Protein Purification Techniques- Chromatographic Techniques

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This presentation is about various chromatographic techniques involved in protein purification at the industrial level, focusing on Ion exchange chromatography, Membrane affinity chromatography and Size exclusion chromatography.

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Protein purification techniques- Chromatographic Techniques By Shradha Menon MSc MBHG 1st year .01
CONTENTS • Chromatographic techniques: 1. Ion exchange chromatography 2. Membrane affinity chromatography 3. Size exclusion chromatography .ißD
PROTEIN PURIFICATION- General Downstream Process PROTEIN PURIFICATION Salt precipitation NaCl Centrifugation to collect precipitate, resuspension Immobi ised Metal Affinity Chromatography (IMAC) Ni2 Ion Exchange Chromatography (IEC) Size Exclusion Chromatography (SEC) Concentrating by centrifugation through a filter 1 Proteins purified
1. Ion Exchange (z Chromatography
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Ion exchange chromatography- Procedure 4. 6. Eluent loading: Equilibration- This involves running the equilibrated buffer via the column. This is done to make the resins attached firmly to the surface of the column. Sample Injection: Loading- This chromatography is based on the attraction between oppositely charged ions, i.e. the sample molecule and the ion exchanger. You can use the ion exchanger as per your desired product where the matrix wili be positivelv or negatively charged, respectively. The aqueous solution containing an impure sample mixed with charged ions is suspended in the ion exchange column. Binding of desired molecules in the sample- Sooner, the oppositely charged molecules will start replacing the covalently attached functional group from the matrix. And after this, the ions of the ion cloud exchange their places with the functional group without altering the properties of the matrix. Washing of the unattached waste molecules- In order to remove the attached free molecules that remain unbonded are washed off usin a washing buffer. This removes all the unwanted elute out while the desired ions still remain intact in the ion exchanger. As the was ing starts, we obtain the first peak on the graph. From the time where the graph starts to form in the graph-we will collect the washouts materials in a different place. Note: Before this peak, there is a baseline that starts just after the equilibration and continues as a straight line till here. Elution of the desired ions- After washing, there is a time of elution. This last step is to get the desired product out as eluents. Elution buffer is used with gradually increasing concentrations to elute the bonded sample. Result Analysis- The purity and yield of the purified protein are assessed by analyzing fractions collected during elution, typically using SDS-PAGE or spectrophotometry.
Equilibration Starting Buffer 000 00 Loading Of proteins Wa Shing Wash 000 fluent With Ionic GradWnt 000 t Buffer With Gradient Ion exchange chromatography- Procedure Negatively Charged Matrix —Target Protein Non-specific Protei
Ion exchange chromatography- How to read the result The charged salt ions compete with bound proteins for the charged resin functional groups. Proteins with few charged groups will elute at low salt concentrations, whereas proteins with many charged groups will have greater retention times and elute at high salt concentrations. Although less common, a pH gradient can also be used for elution. Here, a pH gradient is chosen that approaches the protein of interest's pl. Proteins will elute when the pH gradient reaches their PI, because they will no longer carry a net charge that allows them to interact with the column resin. To elute proteins from an anion exchange resin, a decreasing pH gradient is chosen, while an increasing pH gradient is chosen for elution from cation exchangers. Yellow figure• Salt gradient elution. Elution of proteins (blue trace) with an increasing salt gradient (red trace)
Ion exchange chromatography- Examples • The typical scheme in IEX of binding the target, washing the column and eluting the target is called 'bind/elute mode' and is often applied in intermediate purification steps for example in downstream processing of antibodies. (Purification of monoclonal antibodies for immunotherapy). Anion Exchange Chromatography is an integral part of most plasma protein purification platforms, such as Factor Vlll purification.
I. 2. Ion exchange chromatography- Types Based on the type Of resin used, we can classify this chromatography into two categories: cationic and anionic. Cationic Exchange Chromatography: Here, the cationic exchangers (acidic ion exchangers) are used. In this type of exchanger, the resins attached to the stationary matrix are negatively charged. The functional group associated with the resin is positively charged. This functional group gets replaced by the positive ions of the sample. Anionic Exchange chromatography: Here, the anionic exchanger (basic ion exchange) is used. In this type of exchanger, the resin attached bears a positive charge along with negatively charged functional groups. Thus, the resins attract the negative anions present in the sample, which will get attached to it by replacing the functional group. positively charged proteins Cation exchanger h butter Catim e x charw with cha r ed pa teins exchangable nS Butter charged) Negatively charged proteins butter Anion with charged p rotens buffer
Ion exchange chromatography- Applications 1. 2. 3. 4. 5. A critical tool for pharmaceutical analysis. Ideal for estimating, separating, and purifying biomolecules, including proteins, amino acids, peptides, carbohydrates, vitamins, enzymes, etc. Used for the separation and purification of organic molecules from a natural resource. Vital in modern-day drug discovery methods. Highly preferred in routine analysis of amino acid mixtures.
