Chromatography

Chromatography is a method for separating components of mixtures, It is the most common term used for defining separation techniques used in the chemical and biopharmaceutical industry to describe the extraction and purification of pure chemical compounds. A system consisting of a stationary and a mobile phase is necessary for chromatographic separation. The stationary phase is a substance that binds and shortly releases the molecules moving through the system. The particles can move through the system due to the mobile phase, which can be for example a liquid (eluent) or a gas (carrier gas) that carries the molecules through the stationary phase. The chromatographic separation process is based on the different mobility of different components in the chromatographic system (column, plate, etc.). The compounds that are more like the stationary phase (have higher affinity towards it), move slower that the compounds that are more like the mobile phase. The time spent on going through the chromatographic system is called the retention time (tR). Due to the different mobilities different compounds also have different retention times. The detection of the compounds after the chromatographic separation is carried out visually or by using a special device for detection. The intent of preparative chromatography is to purify materials and use them for additional testing or as final products (1–3). For example, preparative chromatography is used to purify compounds from combinatorial libraries, to obtain material for clinical trials, and in large-scale production of drugs and vaccines (4,5). Consequently, the scales of preparative chromatography vary substantially. Preparative HPLC is used for the isolation and purification of valuable products in the chemical and pharmaceutical industry as well as in biotechnology and biochemistry. Depending on the working area the amount of compound to isolate or purify differs dramatically. It starts in the µg range for isolation of enzymes in biotechnology. At this scale we talk about micro purification. For identification and structure elucidation of unknown compounds in synthesis or natural product chemistry it is necessary to obtain pure compounds in amounts ranging from one to a few milligrams. Larger amounts, in gram quantity, are necessary for standards, reference compounds and compounds for toxicological and pharmacological testing. Industrial scale or production scale preparative HPLC, that is, kg quantities of compound, is often done nowadays for valuable pharmaceutical products. SMB chromatography is a cost effective and environmentally friendly technology, using up to 90% less solvent than similar chromatographic purification schemes. It combines the advantages of high yields, high purity and accelerated scale-up for clinical to commercial-scale API purification. This chiral chromatography based technology works at the molecular level to separate active and non-active enantiomers for active ingredient production. The technique is a valuable tool for the research biochemist and is readily adaptable to investigations conducted in the clinical laboratory. For example, chromatography is used to detect and identify in body fluids certain sugars and amino acids associated with inborn errors of metabolism. Adsorption chromatography that in which the stationary phase is an adsorbent. Affinity chromatography that based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. Any of these substances, covalently linked to an insoluble support or immobilized in a gel, may serve as the sorbent allowing the interacting substance to be isolated from relatively impure samples; often a 1000-fold purification can be achieved in one step. Column chromatography the technique in which the various solutes of a solution are allowed to travel down a column, the individual components being adsorbed by the stationary phase. The most strongly adsorbed component will remain near the top of the column; the other components will pass to positions farther and farther down the column according to their affinity for the adsorbent. If the individual components are naturally colored, they will form a series of colored bands or zones. Column chromatography has been employed to separate vitamins, steroids, hormones, and alkaloids and to determine the amounts of these substances in samples of body fluids. Exclusion chromatography that in which the stationary phase is a gel having a closely controlled pore size. Molecules are separated based on molecular size and shape, smaller molecules being temporarily retained in the pores. Gas chromatography a type of automated chromatography in which the mobile phase is an inert gas. Volatile components of the sample are separated in the column and measured by a detector. The method has been applied in the clinical laboratory to separate and quantify steroids, barbiturates, and lipids. Gas-liquid chromatography gas chromatography in which the substances to be separated are moved by an inert gas along a tube filled with a finely divided inert solid coated with a nonvolatile oil; each component migrates at a rate determined by its solubility in oil and its vapor pressure. Gel-filtration chromatography (gel-permeation chromatography) exclusion chromatography. Ion exchange chromatography that utilizing ion exchange resins, to which are coupled either cations or anions that will exchange with other cations or anions in the material passed through their meshwork. Molecular sieve chromatography exclusion