Antibody Purification

Antibodies are the main contributor of body’s immune system which are proteins in nature and mainly secreted by plasma cells. Antibodies are the main component of the humoral immune response. Antibodies are members of immunoglobulin family. They constitute 20% of the plasma protein. Different population of antibodies are present in different parts of the body. They are host proteins produced in response to foreign molecules known as antigens. When foreign molecules like virus and bacteria invade body’s immune system, antigens or special molecules of the pathogen get recognized by the antibodies and elicit immune response. This antigen-antibody binding is required for the neutralization and clearance of the pathogen from body.  This is the body’s self-defence mechanism against foreign bodies.  

Antibodies are produced in special white blood cells, B-lymphocyte. B- Lymphocytes in the body have cell surface receptors for foreign bodies or antigens. On attachment of these bodies, they differentiate to form lymphoid or plasma cells, which in turn produce millions of antibodies secreting out in blood circulation.  Antibodies generally exists in two forms; a free circulating antibodies present in blood plasma and a bound form attached to B-cell surface.   Science has paved way for the understanding of antigen-antibody interaction and their characteristics. Now scientist can manipulate these characteristics of antibody fragments for different usage.

There are many application of antibodies in diagnosis and therapy. The use of recombinant technology opens up the potential to create an infinite number of combinations between immunoglobulins. We can now manipulate these fragments to our advantage. As the antibody usage has increased, our understanding about these molecules has increased. Various methods of purification technique have been identified and used for commercial applications. All these applications require antibody in a purified state. The state of purity depends on the scale of application. The parameters for purification entirely depend on its intended application.  The parameters can be physical such as size, charge, pI, stability. Purity can be a scale from microgram to a gram or any other weight measurement.

Immunoglobulin Classes

In humans and rodents, there are five immunoglobulin classes or isotypes, which differ in the primary structure, carbohydrate content, and antigenic properties of their heavy chains (Table 1). By contrast, the light chain types are the same for all immunoglobulin classes. Each immunoglobulin molecule contains light chains of one of two types, either lambda (2) or kappa (x). The 2 and light chains have different primary structures and antigenic properties. They are usually free of carbohydrate components. The ratio of x/2 chains in human and swine immunoglobulins is about 60:40, whereas in mouse, rat and rabbit immunoglobulins it is about 95:5. Some other mammals such as dog, cat, and farm animals (ox, sheep, and horse) have mainly 2 chains and chicken immunoglobulins contain only 2 chains.

Structure of Immunoglobulins

Antibodies or iunoglobulin are Y shaped and glycoprotein in nature consisting of one or more units. All immunoglobulins have a common structure with four polypeptide chains; two identical heavy (H) chains almost 50-70kD in molecular weight, each carrying covalently attached oligosaccharide groups; two identical, nonglycosylated light (L) chains of molecular weight 23kD. An inter-chain disulfide bond joins the heavy chain and the light chain together along with non covalent bonds.  Different types of imunoglobulins have different number of interchain disulphide bridge. The heavy chains are also joined by disulfide bonds. The disulfide bonds are present in the flexible region of the heavy chain known as hinge region. In mammal, there are five different types of heavy chains are represented with the greek letter sympbol α, δ, ε, γ, and μ. Whereas, mammalian light chain only consists of two parts known as  lambda (λ) and kappa (κ). All four polypeptide chain s contains constant (C) and variable (V) regions found at the carboxyl and amino terminal portions, respectively. In the tip part of the Y shaped antibody molecule, variable (V) regions are present.

This region is composed of 110-130 amino acids and shown great variance between different immunoglobulins and also specific for antigen binding. The variable region represents end part of both heavy and light chain. On the other hand the constant region ( C)  is important for antigen destruction and shows less variation  . All light chains ( L) have a single V region ,VL and a single constant region CL.  On the other hand, Heavy chain has one variable region VH and contains 3 C region forming CH1, CH2 and CH3. The V regions combine to form two identical antigen binding sites. Immunoglobulins are divided into five major classes according to their H chain components: IgG, IgA, IgM, IgD, and IgE. The light chain molecules have k chains and l chains. Individual molecules have either one of the molecule. The variable region of the immunoglobulin can be further subdivided into two part called hypervariable (HV) and framework (FR). For a given position, HV region will exhibit high ratio of amino acid difference.

Three HV regions, HV1, HV2 and HV3 are exists between heavy and light chain. Whereas, FR regions have comparatively stable amino acid sequence and work to separate HV regions. The function of HV region is to directly contact the antigenic surface and thus called as complementarity determining regions, or CDRs. whereas, the FR part is useful for placing the HV region in position to be in contact with antigen.  Depending on the heavy chain imunoglobulins are further divided into five major classes; IgG , IgA  IgM  IgD and IgE. In each of these class differs from each other by the presence of different heavy chain. IgG , IgA  IgM  IgD and IgE contains the heavy chain γ,α, μ, δ and ε respectively. Whereas for all the imunoglobulin class, the light chain is either κ or λ. 

IgG is most widely found iunoglobulin in serum and expressed on the surface of B cells. IgG can be firther subdivided into IgG1, IgG2, IgG3 and IgG4.  IgA is the second most commonly available immunoglobulin. They are mostly found in secretion such as mucous, tear or saliva and also in milk as secreted form. IgA is further classified to subclass IgA1 and IgA2. The third immunoglobulins which comes in the list of availability is IgG M which is expressed both in immature and mature B cells. It has a pentameric structure and also shows expression in fetus. IgD immunoglobulin works together with for the development of B cell. IgE on the other hand is least available immunoglobulin present in serum and are mostly involved in allergic reaction.

Immunoglobulin G

Immunoglobulin G (IgG) is the major class of immunoglobulins. About threequarters of all serum immunoglobulins belong to this class. IgG molecules consist of two heavy y and two light chains (2y + 2L). Normally each molecule of IgG has two identical antigen combining sites. Upon electrophoresis at alkaline pH, IgG migrates slower than almost all other serum proteins. Each IgG molecule contains about 3% carbohydrates, whereas that of other immunoglobulins is usually much higher (8-12%). After a secondary immunization, B lymphocytes secrete predominantly IgG molecules.

IgG, unlike other immunoglobulins, can cross the placenta barrier and can penetrate into extravascular areas. IgG molecules are able to react with Fcy receptors present on the surface of macrophages and some other cells. The interaction with Fc receptors initiates various effector reactions and particularly facilitates the destruction of potentially harmful germs recognized by IgG antibodies. IgG molecules can also activate complement by the classical pathway. In humans and mice there are four IgG subclasses, which differ in the structure of their heavy chains. Other animals have from one (rabbit) and two (sheep) to four (cattle) and five (horse and swine) IgG subclasses.

Immunoglobulin M

Immunoglobulin M (IgM) is a high molecular weight protein (macroglobulin), consisting of five or rarely of six subunits (IgM monomers). Like IgG molecules, the IgM monomers are composed of two heavy and two light chains, which are linked together by disulfide bridges. IgM appeared early in evolution and primitive vertebrates have macroglobulins whose structure resembles that of mammalian IgM molecules. IgM are the first immunoglobulins synthesized by neonates and are the preponderant class of immunoglobulin molecules appearing during early phases of immune responses. In the monomeric form, IgM functions as an antigen-specific part of the B-cell antigen receptor on the surface of unstimulated B lymphocytes. The antigen receptors with the participation of the/l chains are very important for the normal development of B cells.

The IgM monomers are found at a low concentration in human serum. Each pentameric IgM molecule is composed of 10 heavy ~) chains, 10 light chains and usually one joining (J) chain. The B cells can also secrete functionally active IgM hexamers lacking J chains but the amount of hexamers in serum is no more than 5% of total IgM. The carbohydrate content of IgM is high, about 12%. A pentameric IgM molecule has 10-antigen combining sites and can bind 10 small antigens (haptens). However, due to steric restrictions, only five large antigen molecules can be bound by one IgM molecule. The anntibody activity of IgM is destroyed upon reduction of the intersubunit disulfide linkages. Such a reduction can easily be achieved with very low concentrations of reducing agents, such as dithiothreitol or mercaptoethanol.

Immunoglobulin A

Immunoglobulin A (IgA), the third major class of immunoglobulins, plays the most important role in mucosal immunity. More IgA is produced than all other immunoglobulin isotypes combined. IgA molecules are present in serum, in the gut, and in exocrine secretions, such as saliva, colostrum, breast milk, and tears. The IgA molecules of higher vertebrates are synthesized mainly in gastrointestinal lymphoid tissue. The gut and other mucosal surfaces, which are together 20 times larger than the surface of the skin, are the main sources of the pathogen invasion. On mucosal surfaces, IgA molecules inhibit the binding of microorganisms that try to penetrate through the mucosa, and thus prevent their invasion into the body. There are two IgA subclasses in humans, IgA1 and IgA2, with two allelic variants [A2m(1) and A2m(2)]. IgA1 comprises most of the IgA in the serum (up to 90%) but 60% of IgA molecules in the gastrointestinal tract belongs to the IgA2 subclass. The constant region of IgA1 differs from that of IgA2 at 22 residue and there is a 13-residue deletion in the hinge region of IgA2 as compared with the hinge of IgA1.