Ion Exchange Chromatography • Advantages: 1. Applicable to a wide range of proteins. 2. Low maintenance, cheap and comparatively simpler working mechanism. 3. Separation time is less. Thus, you can separate bulk sample volume in a short time. 4. High resolution and selectivity. • Disadvantages: It remains limited to the separation of 1. charged molecules only. The efficiency of the column diminishes 2. after repeated use. Thus, it becomes difficult to predict the accuracy of results. 3. Instrumentation is expensive and requires a skilled expert to handle the apparatus. The resin can easily get damaged, which 4. will hamper the whole procedure.
2. Affinity Chromatography
Affinity chromatography It is a powerful chromatographic technique that enables purification of desired protein from others with respect to biological function or individual chemical structure Purpose of this technique: Affinity chromatography is employed to purify proteins based on their specific interactions with ligands or antibodies immobilized on a solid matrix. Principle ofthe technique: Proteins selectively bind to the affinity matrix via specific Interactions, while impurities pass through. The bound proteins are subsequently eluted under controlled conditions. Immobilization of the ligand to which the protein binds enables selective adsorption of the desired protein. A biospecific ligand that can be attached to a chromatography matrix covalently is one of the requirements for successful affinity purification. The binding between the ligand and molecules of interest must be reversible to allow the molecules to be removed in an active form. After eluting away the contaminants, the coupled ligand must retain its specific binding affinity for the molecules of interest _ For example, protein A or protein G ligands coupled to an agarose base matrix are used for routine purification of antibodies. The affinity approach is limited to proteins that have a specific binding property, except that proteins are theoretically able to be purified by immunoaffinitv chromatography, which is the most specific of all affinity techniques.
i, in Your specific protein Mobile phase Stationary phase Spacer Affinity chromatography- Introduction
Affinity chromatography- Components • Essential components of Affinity Chromatography system: 1. 2. a. b. Column type(depending on target protein and the ligand) Buffers: Loading buffer Elution buffer
Affinity chromatography- Procedure The operation of affinity chromatography involves the following steps: Sample . • • COIL_mn in Affinity ligands to a 5 is to disrupt interaction between the affinity 3 molecules bind to 1. 2. 3. 4. 5. Choice of an appropriate ligand Immobilization of the ligand onto a support matrix Binding of the molecules of interest with the ligand Removal of non-specifically bound molecules Elution of the molecules of interest in a purified form is thrm.gh tho c 01 umn to punned ta rget is the column
Affinity chromatography- Procedure (alternative diagram)
Affinity chromatography- Result Equilibration Sample application Begi n sample application Elution Re-equilibration Change to elution buffer Time
Affinity chromatography- Direct and Indirect Approach Affinity chromatography can be broadly divided into two method types: via 2. The first method uses a naturally occurring structure or sequence of amino acids on the protein as the binding site. Examples include the affinity of Affi•GeI Blue support binding for albumin's bilirubin•blnding site and the binding of protein A in the Affi•GeI and Affi•Prep protein A supports to the FC region of IgG. An important consideration for antibody purification is to determine the affinity of your target antibody for protein A/G chromatography media, which varies widely. The second method involves binding to a special amino acid sequence engineered into the protein of interest, commonly referred to as a "tag". A number of different tags are available. Two of the most commonly used protein tags are the polyhistidine tag, which binds to certain metal-containing complexes such as those in Profinityw IMAC resins, and the glutathione s•transferase (GST) sequence, which binds to glutathione, found in Bio-Scalew Mini Profinitvw GST media. Theoretically, any protein can be purified using the tagging method; however, many factors must be considered to design a process to purify tagged recombinant proteins.
Affinity chromatography- Applications While the term "affinity chromatography" implies that the protein of interest is being purified via the affinity tag, there are a number of applications that can be done in addition to purificaton. These include: 2. Detection: Specific antibodies are available for most affinity tags, so that tagged proteins can be detected In Western Blots, via immunostaining, in ELISA assays or other antibody-based applications. Immobilization: Affinity tags can be used to immobilize tagged proteins, e.g. on surface plasmon resonance chips, on ELISA plates or other surfaces. The immobilized proteins can then be assessed e.g. for their ligand binding kinetics. Pulldown: Affinity-tagged proteins can be pulled down from complex solutions e.g. via affinity magnetic beads. They can also be immobilized via affinity beads and used to pull down interaction partners from complex mixtures, such as cell tysates.
