158 月/24

KMD Bioscience-Bispecific Antibody Production Service

Antibody Production Platform

Bispecific antibodies (bsAbs) are engineered antibodies that can simultaneously bind to two different antigens or epitopes. They offer unique advantages in therapeutic applications, including enhanced targeting of cancer cells, dual blockade of signaling pathways, and improved immune system engagement. Producing bispecific antibodies involves several key steps, including design, expression, and purification.

Production Methods

Hybrid Hybridoma Technology

Fuse two different hybridomas producing distinct monoclonal antibodies. Select and screen for hybrid cells that produce bispecific antibodies.

Chemical Cross-Linking

Chemically link two different monoclonal antibodies. Requires precise control to maintain functionality.

Recombinant DNA Technology

Use gene cloning to create a single antibody molecule with two different binding sites. Express genes in mammalian or microbial systems to produce the bispecific antibody.

Single-Chain Variable Fragment (scFv) fusion

Link two scFvs with a flexible peptide linker. Allows for the production of smaller, bispecific fragments.

Here’s a detailed overview of the process of Recombinant DNA Technology:

Designing Bispecific Antibodies

Select the Antigen Targets

Identify the two distinct antigens or epitopes that the bispecific antibody needs to target. These could be different sites on the same antigen or entirely different antigens.

Choose a Bispecific Antibody Format

Dual-Variable Domain Immunoglobulins (DVD-Ig): Combines two different variable domains in a single antibody molecule.

Bispecific T-cell Engagers (BiTEs): Usually consist of two single-chain variable fragments (scFvs) connected by a linker. They often target a tumor antigen and CD3 on T cells.

CrossMab: A format where one arm of the antibody has two different variable regions.

IgG-like Bispecifics: Uses modified versions of IgG antibodies, where two different variable regions are introduced into the antibody structure.

Construct the Expression Vector

Design and create expression vectors containing the genes encoding the two different antigen-binding domains. The vectors will need to be optimized for expression in the chosen host cells.

Expression of Bispecific Antibodies

Choose an Expression System

Mammalian Cells: Commonly used for producing bispecific antibodies due to their ability to perform post-translational modifications. CHO (Chinese Hamster Ovary) cells are frequently used.

Yeast or Bacterial Systems: Used for simpler constructs but less common for bispecifics due to complex post-translational needs.

Note: How do different expression systems impact the efficacy of bispecific antibodies?

Different expression systems can significantly impact the efficacy of bispecific antibodies through several factors:

Post-Translational Modifications

Glycosylation Patterns

Mammalian systems (e.g., CHO, HEK293) provide human-like glycosylation, which can affect antibody stability, solubility, and function. Bacterial systems lack glycosylation, potentially impacting efficacy.

Protein Folding and Assembly

Correct Folding

Mammalian cells are better at complex protein folding and assembly, which is crucial for functional bispecific antibodies. Bacteria may produce inclusion bodies, requiring refolding steps.

Yield and Scalability

Production Yield

Yeast and bacterial systems often have higher yields and are more cost-effective but may compromise on proper folding. Mammalian systems generally offer lower yields but better quality.

Immunogenicity

Human-like Modifications

Non-mammalian systems might introduce foreign modifications, increasing immunogenicity risks.

Functional Activity

Binding Affinity and Specificity

Proper modifications and folding in mammalian systems ensure high binding affinity and specificity, critical for bispecific function.

Transfection

Transfect the mammalian cells with the expression vectors. This can be done using methods like lipofection, electroporation, or viral transduction.

Cell Line Development

Cloning: Select and clone cells that produce high levels of the bispecific antibody. This may involve single-cell cloning and expansion.

Stable Cell Lines: Develop stable cell lines that consistently produce the bispecific antibody over time.

Production

Cultivate the cells in bioreactors to produce the bispecific antibody in larger quantities.

Purification of Bispecific Antibodies

Harvesting

Collect the supernatant from the cell culture where the bispecific antibody is secreted.

Purification Techniques

Protein A/G Affinity Chromatography: Useful for initial purification based on the Fc region if the bispecific has an Fc tag.

Ion Exchange Chromatography: Separates proteins based on their charge, helping to further purify the bispecific antibody.

Size Exclusion Chromatography (SEC): Separates proteins based on their size to remove aggregates and obtain the monomeric form.

Affinity Chromatography: Additional affinity tags or specific binding partners can be used to enhance purity.

Characterization

Confirm Identity and Purity: Use techniques like SDS-PAGE, Western blotting, or mass spectrometry.

Assess Functionality: Verify that the bispecific antibody binds to both target antigens effectively using assays like ELISA, surface plasmon resonance (SPR), or flow cytometry.

Validation and Quality Control

Functional Assays

Binding Assays: Assess the binding affinity and specificity of the bispecific antibody to both target antigens.

Cell-based Assays: Test the ability of the bispecific antibody to engage and activate cells or kill target cells (e.g., cytotoxicity assays).

Stability Testing

Evaluate the stability of the bispecific antibody under various conditions to ensure it maintains activity and integrity over time.

Note: The stability of bispecific antibodies is influenced by several key factors

Structural Design

Molecular Architecture: The design of the antibody format (e.g., scFv, diabody) impacts stability.