Immunoglobulin D

Immunoglobulin D (IgD) is a minor class of immunoglobulins. These molecules are monomers composed of two heavy and two light chains (2δ+2L), without any additional chains. They are extremely sensitive to proteolysis. In membrane form, IgD molecules together with IgM monomers are present on the surface membranes of human and murine mature B lymphocytes and serve as an antigen-specific part of the B-cell antigen receptors. The quantity of IgD molecules on the surface of B cells is an order of magnitude higher than that of IgM. Since the discovery of this class of immunoglobulins a number of studies were performed aimed at understanding the biological role of this protein. However, still the precise functions of membrane IgD are still not known. In IgD deficient mice obtained by gene targeting, the number of mature B cells is the same as in normal mice and antibody response is not different from that in wild type animals. IgD molecules can anchor on the cell membrane via a glycosyl phosphatiylinositol linkage.

Immunoglobulin E

Immunoglobulin (IgE) is another minor class of immunoglobulins, molecules of which are present in serum at very low concentrations. IgE molecules exist in a monomeric form consisting of two heavy and two light chains (2ε+2L) and are the most important participants of allergic reactions. Through their Fc portions, IgE molecules bind to the Fcε receptors that are present on the surface of mast cells and basophils. The crosslinking of such membrane bound IgE antibodies by multivalent antigens, triggers the release of chemically active substances, such as histamine, leukotrienes, prostaglandins, and chemotactic factors, from the cells. These substances initiate allergic and inflammatory reactions and serve as chemoattractants for other cells. The serum level of IgE antibodies in patients with allergic conditions (hay fever, for example) or with chronic parasitic infections are usually elevated several hundredfold and can be used for diagnostic purposes.

Detection of Immunoglobulins

In biological fluids and in cell culture media, immunoglobulins are present with many other proteins. To detect immunoglobulins, various immunological methods that are based on their specific antigenic properties or antibody activity, are applied. Different commercial anti-immunoglobulin antisera are used in several variants of semiquantitative precipitation assays and the popular enzyme-linked immunoadsorbent assay (ELISA) technique. To determine the exact quantity of immunoglobulins in a sample, anti-immunoglobulin antibodies can be immobilized on one or another insoluble support, such as cyanogen bromide-activated Sepharose or small particles of modified cellulose. The resulting immunoadsorbents are used to specifically and quantitatively adsorb immunoglobulins from the sample. The amount of adsorbed immunoglobulins can be measured by one of the assays for protein detection. If the immunoglobulin preparation to be studied is radiolabeled, the sensitivity of the immunoadsorbent technique is enhanced greatly. The immunoadsorbent pellet with adsorbed immunoglobulins are put on filter paper disks and the radioactivity is measured in a scintillation counter. To ensure that all immunoglobulins are adsorbed, an additional portion of the immunoadsorbent can be added to the sample after the sedimentation of the first portion.

Isolation of Immunoglobulins

Several methods to isolate immunoglobulins based on their physical and chemical differences from other serum proteins have been devised. One of the most useful techniques to purify IgG involves precipitation with sulfate ions and subsequent chromatography on DEAE-cellulose. The high affinity of the staphylococcal proteins A and G for IgG has been widely used to obtain highly purified preparations of IgG of most mammalian species.

IgG subclasses can be isolated by several methods. For example, IgG3 is the only human IgG subclass that does not react with protein A. Therefore, application of an IgG pool to a protein A-Sepharose column will result in an effluent containing only IgG3. Various immunoglobulin subclasses can be separated on the basis of their differential susceptibilities to proteolysis. For instance, the papain resistance of human IgG2 and trypsin resistance of rat IgG2a facilitates their separation from other IgG subclasses. To isolate large quantities of IgA for passive protection on mucosal membranes is not an easy task. However, a procedure based on classical chromatographic methods was described to isolate IgA from different sources, such as milk, bile, hybridomas and transfected cells, on a laboratory scale as well as to separate different forms of these immunoglobulin molecules.

Chimeric Antibody Molecules and Humanization

Several approaches are used to minimize immunogenicity of rodent monoclonal antibodies. One of them is the construction of chimeric molecules that have rodent variable regions or Fabs joined to constant regions of human immunoglobulins. Chimeric molecules were obtained that have mouse antihapten antibody activity with human effector functions like those of human IgM and IgE . A number of chimeric antibodies, which are able to recognize cancer-specific cell antigens, were developed and clinical trials were performed. As a rule, the chimeric antibodies are less immunogenic than their rodent counterparts and retain the effector functions. A good example is a study of Fab from the 7E3 monoclonal antibody against the platelet receptor that inhibits in vivo platelet thrombus formation.

A significant portion of patients exhibited an immune response after injections of the murine 7E3 Fab fragment. Even though the immune response was mainly directed against the 7E3 variable regions, the anti-7E3 immune reactions were dramatically reduced when the constant parts of Fab were replaced with human ones. Immune response to administered antibodies could be further reduced by the so-called humanization procedure (complementary-determining region [CDR]-grafting,). According to this procedure, CDR parts of the murine or rat variable region taken from antibody molecules with known three-dimensional structure are transplanted on to the human heavy and light chain framework residues.

Minimal Antibody Fragment (Fv)

Another approach is the construction of the minimal Fv fragment with antigen binding activity. There are only rare cases of the Fv fragment being isolated by proteolysis of the whole antibody molecule. If V H and V L fragments obtained as a result of the expression of antibody variable bacterial cells are recombined, the product, the Fv fragment, is unstable because the V L and V H units can easily dissociate. More useful are single-chain Fvs (scFvs), recombinant V L and V H fragments covalently tethered together by a polypeptide link and forming one polypeptide chain. Polypeptides with the average length of 5-18 amino acids are usually used as links. They are rich in serine and glycine residues, to which introduce flexibility and in charged glutamic acid and lysine residues, which improve solubility. Due to small size, rapid clearance in vivo, stability, and easy engineering, scFvs have various potential applications in the diagnosis and treatment of diseases, particularly of cancer. Since then dozens of scFvs with different specificities have been constructed. They are potentially useful in the imaging of tumors after radiolabeling and for genetic fusion to potent toxins (immunotoxins). Anti-idotypic single chain Fv fragments with the internal images of toxins can display a therapeutic activity against fungal pathogens (The monovalency of scFv is a disadvantagevand attempts were made to create constructs with di- or multivalency with increasingvcombining efficiency. 

Antibody fragments

Fab, Fab’, (Fab’)2, and Fv

Antibody fragments are produced either by genetic or chemical procedure. Partial enzymatic digestion of immunoglobulins generates biologically active antibody fragments. These fragments can also be produced using recombinant technology Reducing chemical agents are used to digest the disulphide bond present in the hinge region. Whereas, protein digestion of antibody includes proteases like pepsin or papain. On the other hand when genetic engineering is applied, antibody fragments which are generated has the ability to show unique binding and functional aspect..The most common types of antibody fragments are: Fab, Fab’, (Fab’)2, and Fv.

Fab and Fc fragments: Variable part of IgG ad IgM class of antibodies generates antigen bonding fragments ( Fab) while Fc region are made only from the constant part of the antibody and got separated from Fab.  Antigen binding fragments further includes Fab, Fab’, (Fab’)2, and Fv. All these fragments have the capacity to bind antigen but as they do not the Fc region they lack the domain 2 and 3 of the heavy chain. Digestion by papain digestion creates three molecules. Two antigen binding fragments (Fab) and one crystallizable fragment (Fc). Biochemical methods often used to generate antibody fragments essential for therapeutic purposes. However, the process is labour some and a lot starting antibody material is required.

F(ab’)2 fragment:  Digestion by pepsin creates 4 molecules. One hinge, two Fab units and a fragment containing two antigen bonding sites.

Fv fragment:  When IgG and IgM class of antibodies are digested enzymatically they generate Fv fragements which are smallest among the antibody fragments. Fv fragments, an unstable fragment able to bind to an antigen. An Fv fragment has two V regions, VL and VH. Single chain Fv fragment (scFv): scFv is a stable variant of Fv, commonly produced by recombinant technology, in which a peptide linker connects the two V regions.

scFv, diabody, triabody, tetrabody, Bis-scFv, minibody, Fab2, Fab3

scFv fragement: when genetic engineering methods are applied, a single chain variable fragments are (scFv) produced. scFv fragements are Fv type of fragments where VH and VL domains are presented joined by flexible peptide bond.  A single monomeric unit of scFv fragments contains at least 12 residue long linker polypeptide. When linker polypeptide and orientation of V domains of antibody are manipulated, it will create different Fv forms. When polypeptide linker contains 3 to 11 amino acid residues, the scFv molecule cannot fold into a functional Fv molecule. Then these scFv molecules produce a bivalent diabody by joining a second scFv.

Accordingly, presence of less than three amino acid residues of polypeptide linker will produce triabodies or tetrabodies in association with other scFv molecules. Multivalent scFv has the greater affinity towards antigen binding in comparison monovalent scFv as they can functionally bind to more antigens at the same time. When scFv molecule get attached with CH3 domain, a scFv-CH3 fusion protein or minibodies are produced. On the other hand Bis-scFv is specific fragments, where miniature of scFv fragements are generated to have two different variable domain to bind with different epitope. Additionally, Fab dimers (Fab2) or trispecific Fab trimers (Fab3) antibody fragments are also created using genetic manipulation methods. These fragments are useful to bind 2 or 3 different antigen at a time accprdingly.

dAbs:  dAbs  antibodies are domain antibodies, the smallest functional entity of antibodies. They are able to show specificity for full antigen binding as they have both VH and VL domains. The molecular weight of a dAb is one tenth of a full antibody and has three complementary determining regions (CDRs) out of six region of CDRs which is exhibited by a full length antibody. It is also shows high stability against harsh temperature, pressure or chemicals with denaturing property.

Fd fragment: the N-terminal half of the H chain which remains included in Fab fragment.