chromatography. Paper chromatography a form of chromatography in which a sheet of blotting paper, usually filter paper, is substituted for the adsorption column. After separation of the components as a consequence of their differential migratory velocities, they are stained to make the chromatogram visible. In the clinical laboratory, paper chromatography is employed to detect and identify sugars and amino acids. Partition chromatography a process of separation of solutes utilizing the partition of the solutes between two liquid phases, namely the original solvent and the film of solvent on the adsorption column. Thin-layer chromatography that in which the stationary phase is a thin layer of an adsorbent such as silica gel coated on a flat plate. It is otherwise similar to paper chromatography. The most widely used chromatographic techniques include Gel Filtration Chromatography, Ion Exchange Chromatography, Hydrophobic Interaction Chromatography, Affinity Chromatography, High performance (high pressure) liquid chromatography (HPLC) and Preparative Chromatography. Chromatography may be classified according to its aim, technical details, state of mobile phase or other parameters. The aim of the chromatographic procedure can either be a preparative or an analytical chromatography. Preparative chromatography is used for separation of individual substance, so that the substance can be further used for some purpose (e.g. for use in pharmaceutical preparation). For example, purification of a reaction product from the reaction mixture in order to use it in pharmaceuticals. The aim of analytical chromatography is to detect presence (qualitative analysis) and amount (quantitative analysis) of certain component in the mixture. Chromatographic separation methods are used for commercial scale production of clinical supply molecules through specialized SFC or SMB processes. Other Chromatography technologies such as continuous chromatography with simulated moving beds (SMB) and super critical fluid chromatography (SFC) with solvent recycling are used that lower environmental impact by up to 90%. In some cases, high-volume batch columns are used to manufacture drug substances through chemical purification and chiral separations. All that you have to do is choose an appropriate CMO / CDMO ensures which gives you enhanced support for clinical trials and market supply upon drug approvals. It is important to ensure that the CMO / CDMO is composed of experts in chemical processes and are working at a facility certified to handle highly potent compounds, Highly Potent APIs (HPAPI) to necessary occupational exposure band level. It is extremely essential to ensure that the CMO/CDMO is compliant with United States Food & Drug Administration (FDA) and/or European (EU) current good manufacturing practice (cGMP) regulations for human pharmaceuticals. PharmaCompass gives you access to find FDA approved bulk API manufacturing facilities that perform chromatographic separation of chemicals, biochemical and pharmaceutical compounds. Here you will find the cGMP certified API manufacturing companies that specialise in Chiral Separations, Separation of diastereomers & regioisomers, removal of genotoxic or toxic biochemical and other material separations using high-volume batch columns. The CDMOs/CMOs listed here are categorised according to geographies. The concentration of pharmaceutical companies which supply high performance chromatographic systems and important parts of chromatography systems are in France, Germany, Sweden, Italy, Switzerland and United States. Check the list of API manufacturing companies that support production of pharmaceuticals and/or functional materials with the latest high throughput purification technologies. For chromatographic purification of pharmaceutical compounds, check out all the different API purification equipped contract manufacturing sites that can suit your requirements for the purpose of manufacturing of biopharmaceuticals, chiral compounds, APIs and Highly Potent APIs (HPAPIs). It is very necessary to have an API Purification Plant for pharmaceuticals that is compliant with GMP requirements. There are existing full-scale contract purification services available for high-value-added substances, including complex molecules for medicines and biopharmaceuticals that undergo various phases of preclinical and clinical development. Contract development and manufacturing organizations (CDMOs) can optimize chromatography methods depending on the objective and required purity using high-volume batch columns and perform preparative purification using preparative LC systems and packing materials, developed and produced in-house. Preparative chromatography can be performed with an analytical column (and system) to produce a few micrograms of material, up to process scale, providing a ton quantities of sample, which use 1-m-long, 200-mm i.d columns. The larger the quantity of analyte required, the further the technique is removed from analytical chromatography, both in terms of scale and ideology; the bigger the scale, the more "nonchromatographic" parameters have to be considered. Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. SFC is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Basic separation principles are similar to those of high performance liquid chromatography (HPLC), however SFC typically utilizes carbon dioxide as the mobile phase; therefore the entire chromatographic flow path must be pressurized. Since the supercritical phase represents a state in which liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography. The development of chromatographic methods has brought about a number of refinements and advancements in the process. As is well known, chromatographic separation of active pharmaceutical ingredients (APIs) and highly potent APIs (HPAPIs) require high accuracy, effective dosage forms and quick elution rates so that they are effectively fewer side effects and greater targeted therapeutic effects. So choosing the correct chromatographic method is a function of the types of molecules to be separated. Today, there are small molecules, peptides, antibody drug conjugates, fermented biological compounds and advanced micro-chemicals used for different therapeutic areas. These small molecules present various complexities during drug development and commercial scale production processes, which require chromatographic expertise and cutting-edge technology to ensure the highest quality of molecules produced. Chromatographic methods like continuous chromatography with simulated moving beds (SMB) , super critical fluid chromatography (SFC), gel permeation (or gel filtration chromatography), size exclusion chromatography, Ion-exchange chromatography, Affinity chromatography and thin-layer chromatography methods are utilized in the industry for separation of biomolecules based on batch or continuous flow processes. Gel filtration chromatography is preferred for purifying macromolecules as it allows different sizes and shapes. Ion exchange chromatography is applied for separation of macromolecules based on the net surface charges that can be tuned according to the pH. Affinity chromatography separates on the basis of biological specificity in enzyme-substrate, enzyme-inhibitor, receptor-ligand, antigen-antibody and other interactions. In this method, one interaction partner is immobilised on a solid surface (stationary phase) and can selectively bind its interacting partner from a mixture in the mobile phase. The other components of the mixture can then be removed by replacing the mobile phase (washing). The pure material is then eluted by applying a mobile phase that disrupts the specific interaction. For the purpose of producing clinical drug substances using novel drug development processes, chromatographic techniques are vital for preparation of pure early drug molecules – better known as preparatory chromatography. Since drug manufacturing processes use multi-kilogram active pharmaceutical ingredients (API) for performing toxicology studies, chiral compounds purification is required. Hence, continuous chromatographic separations of intermediates and active pharmaceutical ingredients are an important part of drug production. Challenges faced during separation of biomolecules include: solubility of molecules, asymmetric synthesis, enzymatic/classical resolution and screening of typical chiral stationary and mobile phases. This is complicated further by intrinsic thermodynamic and kinetic constraints. Hence, Affinity Chromatography methods underwent various developments to ensure acid and alkaline resistance in the chromatography media and for the separation of agglutinating antibodies and cost effective, high throughput purification of monoclonal, polyclonal and engineered antibodies. Affinity Chromatography systems are also designed for the large-scale purification of high titre therapeutic antibodies, small molecules and biologically useful chemical compounds. The most important aspect of Pharma product development is ensuring the highest quality of the complex molecules used for clinical supply. The choice of chromatographic technique is a determining factor for ensuring quantitative and qualitative analysis of small molecules and highly potent APIs extensively required for the clinical trial batches of finished drug products. The polarity of molecules is a determining factor in the chromatography process. The most common compounds separated through chromatography are synthetic resins, quinolones, flavonoids, synthetic hormones, antibodies, organic solutes, lipids, anions, carbohydrates, proteins, amines, alcohols and many organic non-polar compounds. The type of interaction between stationary phase, mobile phase, and substances contained in the mixture is the basic component effective on separation of molecules from each other. Chromatography methods based on partition are very effective on separation, and identification of small molecules as lipids, esters, antibodies, complex small molecules, highly potent compounds and speciality chemicals. Affinity chromatography methods (i.e. ion-exchange chromatography) are more effective in the separation of macromolecules as nucleic acids, and proteins. Paper chromatography is used in the separation of proteins, and in studies related to protein synthesis; gas-liquid chromatography is utilized in the separation of all types of complex molecules such as antibodies, proteins, monoclonal antibodies etc. The stationary phase of chromatographic sample is considered as a solid phase or a liquid phase coated on the surface of a solid phase. Along with that, there is a mobile phase flowing over the stationary phase which is referred to as the gaseous or liquid phase. If the mobile phase is liquid it is termed as liquid chromatography (LC), and if it is gas then it is called gas chromatography (GC). The purpose of applying chromatography which is used as a method of quantitative analysis as well as its separation is to achieve a satisfactory