Linker Sequences: Flexible and appropriate linkers can enhance structural integrity.

Expression System

Host Cell Line: The choice of cell line (e.g., CHO, HEK293) affects post-translational modifications.

Production Conditions: Optimization of culture conditions can improve stability.

Purification Process

Aggregation: Minimizing aggregation through optimized purification methods is crucial.

Formulation: Buffer composition and pH should be carefully selected to maintain stability.

Environmental Factors

Temperature: Storage and handling temperatures must be controlled.

Light Exposure: Protect from light to prevent degradation.

Biochemical Properties

Isoelectric Point (pI): Affects solubility and aggregation propensity.

Hydrophobic Interactions: Excessive hydrophobic regions can lead to instability.

Glycosylation

Consistent Glycosylation Patterns: Variability can affect stability and efficacy.

Regulatory Compliance

Ensure that the production and testing meet regulatory requirements if the bispecific antibody is intended for therapeutic use.

Applications and Development

Therapeutic Use

Cancer Therapy: Targeting specific tumor antigens while simultaneously engaging T cells.

Autoimmune Diseases: Blocking multiple disease pathways simultaneously.

Research Tools

Use in basic research to understand dual antigen interactions or complex biological systems.

Diagnostic Use

Develop assays or imaging tools that utilize bispecific antibodies to target multiple biomarkers.

 Challenges

Stability: Ensure the bispecific antibody maintains structural integrity.

Manufacturing Complexity: Requires advanced techniques for consistent production.

Purification: Separating bispecific antibodies from other variants can be difficult.

 Considerations

Specificity and Affinity: Optimize for high binding affinity to both targets.

Safety and Efficacy: Comprehensive testing to ensure therapeutic safety and effectiveness.

Summary

Producing bispecific antibodies involves designing a molecule with two distinct binding sites, expressing it in a suitable system, and purifying it to high purity. The process requires careful planning, from vector design and cell line development to purification and quality control. The choice of format and expression system will depend on the specific application and the desired properties of the final bispecific antibody.

148 月/24

KMD Bioscience-Anti-idiotypic Antibodies Production

Antibody Production Platform

Anti-idiotypic antibodies (anti-Id antibodies) are antibodies that target and bind to the variable region (idiotope) of other antibodies. These can be useful in various research and therapeutic applications, including studying antibody interactions, developing vaccines, and creating therapeutic antibodies. Producing anti-idiotypic antibodies involves several steps:

Immunizing with the Target Antibody

Preparation of Immunogen

Purify the Target Antibody: Start by purifying the antibody you want to generate anti-idiotypic antibodies against. This can be done using affinity chromatography or other purification methods.

Prepare the Immunogen: The purified antibody can be used directly or conjugated to a carrier protein (such as keyhole limpet hemocyanin, KLH) to enhance immunogenicity.

Immunization

Animal Selection: Typically, rabbits, mice, or other animals are used for immunization.

Immunization Schedule: Inject the immunogen into the selected animal(s) multiple times over several weeks to induce a robust immune response. Booster shots may be necessary.

Screening and Hybridoma Generation

Collect Serum

After immunization, collect blood from the immunized animals and prepare serum samples. These sera may contain anti-idiotypic antibodies.

Hybridoma Technology (for monoclonal anti-idiotypic antibodies)

Cell Fusion: Fuse B cells from the immunized animal with myeloma cells to create hybridomas. This is typically done using polyethylene glycol (PEG) or other fusions. Myeloma cells provide immortality, allowing the fused cells (hybridomas) to proliferate indefinitely.

Selection: Use selective media (e.g., HAT medium: Hypoxanthine-Aminopterin-Thymidine) to select only the hybridoma cells that have fused successfully and are producing antibodies. Only hybridomas survive in the HAT medium, as myeloma cells lack the necessary enzyme pathway, and unfused B cells have a limited lifespan.

Screening: Screen hybridoma supernatants to identify those producing antibodies that specifically bind to the idiotope of the target antibody. This can be done using enzyme-linked immunosorbent assays (ELISA), Western blotting, or other assays. Positive clones are identified based on their ability to bind specifically to the target antigen.

Cloning and Expansion

Clone the hybridomas that produce the desired anti-idiotypic antibodies.

Expand the selected clones in culture and then harvest the antibodies from the culture supernatant.

Purification and Characterization

Purification

Affinity Chromatography: Purify the anti-idiotypic antibodies using affinity chromatography. The antibodies can be captured on a column containing the target antibody or idiotype.

Protein A/G Chromatography: Additional purification steps, such as Protein A or Protein G chromatography, can be used to further purify the antibodies if needed.

Characterization

Determine Specificity: Confirm that the purified anti-idiotypic antibodies specifically recognize the idiotope of the target antibody. Techniques like ELISA or surface plasmon resonance (SPR) can be used.

Assess Purity: Analyze the purity of the antibodies using methods such as SDS-PAGE or size-exclusion chromatography.

Application and Validation

Functional Testing

In Vitro Assays: Test the functional activity of the anti-idiotypic antibodies in relevant assays, such as binding studies or functional inhibition assays.