Why antibody fragments are useful?

Fragmented antibodies are preferred over the full length antibodies in some immunochemical assay methods and technical applications due to capacity to retain the functionality while being smaller in size.  There are some instances where uses of antibody fragments are useful. Such as-

  • Full length antibody sometimes shows nonspecific binding as several cells have receptor which can bind with the Fc region and reduce the specificity of antibody interaction.
  • During an imunoprecipitation assay, binding of Fc region to protein A or protein G beads can be controlled.
  • In immunohistochemistry ( IHC), fragmented antibodies are preferred over full length antibodies as they are more efficient to penetrate the tissue sections.
  • Due to the small size of the antibody fragments, they do not show properties like steric hindrance which are common to epitopes of large proteins. As a result there is a high chance of antigen detection when applied to solid phase immunodetection methods
  • Functions associated with Fc regions like complement fixation can be avoided with the use of smaller antibody fragments.
  • Small antibody fragments are very essential tool for detection of antibody structure with the help of X-ray crystallography or NMR techniques.
  • Smaller antibody fragments shows lower immunogenicity compared to full length antibody when used invivo. 

Polyclonal antibodies

In polyclonal antibody, different immunoglobulin molecules are generated from different B-cell clone. A host will produce a large number of antibodies that recognize independent epitopes on the antigen. The serum is the source of polyclonal antibodies. Mostly animals like mouse, goat or rabbit are used as host to generate polyclonal antibodies. An antigen .adjuvant mixture is injected into host which elicit an immune response by recognizing the antigen as foreign substance.  A series of adjuvant-antigen mixture injection is followed over a period of time for the generation of desired antibody. Next blood is extracted and antibody is purified for further applications. As polyclonal antibody has the ability to recognize several  epitopes for a given antigen it has several advantages. Such as-

  • When the target protein expression is low, application of polyclonal antibody will increase the signal as the protein can bind to multiple epitope present in the polyclonal antibody.
  • Polyclonal antibodies shows more tolerance over minor changes in antigen like polymorphism or lower scale degradation
  • They are useful for the detection of proteins which shows high similarity with the protein used for immunization.

However polyclonal antibodies show disadvantages like batch to batch variation or less efficient for the detection of specific antibody domain.

Application of polyclonal antibody-Polyclonal antibody is able to recognize several binding sites at a time for an antigen. It has also the ability to detect antigen for both denaturing and non-denaturing conditions. Polyclonal antibodies can be used in several purposes, such as-

  • ELISA
  • Immunodiffusions
  • To neutralize antibody effect
  • Immunoblotting methods such as western blot
  • Immunohistochemistry

Monoclonal antibodies

Monoclonal antibodies are made from immune cells which are similar in nature and they are the clone of a single parent cell. Monoclonal antibodies are important tool for medicine, molecular biology and biochemical research. Monoclonal antibodies (MAbs) are highly specific antibodies. In contrast to polyclonal antibodies, monoclonal antibodies are monovalent or they will bind to single type of epitope. They are produced from hybridoma cells. These cells are produced by isolating plasma cell precursors and fused with immortal cells.  Hybridoma production includes antigen-specific plasma cells (ASPCs) which is responsible for the production of antibodies which are generated against a specific antigen and then fuse it with myeloma cells. Plasma cell-meeloma hybrids are selected by culturing the hybridoma in presence of   Hypoxanthine-aminopterin-thymidine medium (HAT) which can inhibit synthesis of DNA.

The main advantages of monoclonal antibodies are as follows:

  • They are homogenous in nature and there gives consistent results.
  • When a hybridoma is generated, monoclonal antibodies generation can be renewed.
  • In comparison to polyclonal antibody, the concentration and specificity of the monoclonal antibody is higher
  • They generally show less cross reactivity.

However, like polyclonal antibodies monoclonal antibody also has some disadvantages. Such as-

  • Due to the specific nature of monoclonal antibody the application of monoclonal antibody is limited to certain applications.
  • If there are some changes in the antigen epitope, even a minor change will prevent the activity of monoclonal antibody.
  • Production of monoclonal antibody also required to be highly antigen specific

Application of monoclonal antibody- monoclonal antibodies can be applied on several instances. Such as-

  • Studying the structure of molecules like X-ray crystallography
  • To analyse phosphorylation status.
  • To study protein- protein interaction
  • Treatment option for cancer
  • To prevent allograft rejection

Production of Human Monoclonal Antibodies by Phage-Display and Transgene Technologies

Each hybridoma cell produces only one pair of immunoglobulin chains. Methods were developed that in a relatively short time, allow expression of many variants of immunoglobulin chains in different combinations in bacterial cells. After it was shown that functional, properly folded and assembled Fab and scFv can be synthesized in E. coli, expression of antibody chain libraries in bacterial cells was achieved. To get antibody genes into bacterial cells, bacteriophages are chosen as cloning vectors. Antibody genes contain conserved sequences in the 5′ and 3′ portions of variable and constant region sequences. Polymerase chain reaction (PCR) allows specific amplification of antibody genes using primers directed to these conserved sequences and the construction of libraries of antibody heavy and light chain fragments.

Production of Stable Heterohybridomas Producing Human Monoclonal Antibodies

In many circumstances, it is advantageous to have a continuous source of human antibody of a given specificity and immunoglobulin isotype. Reliance on human volunteers as a source of such antibody is problematic. Therefore, it has been a goal of investigators to establish immortal cell lines that produce the desired human antibody.

Phage-Display Technology

Phage display is a selection technique, according to which an antibody fragment (scFv or Fab) is expressed on the surface of the filamentous phage fd. For this, the coding sequence of the antibody variable genes is fused with the gene that encoded the minor coat phage protein III (g3p) located at the end of the phage particle. The fused antibody fragments are displayed on the virion surface and particles with the fragments can be selected by adsorbtion on insolubilized antigen. The selected particles are used after elution to reinfect bacterial cells. The repeated rounds of adsorbtion and infection lead to enrichment factors of more than a millionfold. Bacterial proteases can cleave the bond between the g3p protein and antibody fragments, which results in the production of soluble antibody fragments by infected bacterial cells. To release the soluble Fabs and scFvs, an excision of the g3p gene is made or an amber stop codon between the antibody gene and the g3p gene is engineered. As a source of the genetic information for development of combinatorial libraries, different tissues containing antibody synthesizing cells are used. Bone marrow, spleen, lymph nodes, tonsils, and peripheral blood are the tissues of choice to isolate mRNA for construction of recombinant antibody libraries.

 Transgenic Animals

Another approach for the preparation of human monoclonal antibodies is generation of mouse strains transgenic for human immunoglobulin genes. Miniloci, yeast artificial chromosomes, or phage P 1 vectors are used to transfer immunoglobulin genes. Miniloci transgenic constructs contain a limited number of variable genes of heavy and light chains, J segment clusters, and several D gene segments, as well as sequences with transcription-enhancer regulatory elements. Much larger contiguous segments of human immunoglobulin loci can be incorporated in yeast artificial chromosomes. After exogenous human transgenes are integrated into mouse germ line, human immunoglobulin genes are rearranged and produce a functional primary antibody repertoire. The transgenic animals can be crossed with mice carrying disrupted endogenous loci (heavy- and light-chain-knockout mice).

Materials

All materials used are of the highest chemical purity, and deiomzed water is of 18-MQ resistance.

  1. L-glutamine (200 n-r&& Sigma, St. Louis, MO)* Add 1 mL of a 100X concentration to 99 mL of medium.
  2. Gentamicin (10 mg/mL, Grbco, Gaithersburg, MD): Add 100 pL to 100 mL of medium to achieve a final concentration of 1 pg/mL.
  3. Sodium pyruvate (100 rnJ4, Cellgro, Herndon, VA): Add 1 mL of a 100X solution to 99 mL of medium.
  4. RPMI-10: RPMI-1640 containmg 10% fetal bovine serum (PBS) and anttbtotics (gentamtcin 1 pg/rnL), 5% human Al3 serum, L-glutamine, and sodium pyruvate. The serum 1s stable for 1 mo, but usually used wtthtn a week.
  5. RPMI-1640: RPMI-1640 (Cellgro) containing 25 mM HEPES, Lglutamine, Na pyruvate, and 100 ~.LL of gentamtcm.
  6. Phosphate-buffered saline (PBS): NaCl (6.8 g/L), Na,HP04 (1.585 g/L), RI&PO, (0.3 15 g/L). Dissolve m 1 L of deionized water. The pH will be 7.4.
  7. Htstopaque (Sigma): Polysucrose (5.7 g/100 mL) and sodium dratrizoate (9.0 g/mL). Use undiluted.
  8. Hank’s Balanced Salt Solution (HBSS): This buffer IS obtained commercially m a 10X stock solution (e.g., Gtbco). To prepare working soluttons, dilute 10 mL of the 10X stock with 90 mL of stertle, high-purity detonized water.
  9. Minimum Essential Medium (MEM)-Eagle: This medium is obtained commercially in a 10X stock solution from multiple companies (e.g., BioWhittaker, Walkersvrlle, MD). MEM contains Earle’s balanced salt solution without L-glutamine and sodium bicarbonate. To prepare working solutions, dilute 10 mL of the 10X stock with 90 mL of sterile, high-purity deionized water.
  10. 1N NaOH: Dissolve 4 g NaOH in 100 mL of deionized water and ftlter-sterilize.
  11. HEPES-MEM: To make 100 mL of a working solution of buffered MEM, add 5.0 mL of 1M HEPES buffer and 3.2 mL of stock 1N NaOH to 91.8 mL of MEM. Add 100 pL of gentamicin.
  12. Sheep red blood cells (SRBC): Obtain sheep blood from a commercial source or directly from animals maintained for research. Collect blood in an equal volume of Alsever’s solution (e.g., Mtcropure Medical Inc., Stillwater, MN, or local supplier). The SRBC are stable in Alsever’s solution for about 2 wk if maintained at 4OC.
  13. Immunization medmm: RPMI-1640 contaming 10% FBS, 40% thymocyte-conditioned medium (TCM), human IL-l (5 U/n.& obtain through commercial sources), L-glutamine, Na-pyruvate, gentamicin, Staphylococcus aureus Cowan 1 (5 x lo4 CFU/mL), and whole killed bacteria (our anttgen preparatton) 1 x 1 O6 CFU/mL.
  14. 50% Polyethylene glycol (PEG 4000, mol wt 3000-4000, Gibco): This is a 50% solution of PEG made in Dulbecco’s PBS without Ca++ or Mg++. This solution is used undiluted.

