Convective Interaction Media® (CIM) – Short layer monolithic chromatographic stationary phases

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Convective Interaction MediaÕ (CIM) – Short layer monolithic chromatographic stationary phases Alesˇ Podgornik* and Alesˇ Sˇtrancar BIA Separations d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia Abstract. Modern downstream processing requires fast and highly effective methods to obtain large quantities of highly pure substances. Commonly applied method for this purpose is chromatography. However, its main drawback is its throughput since purification, especially of large molecules, requires long process time. To overcome this problem several new stationary phases were introduced, among which short layer monoliths show superior properties for many applications. The purpose of this review is to give an overview about short methacrylate monolithic columns commercialised under the trademark Convective Interaction MediaÕ (CIM). Their unique properties are described from different perspectives, explaining reasons for their application on various areas. Approaches to prepare large volume methacrylate monolithic column are discussed and optimal solutions are given. Different examples of CIM monolithic column implementation are summarised in the last part of the article to give the reader an idea about their advantages. Keywords: short layer monoliths, methacrylate monoliths, chromatography, stationary phases, CIM, applications, scale-up, downsream process, purification.

Introduction Chromatography has always been an important purification method in the production of high purity substances. Especially for purification of biological macromolecules, it can be considered as the only method to meet the required demands. As the separation and purification quality mainly depend on the properties of the stationary phase packed in the chromatographic column, preparation of suitable resin, to provide high selectivity combined with high capacity, resulting in high productivity has been the driving force over the entire history of chromatography. A wide use of chromatographic separations of biological macromolecules began with the introduction of the hydrophilic polysaccharide-based stationary phase, like modified cellulose [1] and cross-linked dextran [2]. These stationary phases were very convenient because they were inexpensive and did not damage the very sensitive three-dimensional structure of large molecules. However, softness and swelling or shrinking of these materials impeded faster and better separations, since high flow-rates deformed stationary structure and decreased column performance. The efforts to develop rigid hydrophilic stationary phases resulted in the introduction of the cross-linked agarose [3], modified silica [4] and vinyl polymer gels [5]. They could resist higher pressures and consequently higher *Corresponding author: Tel: +386 1 426 56 49. Fax: +386 1 426 56 50. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 11 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(05)11005-9

ß 2005 ELSEVIER B.V. ALL RIGHTS RESERVED

282 linear velocities and were used in a form of smaller particles, so that faster separations were possible. In comparison with the soft gels, where the separation time often exceeded several hours, more rigid stationary phases enabled separations of macromolecules in less than one hour. In attempts for even faster separations, the mass transfer between the mobile and stationary phases posed the main barrier. Mass transfer occurs in stagnant pores of the particles and is governed by diffusion, which is rather slow especially for macromolecules having low mobility. To avoid slow mass transfer in stagnant pores, which are essential for a high surface area and consequently higher capacity, the pores should be shorter or completely eliminated. One approach was the introduction of nonporous particles (for review see [6]). They exhibit a low surface area and are useful for analytical purposes, where there is no need for a high binding capacity. Low surface area problem was partially overcome by decreasing particle diameter. However, columns filled with these particles exhibit an extremely high pressure-drop. Many theoretical studies in chromatographic supports show that the most suitable solution to enhance mass transfer is convective mass transport [7]. Convective mass transport occurs only in open pores (channels), where the liquid can flow through. Different approaches were used to produce flow-through pores in chromatographic supports. One of the results is the so called perfusion particles [8]. They are similar to convectional particle supports but they contain perfusion pores of large diameter, where the molecules are transported by convection in addition. Similar to the conventional particle supports they also contain small diameter closed pores necessary to increase the surface area. In contrast to non-porous particles they exhibit low backpressure combined with improved hydrodynamic characteristics [9]. However, similar to other particle supports their inherent drawback is a void space between the particles. Due to the lower backpressure most of the mobile phase flows around the particles rather than through the perfusion pores, diminishing the convection effect [10]. This problem was overcome with another type of the convective based supports – membranes. In spite of the excellent hydrodynamic characteristics of the membrane itself [11], their use is limited because of pronounced peak broadening caused by high extra column effects and low-binding capacity related to the low specific surface area [12]. With the goal to overcome most of the abovementioned disadvantages a different type of chromatographic supports, named monoliths, was introduced. Chromatographic monoliths consist of a single block of desired dimensions, containing a flow through pores (channels) and are defined as: ‘‘Monoliths are continuous stationary phases that are cast as a homogenous column in a single piece and prepared in various dimensions with agglomeration-type or fibrous microstructures’’ [13]. Pores are highly interconnected forming a flow through channel network. Such a structure provides many interesting features as it will be described in detail in further sections.

283

Types and properties of the chromatographic monoliths The term ‘‘chromatographic monolith’’ (or ‘‘monolith’’ as it will be used further in text) covers a wide group of stationary phases, which are prepared in different chemistries and according to a variety of procedures. Although the first experiments of the monolith preparation date in the late 1960s and the early 1970s [14,15], the real breakthrough occurred in the 1990s which still continues. Since then there were numerous reports describing preparation and application of the monoliths. The first scientific paper of the second-modern period was published in 1989 by Hjerten et al. who introduced polyacrylamide gel [16]. This work was soon followed by the introduction of methacrylate monoliths [17] and silica monoliths [18,19]. Till then many other monoliths have been described, like silica xerogels [20], monoliths prepared via methathesis polymerisation [21], polymethacrylate monoliths with template pores [22,23], polyacrylamide-coated ceramics [24], continuous urea–formaldehyde resins [25], monoliths prepared from carbon microspheres [26], monoliths cast from cellulose [27], emulsion derived monoliths named also polyHIPE [28] recently extended also to methacrylate chemistry [29], superporous agarose [30], cryogels from polyacrylamide [31] and others. An excellent overview about the chromatographic monoliths can be found in recent literature [32]. The main reason for many different types of chromatographic monoliths lies in their advantageous properties, which can be summarised as follows:






transport based on convection [33], extremely high porosity [34], cheaper preparation [35], simple column filling [35] and high capacity for extremely large molecules [36].

All these properties are strictly related to the monolithic structure. Convection-based transport is an extremely important feature that accelerates separation and purification process, and is especially pronounced for large molecules. Pores in the monoliths are open and highly interconnected forming a network of channels. The mobile phase is forced to flow through them transporting the molecules to be separated onto the active (binding) site by liquid stream – by convection. Since there are normally no dead-end pores in the monoliths (exception are porous silica monoliths mainly intended for separation of small molecules [37]) there are no stagnant regions and the mass transfer between stationary and mobile phase is extremely fast [38]. This is especially beneficial for the purification of very large molecules having small mobility like proteins, polynucleotides or viruses. Fast mass exchange results in practically flow unaffected resolution and dynamic binding capacity [13,39] as shown in Fig. 1.

284 60

3

relative absorbance at 280 nm (mAU)

1

100

2 50

80 40 60

30

40

20

% of buffer A

(a)

20

10 0 0

50

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250

0 300

volume (ml)

(b) 1

C/C0

0.8

0.6

0.4

0.2

0 0

50

100

150

200

250

300

350

volume (ml)

Fig. 1. Effect of the linear velocity of the resolution and dynamic binding capacity. (a) Effect of the flow rate on the separation efficiency. Separation of a protein mixture at six different flow rates (40, 80, 120, 160, 200 and 240 ml/min) normalised to the elution volume. Conditions: Mobile phase: buffer A: 20 mM Tris–HCl buffer, pH 7.4; buffer B: 20 mM Tris– HCl buffer + 1 M NaCl, pH 7.4; Flow rate: 200 ml/min; Gradient: 0–100 % buffer B in 200 ml; Sample: 2 mg/ml of myoglobin (peak 1), 6 mg/ml of conalbumin (peak 2) and 8 mg/ml of soybean trypsin inhibitor (peak 3) dissolved in buffer A; Injection volume: 1000 ml; Detection: UV at 280 nm. (b) Effect of the flow rate on the dynamic binding capacity. Flow rate: 50, 100 and 150 ml/min. Sample: 10 mg/ml of BSA in a 20 mM Tris–HCl buffer, pH 7.4; Detection: UV at 280 nm. (Reprinted from [77].)

