Antibody purification is a crucial process to obtain high-quality antibodies for various applications. However, there is more than one method in antibody purification, from rather crude methods to high precision purification methods.
In this blog post, we will explore different methods for purifying antibodies in the context of antibody production, with a focus on monoclonal antibodies (mAbs) and immunoglobulins (IgGs). We will discuss three important approaches in more detail, but will also mention other important methods in antibody purification.
Antibody purification – what is it and why is it necessary?
The purification of antibodies is a critical step in harnessing their full potential for various applications. During the production of antibodies, whether they are naturally produced or generated through hybridoma technology, they are often accompanied by other cellular components and impurities. This impure mixture can hinder the effectiveness of antibodies in research, diagnostics, and therapeutic applications.
The necessity of antibody purification arises from several key reasons:
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Obtaining high purity: Antibodies, especially monoclonal antibodies (mAbs), need to be purified to achieve high levels of purity. Removing unwanted contaminants ensures that the antibodies exhibit superior specificity and functionality, increasing their reliability in various assays.
Isolating specific antibody subclasses: Different subclasses of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, IgA, and IgE, may be present in the antibody mixture. Purification allows the isolation of specific subclasses, which may possess distinct functions and therapeutic properties.
Separating heavy and light chains: Antibodies are composed of two heavy chains and two light chains. Purification facilitates the separation of these chains, allowing the study of their individual characteristics and functionalities.
ELISA and diagnostic assays: In diagnostic applications like Enzyme-Linked Immunosorbent Assay (ELISA), purified antibodies are required to ensure accurate and reliable results.
Key steps in antibody purification
Antibody purification involves several key steps to obtain pure and functional antibodies, such as centrifugation or filtration. Usually, the antibody-containing sample is loaded onto the purification column. Then, the antibodies selectively bind to the matrix’s ligands or charged groups, while unwanted components pass through as flow-through. The column is washed to remove impurities, and the bound antibodies are eluted in purified fractions. Finally, the purified antibodies are collected and assessed for their quality and functionality. These steps ensure the isolation of high-quality antibodies for various biotechnological and biomedical applications.
Affinity chromatography is a powerful and widely used method for the purification of antibodies, especially monoclonal antibodies (mAbs). This technique takes advantage of the specific binding between antibodies and their corresponding antigens. The principle lies in utilizing a solid matrix with covalently attached ligands that mimic the target antigen, allowing the selective capture and purification of the desired antibodies.
Principle of affinity chromatography
Affinity Chromatography purifies antibodies based on specific binding interactions. Immobilized ligands mimic the target antigen, selectively capturing desired antibodies or Fc-fusion proteins while unwanted components flow-through. Elution releases purified antibodies. It offers high specificity and gentle purification for various antibody formats.
Advantages of affinity chromatography
Advantages of affinity chromatography include:
High specificity: Affinity chromatography ensures the isolation of the target antibodies with exceptional specificity, reducing the presence of non-specific binding.
Gentle Purification: This method is gentle and does not involve harsh conditions or denaturing agents, preserving the biological activity of the antibodies.
High binding capacity: Affinity columns can handle large volumes of antibody-containing samples, making it suitable for purifying antibodies on both small and large scales.
Selective capture of antibody fragments: Besides full-length antibodies, affinity chromatography can also capture Fab fragments, enabling the purification of antibody fragments with desired specificities.
Limitations of affinity chromatography
Affinity chromatography does have some limitations. Here are some downsides of this method:
Cost: The use of affinity chromatography matrices can be expensive, particularly for large-scale purification.
Antigen requirement: The availability of the specific antigen or ligand for immobilization is necessary, which may limit its application for certain antibodies.
Method 2: Size exclusion chromatography
Size Exclusion Chromatography (SEC), also known as gel filtration chromatography, is a widely used method for purifying antibodies based on their molecular size. This technique separates molecules in a sample by their size, allowing the isolation of antibodies from other larger or smaller compounds.
Principle of size exclusion chromatography
Size Exclusion Chromatography (SEC) separates antibodies based on their molecular size. A column with porous beads allows smaller antibodies to enter the pores, resulting in delayed elution, while larger molecules elute first. This gentle method preserves antibody integrity and separates aggregates, ensuring highly pure and functional antibodies for various applications.
Advantages of size exclusion chromatography
Size exclusion chromatography comes with several advantages – here are some of them:
Gentle purification: SEC does not involve harsh conditions or denaturing agents, making it suitable for preserving the structural integrity and bioactivity of antibodies, including the Fc region.
Removal of aggregates: SEC effectively separates aggregated antibodies from the monomeric form, ensuring a high degree of purity.
Versatility: This method can be used for the purification of various antibody formats, including full-length antibodies, antibody fragments, and recombinant antibodies.
Ease of use: SEC is relatively straightforward and does not require extensive optimization, making it an accessible method for many laboratories.
Limitations of size exclusion chromatography
Some disadvantages of size exclusion chromatography include:
Limited binding capacity: The binding capacity of SEC columns may be limited compared to other chromatographic methods, necessitating larger column volumes for large-scale purification.
