Chromatography is a technique employed to separate mixtures of molecules. It involves dissolving the mixture in a solvent or combination of solvents (a so-called mobile phase) to form a solution and subsequently passing the solution over a solid (the so-called stationary phase). The correct combination of mobile phase and stationary phase allow the molecules to be separated by permitting preferential or selective interaction of each molecule with the stationary phase to a differing extent. This differing interaction allows the molecules to be separated and thus isolated, analysed or identified. Both the chromatography mobile phase and the stationary phase are varied to suit (i) the type of molecules being separated, (ii) the scale of the separation (e.g. analytical, preparative or industrial), and (iii) the desired functionality of the separated molecules (e.g. bioactivity). Examples of mobile phases include acetonitrile, water, aqueous salt solution, methanol or mixtures thereof, while common stationary phases include ion-exchange, hydrophobic interaction or affinity interaction resins. Chromatography is employed extensively in the pharmaceutical and food industry for analytical purposes, and also to isolate valuable molecules at preparative and commercial scale.
‘Smart’ or ‘intelligent’ polymers are materials that undergo fast, reversible changes in their structure and function in response to external physical, chemical or electrical stimuli. Temperature is the most widely studied stimulus in ‘smart polymer’ systems and poly(N-isopropylacrylamide) (PolyNIPAAm) is a common and extensively studied temperature-responsive polymer.
Temperature-responsive materials have potential in on exchange chromatography as a versatile separation tool, where the elution of bound target bio-molecules can be induced by a mild physical change, such as an adjustment in temperature. These smart polymer chromatography systems offer promise in the cost-effective isolation of valuable components, particularly from agri-food, pharmaceutical, chemical and water and other complex feeds, in an environmentally-friendly manner.
PolyNIPAAm and related polymers have been used in the separations field to generate temperature-responsive stationary phases for ionic chromatography (Kobayashi et al., Analytical Chemistry, 2003, 75 (13), 3244-3249; Sakamoto at al, Journal of Chromatography A, 2004, 1030, 247-253; Ayano et al., Journal of Separation Science, 2006, 29, 738-749), size exclusion (Hosoya et al., Macromolecules, 1994, 27, 3973-3976; Adrados et al., Journal of Chromatography A 2001, 930 (1-2), 73-78), hydrophobic interaction (Kanazawa, et al., Analytical Chemistry, 2000, 72, 5961-5966), and affinity based chromatography separations (Hoffman and Stayton, Macromolecular Symposia, 2004, 207, 139-151) using a range of different supporting materials.
Further, a pH and temperature responsive copolymer of poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide) grafted onto silica beads has been evaluated as an anionic temperature responsive chromatography medium (Kobayashi et al., Journal of Chromatography A, 2002, 958, 109-119; Kobayashi et al. Analytical Chemistry, 2003, 75 (13), 3244-3249). Effective separation of basic bioactive peptides under exclusively aqueous conditions was attained using anionic temperature/pH responsive polymer-modified surfaces. Similarly, silica beads grafted with poly(N-isopropylacrylamide-co-butyl methylacrylate-co-N,N′-dimethylaminopropylacrylamide) has been evaluated as a cationic temperature responsive chromatography medium (Sakamoto et al., Journal of Chromatography A, 2004, 1030, 247-253; Ayano et al., Journal of Chromatography A, 2006, 1119, 58-65). The medium was designed for efficient separation of bioactive compounds and pharmaceuticals using isocratic aqueous mobile phases.
There are a number of reports showing the use of smart polymers grafted onto silica beads for ion-exchange chromatography. However, the food and other industries tend to avoid silica base matrices due to cost, instability at the high pH and lack of operational robustness under the conditions often used in the food and other industries to clean equipment. In addition, silica based sorbents are generally applicable to analytical separations and lack the flexibility required for process applications in the food and other industries.
Further, previous investigations into the potential of smart polymers in cation exchange chromatography with modified silica beads have only been undertaken using small compounds like amino acids and steroids. There has been little published literature examining the retention and release of large proteins of significance to the food, pharmaceutical, chemical or water industries (e.g. lactoferrin) using temperature responsive ion exchange chromatographic resins.
There is a need for smart polymeric ion exchange resins on non-silica matrices. Specifically, there is a need for smart polymeric ion-exchange media based on a matrix that is compatible with systems currently employed by the food, pharmaceutical and other industries (such as cross linked agarose). Further, there is a need for thermally responsive cation exchange agarose based chromatography resin for application in the isolation of large proteins of commercial importance within the food and other industries (such as lactoferrin).
The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge as at the priority date of any of the claims.
Throughout the description and claims of the specification the word “comprise” and variations of the word, such as “comprising” and “comprises”, is not intended to exclude other additives, components, integers or steps.