285

pressure drop due to porosity (1-ε)2/ε3

pressure drop due to porosity (1-ε)2/ε3

2.5

2

1.5

1

500 450 400 350 300 250 200 150 100 50 0

0.1

0.2

0.3

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0.5

0.6

0.7

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1

porosity (−)

0.5

0 0.5

0.55

0.6

0.65

0.7

0.75 0.8 porosity (−)

0.85

0.9

0.95

1

Fig. 2. Effect of the porosity on the pressure drop calculated according to Kozeny–Carman equation (Eq. 1). Porosity of e.g., 70%, easily obtained by the monoliths, results is in more the 20-fold lower pressure drop in comparison of 40% porosity maximally achieved for particulate beds.

The second important consequence of the monolithic structure is high porosity. Porosity is very important since it strongly influences pressure drop at a given flow rate. This relation is described by Kozeny–Carman (KC) equation: DP ¼ 72k1

ZvL ð1  eÞ2 dp2 e3

(1)

where DP is the pressure drop, Z is the mobile phase viscosity, L is the bed length, dp is the particle diameter, e is the external porosity and the factor k1 is usually assumed constant (2.08 when the KC constant is 150 and 2.5 when the KC constant is 180). As shown in Fig. 2, even a small increase in porosity significantly decreases the pressure drop. The external porosity – porosity consisted of voids between the particles – can in packed beds reach a value of upto 40%, since above this value the bed becomes unstable and collapses. High intraparticle porosity does not contribute to a decrease in the pressure drop since the liquid in such pores is stagnant. The monoliths on the other hand, can exhibit external porosity upto 90% or above [34]. This is possible because the whole bed consists of a single skeleton and no free particles, which might collapse, are present. The next two features are mainly related to the production of the monolithic columns. Particle shaped resins are normally prepared via suspension polymerisation, whereas the monoliths are prepared commonly via bulk polymerisation. Once the beads are polymerised, they have to be sieved to obtain uniform

286 particle size distribution. In the case of monoliths, an equivalent characteristic is pore size distribution, which is already defined by the polymerisation conditions, as described in detail in the next section. In addition, bulk polymerisation can be performed inside the column housing and no packing procedure is required afterwards. The last but not the least beneficial property of the monoliths is the very high binding capacity for extremely large molecules. This high binding capacity is because the entire accessible surface is actually the wall of the interconnected channels through which the sample travels. In the case of porous particles, most of the surface is present in dead-end pores, which might be too small to be accessible for large molecules. For those molecules, such particles behave like non-porous ones and their accessible surface area is drastically reduced. Furthermore, for a given accessible surface area, binding capacity is higher for larger molecules. Assuming a single layer adsorption, the surface covered by a single molecule increases with the square of its diameter, while the molecular mass increases with a power of three as shown in Fig. 3 [36]. Therefore, the bigger the molecule, the higher total mass that can be adsorbed per surface unit. This theoretical prediction was experimentally confirmed recently [40]. The monoliths can be a support of choice for separation and purification of any kind of molecules due to their lower pressure drop, but are especially advantageous for separation of very large molecules since they speed-up separation or purification processes significantly in exhibiting high flow-unaffected dynamic binding capacity.

Capacity [=(1/2√3)(4πρ/3)2/3 (Mw/N )1/3] d = (6Mw/πρN)1/3

Mw = 0.15 MDa C = 4.7 mg/m2

Mw = 100 MDa C = 41 mg/m2

Ahex = (√3/2)d2

Fig. 3. Effect of the molecule size on the binding capacity. Calculation of the effect of molecule diameter (d ) on saturation capacity (C), where Ahex is the area of the molecule on the matrix surface in a face-centered-cubic array, r is the molecule density, Mw is the molecule molecular weight and N is Avogadro’s constant. (Reprinted from [36].)

287

Methacrylate monoliths Methacrylate monoliths were described for the first time in 1990 but their development had started few years earlier [41]. A driving force for their development was the hypothesis that for separation of proteins, only a small part of chromatographic bed is used. This idea was hard to confirm experimentally with conventional particle-based supports, since it was extremely difficult to construct short columns using particulate sorbents due to irregularities in packing density and excessive channelling. Applying a substantial knowledge about the preparation of the methacrylate beads, a Russian–Czech team started in 1987 with the development of a new technology called high performance membrane chromatography (HPMC). In the same year first methacrylate monoliths were successfully prepared and a patent application was filed [41]. Methacrylate monoliths are prepared via bulk polymerisation from glycidyl methacrylate (GMA) as a monomer, ethylene glycol dimethacrylate (EDMA) used as a crosslinker, while cyclohexanol (CyOH) and dodecanol (DoOH) are used as porogens. To start polymerisation normally thermal initiators like AIBN or BPO are applied. As the temperature increases, the initiator decomposes and oligomer nuclei start to form. The solubility of the forming oligomers in the reaction mixture decreases with their growth and they start to precipitate into porogens when they reach a certain molecular weight. The monomers are thermodynamically better solvating agents for the polymer than the porogens. Consequently, the precipitated nuclei are swollen with the monomers. Since the concentration of monomers is higher than in the surrounding solution, the polymerisation in the nuclei is kinetically preferred. In the absence of mixing, due to a higher density, insoluble nuclei sediment and accumulate at the bottom of the mould. Initially, they form a very loose structure which is highly porous. During the course of polymerisation, nuclei continue to grow and crosslink until the final structure is achieved [43]. The IR spectra presented in Fig. 4 show the characteristic fingerprint of methacrylate monoliths. It can be seen that most of the double bonds reacted in between and eventually very few remained (see small unmarked peak on the right of peak at 1730 cm1). Even more important is the finding that there is a substantial amount of epoxy groups which remained intact during polymerisation and can be easily transformed into other desired moieties (see peaks at 908 and 849 cm1). Based on the measurement of mass difference during hydrolysis of epoxy groups using sulfuric acid and confirming complete disappearance of the epoxy peak with IR spectroscopy, we estimated that more than 90% of the groups added into the polymerisation mixture with glycidyl methacrylate remained unreacted during polymerisation. Figure 5 presents the SEM picture of methacrylate monolith skeleton that consists basically of particles linked together. There are larger pores between the clusters of the particles as well as smaller pores between the particles itself. This is clearly seen when pore size distribution is measured using mercury porosimetry

288 80

1390 3445

760 995 849 908

40

1150

2500

2000

30 20

1730

3000

T, %

50 1261

3500

60

1452 2997

4000

70

1500

1000

10 500

Wavenumber, cm−1

Fig. 4. IR spectra of methacrylate monolith. Vibration bands at 908 and 849 cm1 are characteristic for epoxy groups. (Reprinted from [68].)

Fig. 5. SEM picture of the methacrylate monolith at magnification 10,000. Small globules are linked into clusters. Voids between the globules and clusters represent a network of channels.

(Fig. 6). When pore volume is drawn as a function of pore diameter (Fig. 6a) we can see that most of the volume is present in large pores while small pores contribute almost negligible portion of the volume. The situation is quite opposite when the specific surface is compared. While large pores contribute only

289

dV/log (r)

(a)

10

100

1000

10000

Pore diameter, nm

dS/log (r)

(b)

10

100

1000

10000

Pore diameter, nm

Fig. 6. Pore size distribution of methacrylate monolith measured with mercury porosimetry. Volume pore size distribution (a) demonstrates that most of the pore volume is located in large pores with a diameter of around 1500 nm. Surface pore size distributions (b) shows a different picture since most of the surface is accumulated in the pores with a diameter below 100 nm. (Reprinted from [68].)

moderately to surface area, small pores provide more than 80% of the total monolith surface (Fig. 6b). Such monoliths are therefore characterised by the so called bimodal pore size distribution. Even more pronounced example of such pore size distributions are silica monoliths [54] where, however, the small pores are closed (dead-end). For the methacrylate monoliths, in contrast, it was shown that all pores are interconnected and open [38] and an almost flat Van Deemter curve is obtained [58,42]. Pore size distribution of the methacrylate monoliths can be tailored in different ways. Composition of the polymerisation mixture strongly influence pore size and the total porosity, which is roughly determined by the ratio between monomers and porogens. Since porogens do not react during polymerisation, but serve as a precipitation media, the volume of the porogens also roughly represents the void volume of the pores [43,44]. While the total porosity is