Time-consuming: SEC can be relatively time-consuming due to the diffusion-based separation process.
Resolution for similar-sized molecules: When dealing with closely related molecules of similar sizes, such as different antibody formats, SEC may exhibit lower resolution.
Method 3: Ion exchange chromatography
Ion exchange chromatography (IEC) is a widely used method for purifying antibodies based on their net surface charge. This powerful technique exploits the reversible electrostatic interactions between charged antibodies and oppositely charged ligands on a chromatography matrix.
Principle of ion exchange chromatography
Ion Exchange Chromatography purifies antibodies based on their net surface charge. Charged groups on a solid matrix attract antibodies with opposite charges, selectively capturing them. Elution with an increasing gradient releases purified antibodies based on their charge and molecular weight. It offers high resolution and versatility for various antibodies but requires careful optimization to avoid protein aggregation.
Advantages of ion exchange chromatography
Major advantages of exchange chromatography are:
High resolution: IEC provides high-resolution separation of antibodies based on their net charge, ensuring excellent purity.
Ability to handle complex mixtures: IEC can efficiently purify antibodies from complex samples, such as cell culture supernatants and ascites fluids, containing multiple components and solvents.
Applicable to various antibodies: This technique is suitable for a wide range of antibodies from different cell lines, including monoclonal antibodies and antibody fragments.
Limitations of ion exchange chromatography
Exchange chromatography does come with some limitations. Two downsides of this purification method are:
Protein aggregation: Harsh elution conditions may lead to protein aggregation, impacting antibody integrity and functionality.
Salt removal: Additional steps may be required to remove salts and other substances introduced during elution.
List of further antibody purification methods
In addition to these three frequent antibody purification methods, there are several other techniques that researchers and biotechnologists utilize for obtaining pure antibodies. Each protein purification method comes with its unique advantages and is chosen based on the specific requirements of the purification process.
Here is a list of 10 additional antibody purification methods:
Hydrophobic interaction chromatography (HIC): HIC utilizes the hydrophobic properties of antibodies to separate them from other molecules based on their surface hydrophobicity.
Precipitation techniques: This method involves the use of reagents, such as polyethylene glycol (PEG) or ammonium sulfate, to precipitate antibodies, enabling their separation from other components.
Filtration methods: Filtration techniques, like tangential flow filtration (TFF) and diafiltration, efficiently separate antibodies based on their size.
Protein L affinity chromatography: Protein L captures antibodies through their interactions with the light chains, providing an alternative to traditional protein A/protein G methods (proteins usually deriving from Staphylococcus aureus)
Affinity purification with recombinant ligands: This approach employs engineered proteins or recombinant ligands with high affinity for antibodies, allowing for efficient purification.
Antibody purification using IgY capture: IgY capture utilizes avian antibodies from chicken egg yolk, providing a unique method for antibody purification.
Precipitation with caprylic acid: Caprylic acid precipitation is a mild method for removing impurities and inactivating viruses during purification.
IgM-specific purification methods: Specialized techniques for purifying IgM antibodies, typically involving size exclusion chromatography and ion exchange chromatography.
Agarose-based chromatography: Agarose-based matrices provide a high binding capacity for antibody purification, suitable for large-scale applications.
IEX and HIC coupled chromatography: Combining ion exchange chromatography with hydrophobic interaction chromatography can enhance the purification process and improves overall purity.
Optimizing large-scale antibody purification
In biotechnology, antibody production is performed on different scales – be it in the production of polyclonal antibodies for research purposes or recombinant protein production in vaccine manufacturing. In order to optimize the scale-up process, there are some aspects to be considered:
Method selection: Choose efficient methods like affinity chromatography, ion exchange chromatography, and size exclusion chromatography for high yields and selectivity.
Advanced resins: Utilize high-capacity resins with enhanced antibody binding for improved efficiency.
Process automation: Automate purification steps for consistency and reproducibility at a high flow rate.
Buffer and elution: Fine-tune conditions like elution buffer to maximize antibody recovery without denaturation.
Contaminant removal: Implement additional steps for viral inactivation, endotoxin removal, and aggregate reduction.
Continuous processing: Adopt continuous purification for streamlined production.
Quality control: Ensure consistent product quality through robust analytics and assays.
Economic Considerations: Balance performance with cost-effectiveness for successful large-scale purification.
Recombinant antibody purification at evitria
As an important antibody type, rAbs hold immense potential in medicine and research, which is why labs around the globe are in need of high quality recombinant antibodies.
evitria is specialized in providing recombinant antibody expression services based on CHO cells. With this focus, we are able to express and purify antibodies at high throughput and in an outstanding quality – offering purification methods like:
affinity chromatography using protein A and alternative resins
specialized columns like IgM-specific purification or IMAC
size exclusion chromatography
ion exchange chromatography
This is how we help our partners worldwide in succeeding in their antibody projects – by providing them with just the antibody products they need.
