A Beginner's Guide To Custom Antibody Production
The employment of custom antibodies as potent agents in biology and diagnostics has transformed and restructured the world of modern medicine, environmental and “in situ” monitoring, and laboratory-based science. Polyclonal antibodies, monoclonal antibodies (mAbs), antibody fragments, and recombinant antibodies all utilize various purification techniques.
In addition, the ongoing potential of antibody production through recombinant procedures has extensively enhanced purification requirements, reaching up to 100-kilogram batches. Hence, the process requires ingenious purification strategies and methods. To meet current industry standards for purification, investigators commonly utilize affinity chromatography ligands. However, modern trends consider the movement where protein can be used as a bioaffinity ligand. This is due to several restricting elements**, such as** Protein A leakages from the column, low reusability of resin, high cost, and potential antibody aggregation during low pH desorption.
Additionally, a researcher could achieve antibody purification through genetically fused purification tags, the most typical of which is a poly-His or His6 tag utilized in IMAC. In this process, the utilization of ion-exchange chromatography through the platform of multicolumn countercurrent solvent gradient purification (MCSGP) systems has been quoted by scholars as an ideal replacement for Protein A-based chromatography. This arrangement can recycle partially purified fractions, enhancing the overall protein yield. Furthermore, it can simulate experimental models and setups while achieving commercial yield levels with the utmost purity.
Antibodies come in various shapes and sizes.
Gerald Edelman and Rodney Porter initially elucidated the characteristic Y-shaped antibody structure. In 1972, they were awarded the Nobel Prize in Physiology or Medicine for their discoveries. The Y-shaped structures they found were later acknowledged as immunoglobulin G (IgG). IgG is composed of four polypeptide chains – two light chains (25 kDa) and two heavy chains (50 kDa). Additionally, these chains are linked by disulfide bridges and noncovalent bonds.
The primary operational custom antibody elements are the antigen-binding site and the Fc region. This region is further composed of variable light and heavy domains, with the Fc section associated with constant CH1, CH2, and CH3 areas. Consequently, antibodies in modern medicine today occur in all sizes and shapes. Moreover, they have been discovered in mammals and in different formats all over the animal kingdom.
Regardless of naturally occurring formats, recombinant DNA technology has also facilitated the growth of recombinant antibodies**, such as** disulfide-stabilized Fv antibody fragments, fragment variable (Fv), fragment antigen-binding (Fab), and single-chain fragment variable (scFv). It also includes divalent antibody formats like minibodies and diabodies, and multivalent sections like triomabs, tetrabodies, F(ab')2, and triabodies.
What are the conventional methods?
When it comes to specific methods of antibody purification, researchers typically perform them through affinity chromatography or tag-based procedures. Not only do these methods provide significant purity, but they also offer high selectivity. However, the entire process can be expensive if you intend to perform these activities on a large scale. To reduce costs, one can employ physicochemical separation techniques before affinity chromatography to eliminate the bulk of contaminants and avoid fouling. A brief introduction to both processes is mentioned in the summary below.
Affinity chromatography
Monoclonal antibodies are commonly utilized as potent agents in cancer treatment, inflammatory disorders, and autoimmune conditions. Consequently, such entities require the highest purification standards before being administered to patients. Moreover, monoclonal antibodies undergo batch purification from mammalian cells, such as CHO cells. Researchers must prioritize eliminating impurities like endogenous viruses, host cell proteins (HCPs), DNA, aggregates, and endotoxins, which is integral as these can potentially trigger an adverse immune response in a patient.
In this case, investigators usually perform the removal procedure through several steps and techniques, the most prevalent of which is a combined approach. For instance, using a Protein A affinity step followed by anion-exchange chromatography.
In bioscience, studying affinity tags is essential for a researcher due to their importance in the purification process. During the procedure, you can express and encode the tags as fusion partners with a specific protein. In addition, they can exist as single amino acids, short polypeptide chains, or complete proteins. Despite facilitating identification, specified affinity tags can also profoundly increase protein stability and promote native folding.