290 1000

Radius of the pores (nm)

900 800 700 600 500 400 300 200 100 at e ug

EA

E

P

Denotation of the monolith

pe

pt

id

e-

G

M

A

co

nj

D

H

C

-1

8

2 -1 C

/2 -6

-2 C

C -4

C

G

M

A*

0

5%, T1 15%, T1 5%, T2 15%, T2 5%, T3 15%, T3

Fig. 7. Effect of the monomer type and temperature on the monolith pore size distribution. Monoliths were polymerised at three different temperatures and two different concentrations of the added monomer. C–N indicates length of the alkyl chain on the methacrylate while *GMA monolith represents the basic monolith without the substitution of the glycidyl methacrylate with any other monomer. (Reprinted from [44].)

relatively independent of the nature of the monomers and porogens, pore size distribution is a much more sensitive parameter. The reason is that the pore size depends on the onset of the phase separation, which is determined by the compatibility between the porogens and monomers [45]. This is of course affected by the type and amount of each single component in the polymerisation mixture. One example is shown in Fig. 7, in which the effect of the monomer type was investigated [44]. It can be seen that the length of the hydrophobic moiety strongly influences the size of the pores. Another parameter that strongly affects pore size distribution is the polymerisation temperature. The temperature defines a degradation rate of the initiator and, therefore, also the number of nuclei formed in a given time. Since the total amount of the monomers is limited, the lower number of nuclei formed at lower temperatures within a defined volume corresponds to their larger size, and thus, to larger pores between the clusters of growing nuclei. In contrast, at higher polymerisation temperatures, where the initiator decomposition is much faster, the number of growing nuclei is larger and the pores formed are consequently smaller. Temperature effect is extremely strong, since a change in temperature of a few degrees shifts the pore size by almost one order of magnitude [46] (Fig. 8). Being a powerful tool for tailoring of the monolithic structure, temperature also has a strong impact on the design and preparation of

291 T

relative volume

T+8 T+2 T + 6T + 4

10

100

1000

10000

pore radius (nm)

Fig. 8. Effect of the polymerisation temperature on the pore size distribution. At the highest temperature (T+8) the pore radius is 200 nm while at the lowest T the pores are much larger with the pore radius of 850 nm. (Reprinted from [46].)

large volume monolithic blocks, as it is explained in detail in the section ‘‘scaleup of the CIM monolithic columns’’. Since the monoliths consist of a single piece of material they have to possess a uniform structure over the entire monolith volume. In fact, large scale uniformity of the structure reflects in a similar manner the separation efficiency as the quality of packing in the case of particle-shaped supports. Unfortunately, neither the pore size distribution, nor the SEM photographs reveal whether the entire structure is uniform or not [47]. This can be estimated only with pulse response and frontal analysis experiments. Irregularities in the structure reflect in peak doubling or bimodal breakthrough curve, while in the case of uniform monolith a single peak or single steep breakthrough curve is obtained [47]. Similar experiments can also be performed to estimate dispersion of the uniform methacrylate monoliths. Unfortunately dispersion of the monolith cannot be measured directly, since the monolith must be fixed in an appropriate housing that enables connection to the pump and mobile phase flow through. Each system component like tubings, monolith housing and even detector cause additional dispersion and contribute to the total measure dispersion. To accurately evaluate the dispersion contribution of the monolith itself, all the other, so called extra column effects, must be subtracted. This can be done in two different ways. The methods developed by Kalterbruner et al. [48] requires measurement of pulse response dispersion with the columns having a different column length but same extra column dispersion. For methacrylate monoliths, this was done by placing several CIM disks in a single housing, preparing a column with variable bed length [49]. They found that more than 90% of the total dispersion is caused by extra column broadening [38]. An alternative

292 approach is based on numerical deconvolution of experimental data. In this case dispersion of a system with and without the monolith should be measured which is easily performed with CIM disk monolithic columns. Having dispersion of both systems, real monolith dispersion can be calculated using numerical deconvolution [50]. It was found that a Peclet number is around 120, once again confirming extremely low dispersion caused by monolith [50]. Another important factor to be estimated is a pressure drop of the monolith and its comparison to the particle bed. Due to higher porosity and different structure as compared to those of the particle-shaped beds, the application of equations developed for a calculation of pressure drop in particle bed might be questionable. Therefore different approaches were suggested in the literature for various monoliths. Meyers and Liapis used a pore-network modelling approach wherein a number of the so called flow nodes are interconnected by cylindrically shaped pores with variable diameters [51,52,53]. To predict the pressure drop, a detailed knowledge of the structural properties is required, such as pore size distribution and pore connectivity. The latter, however, is very difficult to determine; therefore the lack of accurate experimental data limits wider application of the model. Tallarek and coworkers introduced equivalent particle dimension for silica monoliths [54,55]. This dimension is obtained by dimensionless scaling of macroscopic fluid behaviour, i.e., hydrodynamic permeability and hydrodynamic dispersion in both types of material; particulate and monolithic. As a result there is no need for direct geometrical translation of their constituent unit. This elegant approach can be basically applied to any type of stationary phase. However, since there is no clear correlation to the monolith structural properties, it is difficult to perform an optimisation of the monolithic structure on such a basis. An even more detailed elaboration of the pressure drop prediction on silica monoliths was performed by Vervoort et al. [56,57]. Their calculations were based on computational fluid dynamics simulations using Navier–Stokes equations. The assumption of the tetrahedral skeleton structure enabled the correlation of the pressure drop to the skeleton thickness and column porosity. Using this approach it is possible, on a theoretical basis, to predict the optimal structure for the monolith and can therefore be used as a powerful optimisation tool. However, so far this approach was only applied to the tetrahedral skeleton structure and its application on other types of monoliths having different structure might not be trivial. This is probably the reason why no attempts to describe methacrylate monoliths in a similar manner have been published. Because the structure of methacrylate monoliths resembles the particle beds, attempts have been made to characterise them with the well-known Kozeny– Carman equation (Eq. 1) and calculations of the equivalent particle diameter from the pressure drop data have been made [38,58]. It was noticed that the calculated equivalent particle diameter significantly exceeded the size of the particles determined from SEM pictures [58]. However, in more recent studies published by the same group, the discrepancy was found to be much smaller [38].

293

To compare methacrylate monolithic supports with conventional packed bed, Mihelicˇ et al. [59] recently used a concept of hydraulic radius as a very basic parameter, which can be used for both types of supports. Combining Kozeny– Carman and Happel equations [60] they derived the generalised k1 parameter (see Eq. (1)), which becomes a function of porosity as follows: 0 1 e3 @ 3 þ 2ð1  eÞ5=3 A
h i k1 ¼ (2) 4(1  eÞ 3  9=2ð1  eÞ1=3 þ 9=2ð1  eÞ5=3   3ð1  eÞ2  k1 parameter has a value of 1 for straight uniform pores and higher values for real beds [59] due to friction, pore tortuosity, etc. Based on experimental data of porosity, pressure drop and wetted surface the k1 value for CIM methacrylate monoliths were calculated and surprisingly, the value was found to be below 1. To elucidate this unusual result the authors have proposed an explanation based on the concept of structure self-similarity of parallel pore arrangement [59]. In Fig. 9 three types of pore arrangements are shown. In the first case (A) the structure is made of uniform pores all having equal diameter Da. Structures B and C represent two extreme types of non-uniform pore distribution: B is a structure of parallel type pore non-uniformity and C is a serial type of pore nonuniformity. While the structure B is identical between all the nodes, the structure C is periodically changing. In reality, a combination of both types occurs and it is therefore difficult to predict the overall effect of the structure on the pressure drop. However, it is rather simple to calculate the pressure drop for both extremes. Let us assume that the porosity is in all the three cases the same, meaning that the pore volume should be equal, and nodes should have no volume, and therefore all the volume is in the pores. Let the ratio between pore diameters be N, therefore Db1¼N * Db2 (N>1). Furthermore, structures B and C have also the same hydraulic radius. For the beds of the same length, the pore area should be equal too. The same flow rate is applied to all the structures. Da

Db1 Db2

∆Pa1

Dc1

∆Pb1

∆Pc1

∆Pb2

∆Pc2

Node ∆Pa2 (A)

(B)

Dc2

(C)

Fig. 9. Different configurations of three hypothetical porous structures having the same porosity and hydraulic radius. Structure A represents uniform pore size distribution, while the structures B and C demonstrate parallel (B) and consecutive (C) type of the structure being however identical according to pore size distribution. (Reprinted from [59].)

