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BD Custom Technology TeamDelivering Cell Analysis Services
Custom Products and Services
To provide clients with state-of-the-art tools to advance discovery and development programs, CTT offers a deep knowledge base of advanced methodologies for immunophenotyping, immunogenicity testing of biological therapeutics, immune function testing, and more. Additionally, CTT works closely with highly experienced scientists in the advanced research groups at BD Biosciences. This collaboration allows the team to offer expert guidance on assay selection and advanced custom assay design.
CTT works as an extension of a client’s team to provide custom products that can be quickly and seamlessly implemented once the client brings them in house. CTT solutions expand beyond typical offerings to essential resources such as protocol development, flexible delivery options, and quality documentation.
To meet specific research needs, CTT offers several flexible manufacturing and packaging options for custom products, including manufacturing in a cGMP facility. Lyophilized products uniquely suited for multisite or longitudinal studies are also offered, including antibody cocktails and cells.
For clients with limited capacity or limited experience in flow cytometry and multiplexed protein assays, comprehensive custom services are offered that include sample preparation, analysis, and data reporting. Highly trained members of CTT have processed thousands of clinical trial samples in various studies.
The BD Biosciences Custom Technology Team (CTT) is a specialized contract research group with core competencies in flow cytometry and multiplexed pro- tein assay development. CTT offers custom products, contract manufacturing, assay development, and assay services that enable global biotechnology, pharmaceutical, and contract research organizations to respond efficiently to the rapidly changing landscape of research and development.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
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W O R K I N G
ConceptDevelopment
FeasibilityandOptimization
AssayServices
SamplePreparation
Processingand Analysis
DataDelivery
CustomProduct
CustomProductEvaluationLot
Optimization ProductionScale Up
An overview of the Custom Technology Team process
A goal of seamless implementationThe Custom Technology Team begins all design work with an across-the-board review of technical and logistical project needs. First, the team conducts a detailed review of the study objectives and assay requirements. Then, to understand capacity constraints and in house proficiencies, CTT examines instrument and personnel resource capabilities. Last, the team considers logistics such as custom product or data delivery preferences and information technology infrastructure.
By working to understand client needs and requirements, CTT designs custom products and delivers data that can be seamlessly deployed in house.
Flexible options for feasibility and optimizationCTT can adapt feasibility and optimization to meet specific research needs. Clients can specify the type of sample or cell line to be used and provide external reagents for conducting tests. Additionally, for large-volume production requirements, CTT can accommodate an evaluation and trial period for in-house testing before production is scaled up.
Consultation + Design
Extensive groundwork to facilitate a smooth handover
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C O L L A B O R A T I V E L Y
Serum
Detector Antibody+
+
+
IgMIgG
IgG
IgM
KLH
KLH
TT
TT
SWM
SWM
A.
lgM lgM
lgGlgG
KLHKLH
Myoglobin Myoglobin
anti-human lgG PE FL2-H anti-human lgM FITC FL1-H
FL3-
H10
5
FL3-
H
TTTT
B.
105
104
103
102
104
103
102
102 103 104 105 102 103 104 105
Overview of the CTT capabilities
Examples of assay services:•Immunophenotyping •Intracellularproteomics(BD™Phosflow) •Cellsurfaceproteomics(BDFACS™CAP) •Immunefunctiontesting -Cellular response to antigens -Intracellularcytokineassays -ELISPOTtodetectT-cellresponse -Flow cytometry-based proliferation assays •Receptoroccupancyassays
Examples of custom products: •Antibodycocktailsformulticolorflow cytometry •Premixedformulationsformultiplexed protein quantitation assays (BD™CytometricBeadArray) •Preoptimizedcytokineflowcytometrykits •Lyophilizedflowcytometryreagentsand control cells
A deep knowledge base for advanced concept developmentCTT is conversant in current methodologies for cell and protein analysis. This knowledge base allows CTT to provide expert guidance on the selection and design of state-of-the art assays, helping clients to quickly advance research programs.
With each project, CTT subject matter experts conduct literature reviews, consult with other specialized experts, and review in house data. CTT also can leverage the expertise of BD Biosciences product development researchers to make product and assay recommendations, giving clients the edge that comes with accessing tomorrow’s technology today.
Clients will receive an overview of available methodologies for each project and detailed recommendations on the optimal approach.
