37


With size exclusion chromatography (SEC) stagnant Pm


is the stationary phase


[9]. Small substances diffuse more easily and completely into stagnant Pm


within


pore matrices than large molecules. The longitudinal velocity of a protein or peptide is zero while inside pores. This allows substances in the Pm


to move ahead of


those in pores. In contrast, larger proteins are partially or totally exclude from pore matrices. This means they spend more time in the mobile phase and move through the system faster.


The idea that Pm moves through a particle


bed in a laminar flow profile is derived from hydrodynamic chromatography (HDC). Early HDC studies showed that long packed columns separate colloids of varying size [7]. Larger analytes elute first and smaller ones later, due to preferential sampling of the streamlines of flow in the interstitial medium of the packed column [8]. Because all analyte sequestering transport particles are of the same size and columns are short HDC plays a minimal role in MASC resolution.


Three-Phase System Theory


The theory behind three-phase separation systems comes from literature describing the immobilisation of proteins on the surface of 50 - 100 nm nanoparticles [5,10] to create megadalton (mDa) size immune complexes that remain suspended in solution and retain their biologically activity [11]. In the chromatography application described below these particles will be referred to as an analyte sequestering transport phase (ASTP). Association of an antigen (An


ASTP bearing an antibody is represented in the reaction


Figure 2. A schematic of a longitudinal channel segment in an SEC column being eluted in a three-phase separation mode. Dimensions in the illustration are not to scale. Note that the ASTP is too large to enter pore matrices. The Core of the ASTP is a hydrophilic polymer of at least 2 mDa. A series of antibodies and protein A/G were used as affinity selectors. With the protein A/G affinity selector up to 4 antibodies can be captured with high affinity at one binding site. Support structures are the same as in Figure 1.


The analytical significance of this is threefold. One is that ASTP:An


are soluble in aqueous Pm complexes and sufficiently


small to be transported through a chromatography column. This allows a sorbent particle to be used as a transport phase as opposed to insoluble stationary phases. Obviously an ASTP cannot be used as a transport phase in the remote case where it binds to a very large, multiple protein complex [12] and precipitates. The very high binding affinity of analytes for an ASTP is a second asset. Analyte association with Ps


is precluded. The fact that non- ) with an


analytes (sample impurities) do not associate with an ASTP is still another advantage. This makes it possible to rapidly differentiate between analytes and impurities. The final asset of ASTP:An


complex formation is that


upon binding an analyte will become very different from impurities of very similar


structure (Figure 2). When in an ASTP:An complex the effective molecular weight of an ASTP sequestered analyte is 5X to 10X larger than that of protein impurities.


The association constant (Ka ) for complex formation is expressed by the equation Following analyte sequestration the issue where CASTP and CAn of ASTP and An , respectively and CASTP:An the concentration of the ASTP:An


, respectively. Ideally Ka ratio will be 106


kd and the ka


and /kd


are the concentrations is


complex.


The rates of association and dissociation are represented by the symbols ka


or higher. An association


constant of this magnitude allows the ASTP:An


complex to be transported


through a chromatography column without dissociation.


is how to separate and detect an ASTP:An complex relative to impurities. By design these complexes are soluble in common aqueous mobile phases and of a size amenable to separation by SEC (Figure 2). With an SEC column having 30 nm pores a megadalton size ASTP:An


complex will


elute in the void volume while impurities will be retained by Ps


and elute within a


single column volume. Short columns could be eluted at high flow rates within 60 - 120 sec. Few impurities would coelute with the ASTP:An


complex based on the huge difference in their relative size.


The three phase system described above would allow three layers of selectivity (Figure 2). The first is the very high selectivity of affinity selectors such as antibodies and aptamers. Antibodies can even differentiate between proteoforms as known with isoenzymes [13] and post-translational modification variants of proteins [14]. The second is the very large shift in the effective size of an analyte when it binds to an ASTP. The significance of this is that subsequent to binding small analytes elute in the SEC void volume irrespective of their size. The third is the selectivity of the SEC column in size separating the ASTP:analyte complex from impurities.


Theoretically three-phase chromatography could be carried out in two ways. One would be by continuous addition of ASTP to the SEC mobile phase. In this mode the ASTP:An


complex would be formed in the


column. The concern with this approach is that large amounts of expensive ASTP would be consumed in the absence of An


An alternative would be a zonal mode. In this mode the ASTP:An


is at least 106 complex could


be formed in a sample vial and then injected into the SEC column. Because the association constant Ka


these


two modes will be equivalent in terms of resolution. This allowed all experiments described herein to be carried out in the zonal elution mode to reduce ASTP cost.


ASTP Fabrication


Critical elements in ASTP selection and fabrication are that i) the nanoparticles be hydrophilic and as nearly neutral as possible to preclude aggregation and non-specific binding of sample impurities, ii) the ASTP


.


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