38 February / March 2019 Model Validation


The above three-phase model is the mechanistic basis for mobile affinity sorbent chromatography (MASC). Validation of MASC was achieved in two ways. The first was to show that an ASTP with an immobilised affinity selector would bind a protein and cause it to elute in the void volume of an SEC column (Figure 3). Dextran based ASTP with covalently linked protein A/G (ASTP~PA/G


) was added to a sample


Figure 3. Antibody detection by mobile affinity sorbent chromatography. Detection of the SEC markers was by absorbance at 215 nm (black line) while that of the ASTP~PA/G


:IgG* complex and IgG* was by fluorescence (pink line).


Core material should be amenable to covalent immobilisation of proteins, iii) the ASTP should be of 50-100 nm in size to assure exclusion from pores, and iv) an ASTP should have minimal impact on mobile phase viscosity. Two types of material were evaluated, natural polysaccharides and synthetic inorganic particles. Both worked well, but in the end, very high molecular weight dextran was found to have the fewest limitations. Dextrans used in this work exceed 1 mDa and were natural products of Leuconostoc mesenteroides [15].


Covalent linkage of proteins to dextran core particles was achieved in two ways. One was by oxidative cleavage of vicinal diol residues with periodate to form aldehydes [16]. Amine groups in proteins were coupled to the aldehyde rich dextan by Schiff base formation followed by NaCNBH3


reduction


to a secondary amine linkage [17]. A problem with this approach is extensive hemiacetal formation in the oxidized dextran that makes it difficult to control the number of aldehyde residues available for protein coupling [18].


The second approach was to carboxymethylate the dextran. This was achieved by the addition of chloroacetic acid in 8M KOH [19]. Proteins were amide coupled to the CM-dextran via hydroxysuccinimide assisted water soluble carbodiimide catalysis [20]. This method of ASTP formation was preferred in that it was easier to control the number of functional groups available for protein immobilisation.


bearing an excess of fluorescently labelled IgG - [IgG*; the antibody is covalently labelled with fluorescein isothiocyanate (FITC)]. Protein A/G is a recombinant protein hybrid that contains domains of both protein A and protein G, causing it to bind to the Fc region of most mammalian antibodies [21]. The ASTP~PA/G


:IgG*


complex was subsequently formed and injected into a 4.6 × 300 mm, 5 µm particle diameter, 30 nm pore diameter Sepax SEC column (Figure 3). IgG* was detection by fluorescence at excitation and emission wavelengths of 494 nm and 510 nm, respectively. As judged from the elution of protein standards in the accompanying chromatogram the fluorescence peak eluting at 2.1 min in the void volume clearly contains IgG* transported through the column without entering the pores of the SEC column. Unbound IgG* elutes at 3.2 min. This experiment demonstrated that (i) protein A/G retained IgG binding specificity


when bound to the dextran based ASTP nanoparticle, (ii) analyte IgG* could be isocratically separated from other protein species using a single column volume of mobile phase in the MASC mode, (iii) ASTP~PA/G


bound IgG* behaves as if it


had a molecular weight in the range of 1 mDa, and iv) analyte elution behaviour was independent of analyte molecular weight.


There is concern when producing or buying an antibody that it retains antigen affinity. The experiments described below addressed that issue. A saturating concentration of fluorescent labelled secondary antibody (2


AbmIgG mouse IgG was added to mouse 1


*) targeting Abfitc


solutions of the same volume that varied in concentration. It is assumed in this experiment that 2


AbmIgG * will bind to both


active and inactive forms of the fluorescein isothiocyanate (FITC) targeting antibody. Samples were incubated for 30 min to allow formation of the 1


Abfitc: 2 AbmIgG * complex


and then added to 100 µL of human plasma bearing ASTP~FITC. Following incubation for at least another 30 min to allow formation of the ASTP~FITC: 1


2AbmIgG


Abfitc


: * complex samples were examined


by MASC (Figure 4). The Sepax SEC column used in these experiments was the same as in Figure 3.


The peak in column void volume at 2.1 min is clearly the ASTP~FITC: 1


Abfitc 2AbmIgG : * complex while that at 3.2 min is


Figure 4. A MASC based antibody activity assay. Panel A is the elution profile obtained with a 4.6 × 300 mm, 5 um particle diameter, 30 nm pore diameter Sepax SEC column. Panel B is a concentration profile obtained by measuring the fluorescence of the secondary antibody 2


AbmIgG * targeting mouse IgG.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60