Immobilized Artificial Membrane (IAM) technology is an innovative approach to chromatography in which the chromatographic surface emulates the lipid environment of the cell membrane.
HPLC Separation Tools for Membrane Protein Purification and Drug Membrane Permeability Prediction.
Phosphatidylcholine (PC) is the major phospholipid found in cell membranes. IAM chromatography phases prepared from PC analogs closely mimic the surface of a biological cell membrane. Consequently, IAM phases display a high affinity for membrane proteins and are useful in membrane protein purification and in the study of drug membrane interactions. The IAM surface is formed by covalently bonding the membrane-forming phospholipids to silica.
Types of IAM columns applications:
Membrane Protein Purification
IAM.PC and IAM.PC.MG
• Drug membrane permeability prediction
• Hydrophobic in nature
IAM Fast-Screen Mini Columns
• High throughput estimation of drug permeability
The IAM.PC phase, developed by Dr. Charles Pidgeon of Purdue University, was the first in a line of IAM phases to be manufactured by Regis. Use of this phase has simplified the inherent difficulties of protein isolation and purification, 3-9 allowing for rapid purification of membrane proteins while maintaining biological activity.
The first IAM stationary phase was based on the prevalent membrane lipid, phosphatidylcholine (PC), and consists of monolayers of amphiphilic phospholipids covalently bonded to aminopropyl silica particles through a terminal amide linkage. As a result, the bulky phosphatidylcholine groups shield many of the amine binding sites on the silica surface, preventing amine interaction with the protein molecules.
The membrane nature of the IAM phase imparts surface characteristics which are useful in the chromatography of membrane proteins. These include: high protein loading, increased protein recovery, recovery of functional activity, and selectivity for membrane proteins.
Large membrane proteins can interact with any combination of polar headgroup, hydrophobic chain, or inner amine groups. The subsurface has been shown to interact with certain solutes, and may or may not contribute to the separation of a given biomolecule. The residual amines can be left unaltered on the subsurface or deactivated through an endcapping procedure, which results in increased stability of the bonded phase. The methyl glycolate endcapping, for example, converts residual amines to neutral amides and introduces a hydroxyl group (IAM.PC.MG).
Numerous applications have been developed using IAM.PC columns:
• Purification of Cytochrome P450.
• Isolation of membrane proteins.
• Prediction of solute transport across human skin.
• Prediction of amino acid transport across the blood-brain barrier.
• Binding of solutes to liposome membranes.
• Immobilization of Trypsin and -chymotrypsin for the determination of their inhibitor and substrate activity.
Fast-Screen Mini Column IAM chromatography has recently gained acceptance among drug discovery chemists for estimating the membrane permeability of small molecule drugs.
The interaction between membrane bilayer and drug can be modeled by the IAM column/drug system. KIAM, the equilibrium constant describing the relative concentrations of drug in the membrane and in the external fluid, is analogous to the KIAM.
This IAM technique provides superior correlation with experimentally determined drug permeability when compared to other chromatographic methods. ODS silica, for example, retains analytes solely on the basis of hydrophobicity. IAM more closely mimics the interaction of analytes with biological membranes, where a combination of hydrophobic, ion pairing, and hydrogen bonding interactions are possible. This combination of interactions measured by the IAM column is known as phospholipophilicity.
Intestinal Drug Permeability
The retention factors measured on reversed phase C18 (ODS) columns (a commonly used model to determine drug partitioning) show extremely poor correlation with intestinal drug absorption. For this group of compounds, hydrophobicity alone, as measured by the reversed-phase C18 column, is a poor predictor of drug absorption. Since IAM.PC Drug Discovery columns measure both hydrophilic and hydrophobic interactions between drugs and membranes, the IAM.PC Drug Discovery Column is better suited to the prediction of intestinal drug absorption.
Like the first generation IAM.PC.DD material, the IAM.PC.DD2 is used to predict drug membrane permeability. The ester bonding of the DD2 packing offers more hydrophobicity than the first generation DD phase. This material is a diacylated or double chain ester PC ligand and is endcapped with C10/C3 alkyl chains.
The IAM.PC.DD2 material offers the following advantages:
• Hydrophobic nature
• Greater stability
• Excellent correlation to traditional methods
The IAM.PC.DD2 offers more hydrophobicity than the first generation IAM.PC.DD material. This hydrophobic nature allows for longer retention times to compounds not well retained on the IAM.PC.DD material.
Another distinct advantage of the IAM.PC.DD2 material is its ability to tolerate mobile phases between pH’s 7.0 to 7.5, thus resulting in longer column life under these conditions.
Excellent Correlation to Traditional Methods
The traditional means of predicting membrane permeability include the use of Caco-2 cell line cultures, intestinal tissue, or liposome assays. These methods are laborious and costly to perform.