In Vivo Studies: For therapeutic applications, perform in vivo studies to assess efficacy and safety.

Application

Research: Use anti-idiotypic antibodies to study antibody interactions or map antigen-binding sites.

Therapeutics: Develop anti-idiotypic antibodies as therapeutic agents, such as vaccines or neutralizing agents against pathogenic antibodies.

Vaccine Development: Mimic the structure of the original antigen, potentially acting as a vaccine.

Quality Control

Ensure consistent quality and performance of the anti-idiotypic antibodies through rigorous quality control measures.

Considerations

Specificity: Ensure high specificity for the idiotope to avoid cross-reactivity.

Validation: Confirm the functionality and binding specificity of the antibodies through rigorous testing.

Summary

Producing anti-idiotypic antibodies involves immunizing animals with a target antibody, generating hybridomas, screening for specific anti-idiotypic antibodies, purifying and characterizing the antibodies, and validating their functionality. The process can be complex and may require optimization based on the specific application and target antibody.

108 月/24

KMD Bioscience-Antibody Production Protocol

Antibody Production Platform

 Materials Needed

– Expression Vector: Contains genes for the antibody’s heavy and light chains

– Host Cells: CHO or HEK293 cells

– Transfection Reagents

– Culture Media

– Purification Columns: Protein A/G

Recombinant Antibody Production Protocol

Antigen Selection

The first step is to identify the target antigen that the desired antibody should bind to. This is a critical step as it determines the specificity of the final antibody.

Antibody Library Construction

A library of antibody genes is created, typically by cloning the variable region genes from B cells of an immunized animal or a human donor. This creates a diverse pool of antibody sequences that can be screened.

Antibody Screening

The antibody library is screened to identify the clones that bind the target antigen with high affinity. This is often done using display technologies like phage display or yeast display.

Antibody Engineering and Optimization

The selected antibody sequences can be further engineered and optimized to improve properties like binding affinity, specificity, stability, and effector functions. Techniques like affinity maturation, humanization, and antibody fragment engineering can be employed.

Expression System Selection

The optimized antibody genes are then cloned into an appropriate expression system, such as bacterial (E. coli), yeast (Saccharomyces cerevisiae), or mammalian (CHO, HEK293) cells. The choice depends on factors like post-translational modifications, expression levels, and scalability.

Transient or Stable Expression

The antibody can be expressed either transiently (short-term) or stably (long-term) in the host cells. Transient expression is faster but yields lower amounts, while stable expression takes longer but produces higher quantities of the antibody.

Antibody Purification

The recombinant antibody is purified from the cell culture using techniques like affinity chromatography, ion exchange chromatography, or size exclusion chromatography. This step ensures high purity and homogeneity of the final product.

Antibody Characterization

The purified antibody is thoroughly characterized using various analytical methods, such as ELISA, Western blotting, and flow cytometry, to verify its specificity, affinity, and other key properties.

Scale-up and Manufacturing

Once the optimal antibody candidate is identified, the production process can be scaled up to generate larger quantities for further development and applications, such as therapeutic or diagnostic use.

Polyclonal Antibody Production Protocol

Immunization

Prepare Antigen

– Antigen Purification: Purify the antigen or prepare the antigenic peptide/protein.

– Formulation: Dissolve the antigen in a suitable adjuvant (e.g., Freund’s Complete Adjuvant for initial immunization, Freund’s Incomplete Adjuvant for booster immunizations).

Immunization Protocol

– Animal Selection: Choose an appropriate animal (e.g., rabbits, goats, sheep).

– Initial Immunization: Inject the antigen-adjuvant mixture into the animal (e.g., intramuscularly or subcutaneously).

– Booster Shots: Administer booster injections at regular intervals (e.g., every 2-4 weeks) to enhance the immune response.

Blood Collection

Harvest Blood

– Collection: Collect blood from the immunized animal after the immune response is sufficiently developed (usually 2-4 weeks post-final boost).

– Processing: Allow blood to clot, then centrifuge to separate serum from the clot.

Serum Preparation

– Serum Separation: Collect the serum from the centrifuged blood and store it at -20°C or -80°C for long-term storage.

Antibody Purification

Affinity Purification

– Prepare Affinity Column: Use a column with an antigen-specific ligand or antigen-bound to a solid support.

– Apply Serum: Load the serum onto the column to capture specific antibodies.

– Wash and Elute: Wash the column to remove non-specifically bound proteins, then elute the antibodies using an elution buffer.

Concentration and Buffer Exchange

– Concentration: Concentrate the purified antibodies using centrifugal concentration devices if needed.

– Buffer Exchange: Dialyze or use a desalting column to exchange the buffer to a suitable storage buffer (e.g., PBS with sodium azide).

Quality Control

Test Specificity and Titer

– ELISA: Perform an enzyme-linked immunosorbent assay (ELISA) to test the antibody’s specificity and titer.

Verify Purity

– SDS-PAGE or Western Blot: Check the purity of the antibody preparation.

Monoclonal Antibody Production Protocol

Immunization and Cell Fusion

Immunization

– Antigen Preparation: Prepare and purify the antigen as described above.