Medium for selective growth:

  1. HAT (hypoxanthme, aminopterin, and thymidine) supplement (Gibco, 100X): 10 mM sodium hypoxanthine, 40 @4 ammopterin, 1.6 n&f thymidine. Rehydrate with 10 mL of sterile deionized water, and dilute 1: 100 to prepare a working solution.
  2. HAT medium: Add 1 mL of HAT supplement to 99 mL of RPMI-10 medmm to prepare a working solution.
  3. HT (hypoxanthine and thymidine) supplement (Gibco, 100X): 10 mA4 sodium hypoxanthine, 1.6 mA4 thymidine
  4. HT medium: Add 1 mL of HT supplement to 99 mL of RPMI-10 medmm to prepare a working solution.
  5. Selective medium, 8-azaguanme (Sigma, 50X): The stock solution is 6.6 rnJ4 when reconstituted with 10 mL of RPMI. A few drops of 1M NaOH can be added to the solution if the 8-azaguanme does not go into solution easily.
  6. Selective medmm: Add 2 mL of the 8-azaguanine to 98 mL of RPM1 medium to prepare a working solution.
  7. Clonmg medium: RPMI-10 containing 40% TCM.

Methods

Preparation of TCM As an alternative to feeder cells, we use TCM to avoid potential contamination (

  1. Remove the thymus from 10 Balb/c mice, and place in RPMI-10 medium.
  2. Free thymocytes from the thymus by teasing the lobes apart with forceps and passing the tissue fragments progressively through a 16-gage cannula, an 18-gage needle, and then a 20-gage needle to obtain a single-cell suspension.
  3. Pool the thymocytes, and wash once m culture medium.
  4. Resuspend the thymocytes to a concentration of 3-5 x lo6 cells/ml in RPMI10 medium containing 20% FBS, and culture for 48 h in 7% CO, at 37OC.
  5. Remove the thymocytes from the culture medium by centrifugation (5OOg).
  6. Prepare aliquots of the culture medium, now considered as TCM, at desired concentration, and store at -70°C. 3.2.

Separation of Peripheral Blood Lymphocytes

  1. Collect fresh heparinized blood in either a 15- or 50-n& conical centrifuge tube, and dilute it with an equal volume of PBS at ambient temperature. Mix the diluted blood.
  2. Slowly underlay the blood/PBS mixture with H&opaque (Sigma) by passing it through a sterile pipet placed in the test tube. The working ratio is 3 mL of Histopaque/lO mL of the blood/PBS mixture.
  3. Centrifuge the blood preparation for 30 min at 500g with no brake.
  4. Carefully remove the upper layer (containing plasma and most of the platelets) with a pipet. Using a new pipet, collect the mononuclear cell layer at the interface, and transfer it to a new centrifuge tube.
  5. Wash the mononuclear cells m HBSS by centrifugation for 10 min at 300g at ambient temperature. Repeat the cycle to remove platelets.
  6. After the final wash resuspend the mononuclear cells in RPMI-10, and count the cells with a hemacytometer.

Preparation of Single-Cell Suspensions of Lymphocytes from Tissue

  1. Have the lymphoid tissue removed aseptically from the human donor, and either cut into small pieces or mmce m HEPES-MEM containing antibiotics.
  2. To prepare a single-cell suspension, press the tissue fragments through the cup of a cell selector fitted with a loo-mesh sterile wire screen. Use a glass pestle (Bellco-Cell Selector) to disrupt the tissue. Rinse the wire screen with RPMI-1640, and bring total volume to 45 mL.
  3. Centrifuge the cells at 250g for 10 mm.
  4. Remove the supematant, and resuspend the cell pellet in 5 mL of RPMI-1640.
  5. Underlay cell suspension with Histopaque as described in Section 3.2., step 2. Centrifuge for 30 min at 500g without using the brake.
  6. Remove the mononuclear cell layer at the interface, wash twice in HBSS by centrifugation 300g for 10 min at ambient temperature, and resuspend the pellet m RPMI-10. Count the cells.

Enrichment of B-Cells from Lymphocyte Populations by E-Rosetting

  1. Concentrate the lymphocytes by centrifugation at 300g for 10 min. Resuspend the cells at a concentration of 5 x lo6 cells/ml m HBSS containing 40% FBS.
  2. Wash SRBCs m HBSS twice, and make a 3% suspension of SRBCs in HBSS containing 40% FBS. This is done by adding 3 mL of the packed cells to 97 mL of the HBSS containing 40% FBS.
  3. Mix equal volumes of the lymphocytes and SRBC suspension, and mcubate the mixture over ice for 1 h.
  4. After incubation, place the suspension over 4 mL of Histopaque, and centrifuge at 500g for 30 min to separate E-rosetted T-cells from the nonrosettmg B-cells.
  5. Remove the cells remaining at the interface (B-cells) of the Histopaque/medium and wash twice by centrlfugatlon (300g) in warmed (37°C) serum-free RPMI-10.
  6. Count the cells with a hemocytometer. The cell preparation IS considered to be predominantly B-cells.

Development of a Fusion Partner

To obtain stable heterohybridomas producing human MAbs, a double-fusion (human X [mouse X human]) heterohybridoma is developed using a modification of the technique described by Ostberg and Pursch. The mouse myeloma cell line P3X63Ag8.653 (referred to as P63 in the text) was used to produce the double-fusion hybrid. The method for producing the fusion partner is outlined here as part of the procedure for producing stable heterohybridomas secreting human MAbs

Production of Stable Heterohybridomas

  1. Grow the P63 cells in RPMI- 10 to log phase by splitting the culture the day before the fusion is to take place.
  2. Wash 6 x lo6 P63 cells twice by centrifugation (3OOg), resuspend in serumfree medium, and mix with approx 2.4 x lo7 (a ratio of 1:4) human B-cells in 10 mL of serum-free medium in a conical centrifuge tube.
  3. Centrifuge the cell mixture at 350g for 10 min at ambient temperature to form a tight cell pellet.
  4. Remove all the supernatant from the pellet.
  5. Using a 1-mL pipet, add 1 mL of warm (37OC) 50% PEG over a 1-min period.
  6. Stir the mixture for an additional minute.
  7. Using the same pipet, add another 1 mL of warm serum-free medium to the mixture, and stir for an additional minute.
  8. Repeat step 7.
  9. Add an additional 7 mL of warmed serum-free media over a 2-3 min period with stirring.
  10. Centrifuge the cell mixture at 350g for 10 min. Remove the supernatant, and resuspend in medium (RPMI-10) to give a final concentration of 1 x lo6 cells/O. 1 mL.
  11. Transfer aliquots of the fused cells (400 uL/well) mto each well of a 24-well cluster plate.
  12. Feed the cells with HAT-containing medium on d 1 and 3 following the fusion.
  13. Feed the cells with HT-containing medium every 2-3 d until macroscopic growth is seen. This usually takes about 3 wk. Change to regular medium when growth is evident.
  14. After 3 wk of growth (macroscopic growth of colonies is evident), add 8-azaguanine (1.32 x lOAM) to the medium to select for 8-azaguanineresistant hybridomas.
  15. Grow the cells in the presence of 8-azaguanine for 3 wk. There will be a large die off of the cells, but some wells will have rapidly growing cells. These hybridoma cells are now HAT-sensitive because of the lack of HGPRTase enzyme activity.
  16. These 8-azaguanine-resistant hybridoma cells can be propagated for long periods of time as well as frozen for use in the future as fusion partner cells.

Fusion of the Hybrid Myeloma-Immune B-Cell

The second part of the construction of the murine-human heterohybridoma is the fusion of the immune human B-cell with the mouse-human heterohybridoma fusion partner. The fusion partner should be well established and in log-phase growth when harvested for the fusion. Normally the myelomas is grown to a density of around 1 x lo7 cells/ml and have a viability in excess of 95%.