A novel multiplex protein assay design
Intheassayshown,spectrallydistinctbeadscoatedwithkeyholelimpethemocyanin (KLH) or tetanus toxoid (TT) are incubated with a test serum todetectthepresenceofantibodiestothetwoantigens.Incubatingeachsamplewithamixofanti-IgGoranti-IgMdetectionantibodiesthatarelabeled with unique fluorochromes allows for determination of the isotype ofanti-KLHoranti-TTantibodies.AdditionalbeadscoatedwithIgGorIgMserveaspositivecontrolsandabeadcoatedwithmyoglobinservesasthe negative control. The data shown demonstrates that the test serum ispositiveforTTandKLHspecificIgGantibodies(A),butpositiveonlyforKLHspecificIgMantibodies(B).Allmeasurementsaremadefromonetube, saving time and sample.
C O M P L E T E
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CTT has conducted cell and protein analysis studies for many major biotechnology and pharmaceutical companies. Thousands of samples have been processed in various studies for cancer, autoimmunity,infectiousdisease,andHIVtherapeuticsandvaccines. Expertise in the typical study processes, combined with expertise in cell and protein analysis, can translate into efficient study completion for clients.
Typical assay servicesCTT can:•Develop a proposal that outlines the assays to be used, as well as
validation and qualification studies to be conducted
•Develop study protocols and SOPs that optimize collection and trans-port conditions in coordination with research and logistical teams
•Conduct feasibility studies using known positive and negative controls
•Conduct qualification studies to assess sample-to-sample variation, transport stability, intra-assay variability, inter-technician variability, and normal range expectations
•Receive, accession, and track study samplesusingestablishedSOPs
•Process and analyze study samples following assay protocols
•Submit a cumulative data file and comprehensive bioanalytical study report providing all information necessary to properly interpret study results
Exceeding the standard to deliver highly dependable solutions
Product + Data Delivery
A comprehensive solution for quick implementationThe Custom Technology Team aims to provide a packaged solution to clients to streamline in house implementation. Typical custom products include pre-optimized antibody cocktails or multiplex bead reagents, buffers, and controls. To reduce guesswork, the team can provide a comprehensive study report detailing reagent materials and sample data. Also, the team can recommend protocols, instrumentation, and analysis methods.
Flexible custom product options CTT offers several versatile manufacturing and packaging solutions for custom products to meet specific research needs.Ifrequired,customproductscanbemanufacturedina BD cGMP facility.
Clients can define packaging and labeling of custom products for easy handling and tracking of reagents in the lab. CTT offers unique packaging options such as lyophilization of antibodies, antibody cocktails, immunological proteins, and cells to improve standardization and reduce hands-on time.
CTT provides central order tracking with transparent due dates and tight estimates for delivery to help maximize lab productivity. Standing orders and batch delivery can be set up for high-volume orders to allow clients to effectively manage inventory to reduce costs.
S O L U T I O N S
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BDLyoplate™technologyoffersauniquecustomproductoptionforresearcherslookingforgreaterlab-to-labconsistency and less hands-on time.
Quality systems in place for assaying samplesThe Custom Technology Team ensures that industry best practices for quality are followed by instituting comprehensive quality control systems. The team also participates in the CAP (College of American Pathologists) Proficiency Survey for Flow Cytometry and Hematology.
•All custom products are tested to lot-to-lot specifications to reduce batch variation. Pre- and post-bottle quality testing is employed. The BD Biosciences facility for custom product creation is ISO9001:2000certified.
•Unique protocols for each individual study are tracked, ensuring that every sample is handled consistently and reliably. CTT personnel are trained to handle biohazards, and BD Biosciences maintains personnel training records.
•Instruments are validated for use in human studies and equipment is maintained on a regular maintenance and calibration schedule. Storage devices are monitored daily to ensure proper sample stor-age conditions and maintain sample integrity.
• Data is stored on a secure server and is password protected, and redundant copies of data are archived.
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Surface Pro
teins
Cell Type: A B C D E F
Replicate: 1 2 1 23 1 2 3 1 2 3 1 2 3 1 2 34 Color Key
0–910–1920–2930–3940–4950–5960–6970–7980–8990–100
ImmunophenotypingFeatured Products +ServicesOffered
Multicolor productsThe Custom Technology Team works with clients to make both qualitative and quantitative multicolor antibody cocktails for flow cytometry based immunophenotyping.
These products include conjugated antibodies, compensation controls, buffers, and protocol guidelines. Optimizedmulticolorflowcytometrycocktailscanstreamline sample preparation, acquisition, analysis, and improve standardization between experiments.