– Immunization: Immunize a suitable mouse strain (e.g., BALB/c) with the antigen using the same protocol as for polyclonal antibodies.

Cell Fusion

– Splenocyte Isolation: Harvest splenocytes from the immunized mouse.

– Fusion: Fuse splenocytes with myeloma cells (e.g., NS0, SP2/0) using a fusion agent (e.g., polyethylene glycol).

Selection and Screening

Selection

– HAT Medium: Plate the fused cells in HAT (hypoxanthine-aminopterin-thymidine) selection medium to select hybridoma cells.

Screening

– ELISA or Other Assays: Screen hybridoma supernatants for specific antibody production against the target antigen.

Cloning and Expansion

Cloning

– Limited Dilution: Perform limiting dilution to isolate single hybridoma clones.

– Clone Expansion: Expand positive clones in culture flasks or bioreactors.

Antibody Production

Culture

– Scale-up: Grow the selected hybridoma clones in larger volumes for high-yield antibody production.

– Harvest: Collect and process the culture supernatant to obtain antibodies.

Antibody Purification

Affinity Purification

– Prepare Affinity Column: Use Protein A/G affinity chromatography to purify the monoclonal antibodies.

Concentration and Buffer Exchange

– Concentrate and Buffer Exchange: As with polyclonal antibodies, use concentration and buffer exchange methods as needed.

Quality Control

Test Specificity and Titer

– ELISA and Western Blot: Validate specificity and titer.

Assess Purity

– SDS-PAGE or HPLC: Confirm purity.

The recombinant antibody production protocol leverages the power of genetic engineering and cell culture technologies to create highly specific, customizable, and scalable antibodies. This approach offers several advantages over traditional hybridoma-based methods, including the ability to generate fully human or humanized antibodies, improved production efficiency, and the potential for engineering enhanced antibody properties. Producing antibodies, whether monoclonal or polyclonal, involves several critical steps, from immunization to antibody harvesting.

098 月/24

KMD Bioscience-Antibody Expression and Purification

Antibody Production Platform

Antibody expression and purification is a crucial process in the production of recombinant antibodies for various applications in therapy, diagnostics, and research. The general workflow involves the following steps:

Antibody Expression

Recombinant Antibody Expression

– Antibodies are typically expressed in bacterial (e.g. E. coli) or mammalian cell lines (e.g. CHO, HEK293) using recombinant DNA technology.

– Codon optimization and other techniques are often used to enhance protein production.

– The expressed antibodies can be in the form of full-length IgG, antibody fragments (e.g. Fab, scFv), or engineered formats like bispecific antibodies.

Selection of Antibody Format

– Monoclonal Antibodies: Produced by creating a hybridoma cell line that secretes a single type of antibody.

– Polyclonal Antibodies: Produced by immunizing an animal and collecting antibodies from its serum, which are a mixture of various antibodies.

Cloning the Antibody Gene

– Gene Identification: Identify the gene encoding the antibody of interest, usually from a hybridoma or immune cells.

– Construct Creation: Clone the antibody gene into an expression vector, which is a plasmid or virus designed for high-level protein expression.

Cell Line Selection

– Expression Host: Choose a suitable cell line for expression. Common hosts include:

– Bacteria: E. coli, suitable for simple antibodies but often requires refolding of proteins.

– Yeast: Pichia pastoris, which can perform some post-translational modifications.

– Mammalian Cells: CHO cells, HEK293 cells, which can perform complex post-translational modifications similar to those in humans.

Transformation and Expression

– Transformation: Introduce the expression vector into the chosen host cells.

– Culture Conditions: Grow the cells under conditions that induce high-level antibody production.

Antibody Purification

– The cells expressing the recombinant antibodies are lysed, often using a French press or homogenizer, to release the antibodies.

– Initial purification steps like centrifugation and filtration are performed to remove cell debris and other impurities.

Purification Methods

– Affinity Chromatography: The most common method, using a column with a ligand (e.g., Protein A or Protein G) that specifically binds to the Fc region of antibodies. This allows for the selective capture of antibodies from the mixture.

– Ion Exchange Chromatography: Separates proteins based on their charge.

– Size Exclusion Chromatography: Separates proteins based on their size and shape.

– Protein A/G/Affinity Resins: Specific for antibodies, simplifying the purification process.

Monitoring and Quality Control

– Purity Analysis: Use SDS-PAGE, Western blotting, or other assays to confirm the purity of the antibody.

– Activity Testing: Verify that the antibody retains its biological activity, often through ELISA or other functional assays.

– Concentration Determination: Measure antibody concentration using techniques like UV absorbance or BCA assay.

Removal of Tags and Buffers Exchange

– If the antibody was expressed with an affinity tag (e.g. His-tag, Fc-tag), it may be necessary to remove the tag using a protease cleavage site.

– Dialysis, desalting, or diafiltration are used to exchange the antibody into the desired buffer for the intended application.

 

The choice of expression system, purification strategy, and specific techniques employed will depend on the antibody format, the desired purity, and the intended use of the purified antibody. Careful optimization of each step is crucial to obtain a high yield of pure, functional recombinant antibodies.