  1. Mix the 8-azaguanme-resistant mouse-human heterohybndoma cells with the immunized human B-cells in a ratio of 1:4 heterohybridomas to B-cells, respectively.
  2. Grow the heterohybridoma fusion partner cells to log phase in RPMI-10 by splitting the culture the day before the fusion is to take place.
  3. Wash 1 x lo6 myeloma cells twtce (350g) in serum-free RPMI, and mix with approx 4 x lo6 immune human B-cells in serum-free RPMI-10 medium.
  4. Centrifuge the cell mixture at 35Og for 10 min at ambient temperature to form a tight cell pellet.
  5. Remove all the supernatant from the pellet by careful pipeting.
  6. Using a I-mL pipet, add 1 mL of warm (37OC) 50% PEG over a l-mm period.
  7. Stir the mixture for an additional minute.
  8. Using the same pipet, add an addrtionall mL of warm serum-free medium and stir for another minute. 9. Repeat step 8.
  9. Add an additional 7 mL of serum-free medium over a 2-3 min period with stirring.
  10. Centrifuge the cell mixture at 35Og for 10 min and remove the supernatant.
  11. Count the total cell population, and add sufficient medium (RPMI-10) to give a final concentration of 1 x lo6 cells/O.1 mL.
  12. Add the medium directly to the pellet.
  13. Plate aliquots of the cell mixture (400 pL/well) into each well of a 24-well cluster plate.
  14. Feed the cell cultures with HAT-contaming medium on d 1 and 3 following the fusion.
  15. Feed the cell cultures with HT-contammg medium every 2-3 d until macroscopic growth is seen. This usually takes about 3 wk.
  16. After 3 wk of growth in HT, collect medium from wells containing colonies of cells, and test for antibody production. Set up dilutions of cultures of cells producing antibody using a limiting dilution format.
  17. At this time, screen each well with visible colonies, using an ELISPOT assay. This assay allows the determination of the isotype and specificity of antibodies reactive with the immunizing anttgen, and an estimate of the number of specific antibody-producing cells. We observe some multiple-isotype expression on individual cells within these uncloned cultures of heterohybridomas. At this stage, it is imperative that limiting dilutions be done on the wells containing antibody-producing cells.

Cloning by Limiting Dilution

  1. Suspend cells in wells of interest, and then take a 1-mL aliquot, count the cells, and check cell viability.
  2. Dilute the cells to a concentration of 230 live heterohybridoma cells in 4.6 mL of cloning medium. This dilution yields a concentration of 50 cells/ml of medium.
  3. Plate 36 wells of a g-well plate with 0.1 mL of the cell suspension, which will give approx 5 cells/well.
  4. In the remaining 1 mL of cell suspension, there are about 50 cells. Add 4 mL of cloning medium to the 1-mL suspension, and mix well.
  5. Plate 0.1 n&/well m 36 additional wells of the 96-well plate. This dilution and plating should yield about 1 cell/well.
  6. To the remaining 1.4 mL of cell suspension, add 1.4 mL of cloning medium and mix. Plate 0.1 mL in each of the remaining 24 wells of the 96-well plate. This dilution should give about 0.5 cells/well.
  7. Examme the cultures after 48 h using an inverted phase microscope to identify wells containing single colonies of cells.
  8. At d 5, and again at d 12, feed the cells with 200 p.L of cloning medium.
  9. Following identification of hybrids producing antibody, transfer cells from wells containing a single colony to 24-well plates to expand the culture, and then transfer to 25-cm2 tissue-culture flasks.
  10. Following expansion of the cell line, cryopreserve ahquots of cells, and then grow the cells in large tissue-culture flasks using RPMI-10 culture medium to prepare stocks of antibody. Alternatively, transfer cells to bioreactor casette for production of antibody.

 

Genetically engineered antibody

Nowadays, as use of antibodies are increasing as therapeutic molecule, recombinant technologies are used for the manipulation and preparation of antibodies and fragments with desired characteristics.  Genetic manipulations of antibodies are performed to have increased half-life, lowered immunogenicity or increased binding and neutralizing activity towards antigen. To lower the effect of immunogenicity, chimeric antibodies such as mouse-human is produced where; some part of the constant region of human antibody is added with V region of mouse antibody. Alternatively use of  transgenic mice with human containing human antibody part or antibody phage libraries are in use as an alternative to full human antibody as therapeutic options.

Fusion antibodies

Antibody fusion proteins are important part of research, diagnostic or therapeutic applications. Whole or a part of antibody is fused with a fusion partner which provides the fusion protein to accessibility to several areas of host body such as crossing of blood –brain barrier which otherwise be a constrain for normal protein applications or delivering therapeutic  material to target sites. Fusion antibodies can be divided into two groups, such as-

  • Fusions of ab and F(ab’)2 in which one or more antigen binding sites retained and the fusion partner then replace or get linked to Fc region.
  • Fusion of Fc region also called immunoadhesions, where the antigen recognition site is removed and fusion partner takes over. Whereas, the Fc region remains.

Sample preparation for antibody purification

Antibody sources and their associated contaminants

Antibodies and antibody fragments are produced from a variety of native and recombinant sources. The choice of source material can affect the selection of techniques for sample preparations and purification protocol. Each source contains specific contaminants associated with them. Human serum may contain albumin, transferrin, macroglobulin and other serum proteins as contaminants.  Hybridoma, cell culture supernatant, may contain phenol red, albumin, transferrin, bovine IgG, other serum protein, viruses.  Recombinant antibody sources may contain proteins from the host microbes, hamster ovary, etc,

Antibody extraction

If the antibody to be purified is recombinant, then the extraction procedure is largely dependent on protein source and where it is located. Extraction process will depend on whether the protein is of mammalian or bacterial origin, located as intracellular or intercellular and choice of buffer will varies accordingly. Briefly-

  • The extraction method required to be gentle to avoid proteolytic degradation.
  • Uses of additives are required for stabilization of extracted protein. Additives which are denaturing in nature like Urea (8M) or Gunidine (6M) are applied when the protein of interest is expressed like inclusion body.
  • The whole extraction process is required to perform quickly at suitable temperature, maintaining specific pH and stability of protein of interest.

How to clear sample for antibody purification?

Centrifugation and filtration

During chromatographic methods, lipids or lipoproteins can block the chromatographic columns. Thus it is necessary to remove those before proceed to chromatographic steps. Phenol red which is present in cell culture medium as a pH indicator can also interfere with chromatographic process and it is thus recommended to remove phenol red before protein purification.  To clear sample prior to purification process, filtration and centrifugation are most commonly used. Generally centrifugation removed the particle from the cell extract and when necessary filtration is performed. In addition to centrifugation particulate materials are also get removed by filtration. Polyvinylidene fluoride or cellulose acetate membranes are favourable as it shows lower amount of non-specific binding for proteins.  To remove lipid  from  samples like serum, filters like glass wool is used. To remove phenol red before purification process use of desalting column is recommended.

Precipitation

When low molecular weight impure compounds are present in the sample, use of desalting column is beneficial.

Fractional precipitation: For impurities with high molecular weight compounds, fractional impurities are required. With the increase in salt concentration, increased hydrophibic concentration of protein can observed. Selective precipitation thus occur due to the difference in hydrophobicity. However, use of columns like  HiTrapTM  affinity purification reduce the use of fractional precipitation.

Ammonium sulfate precipitation: This precipitation methods is useful for removal of contaminants such as albumin, transferrin, or other contaminants which are highly soluble. During this method, ammonium sulphate concentration is increased gradually, which results in ‘salting out’ or the proteins become less soluble. Thus, contaminants can be removed from cell extract.

Caprylic acid precipitation: This particular precipitation method is useful protein precipitation from sera and ascites and shows effective results as ammonium sulphate precipitation. Caprylic acid is a fatty acid and used for monoclonal antibody precipitation.

Dialysis

Dialysis use diffusion method to separate small molecules from macromolecules with the help of semi permeable membrane. When sample and corresponding buffers are placed on one side of a membrane, smaller molecules get diffused through the membrane whereas larger molecules remain in the membrane side until an equilibrium concentration is achieved.

Buffer exchange and desalting for chromatography

During buffer exchange, one type of buffer salts get replaced by a second type, whereas excess salts present in the sample get removed by desalting. In both situations, size exclusion chromatography (SEC) principles are followed. At the time of desalting procedure chromatography columns are equilibrated with water, whereas in buffer exchange, column resin is equilibrated with end buffer. In both scenarios, the columns with sample containing buffers will get replaced by the buffer which has been used for pre-equilibration. During buffer exchange and desalting methods, sample containing solutions are flow through resin packed columns. But only small molecules and buffer can enter the porous resin, whereas macromolecules will passed out. Thus macromolecules will be separated first followed by small molecules. As the solution which carries the sample is displacing the equilibration buffer, the macromolecules which left the column will be carried out in equilibration buffer, giving the name buffer exchange.

Diafiltration

Diafiltration is used for separation between macromolecule and micro molecules with the help of semipermeable membrane. Diafiltration use pressure to pass the solution through membrane and based on this several commercially available sample concentrator are available. At the time of separation, solvent mainly water and solutes of low molecular weight are passed through a membrane and collected. Whereas macromolecules stay on the side of the membrane where sample is and becomes concentrated.

 Antibody purification by affinity chromatography: Small scale

Purified antibodies are widely used in several biological applications. Specifity of the antibodies finds its application in this purification process. When proteins are present in crude extract or in combination with other molecules, variety of techniques then applied to purify proteins. Among the purification methods, chromatographic methods in various formats are widely used. During chromatographic separation, a stationary material is used as solid phase and proteins which remain in solution are acting as mobile phase. Now, mobile phase is separated from solid phase by the interaction of chemical and physical material which is used in solid phase. Among the affinity based purification methods, affinity chromatography is used in extensive manner. Affinity chromatography is often the first step in purification for many antibodies and is based on specific molecular binding interaction. The principle of this method is based on the use of specific ligand which can be immobilized on a solid support. Now, when the complex mixture containing the protein of interest is passed over the solid phase, proteins having specific affinity towards the ligand get attached to it.  Once the non-specifically bound molecules are washed out, particular ligand-protein is eluted out.  Affinity chromatography provides highly selective protein purification with higher capacity and sensitivity. Sometimes a polishing process is required after affinity process to get the required homogenicity.  Affinity purification offers high selectivity. More than 95% purity can be achieved in this method. In most applications this level of purity is desired. Affinity purification method follows three general steps:

  • Incubation of crude sample extract with the immobilized affinity support medium or ligand so that the protein of interest can get bound with the affinity medium.
  • Use of proper buffer helps to wash out the non bound samples keeping the ligand-protein of interest interaction intact.
  • Further dissociation of target protein from the ligand-protein complex get dissociated and target protein get eluted with the help of alteration of buffer condition.