294 It can be shown, that the pressure ratio DPa/DPb and DPa/DPc can be described with equations, further details are described in Ref. [59].
 4 2 1 þ ð1=N Þ DPa ¼
(3) 2 DPb 1 þ ð1=N 2 Þ and DPa ¼ DPc

8
2 ð1 þ N4 Þ 1 þ ð1=N 2 Þ

(4)

These two equations are valid for two different pore sizes (n ¼ 2). If there are several pore sizes, having the same diameter ratio N (this is in fact self-similarity level), Eqs. (3) and (4) can be generalised into: P 4i DPa n  n1 N2  1 N2n þ 1 i¼0 N  ¼
¼ n   Pn1 2i 2 N2 þ 1 N2n  1 DPb N i¼0

(5)

and DPa ¼ DPc n1 X

 2 2 N4  1 N 1  !2 ¼ n  4n N  1 N2n  1

n3 4i

N

i¼0



3

n1 X

(6)

2i

N

i¼0

with the limits lim

DPa

N!1 DPb

¼ n and

lim

DPa

n!1 DPb

¼1

(7)

¼0

(8)

and lim

DPa

N!1 DPc

¼0

and

lim

DPa

n!1 DPc

where n stands for the number of self-similar levels. The effect of the structure type on the pressure drop is also demonstrated in Fig. 10. It is clearly seen that the structure B always gives a lower value for the pressure drop in comparison to the uniform pore size distribution, while the structure C always gives a higher pressure drop. In fact, both the limits of DPa/ DPc, when n and N go to infinity, go to 0 which means that DPc goes to infinity. On the other hand, from Eqs. (6) and (7) it is clear that when the pore ratio approaches infinity (N!1) the ratio of the pressure drop DPa/DPb equals the levels of self-similarity n, and consequently when n rises towards infinity, the DPb

295 3,0

2,5

∆Pa/∆Pb,c [/]

2,0

1,5

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0,5

0,0 1

2

3

4

5

6

7

8

9

10

N [/]

Fig. 10. Effect of the pore arrangement (see Fig. 9) on pressure drop for the beds having same porosity. Dependence of DPa/DPb and DPa/DPc ratios on value of N. Solid line represents structure B having two pore sizes (n ¼ 2), dashed line represents structure B having three pore sizes (n ¼ 3) and dotted line represents structure C having two pore sizes (n ¼ 2). (Reprinted from [59].)

tends to zero. Theoretically speaking, with proper pore size distribution and network architecture one can obtain as low pressure drop as desired for a given porosity. These calculations give a clear guideline for the optimisation of the monolithic structure. Although uniform particle size distribution (or monolith equivalent pore size distribution) gives lower dispersion in comparison to a non-uniform one, most of the dispersion, in fact over 90% in the case of CIM methacrylate monoliths, comes from extra column effects [38]. Because of its small contribution to the total dispersion, even an increase of dispersion caused by the monolith itself would not significantly decrease column efficiency. On the other hand, a wide pore size distribution of the pores of parallel type might result in significantly lower pressure drop than the uniform pore size distribution providing also higher specific surface area and consequently higher dynamic binding capacity. This conclusion might be especially important for the design of large volume monolithic columns, where part of separation resolution might be sacrificed on expenses of higher capacity and lower pressure drop.

296 Construction of methacrylate monolithic columns for separation of biomolecules – CIM Convective Interaction MediaÕ We discussed in the section ‘‘types and properties of the chromatographic monoliths’’ about the monolith properties that are especially advantageous for separation and purification of large molecules. Therefore, to benefit most, CIM monolithic columns were optimised for separation and purification of such type of molecules. To obtain efficient separation and dynamic binding capacity, especially the pore size distribution should be properly adjusted. Pores should be large enough to allow penetration of the molecules into them but should not be too wide to allow big molecules to access the surface of the pore so as to decrease resolution. Taking into account these considerations Tennikov et al. [61] found that for medium sized proteins, the optimal mean pore size should be  700 nm. Since commercial CIM methacrylate monoliths are intended for even larger molecules like large proteins, DNA or even viruses, they were designed with even larger pores to achieve optimal performance [62]. Another important aspect of separation and purification of large molecules is their adsorption mechanism. Most of the macromolecules interact strongly with the surface, commonly via several binding sites [49], resulting in extremely steep adsorption isotherms. To elute them, a gradient of mobile phase is commonly applied by changing its composition or pH value. Separation is therefore practically based on selective elution since the strength of attachment differs for various macromolecules. For such a chromatographic principle it was shown that the effect of the column length on the column performance is very small [63–65]. In fact, to achieve optimal resolution, the column length and gradient slope have to be fine-tuned [88]. Since very steep gradients can be applied for the separation of large molecules, even very short columns can be used to achieve efficient separation. This concept was used in the development of CIM methacrylate monolithic columns. Thus, the smallest commercially available units have a specific format of the disks (CIM disk) and fit into specially designed housing (Fig. 11). A single CIM disk has a diameter of 12 mm, a thickness of 3 mm and a volume of 0.34 ml. Monolith is surrounded by a non-porous inert plastic, the colour of which indicates the type of the chemical

Fig. 11. Outlook of the CIM housing (left) and CIM disks (right). Non-porous ring around the monolith prevents bypass, while its color indicates monolith functionality.

297 6 4 CIM disks

Pressure drop (bar)

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4 3 CIM disks 3 2 CIM disks 2 1 CIM disk 1

0 0

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Linear velocity (cm/min)

Fig. 12. Pressure drop on CIM disk monolithic column as a function of flow rate. Upto 4 CIM disks were placed in a single CIM housing (see Fig. 11).

moieties present in the monolith. Such an arrangement allows high flexibility since the CIM disk can be easily exchanged, constructing in this way columns with different chemistries. Furthermore, upto four CIM disks can be mounted into a single housing, thus increasing the column length and volume [49]. In addition, inserted disks might not be of the same chemistry, enabling multidimensional chromatography, called in the case of CIM monoliths Conjoint Liquid Chromatography (CLC) [62]. CIM disk monolithic columns can operate in a range of flow rates between 0.5–10 ml/min at a very moderate pressure drop of few bars as shown in Fig. 12. It is important to notice the linear relation between the pressure drop and the flow rate, i.e., the methacrylate monoliths are very rigid and no compression occurs. Robustness of the product is demonstrated in more than 1000 consecutive injections without significant changes in resolution. As already discussed, one of the main characteristics of the monoliths is the extremely fast exchange between the mobile and stationary phase resulting in flow-unaffected resolution and dynamic binding capacity even for very large molecules. Since the flow rate can be in principle increased unlimitedly, extremely fast separations are possible as shown in Fig. 13, where baseline separation was achieved within 10 s [117]. It was also shown, that for the monolith separations, system limitations like data acquisition rate or detector response time became a bottleneck [58]. Despite a very short column length, CIM disk monolithic columns can be used for fast separation of many compounds achieving very good resolution. This is demonstrated on a separation of oligonucleotides, where 14 oligonucleotides

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49

30 20

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Fig. 13. Extremely fast separation of three standard proteins ribonuclease A, cytochrome C, bovine-serum albumin and chicken-egg albumin on strong reverse phase monolithic column applying linear gradient. (Reprinted from [117].)

were almost baseline-separated in less than 4 min at room temperature using just one disk forming just 3 mm column length (Fig. 14). Further details about the theoretical aspects of separations on short monolithic beds and properties of CIM disk monolithic columns are described elsewhere [46,62,67–69,70–72]. Scale-up of the CIM monolithic columns Development of large CIM monolithic columns was performed following two main criteria:
Since the columns are intended for separation and purification of macromolecules, the idea of short monolithic bed was followed.
To enable easy scale-up of chromatographic methods, the structure of the monoliths was kept constant regardless of the monolith volume. To fulfill the first criterion there are some constraints regarding the design of a column. In fact, from a technical point of view, the only reasonable designs are disk format and cylinder format. Although both designs can, in principle, be applied, cylindrical format seems to be advantageous. In the case of the disk format, only the diameter and the thickness can be varied, while in the case of cylindrical shape, in addition to these two, the cylinder height is also variable. Of course, to follow the idea of short monolithic bed, the mobile phase should flow through the monolith in a direction perpendicular to the cylinder height.