088 月/24

KMD Bioscience-Antibody-Drug Conjugate (ADC) mechanism of action

Antibody Discovery

Antibody-drug conjugates (ADCs) are sophisticated biopharmaceutical agents designed to deliver cytotoxic drugs specifically to cancer cells, thereby minimizing systemic toxicity. The mechanism of action of ADCs involves several critical steps, from targeted binding to the ultimate induction of cell death. Here’s a detailed breakdown of this process:

Target Recognition and Binding

Antibody Component: The antibody in the ADC is engineered to recognize and bind with high specificity to a tumor-associated antigen (TAA) that is overexpressed on the surface of cancer cells but minimally expressed on normal cells.

Binding: Upon administration, the ADC circulates in the bloodstream and binds to the TAA on the cancer cell surface through the antibody component.

Internalization

Receptor-Mediated Endocytosis: The binding of the ADC to the antigen triggers receptor-mediated endocytosis, leading to the internalization of the ADC-antigen complex into the cancer cell.

Endosomal Trafficking: The internalized ADC is trafficked through the endosomal pathway, eventually reaching the lysosome, where the acidic environment facilitates the cleavage of the linker connecting the drug to the antibody.

Drug Release

Cleavage of Linker: The linker is designed to be cleaved in the lysosomal environment, which releases the cytotoxic drug from the antibody. The cleavage can be triggered by lysosomal enzymes, acidic pH, or other specific conditions.

Release into Cytoplasm: Once cleaved, the cytotoxic drug diffuses into the cytoplasm of the cancer cell.

Cytotoxic Effect

Mechanism of the Payload: The released drug exerts its cytotoxic effect through various mechanisms, depending on the nature of the drug. Common cytotoxic agents used in ADCs include:

Microtubule Disruptors: Drugs like auristatin and maytansinoids inhibit microtubule assembly, leading to cell cycle arrest and apoptosis.

DNA-Damaging Agents: Drugs like calicheamicin cause DNA double-strand breaks, leading to cell death.

Topoisomerase Inhibitors: Drugs like camptothecin derivatives inhibit topoisomerase, leading to DNA damage and apoptosis.

Apoptosis and Cell Death

Induction of Apoptosis: The cytotoxic effect of the drug activates apoptotic pathways, leading to programmed cell death.

Bystander Effect: In some cases, the released drug can diffuse into neighboring cancer cells, contributing to the bystander killing effect, where adjacent cells that do not express the target antigen are also killed.

Advantages of ADCs

Targeted Delivery: ADCs specifically target cancer cells, reducing the impact on healthy cells and minimizing side effects.

Enhanced Efficacy: The combination of targeted delivery with potent cytotoxic drugs increases the therapeutic efficacy against cancer cells.

Versatility: ADCs can be designed to target various antigens and utilize different cytotoxic drugs, allowing for customization based on the specific cancer type.

Challenges and Considerations

Target Selection: Identifying suitable TAAs that are highly expressed in cancer cells and minimally expressed in normal cells is crucial for the efficacy and safety of ADCs.

Linker Stability: The linker must be stable in the bloodstream to prevent premature drug release but efficiently cleavable in the target cell environment.

Resistance Mechanisms: Cancer cells may develop resistance to ADCs through various mechanisms, such as downregulation of the target antigen or efflux of the cytotoxic drug.

 Conclusion

Antibody-drug conjugates represent a promising class of targeted cancer therapies that combine the specificity of antibodies with the potency of cytotoxic drugs. By leveraging the unique mechanism of action of ADCs, it is possible to achieve enhanced therapeutic outcomes while minimizing systemic toxicity, thereby offering a valuable treatment option for patients with various types of cancer.

078 月/24

KMD Bioscience-Antibody Discovery Process

Antibody Discovery

The antibody discovery process involves several key stages designed to identify and develop antibodies with high affinity and specificity for their target antigens. This process can be broadly categorized into the following steps:

Antigen Selection and Preparation

Target Identification: The first step is to identify and select the target antigen, which is often a protein associated with a disease state, such as a cell surface receptor, pathogen protein, or a specific biomarker.

Antigen Preparation: The selected antigen is produced and purified. This can be done using recombinant protein expression systems, peptide synthesis, or isolating native proteins from biological sources.

Immunization

Host Immunization: Animals (such as mice, rabbits, or other suitable hosts) are immunized with the target antigen to elicit an immune response. This typically involves administering the antigen along with an adjuvant to enhance the immune response.

Booster Injections: Multiple booster injections are often given over a period of weeks to ensure a robust immune response and the generation of high-affinity antibodies.

Hybridoma Technology (for Monoclonal Antibodies)

B Cell Isolation: B cells producing antibodies against the target antigen are isolated from the immunized animal’s spleen or lymph nodes.

Fusion with Myeloma Cells: These B cells are fused with immortal myeloma cells to create hybridoma cells, which can proliferate indefinitely and produce monoclonal antibodies.

Selection and Screening: Hybridoma cells are screened for the production of antibodies that bind specifically to the target antigen. Positive clones are selected and expanded.

Phage Display (Alternative Method for Monoclonal Antibodies)

Library Construction: A diverse library of antibody fragments (such as single-chain variable fragments, scFvs) is constructed and displayed on the surface of bacteriophages.