Binding with ligands: Immobilization or coupling of ligand on solid support occurs through the covalent bond formation between ligand primary group and support’s reactive group. Additionally  indirect coupling also possible like  glutathione S-transferase (GST)-tagged fusion protein s which are attached to a glutathione support with the help of  glutathione-GST affinity. Thereafter affinity purification of GST tagged protein is performed for the purification of fusion protein

Bonding and elution buffer: During affinity purification binding of protein with ligand occurs generally in PBS buffer with ionic bond formation atphysiologic pH. After specific binding non-specific bindings are removed by washing however non-specific bindings are reduced by using mild detergents or adjusting salt concentration in wash buffer. Further elution buffer is used for breaking of the ligand-protein interaction and releasing the target protein molecule. The dissociation occurs mostly in the presence of extreme pH , high concentration of salts, denaturing buffer or counter ligand. Lastly a desalting or dialysis process is followed to remove the elution buffer and present target protein in suitable buffer condition for storage or downstream applications.

Solid support material:  Any material where the ligand is able to bind covalently and does not show solubility to the system can be used as solid support matrix.  So far several matrixes has been reported to use as matrices for affinity purification such as cellulose, agarose, latex or polyacrylamide. Support material generally shows characteristics such as-high ratio of surface to volume, chemical groups which can be manipulated easily for ligand attachment, lower non-specific binding, better and stability and with increased flow characteristics.

Porous support material- porous supports such as hydrated sugar beads with a diameter of 50-150 µm are used as wet slurry which can be used to fill up the columns. Due to the large size of the pores of the beads biomolecules like protein can easily passed through. When ligands molecules get attached to the support beads they produce a loose matrix through which biomolecules flow though and molecules of interest get bonded with the ligand. Among the porous material, cross-linked agarose beads are extensively used.  Alternatively, polyacrylamide based resin beads are also used in affinity chromatography.  

Magnetic particle as support material- Magnetic particle are alternative to beaded agarose or other types of porous resins. They are non-porous in nature and smaller in diameter, 1-4 µm in comparison to porous beads. The small size is beneficial for immobilization of ligands and further affinity purification. Superparamagnetic iron oxide particles are coated with silane derivatives to produce magnetic beads.  As a result the beads become inert which permit the attachment of specific ligands onto it.  Instead of affinity columns, few microliters of magnetic beads are mixed with larger volume of sample to make loosely formatting slurry. In the slurry environment, suspended beads allow the affinity interaction to take place with the ligand. After the completion of the reaction, magnets are used to separate out the beads from the mixture. In comparison to agarose beads, magnetic beads give lower non-specific binding and can be used for all formats of cell separation techniques such as immunoprecipitation or pulldown.

Different types of affinity purification

In principle, different types of purification process exist according to desired targets, purification scale or technical support.

Antibody purification:  Antibody purification is particularly used with small sample volume such as supernatant of the cell culture or serum. Depending upon the antibody use and detection methods, antibody purification can be divided into several categories like-

Amonium sulphate precipitation- Useful as crude purification method of total imunoglobulins.

immobilized Protein A, G, A/G or L-  The most predominately found Imunoglobulin IgG is able to purify by using these proteins. They are found in different size, format and ready to use.

Immobilized antigen- Purified antigens like haptens which are useful for the production of antibody can also be used as affinity support to allow specific antibody purification.

Antigen purification:  As antibodies can bind to specific antigen to detect them in assay, they can also be useful for antigen purification. However these particular antibody purification methods mostly use for small scale purification like immunoprecipitation due to higher production costs of antibody.

Immunoprecipitation / Co-Immunoprecipitaiton:   Antigens are purified in small scale with the help of immunoprecipitation. Breifly, immobilized proein A or G agarose beads are used to capture antigen-antibody complex and purified antigens are isolated. On the other hand  Co-Immunoprecipation not only purified antigen of interest but at the same time also pulled down all the associated interacting partners of the antigen.

Pull down assay:  Pull down assay is useful to detect protein protein interaction.  In this method a purified protein called bait is use to isolate and pull down the ineracting partner of bait protein known as prey protein. The bait protein is generated either by fusion protein expression or cloning or through covalent modification like biotin.  Next, bait with a tag is attached on tag specific affinity matrix like streptavidin for biotin tagged protein. When protein solution containing prey protein is interacted with the bait, prey get bound with the bait and can be isolated out.

Purification of tagged protein: To increase the purification of recombinantly expressed proteins sometimes tags which are short amino acid sequence or protein domain are used. These tags or domains get attached with the DNA sequence which codes for the native protein. Fusion tags like histidine or poly-His tag or glutathione S-transferase (GST) are commonly used for purification. These tagged proteins are useful for large scale purification process as they require less expensive affinity matrix like glutathione  agarose for GST. On the other hand tags like HA, Myc or FLAG are mostly used in small scale affinity purification. Use of fusion tagged protein give access to easy control of purification in laboratory settings.

Avidin-Biotin interaction:  Biotin or vitamin H is a small molecular weight compound present in living cells. As biotin shows strong interaction towards avidin or streptavidin, molecules attached with biotin can be used for immobilization, capture , detection or affinity purification in presence of avidin/ streptavidin.

Affinity purification for contaminant removal: In some instances affinity purification is working to remove particular component from cell suspension rather than purification. By throwing out the ligand bound matrix and working with the flow through, the solution can get removed from the bound target molecule. Although gel filtration  is the technique of removal or small  molecules from macromolecular compounds, affinity purification is useful in situation where the size of the molecules to be removed do not show differences in size. Normally removal of contaminants carried out at the end of the reaction with the help of detergent based resisns.   

Affinity ligands for antibody purification

In several species, it has been found that protein A and protein G has high affinity towards Fc region of IgG. Thus when protein A and protein G is bound to a matrix, it can be used to isolate IgG from several samples like serum or supernatants obtained from cell culture.

Protein A and Protein G

High affinity of these two proteins for the fc region of polyclonal and monoclonal IgG type antibodies forms the basis of purification of IgG, IgG fragments containing the Fc region and IgG subclasses.  Protein G and protein A are bacterial proteins from group G streptococci and staphylococcus aureus, respectively. They have binding sites for mammalian immunoglobulin.  These proteins when coupled with sepharose forms a very good media for purification of antibodies. IgG class antibodies and fragments are purified by this media. Binding strengths obtained from individual proteins can be used as guidelines in purification process. Single-step purification of sample from native sources will purify host IgG and trace amount of proteins. In cases like this immunospecific affinity using anti-host IgG native bodies is coupled to media for effective seperation. Other techniques like Ion Exchange chromatography and hydrophobic interaction chromatography can also be used.  When antibody binds to protein A, the binding is influenced by neutral or alkaline pH of the media and hydrophobic reactions occurring at conserved histidine site of IgG where protein A can bind. On the other hand, antibody is recovered or eluted from protein A with the help of acidic buffer by lowering the pH. Next purified antibodies are further neutralized in presence of base or desaltation carried out with the help of gel-filtration column. This will help to reduce the effect of hydrolysis and denaturation influenced by acids.  On the other hand protein G shows higher binding affinity in comparison to protein A. Acidic pH provides optimal binding condition for protein G and it also requires harsh elution buffer due to stronger affinity. However, low pH such as 3.0 or less is detrimental for the purified antibody function.  To reduce the effect, rapid neutralization following purification is recommended. Additionally, IgG also get carried over due to strong protein G-IgG interaction.  This carryover also results in shorter life for the protein G columns. Further to avoid the effect of cross contamination, use of antibody specific protein G columns is preferred.

 Protein L binds to Fv of kappaight chain

Protein L, containing 719 amino acids is first isolated from the surface of bacterial species Peptostreptococcus magnus.  Protein L is devoid of any disulphide loops or subunits. It binds with immunologlobulins through the light chains interaction. Protein L binds with a wide range of antibody classes. As protein L does not show binding interaction with antibody heavy chain, it has the ability to binds with almost all antibody classes better than protein A and protein G. In addition protein L is also binds to Single chain variable fragments (scFv) and Fab fragments of antibody. However, like protein A and protein G, protein L is not considered and used widely because of it’s inability to bind with kappa light chains containing antibodies. As most of the human and mice antibodies contain kappa (κ) light chain and remaining lambda (λ) light chains, thus protein L can only working for some subtypes of kappa (κ) light chain.  In most of the cases protein L is used for purification of monoclonal antibodies isolated from cell culture with kappa (κ) light chain or monoclonal antibodies with VLκ fragments from bovine imunoglobulins.  

 Ligands that bind with Fc of Fab kappa or lambda light chain

Recombinant protein of MW 13000 is commercially available in market. They bind with both light chain regions. It is produced in S. cerevisiae.

Optimization of parameters

Some parameters for antibody purification can require optimization to obtain the optimal result. Pretreatment, buffer solution, quantity of antibody to be purified, number of washes are some optimizable parameters.