Graphical representation of BD FACS CAP screen showing relative expression of cell surface markers on multiple cell types from multiple donors. Color key represents percent positive cells.
Cell surface marker profilingFor researchers using stem cells in drug discovery or cell therapy, CTT offers cell surface marker profiling to identify cell state variations indicative of donor to donor variations, cell type specific markers, or treatment-specific changes. The team leverages the novel proteomics tool BD FACS CAP (Combinatorial Antibody Profiling), a flow cytometry based method that uses a broadscreeningpanelofmorethan200highquality fluorescently labeled BD antibodies.
BD FACS CAP services are ideal for:•Donorqualification •Subpopulationanalysis •Qualityassurancetesting •Processdevelopment •Expression/functioncorrelations•Mediaoptimization •Cellsorting •Discoverybiology •Cellpurification
M U L T I C O L O R
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pStat1 pStat3 pStat5 pp38
in vivo
B cells(B220+)
unstimIFN-γGM-CSFIL-10LPS
unstimIFN-γGM-CSFIL-10LPS
unstimIFN-γGM-CSFIL-10LPS
T cells(TCRβ+)
Neutrophils(CD11bhi)
ex vivo in vivo ex vivo in vivo ex vivo in vivo ex vivo
FoxP3 Alexa Fluor® 488100 101 102 103 104
CD
25 P
E
100
101
102
103
10457.9 7.35
34.2 0.51
Intracellular proteomics (BD Phosflow)CTT offers assay optimization, sample processing, and data delivery for BD Phosflow analysis. An intracellular phosphoprofiling tool, BD Phosflow allows scientists to examine cellular subpopulations in complex samples, such as primary cells, whole blood, or peripheral blood mononuclear cells (PBMCs). Unlike traditional methods such as Western blotting, BD Phosflow enables researchers to analyze phosphoprotein signaling in single cells through the use of multiple cell surface markers. Using BD Phosflow alone, researchers can uncover rare cell subtypes with different signaling mechanisms.
Multiple phosphoproteins in different cell types studied by BD Phosflow
This figure illustrates how BD Phosflow makes it possible to conduct the simultaneous analysis of multiple phosphoproteins in complex cell mixtures. Inthisexperiment,BDPhosflowwasusedtoanalyzetheeffectoffourdifferent stimuli on cell signaling pathways in mouse splenic cell subsets (Bcells,Tcells,andCD11bhigh cells). Differences in signaling pathway responses were uncovered between mouse splenocyte cultures stimulated in vitro and splenic cells stimulated in vivo, underscoring the importance of conducting studies in close to native conditions. Data was analyzed in Cytobank software (www.cytobank.org). Histograms are colored according to fold change in phosphorylated protein relative to unstimulated samples.
Data courtesy of Dr. Peter Krutzik and Dr. Matt Hale, Stanford University.
Immunophenotyping of regulatory T cellsThe data shown demonstrates the characterization of regulatory T cells through multicolor flow cytometry. Human PBMCs were surface stained for CD4 and CD25,fixed,permeabilized, and stained intracellularly for FoxP3. The data shown werederivedfromanacquisitionof50,000
events in a lymphocyte gate, followed by CD4+ gating by fluorescence. A compound gating strategy by morphology, then side scatter vs. fluorescence, was used to identify FoxP3+ Treg cells shown in a final plotrepresentingCD25vs.FoxP3.
N O V E L
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Immunogenicity of biological therapeuticsThe Custom Technology Team specializes in developing novel multiplexed protein assays for immune monitoring. These assays have been designed to measure humoral immune response to biological therapeutics and vaccine antigen, vaccine carrier proteins, as well as infection.
CTT uses the BD Cytometric Bead Array as an assay platform for immune monitoring. This bead-based flow cytometry application allows users to quantify multiple proteins simultaneously. CTT has extensive experience working with pharmaceutical companies developing CBA-based antibody detection assays.
Immune Function TestingFeatured Products +ServicesOffered
Intracellular cytokine assaysCTT offers flow cytometry-based intracel-lular cytokine assays and sample processing services for immune monitoring relevant forstudiesofautoimmunedisease,HIVandtumor vaccine development, allergy, and viral and infectious diseases. These custom premixed cytokine assays speed immune monitoring by providing data on multiple cytokines simultaneously on specific cellular subsets in complex samples. CTT’s intracel-lular cytokine assays also save precious sam-ple,usingonly0.5mLofbloodformultiplecytokine results.