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% buffer A

relative absorbance at 260 nm (mAU)

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Fig. 14. Separation of oligomers using optimised gradient conditions. Conditions: Mobile phase: buffer A: 20 mM Tris–HCl buffer, pH 8.5; buffer B: 1 M NaCl in 20 mM Tris–HCl buffer, pH 8.5; Flow rate: 4 mL/min; Stationary phase: CIM disk monolithic column comprising of a single disk; Sample: oligonucleotides of different lengths – number near the peak represents the oligonucleotide length; Gradient: as shown in figure; Injection volume: 20 mL; Detection: UV at 260 nm. (Reprinted from [49].)

Therefore, the so called radial operation mode should be applied. It can be shown, that for a given volume of the column, a properly designed cylindrical monolith exhibits a lower pressure drop than the corresponding disk-shaped unit [76]. Furthermore, the cylindrical format is much more compact and mechanically stable; and the by-passing of mobile phase is less common in comparison to the disk-shaped monoliths. To answer the second criterion, that is, to prepare a large monolith with a uniform structure, the polymerisation mechanism should be well understood. As already described in the section ‘‘Methacrylate monoliths’’, methacrylate monoliths are prepared via bulk polymerisation and the polymerisation temperature significantly influences the pore size distribution. It was shown that the change of the polymerisation temperature for 8 C shifts the pore size by almost one order of magnitude [46]. Although an excellent tool for tailoring the monolith structure, temperature also represents one of the main problems in the preparation of large volume methacrylate monolithic columns. Methacrylate polymerisation is a very exothermic reaction releasing particularly in the case of the methacrylate monoliths around 190 J/g of heat [73].

300 Since the preparation of monoliths proceeds through bulk polymerisation, the heat generated cannot be dissipated fast enough, therefore an increase in the temperature inside the polymerisation mixture during polymerisation occurs. At the maximal polymerisation rate, the increase can be as high as 80 C [46]. Taking into account that already one-tenth of this value dramatically changes the pore diameter, it is clear that such an increase results in extremely non-homogeneous pore distribution [74]. Two approaches are described to overcome this problem. Peters et al. [74] suggested slow and gradual addition of the polymerisation mixture to the reaction vessel in which the polymerisation proceeds continuously. In this way heat release is minimised causing much smaller temperature increase and consequently facilitating the production of large volume monoliths having a uniform structure. Another approach is based on the estimation of maximal monolith thickness to obtain a uniform structure during the conventional batch polymerisation. The idea is to solve a mathematical model based on the heat balance equation [75]:       ðt   @T aðxÞ @ @T 1 @ ¼ r DHr 1  exp A exp Ea;app =RT d t (9) þ @t r @r @r cp ðxÞ @t 0 where T is the temperature, t is the time, a(x) the thermal diffusivity, r the cylindrical coordinate, cp(x) is the specific heat capacity, DHr is the heat of reaction, A is the pre-exponential factor, Ea,app is the apparent activation energy and R is the gas constant. For a cylindrical format the following initial and boundary conditions have to be applied [76]: Initial condition: T ¼ T1

R1  r  R2

t¼0

(10)

Boundary conditions: T ¼ T1

r ¼ R1

t 0

(11)

T ¼ T1

r ¼ R2

t 0

(12)

This model was verified with experimental data and a good correlation was found [75]. Based on this it is possible to predict temperature increase for a cylindrical shape of specific diameter and thickness as shown in Fig. 15. From these data it is clear that only limited thickness of the monolith can be prepared since already at a thickness of 3 cm, a temperature increase of  70 C occurs. However, preparation of the cylinder-shaped monolith of a desired thickness is easy and achieved just by polymerising several cylinders of appropriate dimensions and inserting one into another (the so called ‘‘tubein-tube’’ approach [77], as shown in Fig. 16) and perform subsequent polymerisation to fill the gaps [78]. On this basis large-volume monolithic columns were constructed, having volumes of 880, 800 an 8000 ml [79]. Monoliths are

301

Temperature increase, °C

100

80

60

40

20 30 0

m ,m

20 10

10

20 30

rin , m

40

m

s es

n

ick

th

0

Fig. 15. Simulation of the maximum temperature increase inside the polymerisation mixture placed in an annular mold during polymerisation of the methacrylate monoliths as a function of both tube inner radius (rin) and thickness. (Reprinted from [76].)

1 2

3

4

Fig. 16. Construction of a tubular large volume CIM monolithic unit of desired volume. The monolithic unit (4) consists of three monolithic annuluses (1, 2 and 3). Total thickness of unit 4 is a sum of the thicknesses of the monolithic annuluses 1, 2 and 3. (Reprinted from [77].)

302

Fig. 17. Large scale tubular CIM monoliths of volume 8, 80 and 800 ml.

shown in Fig. 17. More details about large-scale CIM monolithic columns and modelling can be found elsewhere [46,62,73,75–79, 85]. Characterisation of CIM monolithic columns To properly characterise monolithic columns, some new concepts have to be derived. Particle shape supports are normally prepared in large batches. For their characterisation, a small representative sample is taken and all tests, either nondestructive or destructive can be performed. The results are assumed to be valid for the entire batch. This approach cannot be implemented to the monolithic supports as each monolith is prepared in a single mould. Even if several moulds of identical material and dimensions, containing the same polymerisation

303

mixture, are placed simultaneously in the same thermostated media, there is still a possibility that that due to some factors, e.g., local temperature gradients, the monoliths can differ in their structure. From this point of view, each monolith should be considered as a single batch and consequently its properties should be checked. This principle is adopted for the production of CIM monolithic columns. To control the reproducibility of the monolithic columns, two main monolith properties should be checked: monolith pore size distribution with total porosity and ligand density. Pores size distribution together with a total porosity defines the permeability of the matrix, pressure drop and specific surface area, while the ligand density determines the strength of interaction between the sample molecule and the matrix. In combination with the specific surface area, the dynamic binding capacity can also be estimated. Knowing this data, chromatographic properties of the monolith can be defined to a large extent. Porosity and pore size distribution can be estimated from the pressure drop data. However, this relation is not uniquely determined since the pressure drop might remain constant if for example, pores are larger but porosity is accordingly smaller (see Eq. (1)). To properly evaluate these two basic structural properties, a small piece should be cut from each monolith and evaluated using mercury porosimetry. Data about pore size distribution (Fig. 6) and porosity are obtained. In addition, the available surface area is calculated on the basis of pore size distribution. Although the value of the surface might not be absolutely correct, since certain assumptions related to the structure topology are assumed during calculation, for materials of similar structure, an accurate comparison can be made. Ligand density can be, in principle, derived from the measurement of capacity, taking into account specific surface area from mercury porosimetry data. Since CIM monolithic columns are intended for separation and purification of large molecules, the most appropriate measure would be for example, protein capacity. Besides being an expensive method, loading the column with the proteins inevitably leads to column contamination, which is unacceptable for industrial purposes. An alternative method, commonly applied for the determination of ligand density, in particular of ion-exchange groups, is using NaNO3 [80], which is however limited to anion exchangers. The third commonly applied method is titration of the groups with acid or base. This method is extremely time-consuming, in some cases several days are required to achieve the equilibrium [81]. Furthermore, long duration exposure of the resin to harsh conditions might cause its partial degradation. Another alternative would be, similar to the measurement of pore size distribution, to cut a piece of the monolith and measure separately one of the above described properties. To do so, the piece should be immersed in the liquid containing a predefined solution and allowed to stand till the equilibrium is established. In this case also, the entire procedure would be time

