Isolasi dan Pemurnian Antibodi

Meiriza DjohariMata Kuliah Immunologi

STIFAR

Structure of Antibody

Fragmen antigen Binding

Fragmen crystallizable

Antibody purification is a multi process by which antibodies with high purity can be achieved--> for immunochemical techniques within general research and for therapeutic and diagnostic applications

What is Antibody Purification?

• Serum (polyclonal antibodies),

• Ascites fluid

• Cell culture supernatant of a hybridoma cell line (monoclonal antibodies).

Source of Antibody

To remove possible contaminants serum protein such as albumin, transferrins, cell degradation products like DNA and cellular proteins

Why to Purifiy Antibody

The choice of a purification method based on these factors :

Nature of antibodyNature of feed stockScale of ProductionEconomics-- cost and other factorsProcess timing Desired purifity

Purification Methods

Sample preparation

Capture

Initial purification

Secondary Purification

Polishing/Formulation

Antibody Purification Process

It is the initial step in which crude protein sample is conditioned or making it ready for the initial capture stepInvolves changing Ph or Ionic strength, dilution of the crude sample or addition of salts for the ionic changing (cost ) --> Buffer exchange by size exclution chromatography or to use ultrafiltration or diafiltration

Antibody PurificationStep 1 : Sample Preparation

Physicochemical fractionation

Class-specific affinity

Antigen-specific affinity

Antibody PurificationStep 2 : Capture

On the nature and the optimization requirement of the crude antibody source.In addition to protein contaminants, other impurities such as DNA, Endotoxins, viruses, and aggregate need to be removed. In such case multistep procedure are inevitable.

Antibody PurificationStep 3 : Intermediate Purification

• Final polishing/formulation step can be considered as a part of purification in which it removes condition that would impair the stability or utility of the antibody in its intended use.

• Ultrafiltration• SEC, Dialfiltration• Lyophilization

Antibody PurificationStep 4 : Polishing/Formulation

Antibodies that were developed as monoclonal antibody can be fully purified without using an antigen-specific affinity method because the target antibody is (for most practical purposes) the only immunoglobulin in the production sample. By contrast, for polyclonal antibodies (serum samples), antigen-specific affinity purification is required to prevent co-purification of nonspecific immunoglobulins.

Physicochemical fractionation

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation

Ion Exchange Chromatography

Immobilized Metal Chelate Chromatography

Thiophilic Adsorption

Melon Gel Chromatography

Differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on :

• Size

• Charge

• Other shared chemical characteristics of antibodies in typical samples.

This isolates a subset of sample proteins that includes the immunoglobulins.

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation

Ion Exchange Chromatography

Immobilized Metal Chelate Chromatography

Thiophilic Adsorption

Melon Gel Chromatography

Dialysis, desalting and diafiltration can be used to exchange antibodies into particular buffers and remove undesired low-molecular weight (MW) components. Dialysis membranes, size-exclusion resins, and diafiltration devices that feature high-molecular weight cut-offs (MWCO) --> can be used to separate immunoglobulins (>140kDa) from small proteins and peptides.

However, except with specialized columns and equipment, these techniques alone cannot purify antibodies from other proteins and macromolecules that are present in typical antibody samples. More commonly, gel filtration and dialysis are used following other purification steps, such as ammonium sulfate precipitation

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation

Ion Exchange Chromatography

Immobilized Metal Chelate Chromatography

Thiophilic Adsorption

Melon Gel Chromatography

Ammonium sulfate precipitation is frequently used to enrich and concentrate antibodies from serum, ascites fluid or cell culture supernatant. As the concentration of this lyotropic salt is increased in a sample, proteins and other macromolecules become progressively less soluble until they precipitate; the lyotropic effect is called "salting out." Antibodies precipitate at lower concentrations of ammonium sulfate than most other proteins and components of serum.

The selectivity, yield, purity and reproducibility of precipitation depends on :

Time

Temperature

PH

Rate of salt addition .

Ammonium sulfate precipitation provides sufficient purification for some antibody applications, but most often it is performed as a preliminary step before column chromatography or other purification method

Other antibody precipitation reagents that have been used for special antibody purification situations include using caprylic/octonoic acid, polyethylene glycol and ethacridine .

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation

Ion Exchange Chromatography

Immobilized Metal Chelate Chromatography

Thiophilic Adsorption

Melon Gel Chromatography

Uses positively or negatively charged resins to bind proteins based on their net charges in a given buffer system (pH).

IEC is a cost-effective, gentle and reliable method for antibody purification.

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation

Ion Exchange Chromatography

Immobilized Metal Chelate Chromatography

Thiophilic Adsorption

Melon Gel Chromatography

Uses chelate-immobilized divalent metal

ions (usually nickel, Ni2+) to bind

proteins or peptides that contain clusters

of three or more consecutive histidine

residues.

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation

Ion Exchange Chromatography

Immobilized Metal Chelate Chromatography

Thiophilic Adsorption

Melon Gel Chromatography

Combines the properties of hydrophobic

interaction chromatography (HIC) and

ammonium sulfate precipitation (the

lyotropic effect).

Size Exclusion Chromatography (SEC)

Ammonium Sulfate Precipitation

Ion Exchange Chromatography

Immobilized Metal Chelate Chromatography

Thiophilic Adsorption

Melon Gel Chromatography

A proprietary resin chemistry (and optimized buffer system) for purifying antibodies by chemical-based fractionation. In the specified mild buffer condition, Melon Gel resin binds most non-IgG proteins found in serum, ascites fluid and culture supernatants, while allowing purified IgG to be collected in the flow-through fraction.

Class-specific affinity

Solid-phase binding of particular antibody classes (e.g., IgG) by immobilized biological ligands (proteins, lectins, etc.) that have specific affinity to immunoglobulins. This purifies all antibodies of the target class without regard to antigen specificity.

Protein A, G and L Antibody-binding Ligands

Antibody Purification with Protein A, G and L

IgM Purification

IgA Purification

Antigen-specific affinity

Purification of antigen-specific antibodies is often required.

Can be accomplished by immobilizing the particular antigen used for immunization so that only those antibodies that bind specifically to the antigen are purified in the procedure.

Antigen Immobilization and PresentationPeptide Antigens and Affinity LigandsProtein Antigens and Affinity LigandsBinding and Elution Conditions

Tugas• Cari jurnal tentang purifikasi antibodi• Dibagi 6 kelompok

klp 1 : Size Exclusion Chromatography klp 2 :Ammonium Sulfate PrecipitationKlp 3 :Ion Exchange ChromatographyKlp 4 :Immobilized Metal Chelate ChromatographyKlp 5 :Thiophilic AdsorptionKlp 6 :Melon Gel Chromatography

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