitive
  • Quantitative Western Blot
    • Western blot analysis is an immunological testing method that can detect a protein of interest from a mixture of a great number of proteins. Western blotting generates information about the size of a protein and how that protein is expressed when compared directly to a control sample. Samples for analysis can include cells, tissues, and synthesized recombinant proteins. The success of the western blot method depends upon the quality and specificity of the antibodies used to detect a response against a specific protein of interest.

    • Absorption Systems can generate a method for a western blot utilized in either R&D applications or in a GLP/cGMP study. There is an additional charge for GLP and cGMP methods as compared to R&D projects. Using the Syngene System, western blots can be quantitated through the GeneSnap Software and particular methods can be validated for use in clinical trials.

  • Enzyme Activity Assays
  • ELISpot and FluoroSpot
    • Assays in 96-well plates allow rapid analysis of large numbers of samples
    • Quantification of the number of cytokine (for example IFN-Îł or TNF-α) secreting cells.
    • The ELISPOT assay can be used to determine effects of drugs or other compounds on cytokine secretion in vitro.
    • Monocytes/macrophages/dendritic cells characterization
    • Apolipoproteins analysis
    • The ELISPOT assay can be used to determine the frequency of antigen-specific cytotoxic T cells (CTL) in fresh unstimulated peripheral blood.
  • Luminex Multiplex Technology
    • Luminex technology is based on the principles of Flow Cytometry. It uses a single 5.6 micron size microspehere that are dyed with red and infrared dyes in different combinations to create 500 uniquely colored microspheres.
    • Luminex bead array assays are widely used for rapid biomarker quantification
    • Assessment of serological immunity after successful vaccination
    • Assessment of immunogenicity
    • Cytokines, chemokines and growth factors
    • Cell signaling pathways
    • Adverse effect diagnosis, for example, cytokine storm, by comparing a baseline samples to various time points after administration of vaccines
    • Ready to use kits from Vendors (Affymetrix, Millipore, Bio-Rad etc) or use in-house developed kits.
    • Fluorescent/Luminescent Assays
  • PCR, qPCR, and ddPCR
    • Transduction/Transfection efficiency
    • Retroviral vector-based gene therapy products testing
    • Analysis of patient PBMC for RCR-specific DNA sequences
    • Determination of efficacy of drugs on target genes
    • Analysis of the spread of diseases virus, bacterial, parasites
    • Viral Shedding
    • Gene Expression
    • Mutation Detection
    • Genetic Variation

Biologics & Gene Therapy In Vivo Services

Absorption Systems offers the client a wide range of support for in vivo animal studies for research and clinical applications.

Working as an extension of our client’s laboratory, we offer the customer flexibility in the planning of their studies as well as progress reports at each phase. Absorption Systems is AAALAC accredited and USDA registered and maintains an internal Institutional Animal Care and Use Committee (IACUC) in accordance with USDA and HHS regulations and guidelines.

We work with Mouse, Rat, and Guinea Pigs using ventilated caging systems.
We value a close relationship and communication with our clients, you’ll always have a dedicated team who’ll answer your questions within 24 hours and you will know in real-time what is going on with your study.

Core Experience and Technology:

  • In Vivo Studies
    • Prepare and approve IACUC protocols in accordance with USDA and HHS regulations and guidelines.
    • Development of vaccine immunization schedules, dosing and adjuvant screening
    • Dose tolerability and Immunogenicity evaluation
    • PK and PD rodent studies
    • Extensive experience with Gene therapy applications and AAV testing
    • Dermal and metabolism (i.e. DIO) model
    • Injection Site Reaction (ISR) studies
    • Xenograft tumor models
    • Transgenic breeding programs
    • Biodistribution studies using Li-Cor Fluorescent Imaging system for tissue section imaging (including full body image).
    • Bioanalytical support for dose-ranging and bioavailability studies
    • Tissue homogenization, protein and nucleic acids extraction
  • Diagnostic Analysis of Samples:
    • Complete Chemistry Analysis: Albumin, ALT, BUN, Calcium,
    • Cholesterol, Creatinine, Glucose, Protein, Uric Acid, etc.
    • Complete Clinical Analysis: Blood gases, acid-base balance, pH, electrolytes, etc.
  • Vivarium and Laboratory Space Rental

In Vivo Services

  • Biodistribution & Shedding
  • Toxicology
  • Immunogenicity
  • Small and Large Animal Models
  • Regenerative/Stem Cell Testing
  • Full Spectrum Ocular Services
  • Custom Animal Models
  • Immunocompetent & Immunocompromised Models