Advanced therapy and medicinal products (ATMPs) are medicines based on genes, tissues, or cells. They offer groundbreaking new opportunities for the prevention or treatment of disease and injury. For example, they might contain genes that lead to a therapeutic, prophylactic, or diagnostic effect and work by inserting recombinant DNA into cells.

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Bind & elute is the most typical application mode of chromatography. Solutes, especially the target, bind to the chromatography matrix. Impurities are washed out by changing the composition of the mobile phase (eluent) to discourage their binding (desorption), and the target is recovered. Desorption (elution) can be with a linear gradient or a step-change in conditions.

Breakthrough is the point in a chromatography run where a target or impurity first appears in the effluent. Breakthrough often represents a limit beyond which a chromatography matrix volume or device size is unable to handle more incoming target or impurity (see also dynamic binding capacity).

Capture is a chromatography stage where the focus is on target binding. It is usually an early unit operation. Ideally, capture chromatography will achieve high levels of purity so that subsequent unit operations are only required for particular safety concerns or polishing (e.g., removing viruses, leakage contaminants, and trace HCPs).

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A contaminant is an impurity arising from an unintended source. Contaminants might include ligand leakage from an affinity matrix, endotoxins from unwanted bacterial infection, viruses, or other materials introduced due to the bioburden.

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Microbiological Contaminants

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Robust Parvo Virus (MVM) Clearance on Sartobind^® Membrane Adsorbers

Convective chromatography descrives any type of chromatography in which the matrix contains large pores or channels instead of small micropores. Mass transport to the ligands takes place purely by convective flow, so separation of components is achieved without the need for diffusion into small pores. Capacity and resolution are generally unaffected by flow rate since there is no limitation due to diffusion.

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Critical quality attributes (CQAs) are defined characteristics of the purification target that are demanded and monitored in the final product.

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Dynamic binding capacity (DBC) describes the capacity of a chromatography matrix to bind a target or impurity under operating conditions (i.e., with a flowing mobile phase - eluent - containing the target/specific impurity with or without other impurities present). It is usually defined for a device of specified dimensions, eluent flow rate, and target|impurity composition. The results are expressed as mass (of target or impurity) per device unit/column volume at a % (typically 1% or 5%) of maximum effluent concentration of the target or impurity.

The effluent is the mobile phase (eluent) that leaves the chromatography column or device.

The eluent is the mobile phase that flows through a chromatography matrix. It is frequently used to describe the mobile phase at any stage in chromatography, whether entering, passing through, or leaving the matrix during sample application or desorption (elution).
 

Elution is the flow of eluent through a chromatography matrix. It is often used to describe the desorption phase when the target is recovered in bind & elute mode.

Exosomes are membrane-bound extracellular micro-vesicles (EVs) that are often produced in the endosomal compartment of eukaryotic cells. Exosomes can contain proteins, lipids, or nucleic acids.  They are currently being developed as potential therapeutics with the ability to elicit cellular responses in vitro and in vivo.
 

Flow-through is a mode of chromatography where the target flows through the matrix without binding, or with little retention, and impurities are bound (negative chromatography). This mode is especially useful when the target has a high concentration in the sample, and the impurities are present in relatively small amounts (scavenging).

Flow velocity is an expression of flow rate, typically expressed in a scalable unit such as centimeters per hour (cm/hr). It is most often calculated by dividing the volumetric flowrate by the column cross-sectional area.

Format refers to the chromatography device - whether it is a column (either pre-packed or self-packed), a monolithic matrix device, or a membrane cartridge. A wide range of materials is available, including empty glass, plastic, or stainless steel columns for packing beaded resins, columns that allow automatic packing, pre-packed columns of all sizes, radial flow devices, and membrane cartridges for small lab applications to large-scale production.

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Particularly important when the target sources are mammalian cells, host cell proteins (HCPs) refer to all protein impurities present in the initial feed. Commonly, assays are developed and employed to track the removal of these impurities without defining individual components.

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Impurities are unwanted solutes or other materials that originate from the target source. For instance, impurities from bacterial fermentation, cell-culture supernatant, synthetic sample, or tissue extract.

Intermediate purification is a stage of chromatography between target capture and polishing. Typically, the selected chromatography mode is different from the mode used in capture and polishing. In an ideal scenario, an intermediate purification step would not be required.

Laminar flow is a smooth type of liquid flow in which the fluid behaves as a system of orderly parallel layers with no eddies or irregular regional fluctuations in velocity and pressure. Laminar flow is gentle for molecules that are sensitive to shear stress, such as mRNA. Monolithic chromatography matrixes that have regular-sized interconnected channels offer good laminar flow properties. Beaded resins or irregular particle resins tend to have irregular flow with eddies and turbulence.

A chromatography ligand describes any functional group attached to the matrix that helps to isolate a target or impurity. Commonly used ligands include amine groups, protein A, oligo dT, and enzyme substrates.