Panning and Selection: The phage library is screened against the target antigen. Phages displaying high-affinity antibodies are isolated and amplified through iterative rounds of panning and selection.

Sequence Identification: The DNA sequences encoding the selected antibody fragments are identified and used to produce full-length antibodies.

Antibody Characterization

Binding Affinity: The binding affinity of the identified antibodies to the target antigen is determined using techniques such as ELISA, surface plasmon resonance (SPR), or biolayer interferometry (BLI).

Specificity Testing: The specificity of the antibodies is tested against a panel of related and unrelated antigens to ensure that they specifically bind to the target antigen.

Functional Assays: Functional assays are performed to assess the biological activity of the antibodies, such as their ability to neutralize pathogens, block receptor-ligand interactions, or mediate cell killing.

Antibody Engineering

Humanization: If the antibodies are derived from non-human sources, they may need to be humanized to reduce immunogenicity in humans. This involves grafting the antigen-binding regions onto human antibody frameworks.

Affinity Maturation: Antibodies can undergo affinity maturation to improve their binding affinity and specificity. This can be achieved through directed evolution techniques or rational design.

Production and Purification

Expression Systems: The selected antibody genes are cloned into expression vectors and produced in suitable expression systems, such as mammalian cells, yeast, or bacteria.

Purification: The antibodies are purified from the expression system using techniques such as protein A/G affinity chromatography, size-exclusion chromatography, and ion-exchange chromatography.

Preclinical and Clinical Development

In Vivo Testing: The efficacy and safety of the antibodies are tested in preclinical animal models to evaluate their therapeutic potential.

Clinical Trials: Promising antibody candidates undergo clinical trials to assess their safety, efficacy, pharmacokinetics, and pharmacodynamics in humans.

Regulatory Approval

Submission to Regulatory Authorities: Data from preclinical and clinical studies are compiled and submitted to regulatory authorities (such as the FDA or EMA) for approval.

Manufacturing and Quality Control: Large-scale manufacturing processes are developed, and stringent quality control measures are implemented to ensure the consistency and safety of the antibody product.

Conclusion

The antibody discovery process is a multi-step journey that involves the identification and preparation of the target antigen, immunization of hosts, screening and selection of high-affinity antibodies, extensive characterization and optimization, and rigorous preclinical and clinical testing. Each step is critical to ensuring the development of effective and safe therapeutic antibodies for clinical use.

068 月/24

KMD Bioscience-Advantages of Polyclonal Antibodies

Antibody Production Platform

Polyclonal antibodies (pAbs) are antibodies derived from multiple B cell clones. These antibodies are a mixture of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope. Polyclonal antibodies offer several advantages, making them valuable tools in research, diagnostics, and therapeutics.

Broad Epitope Recognition

Comprehensive Targeting: Polyclonal antibodies recognize multiple epitopes on the same antigen, which increases the likelihood of binding to the target even if some epitopes are mutated or masked.

Higher Affinity: The presence of antibodies against multiple epitopes can enhance the overall binding strength (avidity) to the antigen, making them highly effective in detecting and capturing targets.

Increased Sensitivity

Enhanced Signal Detection: Because pAbs bind to multiple epitopes, they can amplify the detection signal in assays, making them particularly useful in applications where high sensitivity is required, such as ELISA, Western blotting, and immunohistochemistry.

Robustness and Versatility

Tolerant to Variations: Polyclonal antibodies are generally more tolerant of minor changes in the antigen structure, such as conformational changes or slight sequence variations, ensuring reliable performance across different samples and conditions.

Versatile Applications: They can be used in a wide range of applications, including immunoprecipitation, flow cytometry, and immunoassays, due to their ability to recognize multiple epitopes.

Cost-Effective Production

Relatively Inexpensive: The production of polyclonal antibodies is generally less costly and time-consuming compared to monoclonal antibodies. This is because the process involves immunizing animals and collecting serum, without the need for hybridoma technology.

Scalable: Large quantities of polyclonal antibodies can be produced relatively easily, making them suitable for applications requiring bulk antibodies.

Quick Development

Rapid Generation: Polyclonal antibodies can be generated quickly compared to monoclonal antibodies. The process from immunization to antibody collection can be completed in a few months.

Suitable for Urgent Needs: This rapid development timeline is advantageous for urgent research needs, new antigen discovery, or response to emerging infectious diseases.

Enhanced Immunogenicity

Effective Against Complex Antigens: Polyclonal antibodies are effective in recognizing complex antigens with multiple epitopes, such as whole-cell antigens, viral particles, and large proteins, making them ideal for detecting pathogens and other complex targets.

Natural Immune Response Mimicry

Physiological Relevance: Polyclonal antibodies more closely mimic the natural immune response to an antigen, providing a more comprehensive and physiologically relevant antibody profile for research and diagnostic applications.

Higher Yield

Sufficient Quantity: The amount of antibody produced from immunized animals is typically high, ensuring an adequate supply for extensive experimental use and large-scale applications.