Purification using Protein G Sepharose media

Protein G is generally used for the media as it shows a greater affinity for most IgG from a lot of eukaryotic species. Protein A produces highly purified antibodies. The binding strength of protein G for IgG depends on the source species and subclass of the immunoglobulin. The dynamic binding capacity depends on the binding strength and also on several other factors, such as flow rate during sample application. If harsh elution system are used leakage of ligands may also happen. Thus protein G attached at multiple points shows a very less leakage over a wide range of elution condition. Polishing is done by SEC (Size Exclusion Chromatography) and IEX (Ion exchange chromatography). Protein G can be operated in a wide range of pH. Affinity strength is maximum at physiological pH and ionic strength. Avoid excess washing, if ligand is weak.

  1. Equilibrate all the material used in the reaction in reaction temperature and de-gas the media slurry.
  2. Eliminate any air from the columns by flushing with the operating buffer.
  3. Re-suspend the slurry and pour the slurry in one single motion. Air bubbles should not enter the column.
  4. Open the bottom outlet of the column and set the pump to run at the desired flow rate.
  5. Maintain packing flow rate for at least 3 bed volumes after a constant bed height is reached. Mark the bed height on the column.
  6. Stop the pump and close the column outlet.
  7. Connect the column to a pump or a chromatography system and start equilibration.

Sample preparation

The sample which is used for affinity chromatography required to be clear and without the presence of solid particles. Clearing of samples obtained by centrifuging samples at 10000 g for 10 minutes to remove cells and debris. When required sample Filter through a 0.45 µm pore size filter is also used. During sample preparation, pH and salt concentration is known to affect the solubility and stability of the desired protein product.

Buffer preparation

Binding buffer:0.02 M sodium phosphate, pH 7.0

Elution buffer: 0.1 M glycine-HCl, pH 2.7

Neutralizing buffer: 1 M Tris-HCl, pH 9.0

Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.45 μm filter before use.

 Purification

  1. Prepare collection tubes by adding 60 to 200 μl of 1 M Tris-HCl, pH 9.0 per milliliter of fraction to be collected. pH of the solution should be 7.0.
  2. If the column contains 20% ethanol, wash it with 5 column volumes of distilled water. Use a linear flow rate of 50 to 100 cm/h.
  3. Equilibrate the column with 5 to 10 column volumes of binding buffer at a linear flow rate of 150 cm/h.
  4. Apply the pretreated sample.
  5. Wash with binding buffer until the absorbance reaches the baseline.
  6. Elute with elution buffer using a step or linear gradient. For step elution, 5 column volumes of elution buffer are usually sufficient. For linear gradient elution, a shallow gradient over 20 column volumes allows separation of proteins with similar binding strengths.
  7. After elution, regenerate the column by washing it with 5 to 10 column volumes of binding buffer. The column is now ready for a new purification. Desalt and/or transfer purified IgG fractions to a suitable buffer using a desalting column

Storage

Proteins are heterogeneous in nature.  There are possibilities of protein instability when the loose the native confirmation. Depending upon the source of protein, different proteins may require specific environment to be stable. As a result of proteolysis, improper storage condition or protein aggregation increases the degradation rate of proteins. Thus it is important maintain proper storage conditions for long term storage of purified proteins.  The shelf life of proteins thus shows differences depending upon protein nature and storage conditions. The general condition for protein storage is to store in 20% ethanol at 2°C to 8°C.

Parameters for protein storage

Temperature- The recommended condition for longer period of protein storage is less than or equal to 4°C temperature and use of polypropylene tubes or glassware which has been autoclaved. Proteins kept in room temperature leads to microbial growth and degradation. Storage for lesser period of time such as 1 day or 1 week, proteins can be kept at 4°C. For longer periods, such as 1 year or more, proteins are stored in cryotubes and placed in liquid nitrogen. To reduce protein loss due to repeated free and thaw, proteins are recommended to store in aliquots. 50% glycerol or 20% ethanol are added to avoid protein freezing.

Concentration- When proteins are stored in lower concentration; there are high chance of deactivation and loss. However, a higher protein concentration or addition of carrier protein like BSA could reduce the risk of protein degradation.

Additives: there are several compounds which could be used to increase the protein shelf life known as additives. Such as-

  • Cryoprotectants like glycerol at a final concentration of 20-50% is helpful to provide stability to protein structure.
  • Protease inhibitors like Phenylmethylsulfonyl fluoride or PMSF, Pepstatin A or leupeptine can be used to inhibit proteolytic degradation of proteins.
  • To inhibit microbial growth, Metal chelators like  EDTA with the final concentration os 1 to 5nM is added to proteins to retain the reduced protein stage as well as to reduce oxidation of –SH groups induced by metal.
  • Addition of reducing agents like dithiothreitol or DTT, 2-mercaptoethanol  or 2-ME is also added to stored protein for the maintenance of reduced state of proteins and prevention of cysteine oxidation.

 Affinity of other antibodies

Protein A can interact with human colostral IgA as well as human myeloma IgA2 but not IgA1. Polyclonal IgA from pig, dog, and cat and monoclonal canine IgA have also exhibited binding affinity for protein A. Protein L has strong affinity for human IgA. Protein L has strong affinity for human IgD.  Protein L has strong affinity for human IgE. IgM present in human and mouse serum binds weakly to protein A. However protein L has strong affinity for human and mouse IgM.

Making Immunospecific purification media with custom ligands

A ligand, a pure antigen, can be covalently coupled to a suitable matrix to create an immunospecific medium for purification. This methodology is particularly useful when the target molecules does not bonds or bonds weakly with protein A and G. It can also be used for removing contaminants. The ligand is coupled through its active amine group to a pre-active media. The pre-active media is hydrophilic in nature which contributes to the reduction of non-specific bonding of proteins.

Coupling ligands to HiTrap NHS-activated HP columns

HiTrap NHS columns are prepaked column of NHS activated Sepharose High Performance.  This column is useful for easy coupling of primary amine containing ligands. This column is available for 1 ml and 5ml size. The protocol below describes the preparation of a prepacked HiTrap NHS-activated HP column and a recommendation for a preliminary purification protocol. Many of these details are generally applicable to NHS-activated sepharose media. Coupling can take place within the pH range of 6.5 to 9.0 with a maximum yield achieved at around pH 8.0.

Buffer preparation

During the preparation of buffers high quality water and chemicals are required. The column can be used by several methods such as syringe, chromatographic methods or with peristaltic pump. 100% isopropanol is used as prevention method so that NHS group can not get deactivated.

Acidification solution: 1 mM HCl (kept on ice)

Coupling buffer: 200 mM sodium hydrogen carbonate, 500 mM sodium chloride, pH 8.3 Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.45 μm filter before use.

 Ligand and HiTrap column preparation

  1. Dissolve the desired ligand in the coupling buffer to a final concentration of 0.5 to 10 mg/ml (for protein ligands) or perform a buffer exchange using a desalting column. The optimal concentration depends on the ligand. Dissolve the ligand in one column volume of coupling buffer.
  2. Remove the top cap and apply a drop of acidification solution to the top of the column to avoid air bubbles.
  3. Connect the Luer adapter (or tubing if using a pump or system) to the top of the column.
  4. Remove the snap-off end at the column outlet.

 Ligand coupling

Ligand coupling can be followed with the help of syringe. For 1ml HiTrap column, a syringe with a volume of 1 or 2 ml can be used. Whereas use of 5 or 10 ml syringe is beneficial for 5 ml HiTrap column

  1. Wash out the isopropanol with acidification solution. Use 3 × 2 ml for HiTrap 1 ml and 3 × 10 ml for HiTrap 5 ml. Do not exceed flow rates of 1 ml/min for HiTrap 1 ml columns and 5 ml/min for HiTrap 5 ml columns at this stage to avoid irreversible compression of the prepacked medium.
  2. Immediately inject 1 ml (HiTrap 1 ml) or 5 ml (HiTrap 5 ml) of the ligand solution onto the column.
  3. Seal the column and leave for 15 to 30 min at 25°C or 4 h at 4°C*.

In a situation of larger ligand volume, recirculation of the solution is required. To perform this connection of a second syringe at the column outlet is required with a gentle pumping of solution for 10 o 30 minutes at back and fourth direction.

 Washing and deactivation

Deactivate any excess active groups that have not coupled to the ligand, and wash out the non-specifically bound ligands, by following the procedure below:

Buffer A: 500 mM ethanolamine, 500 mM sodium chloride, pH 8.3

Buffer B: 100 mM acetate, 500 mM sodium chloride, pH 4.0

  1. Inject 3 × 2 ml (HiTrap 1 ml) or 3 × 10 ml (HiTrap 5 ml) of Buffer A.
  2. Inject 3 × 2 ml (HiTrap 1 ml) or 3 × 10 ml (HiTrap 5 ml) of Buffer B.
  3. Inject 3 × 2 ml (HiTrap 1 ml) or 3 × 10 ml (HiTrap 5 ml) of Buffer A.
  4. Leave the column for 15 to 30 min at room temperature or approximately 4 h at 4 °C.
  5. Inject 3 × 2 ml (HiTrap 1 ml) or 3 × 10 ml (HiTrap 5 ml) of Buffer B.
  6. Inject 3 × 2 ml (HiTrap 1 ml) or 3 × 10 ml (HiTrap 5 ml) of Buffer A.
  7. Inject 3 × 2 ml (HiTrap 1 ml) or 3 × 10 ml (HiTrap 5 ml) of Buffer B.
  8. Finally, inject 2 ml (HiTrap 1 ml) or 10 ml (HiTrap 5 ml) of a buffer with neutral pH to adjust the pH.