Affinity chromatography- Immunoaffinity Chromatography Immuno-affinity chromatography (IAC) • A sub category of affinity chromatography A biologically related binding agent is used for the selective purification or analysis of a target compound stationary phase consists of an antibody or antibody- related reagent The selective and strong binding of antibodies for their given targets has made them of great interest as immobilized ligands in affinity chromatography
Affinity chromatography- Benefits and Limitations Benefits: 1.High sensitivity 2.High Specificity 3.Affinity chromatography doesn't rely on ionic strength, pH, temperature, and composition of the buffer. 4.High degree of purity can be obtained by Affinity Chromatography. 5. This is a very reproducible process. Limitations: l.lt takes a lot of skill to handle it. 2. The volume of the sample is limited. 3. The ligands used in affinity chromatography are costly. 4.Metal-ion transfer and metal ion leakage lead to loss of protein. 5.Affinity chromatography is non- specific for adsorption than other chromatography methods.
3. Size Exclusion Chromatography
Size Exclusion Chromatography Size exclusion chromatography (SEC), otherwise known as gel filtration chromatography, is one of the simplest and mildest methods of protein separation which is too often thought of as merely a final polishing step in a purification protocol. Purpose of this technique- Size exclusion chromatography, also known as gel filtration chromatography, is utilized to separate proteins based on their size or molecular weight. Principle of technique- SEC separates molecules by differences in size as they pass through a SEC resin packed in a column. A very effective method for protein analysis and it allows true size profiling of protein samples due to the mild separation conditions that can be used to obtain high-resolution separations. This is a great advantage compared to other size-separation techniques, such as ultrafiltration or dialysis.
Size Exclusion chromatography- Instrumentation Pump c on taüler Standards etecto Columns array) Sample Injection Data Station 'or all eluents for all dirtmsk•ns 1000m") : concentration: R'. UV, etc. IR, MS, viscometer, light scattering. etc.
Size Exclusion chromatography- Procedure 1. Firstly, a column of spherical gel beads is prepared for the gel filtration chromatography. 2. The packed bed is equilibrated with a buffer. 3. Then, the test solution (mobile phase) is eluted through the column. After that, the particles in the test sample will enter into or diffuse out of the porous gel matrix (stationary phase). 4. 5. The molecules with a small molecular size will enter the gel pores, i.e. small molecules will cover a longer path or stay longer on the column. 6. Large molecules with large molecular sizes cannot get into the pores of gel beads or easily pass through the column. 7. Thus, the separation of the particles occurs at different intervals by following isolation and identification of the components separated. 8. Fractionation and desalting are the methods that facilitate the separation of components in size exclusion chromatography. a. b. Desalting elutes the heavy molecules by keeping the exclusion limit of the gel smaller. The smaller molecules enter the gel pores while the larger particles leave the column. Fractionation isolates the target molecules within the gel matrix, whose molecular size must be within the gel's fractionation range.
Size Exclusion chromatography- Procedure 00 o 09 00
SEC target "tein Size Exclusion chromatography- Result and how to read it SOS Results from SEC are usually expressed as a chromatogram (elution profile) that shows the variation in concentration of sample components as they elute from the column in order of their molecular size.
Size Exclusion chromatography - Examples • Figure- Size Exclusion Chromatography of Protein Biopharmaceuticals (monoelond
Size Exclusion Chromatography- Applications *Proteins fractionation + Purification acids) + Molecular weight determination(globular proteins). + Separation of sugar. proteins, peptides, rubbers' and others on the basis of their size. + This technique can be determine the quaternary e Structure Of purified proteins.
Size Exclusion Chromatography 1. 2. 3. 4. 5. 6. Advantages: Identify high mass components even in low concentrations. Absolute molecular weight can be obtained. Short analysis time and well-defined separation. Narrow bands, which leads to good sensitivity. There is no sample loss as the solute does not interact with the stationary phase. A small amount of the mobile phase is required. 2. 3. 4. 5. Disadvantages: Filtration of the sample should be done before injecting into the column to prevent dust and other particles from ruining the column and interfering with the detector. A limited number of peaks that can be obtained within the short time scale of the GPC run. Bad response for very small molecular weights. High investment costs may be required. Inapplicable to sample with similar sizes, such as isomers. A 10% difference in molecular mass is required for a reasonable resolution.
CHROMATOGRAPHIC TECHNIQUES- SUMMARY CtM ?????? ???
PROTEINS PURIFIED Technique 1. Membrane Affinity Chromatography 1 2. Ion Exchange Chromatography 3. Hydrophobic Interaction Chromatography 4. Size Exclusion Chromatography Protein purified (a)Polyclonal antibodies (b)Human Chorionic Gonadotrophin (a)Vibronectin-binding surface protein (b)Tubulin a- N- acetyl galactosamidase Monoclonal antibodies
Papers for Reference I. Ion Exchange Chromatography- https://mvw.ncbi.nIm.nih.gov/ømc/articIes/PMCg83S05g/ 2. Affinity Chromatograph'f httos://onIineIibrarv.wiIev.com/doi/fuII/IO.1002/anie.20220007g 3. Size Exclusion Chromatograph•r httos://www.ncbi_nIm.nih.aov/omc/articIes/PMC76g3800/

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Protein Purification Techniques- Chromatographic Techniques

Shradha M / Dubai

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