separation within a suitable time frame. Agarose-gel chromatography is used for the purification of RNA, DNA particles, and viruses. Various other API manufacturing companies and CROs use High performance liquid chromatography (or high pressure liquid chromatography HPLC) in the process of drug discovery. High-performance liquid chromatography is a technique in analytical chemistry and bulk drug manufacturing that is used to separate, identify, and quantify each component in a mixture. A well-known highly qualified team of experts in chemical processes which is cGMP certified for handling highly potent compounds to occupational exposure band five (OEB 5) compounds can be a suitable candidate for commercial scale drug manufacturing used in clinical trials for human pharmaceuticals. CRO (Contract Research Organizations) offers fully customizable purification services for isolation of recombinant and native proteins using manual, FPLC, and HPLC chromatography methods. Production scales range from less than a milligram up to multiple gram quantities. Our scientists have decades of protein purification experience and can either develop a de novo process or perform an established protocol. All CDMOs establish quality drug manufacturing through the utilization of existing configuration of high-volume batch columns to manufacture drug substances. It is important to have an API manufacturing facility that can perform chemical purification of at least 50kg (kilograms) per day as well as chiral separations of 10kg per day to support clinical trials and market supply upon drug approvals. The substances having the greater interaction with the stationary phase are retarded to a greater extent and consequently separate from those with smaller interaction. As each component leaves the column with the carrier, it passes through a detector and then either goes to a fraction collector or is discarded. Ion- exchange chromatography uses electrostatic interactions between charged protein groups, and solid support material (matrix) for the separation of biomaterials. The matrix has an ion load opposite to that of the protein to be separated, and the affinity of the protein to the column is achieved through ion exchange complexes. Proteins are separated from the column either by changing pH, concentration of ion salts or ionic strength of the buffer solution. Positively charged ion-exchange matrices are called anion-exchange matrices because they adsorb negatively charged proteins. Whereas, matrices bound with negatively charged groups are known as cation-exchange matrices since they adsorb positively charged proteins. Affinity chromatography is the best separation technique for the purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins. It consists of a ligand which can make a complex with specific protein such as a dextran, polyacrylamide, or cellulose etc. that binds the filling material of the column. The specific protein which makes a complex with the ligand is attached to the solid support (matrix), and retained in the column, while free proteins can simply leave the column. The process ends when the bound protein leaves the column by means of changing its ionic strength through alteration of pH or addition of a salt solution. Continuous chromatography reduces costs and increases overall facility throughput using inherent unit operation efficiency rather than dramatic process change. Unlike batch chromatographic processing in which only 60–70% of the total binding capacity is used, with simulated moving bed (SMB) technology, a series of multiple, much smaller columns work together to improve process efficiency and make use of chromatographic resins more effectively. High Pressure Liquid Chromatography (HPLC) technique makes it possible to perform structural, and functional analysis, and purification of many molecules within a short time. This technique yields perfect results in the separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active molecules. In HPLC, mobile phase passes through columns under 10–400 atmospheric pressure and with a high (0.1–5 cm/sec) flow rate. HPLC is the most preferred method for the separation, purification and quantitative analysis of small molecules. During the HPLC process, high pressure is applied on the fast flowing solvent which increases the separation power, which ensures that the HPLC analysis is completed within just a few minutes. The essential components of a HPLC device are solvent depot, high- pressure pump, commercially prepared column, detector, and recorder. Duration of separation is controlled with the aid of a computerized system, and material is accrued. Ever since continuous chromatography was made possible for clinical manufacturing, more efficient facilities with higher flexibility that support biopharmaceutical and drug manufacturing have come up. Continuous technologies that are designed as single-use systems help to greatly facilitate process intensification, delivering further efficiencies with reduced set-up times and elimination of the need for cleaning and cleaning validation. The industry has also developed combat counterfeiting techniques to prevent health risks due to improper manufacturing of drug products and to prevent patient populations from consuming fake medicines. There are guidelines present to control the quality of raw materials used by API manufacturing facilities to ensure product quality and clearance from inspection policymakers, periodic reviews and quality performance indicators.