 Storage

Store the column in a solution that maintains the stability of the ligand and contains a bacteriostatic agent, for example phosphate-buffered saline (PBS), 0.05% sodium azide, pH 7.2.  pH stability of the media when coupled to the selected ligand depends on the stability of the  ligand. Sodium azide can interfere with many coupling methods and some biological assays. It can be removed using a desalting column.

Performing a purification on a coupled HiTrap NHS-activated column

Use high quality water and chemicals. Filtration through 0.45 μm filters is recommended. Optimal binding and elution conditions for purification of the target protein must be determined separately for each ligand. The general protocol given here can be used for preliminary purification. For the first run, perform a blank run to ensure that any loosely bound ligand is removed. Samples should be centrifuged immediately before use and/or filtered through a 0.45 μm filter. If the sample is too viscous, dilute with binding buffer. Sample binding properties can be improved by adjusting the sample to the composition of the binding buffer. Perform a buffer exchange using a desalting column or dilute the sample in binding buffer.

Prepare the column by washing with:

(i) 3 ml (HiTrap 1 ml) or 15 ml (HiTrap 5 ml) binding buffer.

(ii) 3 ml (HiTrap 1 ml) or 15 ml (HiTrap 5 ml) elution buffer.

  1. Equilibrate the column with 10 column volumes of binding buffer.
  2. Sample preparation. The sample should be adjusted to the composition of the binding buffer. This can be done by either diluting the sample with binding buffer or by buffer exchange or desalting. The sample should be filtered through a 0.45 μm filter or centrifuged immediately before it is applied to the column.
  3. Apply the sample, using a syringe fitted to the Luer adapter or by pumping it onto the column. Recommended flow rates: 0.2 to 1 ml/min (HiTrap 1 ml) or 1 to 5 ml/min (HiTrap 5 ml)*. The optimal flow rate is dependent on the binding constant of the ligand.
  4. Wash with binding buffer, 5 to 10 column volumes or until no material appears in the effluent. Excessive washing should be avoided if the interaction between the protein of interest and the ligand is weak, since this can decrease the yield.
  5. Elute with elution buffer; 1 to 3 column volumes is usually sufficient but larger volumes might be necessary.
  6. The purified fractions can be desalted.
  7. Re-equilibrate the column by washing with 5 to 10 column volumes of binding buffer. The columns is now ready for a new purification of the same kind of sample. 1 ml/min corresponds to approximately 30 drops/min when using a syringe with a 1 ml HiTrap column; 5 ml/min corresponds to approximately 120 drops/min when using a syringe with a 5 ml HiTrap column. To preserve the activity of acid-labile IgG, add 60 to 200 μl of 1 M Tris-HCl pH 9.0 to collection tubes, which ensures that the final pH of the sample will be approximately neutral.

Elution buffers

Immunospecific interactions can be very strong and sometimes difficult to reverse. The specific nature of the interaction determines the elution conditions. Always check the reversibility of the interaction before coupling a ligand to an affinity matrix. If standard elution buffers do not reverse the interaction, alternative elution buffers that can be considered are listed below:

  • Low pH (below pH 2.5)
  • High pH (up to pH 11.0)
  • Substances that reduce the polarity of a buffer can facilitate elution without affecting protein activity: dioxane (up to 10%), ethylene glycol (up to 50%).

Adding a polishing step after initial purification

One-step affinity purification generally achieves satisfactory purity of the target antibody. To achieve adequate homogeneity of the purified antibody, however, an additional polishing step using size exclusion chromatography (SEC) is recommended.  This chapter also describes methods for removal of specific contaminants remaining from purification of IgG from native source or serum as well as cell culture.

What are other chromatographic method ?

Gel filtration chromatography

Gel filtration chromatography is also known as size exclusion chromatography or SEC. This method is based on the separation of molecules according to size with the help of porous resins. When a solution with different sized molecules passed through porous resins, small molecules get entrapped inside the pores of the resin whereas larger molecules get excluded and get eluted quicker than the small molecular weight compounds.

Ion Exchange Chromatography

MAb has higher pI than most host cell proteins. That forms the basics of Ion exchange chromatography for purification. Proteins are thus get isolated based on their ionic interaction  with  groups attached with resin positively or negatively. Further when buffer condition is manipulated such as change on the pH or ionic buffer condition, bound proteins can be isolated.Most impurities like DNA, endotoxins, HCP flow through the column or washed away by buffer. Other approach is MAb is allowed to pass through while impurities gets attached to the protein A in column media.

Immobilized Metal Ion Affinity Chromatography

A specialized sub category which is based on peptide tagging affinity chromatography is called Immobilized Metal Ion Affinity Chromatography (IMAC). This chromatographic method is dependent on the interaction between His tag and divalent metallic ions such as Ni2+, Cu2+. During this process the proteins got separated based on their difference in affinity towards metal ions. At a neutral pH value, amino acids like histidine, tryptophan or cysteine has the ability to complex formation with chelates metal ions such as – Zn2+, Ni2+ or Cu2+. Further the elution process can be carried out through reduced pH or increased ionic strength of the mobile phase.

Hydrophobic interaction chromatography

Many antibodies form dimers or aggregates, in particular at high expression levels. The aggregates are more hydrophobic in nature. It makes the aggregates bind more strongly to  HIC media compared with the corresponding monomer. Therefore, HIC is an efficient tool for aggregate and dimer removal in flowthrough mode. Aggregates bind to the medium while the antibody passes straight through. HIC is also useful for removing HCP and endotoxins. During HIC chromatography, beads are prepared by modifying the surface with groups such as alkyl or aryl which are hydrophobic in nature. When sample molecules both with hydrophobic and hydiphilic group  are passed through HIC columns , high salt concenraion of the buffer is used to reduce the solvation  property of the molecules. Low solvation results in exposure and media absorbance of the hydrophobic molecule.  Molecules with low hydrophobicity requires lower amount of salt for binding and usually a elution buffer with decreasing salt gradient is useful to elute molecules according to increased hydrophobicity,

Multimodal chromatography media

Multimode chromatography represents multiple types of chromatographic formats in single resin. This will induce the selective nature of resin as in this method separation of proteins depends on several characteristics instead of single one.New types of chromatography media that have a ligand with additional interaction mechanisms in combination with ion charges, commonly called multimodal media, are now also available. Capto adhere is a multimodal strong anion exchanger, and is designed for operation in flowthrough mode for the MAb. Multimodal media can also be operated in bind-elute mode for cases where a flowthrough step does not remove impurities such as antibody fragments.

Melon gel chromatography

During melon gel chromatography, antibodies are purified by fractionation based on chemicals. In presence of specific mild buffer melon gel resin will attach with most of the immunoglobulins of serum or cell culture supernatants, which is non IgG in nature. This pure IgG will get separated at the flow through part.  Various types of melon gel kits are commercially available for IgG purification. This method is relies on negative selection and do not have elution steps. Melon gel chromatography is also useful for Bovine serum albumin removal from the stock solution of antibody. As a result BSA cannot interfere with antibodies during antibody labelling techniques. A ammonium sulphate precipitation is usually followed for cell culture supernatants before going to melon gel chromatography.

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Factors affecting chromatography

There are some basic parameters which need to take care during antibody purification, such as-

Column size- Backpressure is one of the important aspects of selection of columns for chromatography. A high backpressure will distort the tubing and connections. Thus in respect to backpressure formation a short thick column is preferred over a thin tall column.  A short and wide column will give higher flow rate. The amount of resin to be used for columns is dependent on which type of chromatography is going to perform and also binding property of resins.

Flow rate- the flow rate of the open columns is dependent on gravity. However, if the buffers are pumped out through the columns, then the flow rate  will show variation. A higher flow rate will cause excess resin packing and generation of backpressure.

Buffer .during protein purification maintenance of protein stability is important. Thus a well-balanced buffer with a pH which is able to keep the protein structure, function and solubility.  The buffer of choice should be based on the characteristics such as-  i) Solubility to water ii)Chemically stable in nature iii)Capable of high buffering capacity or shows compatible nature with other solutions present in the chromatographic method.

Sample loading: samples to be loaded onto chromatographic tube should be devoid of any contaminants and precaution should be taken to check whether they could interfere with the purification process. Such as EDTA interference with Nickel column.  In connection with His tagged protein, EDTA will compete for binding site with the metal nickel. Additionally salt concentration is also of great importance in respect to protein purification.

Troubleshooting  purification

Target protein fails to bind with the column- there could be several reasons such as -less optimal condition for protein and binding or elution or both, the antibody has less affinity for ligand molecules and column storage was not proper. To reduce the problem several remedies can be taken like-i) Optimization is necessary for pH, flow rate, temperature, ion or salt concentration ii) Re packing of column can be beneficial or iii) Addition of protease inhibitor to sample buffer.

 Eluted protein gets degraded- Protein of interest is not stable in elution buffer. To better the yield, Neutralization followed by elution of the antibody fragments.

Antibody cannot be detected after elution- The IgG might not getting attached with resin or Column packing, storing and working temperature might be different.To reduce the problem-i)Use of  another affinity column for the purification process is recommended. ii) Equilibrate the column temperature to make a balance between storage and usage temperature.

Presence of Bubbles at prepack column- that might be due to presence of bubbles which is already present in sample or buffer.  De gassing of sample or buffer might be helpful to overcome the problem.

Flow rate of column is slow- When bubbles present in the sample blocks the pores of the affinity column, flow rate might get reduced. To overcome the problem degassing of sample or buffer is useful.