304 consuming due to diffusional restrictions and might again require several days to achieve equilibrium [82]. To determine the amount of ion-exchange groups on the monolith, an alternative method was recently developed [83]. The main advantage of the method is that it is performed in a flow-through mode and it is very fast. In addition, only biologically compatible buffers, like e.g., phosphate buffer, are used; therefore there is no risk of any kind of contamination of the matrix. This method is based on the formation of a pH transient when a high ionic strength buffer is momentarily switched to a low ionic strength buffer, both having same pH value. The phenomenon is explained by the local equilibrium theory [84]. Based on this theory we derived the equation showing that the time required for the pH stabilisation is proportional to the amount of the ion-exchange groups [83]: tðpHÞ ¼

L vfluid



  ð1  aÞ L  qt  f 0 ðCA Þ ¼  K  qt a vfluid

(13)

where t(pH) is the time required for pH stabilisation, L is the column length, v is the mobile phase linear velocity, a is the porosity, f 0 (CA) is the first derivative of adsorption isotherm and qt is the amount of ion-exchange groups. We also showed that instead of pH value, the absorbance can be monitored and a typical break-through curve can be obtained as shown in Fig. 18. A good

140 Absorbance, mAU

40

Absorbance, mAU

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Time, min

40 20 5

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Fig. 18. Absorbance breakthrough curve obtained by the step change from high to low concentration buffer solution on a CIM DEAE 80 ml tube monolithic column. Method: 1.0M phosphate buffer solution, pH 6.8 (5 min), 20 mM phosphate buffer solution, pH 6.8 (60 min). Flow rate: 80 ml/min. Detection: UV at 210 nm. (Reprinted from [83].)

305

correlation between the proposed method and estimation of amount of the groups was found for the anion and for cation exchangers. To complete the characterisation of monolithic columns, a uniformity of structure should be checked. This is easily performed in a flow through mode by pulse response experiments. Again, biological buffers of different concentration can be used for injections. From the description of the above characterisation, it can be concluded that although each single monolith represents a separate batch, monolithic columns can be extensively and non-invasively characterised. In this way, a good reproducibility can be achieved. Having in hand methods for quality control of CIM monoliths, the mechanical and chemical properties of the produced monolith can also be investigated. One of the main properties for a resin to be used on an industrial scale is its cleanability or regeneration method. This is commonly done with the so-called cleaning-in-place (CIP) procedures. Various CIP procedures are routinely applied, all containing a reaction with NaOH. In our experiment, CIP of exchanging low and high salt concentration buffers and NaOH was applied [79]. Besides chromatographic stability, we investigated if changes in the mobile phase composition, resulting in swelling and shrinking of the monolith, cause any mechanical damage. Results of the CIP procedure on dynamic binding capacity of BSA are shown in Fig. 19. Two hundred CIP cycles were performed with a single 80 ml CIM DEAE monolithic column still preserving initial bovine serum protein (BSA) dynamic binding capacity [79]. The inspection of the monolith showed no damage. It can therefore be concluded that chemical and

Dynamic binding capacity [mg BSA/ml CIM]

30 25 20 15 10 5 0 start

16

32

48

70

88

116 130 145 160 176 191

No. of CIP cycles

Fig. 19. Effect of the CIP procedure on BSA dynamic binding capacity. Even after 200 CIP cycles there was no change in dynamic binding capacity. (Reprinted from [79].)

306 mechanical stability of the CIM monoliths is high enough to be used on industrial scales. A set of CIM products consists of CIM disk monolithic columns operating in an axial mode (smaller units) and CIM tube monolithic columns operating in a radial mode [62]. Since the operating principle is different, the transfer of chromatographic methods does not seem to be trivial. Commonly applied scaleup criteria are constant column height or length, constant residence time, constant L/dp ratio, etc. [85]. They all consider geometrical similarity of the columns which is especially important for the separation of molecules based on selective migration. For separation based on selective elution, common for large molecules, the column length does not play such an important role as already discussed in details in the section. Because of that, scale-up criteria is based on a different principle introduced by Yamamoto [86]. He derived an equation to obtain constant resolution on different types of columns and resins, which is valid when steady-state conditions in the column are achieved [87]. Establishment of the steady-state conditions depends on linear velocity, concentration of displacer salt at which protein elutes, Z factor as a ratio of the protein charge and displacer charge and gradient steepness, from which a critical distance is calculated [88]. On the basis of these two works, a very simple equation for the transfer of chromatographic methods between the disk and the tube CIM monolithic columns was derived [76,89]:

tg;2 ¼ tg;1 

      VV;2 F1 L1   VV;1 F2 L2

(14)

where V represents the column void volume, F is the flow rate, L is the column length (or thickness in the case of tubular shape CIM columns) and t is the gradient time. Equation (14) was verified on standard protein solution, as well as on real samples [89] as shown in Fig. 20. For all the columns, a comparable resolution was obtained confirming adequateness of derived approach.

Application of the CIM monolithic columns Since their introduction in the market in 1998 [90] CIM monolithic columns have been implemented in various applications in different interaction modes. However, already before the commercial exploitation, several applications of disk and tube methacrylate monolithic columns have been described. In the following section, a comprehensive overview about the implementation of methacrylate monoliths, structured according to the separation mode and sample molecule will be given.

absorbance at 409 nm (mAU)

absorbance at 409 nm (mAU)

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absorbance at 409 nm (mAU)

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Fig. 20. Chromatograms of LiP isoforms obtained on axial and radial monolithic columns using equation 14. Conditions: Sample: lignin peroxidase H2 (1st peak), lignin peroxidase H6 (2nd peak), lignin peroxidase H8 (3rd peak), lignin peroxidase H10 (4th peak); Mobile phase: buffer A: 10 mmol/L sodium acetate buffer, pH 6; buffer B: 1 mol/L sodium acetate buffer, pH 6; Columns: CIM disk monolithic column of volume A – 0.34 mL, B – 0.68 mL, C – 1.02 mL and CIM tube monolithic column of volume D – 8 mL and E – 80 mL; Detection: spectrophotometric at 409 nm. (Reprinted from [89].)

Ion-exchange, hydrophobic and reverse phase chromatography Small molecules Although CIM monolithic columns were mainly developed for the separation of large molecules, a few successful separations of small molecules were also reported. By stacking several weak anion exchange CIM disks into a single

308 4

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5 relative absorbance at 210 nm

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Fig. 21. Separation of pyruvic (1), malic (2), tartaric (3), a-ketoglutaric (4), fumaric (5), citric (6) and isocitric (7) acids on a CIMÕ column comprising of four CIMÕ QA disks. Conditions: Mobile phase: 100 mM NaCl in 50 mM phosphate buffer, pH 8.0; Flow rate: 5 ml/min; Sample: pyruvic acid – 0.03 g/L, malic acid 0.5 g/l, tartaric acid 0.5 g/l, a-ketoglutaric acid 0.2 g/l, fumaric acid 0.01/l g, citric acid 2g/l and isocitric acid 2 g/l ; Injection volume: 20 ml; Detection: UV at 210 nm. (Reprinted from [91].)

monolithic housing, it was possible to separate upto six organic acids, namely citric, isocitric, pyruvic, fumaric, malic and alpha-ketoglutaric acid in less than 7 min (Fig. 21) [91]. A chromatographic method was applied for monitoring the organic acid formation during the fermentation of yeast Yarrovia lypolitica. A similar column was also used for the determination of Mn3+ tartrate complex in a fermentation media of Phanerochaete chrysosporium [92]. Another usage of CIM monolithic columns describes the determination of organic acids and sugars in soft drinks by sequential injection Fourier transform infrared (FTIR) spectroscopy [93]. In this case the monolithic column carrying the quaternary amino moieties was added as a solid-phase extraction column to the flow system. Upon injection of a sample the organic acids were completely retained on the CIM column whereas sugars passed to the flow cell. The organic acids were subsequently eluted by injection of an alkaline (pH 8.5) 1 M sodium chloride solution. Authors stated that ‘‘. . . the developed method is characterised by its short analysis time, experimental simplicity and its potential application in routine analysis and process control’’ [93]. An interesting application comes from the environmental protection field. Efficient separation of Zn complexes of citrate, oxalate and EDTA as well as hydrated Zn2+ species was performed in a very short time and found to be more

