Purification is the process of isolating and refining a desired component from a complex mixture. Whether used in industrial manufacturing, pharmaceutical development, or scientific research, purification ensures that the target material is separated from impurities, including salts, proteins, solvents, or other unwanted substances. Methods such as chromatography, precipitation, and filtration, are used to achieve high purity levels, tailored to the specific properties of the substance being purified.
In antibody production, purification is critical for obtaining functional, high-quality antibodies suitable for research, diagnostics, or therapeutic applications. The following sections detail the techniques and principles behind purification, with a focus on antibodies.
Antibody Purification: A Key Step in Antibody Production
Antibody purification is the process of isolating monoclonal or polyclonal antibodies from complex biological samples, such as serum, culture supernatants, or ascites fluid. This step is to ensure the antibodies are free from contaminants, functional, and ready for use in applications like ELISA, Western blotting, or therapeutic interventions.
A typical monoclonal antibody recovery process. DOI:10.4161/mabs.2.5.12645
Phases in Antibody Purification
Capture Phase: The initial step focuses on isolating antibodies from the mixture. This is often achieved using affinity chromatography, which leverages the specific interactions between antibodies and ligands like Protein A, G, or L.
Intermediate Purification Phase: This phase removes major contaminants, such as host cell proteins and nucleic acids, to improve antibody purity. Methods like ion-exchange chromatography are commonly employed.
Polishing Phase: The final step ensures the removal of aggregates, endotoxins, and trace impurities. Techniques such as size-exclusion chromatography (SEC) or hydrophobic interaction chromatography (HIC) are effective at this stage.
Antibody Purification Techniques
Affinity Chromatography
Affinity chromatography is the most widely used method for antibody purification. It utilizes immobilized ligands such as Protein A, Protein G, or Protein L, which bind specifically to antibody domains.
Protein A: Binds to the Fc region of IgG antibodies from several species, particularly useful for rabbit, pig, and cat IgG.
Protein G: Broader binding capacity, effective for human and mouse IgG subclasses.
Protein L: Targets the kappa light chains in the Fab region, enabling the purification of various immunoglobulin classes
This method often achieves high purity (>95%) in a single step. However, additional polishing can be done for therapeutic-grade antibodies.
Ion-Exchange Chromatography (IEX)
IEX separates antibodies based on their net charge. Columns are designed for either:
By adjusting the pH or ionic strength of the elution buffer, antibodies are selectively released, enabling high-resolution purification. IEX is often combined with other methods to achieve optimal purity.
Size-Exclusion Chromatography (SEC)
SEC, also known as gel filtration, separates molecules based on size. Larger antibodies elute first, while smaller contaminants take longer to pass through the porous beads. This technique is particularly effective for removing aggregates and ensuring a monomeric antibody product, making it an essential step in polishing.
Hydrophobic Interaction Chromatography (HIC)
HIC separates antibodies based on their hydrophobicity. Under high-salt conditions, hydrophobic regions of the antibody interact with the hydrophobic ligands on the chromatography resin. This method is commonly used in combination with other techniques for aggregate clearance and host cell protein reduction.
Optimizing Large-Scale Antibody Purification
Scaling up antibody purification involves several key considerations:
Resin Selection: Use high-capacity resins to maximize yield without compromising purity.
Process Automation: Automation ensures consistency and reproducibility across large batches.
Buffer Optimization: Tailor buffer conditions to enhance antibody binding and elution without denaturation.
Contaminant Removal: Incorporate steps for viral inactivation, endotoxin removal, and aggregate clearance.
By streamlining these processes, manufacturers can meet the high-quality standards required for therapeutic antibodies.
At Biointron, we specialize in recombinant antibody expression and purification services. Our services cater to research and therapeutic needs, enabling our partners to focus on innovation while we deliver high-quality antibodies tailored to their specific applications.
