Advanced synthetic polymers represent important group of modern, high-performance materials. Here belong complex polymers (COP) and complex polymer systems (CPS). COP exhibit more than one dispersity (distribution) in their molecular characteristics - molar mass, chemical structure and physical architecture. Their typical representatives are various kinds of copolymers and functional macromolecules. CPS comprise the two and multicomponent blends of macromolecules and block copolymers containing their parent homopolymers. Molecular characterization of both COP and CPS is an exacting analytical challenge because the analytes must be separated. At present, size exclusion chromatography/gel permeation chromatography (SEC/GPC) dominates the area. Though SEC/GPC is a marvelous method, it cannot give quantitative data on molecular characteristics of COP and of most CPS because the size of macromolecules in solution depends on all molecular characteristics of macromolecules to be separated. Moreover, the separation selectivity of SEC/GPC is fairly limited and both low detector sensitivity and sample capacity of SEC/GPC does not enable to characterize minor ( solve the problem, coupled methods of polymer HPLC were introduced, which combine exclusion – entropic, and interaction – enthalpic retention separation mechanisms. Two of the coupled methods of polymer HPLC, namely liquid chromatography under critical conditions (LC CC) and liquid chromatography under limiting conditions (LC LC) of enthalpic interactions will be presented, compared and critically evaluated in the contribution with the conclusion that LC CC is preferable for oligomers and for binary blends of macromolecules with molar mass below about 10 kg/mol whereas LC LC has no upper molar mass limit but its performance may be not sufficient for low oligomers. The sample recovery represents an important limitation of LC CC of macromolecules with high molar mass. In contrast to LC CC, LC LC is robust and user-friendly. LC LC enables to separate even four-component CPS in one single step and can be easily engaged in the two-dimensional polymer LC.
While traditional drugs are usualy small molecules, biopharmaceuticals, like proteins, plasmid DNA, viruses or virus-like particles, are large complex bioparticles, with heterogeneus physicochemical properties, sensitive to the environmental conditions (pH, I) and shear forces. Therefore, during manufacturing process of biological nanoparticles there are several issues which have to be considered. To satisfy the severe requirements placed on the purity of biopharmaceutical products demanding several-step purification processes have to be developed. An integral part of downstream processing is the chromatographic unit operation.
To gain as high productivity as possible chromatographic resin should exhibit high dynamic binding capacity and high purification yield meaning high selectivity and low degradation of target macromolecule should be achieved preferably at high linear velocity and low pressure drop. Convection-based transport is an extremely important feature that accelerates the separation and purification process, especially of large biomolecules with low mobility. In recent years chromatographic monoliths, also called Convective Interaction Media (CIM), appeared as a resin of choice for biopharmaceutics’ purification. Their specific porous structure enables the convective mass transport and high surface accessibility to large molecules. Therefore they allow very fast separations and exhibit very high binding capacities for extremely large bioparticles.
In this work key features of large biological nanoparticles that influence the downstream process of target compound will be discussed. The differences between classical matrixes for liquid chromatography and for biochromatography will be exposed and case studies of several biopharmaceuticals of different classes using special monolithic chromatography columns will be presented.
1 Bioprocess Engineering Laboratory, School of Engineering and Graduate School of Medicine, Yamaguchi University, Ube 755-8611, Japan
Numerous techniques have been developed over the past decades to enhance the in-vivo stability of therapeutic proteins. In particular, PEGylation, known as the covalent binding of PEG chains to proteins, has become a technique of choice to extend their in-vivo circulation half-life. However, the side directed PEGylation reaction needed for human treatment still represents a challenge, due to consecutive competitive reactions with activated PEG, native and PEGylated protein. As a consequence, PEGylation produces positional isoforms and isomers which have different PEG chain number and further purifications steps are needed to isolate target positional isoform. For efficient development and production of PEGylated protein drugs, it is therefore essential to have fast analytical method for monitoring of PEGylation reaction either during development phase (QbD) or as a part of production process (PAT) where high frequency data are required for process control. As PEGylation alters surface protein charge distribution, ion-exchange chromatography seems to be a method of choice. Among different types of stationary phases monoliths were selected due to predominant convective transport providing efficient separation in short time at low pressure drop for such large protein molecules as PEGylated proteins.
In this work we developed different chromatographic methods for separation of PEGylated protein positional isoforms and isomers using a strong cation exchange SO3 CIM disk monolithic column (diameter =12 mm, thickness = 3 mm, volume 0.34 mL) and implementing either salt or pH gradient. While optimal separation using salt gradient was achieved with citric buffer pH 4.5, for pH gradient carbonate buffer from pH 10 to 12 provided optimal results. Despite different elution mechanism both methods provided separation of mono, di and tri PEGylated proteins as well as positional isomers but differing in resolution and elution order. Under optimal conditions, the analysis was completed within 3 min at flow rate of 10 mL/min and pressure drop below 1 MPa (10 bar).
Emulsion templating method is frequently used for the preparation of macroporous polymeric monoliths (1). While the morphology of so prepared polymers (termed polyHIPEs) is generally composed of spherically shaped primary pores several micrometer in diameter and a number of secondary pores connecting primary pores, there are some methods enabling the manipulation of mesoporous and microporous structure of the polymer film in the monoliths. The lecture will focus on presentation of a combination of emulsion templating and post polymerisation hypercrosslinking in order to produce polyHIPE monoliths with a distinct bimodal porous structure. Furthermore, examples of chemical functionalization and solid phase extraction from aqueous sources will be given.
1 I. Pulko, P. Krajnc, Macromol. Rapid Commun. 2012, 33, 1731
Jiři Urban, Radovan Metelka, Simona Janků, Martina Komendová
One of the main advantages of organic polymer-based monoliths is their simple and straightforward preparation and subsequent surface modification. In this work, we describe our recent attempts in the development of a single monolithic capillary column that offers integrated sample focusing, separation, and electrochemical detection of monoamine neurotransmitters. Fast, simple, and efficient quantification of neurotransmitters plays a crucial role in a proper diagnosis and treatment control of several neurological diseases.
At first, we have used 4-vinylphenylboronic acid monomer to prepare monolithic stationary phase that selectively entraps neurotransmitters on the basis of cis-diol functionality. We have optimized its online coupling with another monolithic capillary column providing neurotransmitters separation. In the next step, we have tested several approaches how to prepare monolithic capillary column which integrates sample focusing with separation. This included coupling of columns with a zero volume union, copolymerization of polymerization mixtures, and surface-initiated grafting.
To incorporate electrochemical detection at the end of monolithic capillary column, we have optimized experimental conditions used to fix carbon microfibers (7 µm diameter) by a monolithic stationary phase formed at the end of the capillary. In parallel, we have tested an outer fixation of a tetrode fiber (with an outer diameter of 100 µm) consisting of four platinum/tungsten alloy electrodes isolated by quartz glass. The application of a tetrode is advantageous since it could provide two-electrode system by interconnecting of electrodes to operate in biamperometry mode, when there is no need to use laborious and potentially unstable reference electrode of small dimensions. Linear dependence of peak heights on the concentration of catecholamines was observed in the concentration range of 5×10−4 – 1×10−5 M.
The financial support of GACR project 14-22426S is gratefully acknowledged.