309

efficient than with a conventional column [94]. Another, validated method was developed by the same group for speciation of airborne chromium [95]. Besides good reproducibility, speed was again found to be the main advantage. Very recently CIM monolithic columns were also applied for the separation of inorganic anions [96]. Peptides and proteins Since there are several applications of CIM monolithic columns for the separation of small molecules it is not surprising that there are also reports about the separation of peptides. Vlakh et al. [97] used strong cation exchange CIM disk monolithic column for the separation of synthesised linear lysine homologues in gradient chromatographic mode. Baseline separation of three peptides was achieved and the homogeneity of each peptide could be evaluated from a chromatogram. The possibility of the application of CIM monoliths for metal affinity chromatography was investigated by Ren et al. [98] They prepared metal–chelate monolithic column by reacting iminodiacetic acid with epoxy groups. Four different resins HiTrap Chelating HP (agarose), TSK Chelate-5PW, Poros 20MC and CIM disk columns were compared using tryptic digests of transferrin and b-galactosidase as model samples. CIM units showed a good performance with low non-specific binding and gave comparable results to TSK Chelate-5PW, but were less retentive. Obviously, there are many more publications of protein separation. A wellexplored area is the application of methacrylate monolithic columns for the separation and purification of plasma proteins. Already in 1992 methacrylate monoliths were applied for the separation of rat serum plasma proteins and kidney plasma membrane proteins using weak anion exchanger (DEAE) [99]. Good recovery and separation was achieved with the separation time  20 min. Anion-exchange monolithic columns were also used for the separation of annexins from rat liver and Morris hepatoma 7777 [100]. Baseline separation was completed in 10 min under non-denaturating conditions obtaining highly purified proteins. First ultra-fast separation of plasma proteins was demonstrated in 1996 (Fig. 22) [66]. Authors were able to monitor Factor IX and a1-antitrypsin purification process with the separation completed in less then 1 min. A similar analysis time was also achieved in monitoring of glucose oxidase immobilisation [66]. The first large-scale purification of plasma proteins using 8-ml methacrylate monolithic column was reported in 1997 [101]. The radial operation mode was used to further accelerate the purification process. In this way the purification of clotting factor VIII was 5 times faster than the corresponding column chromatographic method giving a similar yield and purity (Fig. 23). Smaller monolithic units were used for monitoring the same purification process. A detailed study of the purification of different concentrates of clotting factor IX was performed by Branovicˇ et al. [102]. They tested weak and strong

310 160 140 3 RELATIVE ABSORBANCE

120 1 100 2 80 60 40 20

A

0 B −20 0

20

10

30 TIME [s]

40

50

60

Fig. 22. In-seconds separation of plasma proteins during a1-antitrypsin (AAT) production using weak anion-exchange methacrylate monolithic column. (Reprinted from [66].) 600.00

40.00

35.00 500.00

400.00 25.00

300.00

20.00

15.00 200.00

RELATIVE ABSORBANCE

CONCENTRATION (U/ml)

30.00

10.00 100.00 5.00

0.00

0.00 0

2

4

6

8 10 TIME (min) vWF

12

14

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18

F VIII

Fig. 23. Purification of FVIII using radial methacrylate monolithic column. Full bars indicates von Wilebrand Factor (vWF) while empty bars represent factor VIII (FVIII). (Reprinted from [101].)

311

anion-exchange methacrylate CIM monolithic columns on dynamic binding capacity, regeneration procedure with 1 M NaCl and 0.5 M NaOH and optimising chromatographic conditions. They were able to baseline separate vitronectin from FIX. Similar results were obtained on CIM disk monolithic columns and on CIM 8-ml tube monolithic columns. Further, they demonstrated the applicability of CIM monolithic columns for the purification process of factor IX from human plasma [103]. Starting with the eluate after solid-phase extraction with DEAE-Sephadex, the use of monolithic columns has allowed much better purification than that achieved with conventional anion-exchange supports and the separation time was also significantly reduced. In upscaling experiments, separations were carried out with 8, 80 and 500-ml columns approaching a pilot scale level with the largest one. The results after upscaling were comparable to those obtained with the 8-ml column on a laboratory scale and the authors reported ‘‘The use of CIMÕ monolithic columns considerably reduces the period of time required for separation. The specific activity of factor IX is increased for almost one order of magnitude’’. Another well-studied system was the monitoring and purification of extracellular ligninolytic enzymes from the fermentation broth of fungus P. chrysosporium [89,104–107]. For this purpose, CIM anion-exchange disk and tube monolithic columns were used. The different enzyme isoforms present in a fermentation broth have similar molecular weight. An efficient separation of lignin peroxidases (LiPs) was achieved in a few minutes, thus, reducing a required separation time by one order of magnitude as compared to a conventional column [104]. The purity of the isoforms was comparable using monolithic vs. conventional columns. The chromatographic method developed was also used for monitoring the isoenzyme profiles during the fermentation process [107]. Recently, very fast separation of managan peroxidases (MnP) was performed on CIM QA monolithic column, using a combination of salt and pH gradient [106]. This enzyme system was also used as a test for the transfer of chromatographic methods between differently sized CIM columns [89]. CIM monolithic columns were used for isolation of different other enzymes. Due to the short separation time, it was possible to separate two xylanolytic enzymes from bacteria Butyrivibrio sp. strain Mz5 using CIM DEAE 8-ml column [108]. Speed was crucial in this case since these enzymes tend to agglomerate frequently. The same type of column was used also during the isolation and characterisation of a new type of thermostable NAD+-dependent R-specific secondary alcohol dehydrogenase from cholesterol-utilising Burkholderia sp. AIU 652 [109]. This isolation is important since the enzyme might be applicable as an effective biocatalyst for the production of chiral alcohols. Methacrylate monolithic columns were further applied for the purification of human tumour necrosis factor (TNF) [110]. A purification of recombinant human TNF from Escherichia coli extract was performed on anion-exchange and hydrophobic interaction chromatography. Separation on particulate based

312 support and methacrylate monolith was compared. Another application describes the purification of GTPgammaS binding proteins from membranes of porcine brain [111]. A comparison with conventional media was performed and similar results in terms of purity and yield were obtained. However, the separation on the CIM column was performed on a second time scale. Very recently, CIM monolithic columns were used for the separation of pegylated proteins, more particularly in the separation of myelopoietin from its pegylated form [112]. Baseline separation was achieved. Results were compared to Q- and SP-Sepharose high performance chromatography for preparative purification and to Q and SP-5PW chromatography for analysis. The use of either the monolithic or the Sepharose based supports for preparative chromatography produced highly purified pegylated MPO, but with the monolithic media run times as much as fivefold shorter were achieved. The monolithic disks also resulted in tenfold shorter run times for the analytical chromatography of pegylated proteins, however, their chromatographic profiles and peak symmetry were not as sharp compared to their Q-5PW and SP-5PW counterparts which can probably be explained by the not ideal disk format for analytical purposes. All applications for protein separation described above used CIM columns in a single separation mode. Due to the monolithic structure however, a multidimensional approach can be easily realised as described in detail in the section ‘‘Construction of methacrylate monolithic columns for separation of biomolecules – CIM Convective Interaction MediaÕ ’’. This feature was explored during the development of the chromatographic method for quality control of immunoglobulin G (IgG) concentrates [113]. The established method combined two different chromatographic modes in one step: affinity and ion-exchange chromatography (IEC) placed in the same column housing. Two CIM Protein G and one CIM quaternary amine (QA) monolithic disks were placed in series in one housing forming a CLC monolithic column. Binding conditions were optimised in a way that immunoglobulins were captured on the CIM Protein G disks, while transferrin and albumin were bound on the CIM QA disks. Subsequently, transferrin and albumin were eluted separately by a stepwise gradient with sodium chloride, whereas immunoglobulins were released from the Protein G ligands by applying low pH (Fig. 24). A complete separation of all three proteins was achieved in less than 5 min. The method permitted the quantification of albumin and transferrin in IgG concentrates and has been successfully validated. Further details about peptide and protein separations on CIM and other monoliths can be found elsewhere [114]. Oligo- and polynucleotides There are several studies on the separation and purification of oligo- and polynucleotides. Fast and efficient separations of oligonucleotides were reported using CIM DEAE disk monolithic columns [49,115,116]. A separation was