N-terminal GST
The N-terminal glutathione S-transferase (GST) tag is typically utilized as an affinity tag for recombinant antibody purification. GST is a dimeric protein initially discovered in Schistosoma japonicum. When it comes to the elution conditions, they are mild**, as reduced** glutathione can be utilized within a competitive elution protocol.
However, the purification process of highly concentrated GST-fused proteins can lead to protein aggregation, which may be problematic depending on the chosen application. In addition to these specific applications, high-molecular-weight fusions can lead to fully or partially insoluble aggregates.
Physicochemical Fractionation: Membrane Filtration and Precipitation
While recovering the initial antibodies, it is essential to reduce turbidity and facilitate impurity removal. Turbidity, emerging due to the presence of cell debris during tissue preparation or cell lysis, should be removed before adding the mixture to chromatographic columns, as it may clog the column bed.
Moreover, the physical procedures of primary antibody recovery, such as filtration and centrifugation, are routinely performed in the initial stages of the purification process. When researchers perform clarification through centrifugation, particle separation is based on density and centrifugal force.
Centrifugation expedites the separation of antibody-containing supernatant from cellular debris and cells. However, factors like residence time, g-force, and discharge frequency can influence clarification efficacy and vary considerably while scaling up from small-scale to large-scale centrifugation.
Membrane filtration—including ultrafiltration, microfiltration, and depth filtration—allows molecular separation by size, whereas charged ultrafiltration can detach proteins through factors like charge and molecular weight. For instance, investigators use cellulose-based ultrafiltration membranes to separate various therapeutic monoclonal antibodies from CHO cells. Regarding microfiltration membrane chromatography, it is important to know that the process can remove biological entities like viruses, DNA, and protein contaminants from fluids utilizing microporous membrane filters, such as those made from polyester or polypropylene.
What’s depth filtration?
Depth filtration is the primary workhorse of cell clarification. An investigator can utilize depth filtration before complex media are applied to chromatography columns. Depth filtration is used when there is a high propensity for the medium to plug or clog polishing membrane filters. To put it simply, depth filters are usually composed of filter aids and cellulose fibers bound with a positively charged polymer resin. During the depth filtration procedure, contaminant particles are retained within the filter matrix instead of accumulating on the surface.
Based on particle retention theory, separation occurs through a combination of size exclusion and adsorption via ionic, hydrophobic, and other interactions. In addition, chemical processes and techniques for reducing turbidity comprise precipitation, flocculation, and crystallization.
Crystallization is a highly beneficial tool. It can contribute to the concentration, stabilization, and purification of the entire antibody product without the requirement for affinity tags or their subsequent removal. Advancements in such methodologies are discussed in considerable detail in the “non-chromatographic techniques” section.
Antibody production
When it comes to the term "antibody production," it has both specific and general denotations. In a general sense, it refers to the entire process of developing a particular antibody for use in several forms of experiments. This may include immunization steps, immunogen preparation, collection, hybridoma creation, purification, screening, isotyping, and labeling for consistent use in various applications.
In a more specific sense, the production of antibodies also comprises antigen sample preparation and secure injection into farm or lab animals. This is because host animals produce high titers of antigen-specific antibodies within their serum.
In the case of polyclonal antibody production, researchers can recover antibodies directly from blood bleeds. On the other hand, monoclonal antibodies are made by fusing antibody-secreting cells with myeloma cells to develop hybridoma cell lines that express specific antibodies. Some bioscience departments also offer specific protein expression services and antibody sequencing services.