 

 Conclusion

Polyclonal antibodies offer distinct advantages, including broad epitope recognition, increased sensitivity, robustness, cost-effectiveness, quick development, and versatility. These attributes make them indispensable tools in various fields, from basic research to clinical diagnostics and therapeutic applications. The ability to generate a robust and diverse immune response against antigens ensures that polyclonal antibodies remain a valuable resource for scientists and healthcare professionals.

028 月/24

KMD Bioscience-Advantages of Monoclonal Antibodies

Antibody Production Platform

Monoclonal antibodies (mAbs) are laboratory-made molecules engineered to serve as substitute antibodies that can restore, enhance, or mimic the immune system’s attack on unwanted cells, such as cancer cells. Here are some of the key advantages of monoclonal antibodies:

Specificity

Targeted Therapy: Monoclonal antibodies are designed to bind to specific antigens found on the surface of cells, allowing for highly targeted treatment. This specificity reduces damage to healthy cells and minimizes side effects compared to traditional therapies.

Consistency

Uniformity: Since monoclonal antibodies are produced from identical immune cells cloned from a single parent cell, they are highly uniform in structure and function. This consistency ensures predictable therapeutic outcomes and reliable results in research and diagnostic applications.

Versatility

Wide Range of Applications: Monoclonal antibodies can be used in a variety of ways, including cancer therapy, autoimmune disease treatment, infectious disease management, and more. They can also serve as tools in diagnostic tests and research.

Immunotherapy

Enhanced Immune Response: Some monoclonal antibodies are designed to enhance the body’s immune response against cancer cells. They can work by blocking inhibitory signals on immune cells or by recruiting other components of the immune system to attack cancer cells.

Combination Therapy

Synergistic Effects: Monoclonal antibodies can be combined with other treatments such as chemotherapy, radiation therapy, or other immunotherapies to enhance their effectiveness and improve patient outcomes.

Reduced Toxicity

Fewer Side Effects: Due to their targeted nature, monoclonal antibodies often have fewer and less severe side effects compared to conventional treatments like chemotherapy and radiation, which can damage both healthy and cancerous cells.

Versatile Engineering

Modification and Optimization: Monoclonal antibodies can be engineered to improve their efficacy, stability, and half-life. Techniques such as humanization, conjugation with drugs or radioactive substances, and modification of Fc regions can optimize their performance.

Diagnostic Use

High Sensitivity and Specificity: Monoclonal antibodies are widely used in diagnostic tests due to their ability to specifically bind to unique biomarkers. This high sensitivity and specificity make them valuable in detecting diseases at an early stage.

Therapeutic Monitoring

Track Treatment Progress: Monoclonal antibodies can be used to monitor the presence and progression of disease, allowing for adjustments in treatment regimens based on real-time feedback.

Development of Biosimilars

Cost-Effective Alternatives: The development of biosimilar monoclonal antibodies provides cost-effective alternatives to existing therapies, making treatments more accessible to a broader population.

Monoclonal antibodies have revolutionized the fields of oncology, immunology, and beyond, offering precise, effective, and personalized treatment options that improve patient outcomes and advance medical research.

297 月/24

KMD Bioscience-Antibody Production Guide

Antibody Production Platform

Introduction

Antibody production is a fundamental process in immunology and biotechnology, essential for research, diagnostics, and therapeutic applications. Antibodies, or immunoglobulins, are proteins the immune system produces to identify and neutralize pathogens like bacteria and viruses. The production process involves several steps, from antigen selection to purification and characterization of the antibody.

Types of Antibodies

Polyclonal Antibodies (pAbs)

Source: Produced by different B cell lineages.

Characteristics: Recognize multiple epitopes on a single antigen.

Applications: Widely used in research for detecting proteins in various assays, including ELISA, Western blot, and immunohistochemistry.

Monoclonal Antibodies (mAbs)

Source: Produced by identical B cells cloned from a single parent cell.

Characteristics: Recognize a single epitope on an antigen.

Due to their specificity and consistency, applications are used in diagnostics, treatments, and research tools.

Antibody Production Process

Antigen Preparation

You can select an appropriate antigen to elicit an immune response. This can be a whole protein, peptide, or hapten conjugated to a larger carrier molecule.

Immunization

Polyclonal Antibodies

Animals such as rabbits, goats, or mice are immunized with the antigen. A series of booster injections are given to enhance the immune response. Blood is collected, and serum containing the antibodies is separated.

Monoclonal Antibodies

Mice or other host animals are immunized with the antigen. Spleen cells producing antibodies are harvested and fused with myeloma cells to create hybridomas. Hybridomas are screened to identify those producing the desired antibody.

Hybridoma Technology (for mAbs)

Fusion: Spleen cells from the immunized animal are fused with immortal myeloma cells.

Selection: Hybrid cells are selected and grown in a selective medium (HAT medium) to ensure that only fused cells survive.

Screening: Hybridomas are screened for antibody production, typically using ELISA or other binding assays.

Cloning: Positive hybridomas are cloned by limiting dilution to ensure monoclonality.

Antibody Purification

Polyclonal Antibodies

The serum is subjected to affinity chromatography using antigen-bound columns to isolate specific antibodies.

Monoclonal Antibodies

Supernatants from hybridoma cultures are collected. Antibodies are purified using Protein A or Protein G affinity chromatography.