Custom, premixed CBA kitsTo help improve lab efficiency and standard-ization, CTT can provide researchers with premixed formulations of off-the-shelf CBA reagents and detection beads. CBA reagents are available for the detection of human, mouse, or rat cytokines and chemokines and growth factors from serum, plasma, or tissue culture supernatant samples.
A S S A Y S
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1 3
Mean ± SDNumber of dots(in triplicate)
2
2 0 1 1 ± 1
284 326 329 313 ± 25
43 21 22 29 ± 12
20 31 22 24 ± 6
Serum
Detector Antibody+
+
+
IgG
IgG
EPO
EPO
SWM
SWM
Custom CBA assay to detect anti-epoetin alfaCTT used the BD Cytometric Bead Array platform to design custom assays to detect antibody response to epoetin alfa in persons receiving therapy for the treatment and prevention of anemia for a leading pharmaceutical company.Inthisstudy it was postulated that early detection of anti-epoetin alfa might help prevent an adverse event resulting from this protein therapy treatment.
The custom assay included positive and negative control beads for each specimen processed to ensure the specificity of the signal when detected. The pharmaceutical company examined the assay’s detection capability and found that it coulddetectarelativeantibodyconcentrationof50ng/mL.1
ELISPOT servicesCTT’sELISPOTassayservicesallowresearchersto monitor cellular immune response to vari-ousantigens.ShownhereisanELISPOTassayforhumanIL-5productiondemonstratingaresponse to stimulation with cytomegalovi-rusantigensfromalysate(CMV-L)orCMVpeptidemix(CMV-P) (shown in triplicate). Staphylococcal enterotoxin B (SEB) serves as a positive control.
Additionally, CTT actively participates in anNIH-sponsoredprogramtomonitorinter-laboratory standardization protocols for the ELISPOTandColonyFormingCellassays,in-volvingmorethan14laboratoriesworldwide.
A custom CBA assay designed by CTT to detect anti-epoetin alfa.
A multiplexed protein assay designed to detect antibodies to epoetin alfa using four beads of distinct fluorescence intensity coated with epo-etinalfa(EPO),spermwhalemyoglobin(SWM,anegativecontrol),orhumanIgG(IgG,apositivecontrol).Afourthbeadwasleftblankandserved as a reference bead control.
1 Ferbas J, Thomas J, Hodgson J, Gaur A, Casadevall N, Swanson SJ. Feasibility of a multi-
plex flow cytometric bead immunoassay for detection of anti-epoetin alfa antibodies.
Clin Vaccine Immunol.2007;14:1165-1172.
Unstimulated
SEB
CMV-L
CMV-P
1 2 3
Custom Lyophilized ProductsFeatured Products +ServicesOffered
Standardize and simplify multisite, longitudinal studiesThe Custom Technology Team offers lyophilized products for flow cytometry, including reagents and control cells. Designed for specific experiment requirements, lyophilized reagents in pre-aliquoted formats can reduce variability between experiments, making these reagents ideal choices for multisite or longitudinal studies. Lyophilized reagents also have an extended shelf life compared to liquid reagents.Inaddition,researcherscaneasilyincorporatelyophilized reagents into automated workflow processes, providing greater consistency and improved lab efficiency.
Researcherssimplyaddsample—cells,blood,orotherliquids—torehydratethebiomolecules,andimmediatelybeginexperimental analysis. Lyophilized products can be packaged in many formats such as tubes, plates, and single vials.
BD Lyoplate technologyBD Lyoplate products offer unique custom options for researchers looking for greater lab-to-lab consistency and less hands-on time. Lyophilized formats also provide enhanced stability.
S T A N D A R D I Z E
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Function equivalent to liquid reagentsStudies have also demonstrated that lyophilized reagents retain functionality equivalent to liquid reagents. Using standard protocols for antigen-specific activation of peripheral blood lymphocytes, and assessment of response using cytokine flow cytometry, a strong correlation in activity was found between lyophilized antibody cocktails with liquid cocktails (right).
Lyophilized cells as controlsLyophilized cells are excellent as staining, instrument setup, or gating controls.Inthisexperiment,freshlyfixed,permeabilized,andstainedcells were compared to lyophilized cells that were stained with con-jugatestoCD69andIFN-g (left and middle). Freshly prepared cells were also compared to cells that were stained and then lyophilized (right). There was virtually no difference in signal intensity or cellular events between these three preparations.
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