Successful antibody development relies on straightforward implementation and planning of several essential considerations and techniques:
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Purifying target antigens
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Selecting a suitable immunogenic carrier protein
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Conjugating the carrier and antigen protein to produce immunogens
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Immunizing target species utilizing suitable adjuvants and dosing schedules
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Screening serum or hybridoma for antibody titers and isotypes
Antibody purification
The steps involved in antibody purification comprise antibody isolation from polyclonal antiserum, culture supernatant, or ascites fluid. In addition, purification techniques range from crude to refined methods as outlined below:
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Crude: Fractional precipitation of total serum proteins, including immunoglobulins.
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General: Group-specific antibody affinity purification (e.g., Protein A/G) without antigen specificity.
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Thorough: Antigen-specific affinity purification of selected antibodies that interact only with specific antigen molecules.
Antibody characterization
This process comprises three types of activities usually performed at multiple stages throughout the antibody production and purification procedure:
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Screening: Identifying antibody samples with antigen-binding specificity.
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Titering: Determining antibody functional assay titers and concentrations.
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Isotyping: Determining monoclonal antibody classes and subclass identities.
The screening step is one of the most important stages in identifying which hybridoma clones or animals are producing the highest levels of antigen-specific antibodies. Such a step is part of antibody validation, which is typically accomplished utilizing ELISA methodologies. Moreover, researchers estimate antibody concentration by using either standard protein assays or species-specific assays, such as specialized microagglutination assay platforms. While antibody titer is correlated to concentration, it refers to the functional potency of specific antibody samples.
Studies have stated that measuring titers involves determining the functional dilution of antibody samples essential for detection in a specific assay, like ELISA. Additionally, isotyping comprises identifying the subclass and class of monoclonal antibodies. It is vital to know that this is an integral step in producing antibodies, whether polyclonal or monoclonal. This also informs the selection of suitable modification and purification processes for a specific molecule. In the end, isotyping is relatively easy to determine with ready-to-use commercial antibody kits.
Antibody fragmentation
Investigators have previously modified antibodies for specific uses through various methodologies involving fragmentation into smaller antigen-binding units. While most antibodies are utilized in their whole-molecule forms, the performance of certain experiments and techniques can be enhanced by employing fragments whose non-binding portions have been eliminated.
Antibody fragmentation refers to processes used to cleave the entire antibody molecule and remove portions that are not necessary for binding. F(ab')2 and Fab are the antibody fragments most frequently developed and used by investigators.
Antibody immobilization and labeling
If you’re considering how antibodies are produced and purified for use as antigen probes, their application in any technique (Western blotting, ELISA, immunohistochemistry, or cellular imaging) relies on incorporating a mechanism to detect the antibodies.
Methods that utilize antibodies for immunoprecipitation or various types of affinity purification depend on specific coupling mechanisms for binding and immobilizing them to beaded agarose resin. Processes for achieving such results involve similar considerations and chemical techniques to those used in antibody labeling.
Features and elements of monoclonal antibodies
Studies have shown that you can characterize monoclonal antibodies thoroughly prior to use. Such characterization must include the biochemical and biological properties of the antibodies. Through this process, a researcher can assess the reactivity and specificity of the antibodies.
Biochemical characterization
When it concerns biochemical properties, one should describe them in detail. At a minimum, they should be classified by an investigator while proceeding with experiments. Moreover, you should describe the immunological nature of the antibodies in detail, including elements such as antigenic specificity, the antibody epitope, the ability to activate complement, binding capacity, antibody-dependent cellular cytotoxicity (ADCC), cytotoxic properties, antigen relevance, and the potential to interact with immunocompetent cells.
Single harvest production
It is important to note that the maximum permissible generation limit for production is defined based on cell line stability and production levels. On the other hand, for multiple harvest production, this includes recurring cultivation that a researcher can use to maintain the system over an extended period.
The Bottom Line
Since biochemistry and the biosciences have significantly transformed in the past few decades, investigators and researchers have found more methods to support their experiments. Regarding custom antibody production, several companies now offer protein expression and purification services, peptide synthesis, and gene synthesis, alongside protein production services that account for even the minutest factors involved.