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The backbone material used as the support for chromatography is called the matrix. The chromatography matrix typically consists of beaded resin particles packed in a column, layers of membranes arranged in a cartridge, or a monolithic structure of interlinked channels.

The liquid that flows through the chromatography matrix is called the mobile phase. Initially carrying the sample feed, the mobile phase is typically followed by washing out non-bound solutes. Then, the eluent conditions are changed to desorb bound solutes.

Modality is the term used to describe all types of purification targets. These include protein biomolecules, nucleic acids, vectors, viruses, vesicles, exosomes, and other ATMPs.

A chromatography mode is a group term that describes different separation mechanisms such as ion exchange, hydrophobic interaction, affinity, multi-modal, and reversed-phase chromatography. It is also used to describe flow-through and bind & elute techniques.

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Oligo dT is a short fragment of single-stranded DNA consisting of thymine (T) bases. The sequence can bind the poly(A) tail present in mRNA molecules. Oligo dT is frequently used as the ligand in affinity purification of mRNA.

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Polishing is a chromatography stage that typically removes small amounts of remaining impurities and contaminants following earlier purification steps that have removed the bulk of the impurities. Polishing chromatography demands a chromatography matrix with high efficiency and selectivity to achieve the required level of purity of the target product. It is usually the final chromatography unit operation and is often needed to assure safety and efficacy. 

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Protein A was originally derived from the cell wall of the bacteria Staphylococcus aureus. Now frequently genetically engineered, Protein A is a widely used ligand in affinity chromatography for the purification of monoclonal antibodies (mAbs), as it exhibits highly specific binding to the Fc region of IgG.

A resin is a solid, usually organic, polymeric structure (sometimes it refers to inorganic materials such as silica or hydroxyapatite) that forms the backbone of particulate and beaded chromatography matrixes. Such matrices are most commonly porous with a stationary liquid phase within pores. Mass transport (exchange) of substances from the mobile phase, which flows past resin particles to the pores, mainly occurs by diffusion.

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Reversed-phase chromatography (RPC) is a chromatography mode based on a hydrophobic stationary phase. The matrix often consists of alkyl chains covalently bonded to silica or might be a matrix with inherently hydrophobic properties, such as various plastic polymers. Solutes bind due to their hydrophobic or non-polar properties. They can be eluted according to differences in these properties using a gradient containing an organic solvent, such as acetonitrile or ethanol. RPC is most often used in the lab as HPLC to separate and analyze organic molecules or closely similar peptides. At large scales, it is used in the production of certain peptides and polypeptides (notably insulin), where it requires an explosion-proof environment and high-pressure stainless steel equipment.

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Salt tolerant describes a form of mixed-mode ligand and - when attached to a matrix - chromatography, where the binding characteristics allow ion exchange at an elevated ionic strength. This is accomplished by mixed-mode ligands with both ion exchange and additional binding mechanisms that support and enhance the ionic binding of targets and/or impurities, despite elevated concentrations of salts in the sample and mobile phase.

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Ion Exchange Chromatography (IEX)

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Size-exclusion (SEC) is a chromatography mode that separates molecules purely on their size. The components of a mixture do not bind to the stationary phase in SEC. Instead, they pass through the stationary phase at different rates depending on their size. 

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Simulated moving bed (SMB) is a form of continuous chromatography that uses several columns (or other chromatographic devices) with valves on the inlet and outlet sides. The sample is applied continuously to the columns in sequence so that it appears that the bed is moving past the sample inlet. As with simpler arrangements of continuous chromatography, SMB reduces matrix and eluent consumption and can allow major cost and time savings compared with batch chromatography. However, it puts high demands on in-process control and valve performance.

The stationary phase describes stationary regions in a column or other device where partitioning of solutes occurs with the mobile phase. In beaded and particulate resins, the partitioning region is inside the liquid-containing micropores of the chromatography matrix. Mass transfer of solutes and exchange with fresh, incoming eluent occurs by diffusion from the mobile phase to the micropores, rather than convective flow. Solutes that have diffused into the micropores can then bind to groups attached to the stationary matrix. In monoliths and membrane matrixes,  solutes in the mobile phase have direct access to the stationary phase binding sites, and mass transfer is by convection.

The purification target is the molecule, particle, vesicle, virus, viral particle, or vector which requires purification. 

The target product profile (TPP) is a description of the product containing the defined target of the purification and other components. These might include its solvent environment or lack of specified, harmful impurities.

Turbulent flow is the fluid motion characterized by chaotic changes in pressure and flow velocity. This is in contrast to laminar flow, which occurs when a fluid flows smoothly with no disruption between parallel layers. Turbulent flow increases with flow rate, especially when the liquid has to follow irregular paths, for example, around resin beads. Turbulent flow can damage shear-sensitive purification targets such as mRNA and leads to zone broadening and loss of resolution.