Characterization

Specificity: Tested using Western blot, ELISA, or immunoprecipitation to confirm the antibody binds to the intended antigen.

Affinity: Determined by surface plasmon resonance (SPR) or other binding assays.

 Isotyping: Identifying the antibody class (IgG, IgM, etc.) and subclass.

Applications of Antibodies

Research

Detection and quantification of proteins in various assays. Study of protein-protein interactions, cell signaling pathways, and cellular localization.

Diagnostics

Disease diagnosis through the detection of specific biomarkers. Use in assays like immunohistochemistry, flow cytometry, and ELISA.

Therapeutics

Treatment of diseases such as cancer, autoimmune disorders, and infectious diseases. Development of therapeutic monoclonal antibodies, such as checkpoint inhibitors in cancer therapy.

Industrial

Quality control and testing in biopharmaceutical manufacturing. Development of biosensors for detecting contaminants.

Conclusion

Antibody production is a critical technology in modern bioscience, enabling advances in research, diagnostics, and therapy. Understanding the intricacies of producing both polyclonal and monoclonal antibodies allows for their effective application across various fields, driving innovation and improving healthcare outcomes.

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Antibody Engineering and Therapeutics-KMD Bioscience

Antibody Production Platform,

Antibody engineering has revolutionized the field of biomedicine, offering highly specific and effective therapeutic options for a wide range of diseases. This article explores the principles of antibody engineering, its various applications in therapeutics, and the advancements that continue to shape this innovative field.

Principles of Antibody Engineering

Antibody engineering involves the modification and optimization of antibodies to enhance their efficacy, specificity, and safety for therapeutic use. The primary techniques in antibody engineering include:

Humanization

Reduce the immunogenicity of non-human antibodies.

Method: Replace mouse antibody regions with human antibody regions while retaining the antigen-binding sites.

Affinity Maturation

Increase the binding affinity of antibodies for their target antigens.

Method: Introduce mutations in the antibody’s variable region and select for higher-affinity variants using techniques like phage display.

Bispecific Antibodies

Enable one antibody to bind to two different antigens simultaneously.

Method: Engineer antibodies with two different binding sites, often by combining parts of two different antibodies.

Antibody Fragments

Create smaller antibody fragments that retain antigen-binding ability but have improved tissue penetration and reduced immunogenicity.

Method: Generate Fab (fragment antigen-binding), scFv (single-chain variable fragment), or other antibody fragments through genetic engineering.

Glycoengineering

Enhance the therapeutic properties of antibodies by modifying their glycosylation patterns.

Method: Alter the glycosylation machinery in the production host cells to produce antibodies with desired glycoforms.

Therapeutic Applications of Engineered Antibodies

Antibody engineering has led to the development of a wide array of therapeutic antibodies used in various medical fields:

Cancer Therapy

Monoclonal Antibodies (mAbs): Target specific cancer cell antigens (e.g., Herceptin for HER2-positive breast cancer).

Bispecific Antibodies: Engage both cancer cells and immune cells to enhance anti-tumor response (e.g., Blincyto for acute lymphoblastic leukemia).

Antibody-Drug Conjugates (ADCs): Deliver cytotoxic drugs directly to cancer cells (e.g., Kadcyla for HER2-positive breast cancer).

Autoimmune Diseases

TNF Inhibitors: Block tumor necrosis factor (TNF) to reduce inflammation (e.g., Humira for rheumatoid arthritis).

Checkpoint Inhibitors: Inhibit immune checkpoints to boost the immune response against autoimmune attacks (e.g., anti-PD-1 antibodies).

Infectious Diseases

Neutralizing Antibodies: Bind to and neutralize pathogens such as viruses and bacteria (e.g., REGN-COV2 for COVID-19).

Passive Immunization: Provide immediate protection by administering pre-formed antibodies (e.g., Palivizumab for respiratory syncytial virus).

Cardiovascular Diseases

PCSK9 Inhibitors: Lower LDL cholesterol levels by inhibiting PCSK9 protein (e.g., Repatha).

Anti-Inflammatory Antibodies: Target inflammatory pathways involved in cardiovascular diseases.

Advancements in Antibody Engineering

Recent advancements in antibody engineering continue to push the boundaries of what is possible in therapeutic applications:

Next-Generation Sequencing (NGS)

Enables the rapid identification of antibody sequences with high specificity and affinity.

Facilitates the discovery of novel antibodies from diverse sources.

Synthetic Biology

Utilizes synthetic genes and pathways to create entirely new antibodies with tailored properties.

Allows for the design of antibodies with improved stability, reduced immunogenicity, and enhanced functionality.

Gene Editing Technologies

CRISPR/Cas9 and other gene-editing tools enable precise modifications of antibody genes.

Facilitates the creation of transgenic animals and cell lines for antibody production.

 Conclusion

Antibody engineering has significantly impacted the landscape of therapeutic development, providing targeted, effective, and safe treatments for a wide range of diseases. With continuous advancements in technology and a deeper understanding of antibody biology, the future of antibody therapeutics looks promising, offering hope for better management and treatment of complex medical conditions.