313 0.019

3

9

7

1 0.009

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2 0.004

−0.001. 0

pH

Absorbance (280 nm)

0.014

3

1 0.5

1

1.5

2 Time (min)

2.5

3

3.5

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Fig. 24. Separation of IgG (peak 3), transferrin (peak 1) and albumin (peak 2) on monolithic CLC column consisting of two CIM Protein G disks and one CIMÕ QA disk. Buffer A: 20 mM Tris–HCl, pH 7.4; Buffer B: 20 mM Tris–HCl, 1 M NaCl, pH 7.4 Buffer C: 0.1 M Gly – HCl, pH 2.6; Flow: 4 ml/min; Injection volume: 250 ml. (Reprinted from [113].)

performed either in isocratic or in gradient mode with the analysis time of a few minutes. Efficient separation of these rather small molecules can be attributed to significant charge differences between differently sized oligonucleotides, which for oligonucleotides with the length of at least upto 12 nucleic bases equals the number of charged phosphate groups [115]. Because of that it was not only possible to purify a target oligonucleotide but also monitor its impurities as a result of the synthesis process [46]. Using a reverse phase CIM SDVB disk monolithic column, it was possible to separate trityl-on oligodeoxynucleotide from trityl-off oligodeoxynucleotide in less than 1 min at room temperature [117]. CIM monolithic columns were found to be very efficient media also for the separation of polynucleotides. In 1998 Giovannini et al. [118] reported partial separation of different plasmid DNA isoforms using anion-exchange CIM monolithic column. An extensive investigation about the suitability of the CIM monolithic columns for the purification of plasmid DNA was performed by Sˇtrancar et al. [46]. It was found that the capacity for a tested plasmid was very high, around 8 mg/ml of support with the recovery of 100%. No damage of pDNA occurred as concluded from unchanged percentage of the supercoiled form (Fig. 25). Furthermore, the endotoxin content was significantly reduced. In a recent work it was also demonstrated that efficient separation of pDNA and RNA can be achieved without the addition of RNAse [119]. The method developed was used for in-process control of the purified plasmid. Another extensive study of the separation and purification of different polynucleotides was recently reported by Bencˇina et al. [120]. Besides plasmid DNA, uniformly sized lambda DNA of 50 kbp and genomic DNA with the size upto 200 kbp also were used. Effects of the pore diameter, ligand density and mobile phase on the recovery and dynamic binding capacity were investigated. Under optimal conditions the capacity exceeded 9 mg mL1 for all types of

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DNA. Successful purification of pDNA and gDNA was demonstrated by isolation from microorganisms (Fig. 26). Another interesting study describes the application of CIM anion-exchange columns for detection of genetically modified corn in thermally treated food [121]. Authors reported that the new method is faster and more sensitive. Mass transfer study of plasmid DNA in CIM monoliths was recently performed by Zo¨chling et al. [38]. Similar to other molecules (e.g., proteins) it was found that there is no detectable mass transfer limitation. However, they observed a slight decrease of capacity with the increase of linear velocity. The reasons are not yet clear and are under investigation. Nevertheless, capacity was still found to be very high in comparison with most of the other supports. Due to the outstanding capacity of CIM for plasmid DNA, combined with good resolution and high throughput, development of an industrial scale pDNA production process has been permitted, which is an order of magnitude more efficient than the previous bead-based processes [122].

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Further details about oligonucleotide and polynucleotide separations on CIM and other monoliths can be found elsewhere [123]. Viruses Purification of viruses is becoming a very important topic because of the increasing demand for the production of high purity vaccines and usage of virus vectors in genetic therapy. Since viruses are very large, some of them exceeding 200 nm, chromatographic supports having large pores are required for their efficient purification. Channels in CIM monolithic columns ranges around 1500 nm and could therefore be suitable for this purpose. In fact, Branovicˇ et al. [124] described the improvement of detection of measles and mumps viruses using PCR by enrichment of virus RNA on CIM monolithic columns. They were also able to load and elute the entire measles and mumps viruses. Recently, Kramberger et al. [125] used CIM QA disk monolithic columns for the concentration of intact rod-shaped tomato mosaic virus (ToMV). No problems about the blocking of the column were reported and the virus preserved its infectivity. The new method was much faster than the existing ones. Based on this preliminary work it can be anticipated that new reports will appear soon. Affinity chromatography Methacrylate based monoliths disks are very suitable for the immobilisation of various ligands, since they inherently contain epoxy groups, which form very

316 stable covalent bonds with the amino- or sulfhydryl groups of the ligand. Therefore it is not surprising that a high number of reports can be found in the literature. Since there are several excellent reviews covering this field [114,126–128], only a brief overview is given here. Low molecular mass ligand Soon after the introduction of methacrylate monoliths, the first report about the immobilisation of a small affinity ligand on them was published [147]. Immobilised p-(amino methyl) benzol sulfonamide, a carbonic anhydrase inhibitor, was used for the purification of carbonic anhydrase, which was subsequently immobilised on epoxy monolith and used as a bioreactor. In another study, three specific peptides, bradykinin, and peptides with 15 and 16 amino acids were used for isolation of various antibodies [129]. Immobilisation was performed on epoxy groups without any additional spacers and immunoaffinity properties were investigated determining adsorption isotherms. The prepared units enabled high purity isolation of antibodies and were stable over one year. Therefore, they could be efficiently used for monitoring and purification of target antibodies. The fact that immobilisation of peptides without a spacer gave good results is quite surprising since it is known that for small ligands, distance from a skeleton might play a crucial role. Because of that Jungbauer et al. [130,131] performed a detailed investigation of ligand utilisation immobilised on different types of support. They immobilised a model peptide for lysozime on different beaded material like Sepharose and Fractogel, having spacers of different length, and on monolithic CIM epoxy column [130]. Although the longest spacer, synthesised on Fractogel, had 16 C atoms, maximal ligand utilisation was achieved on the CIM monolithic support. A similar conclusion was obtained with immobilisation of six synthetic peptides prepared for the binding of FVIII [131]. In comparison with the conventional beaded material, a much better performance with respect to ligand utilisation, capacity and selectivity has been observed. The authors speculate that the short spacer inherently present on CIM monoliths provides enough accessibility also in the case of small molecules. With further optimisation using rational substitution of amino acids by spot synthesis, the best peptide against FVIII was selected [132]. With its immobilisation on CIM epoxy column, it was possible to capture FVIII from diluted plasma. To further improve the performance of the immobilised peptides two other approaches have also been proposed. Instead of immobilising the ligand on a formed monolithic skeleton, ligand conjugation can be performed with a monomer, which is used in the polymerisation mixture [133]. Thus the ligand is oriented to the matrix, the amount of the ligand can be precisely controlled and a uniformity of the ligand is assured. It was found that such an approach provides even better ligand utilisation and binding capacity for a lysozyme model peptide than conventional immobilisation (Fig. 27). The second proposed approach was to attach to the peptide a large molecule like polyethyleneglycol (PEG) [134].

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Because of the size, ligand–PEG conjugate cannot penetrate the small pores inaccessible for a target protein, but is attached only to the large pores. After the immobilisation is completed, PEG is cleaved off. For a model peptides against lysozyme and FVIII, it was shown that even better ligand utilisation is achieved on both, beaded and CIM monolithic supports. Short hydrophilic octapeptide (Flag) was immobilised on CIM epoxy column to isolate Flag-human serum albumin from a clarified yeast culture supernatant [135]. The performance of the monolithic column was compared to gigaporous glass bead support. Both columns were found to be suitable for purification of protein within few minutes and CIM column exhibited flow-unaffected dynamic binding capacity in addition. Another interesting study was recently reported by Vlakh et al. They prepared various synthetic linear or branched oligo/poly-L-lysines as a ligand to capture tissue plasminogen activator (t-PA) [97]. CIM epoxy disk monolithic columns were selected as a matrix since negligible mass transfer resistance enables direct evaluation of immunoaffinity properties. The efficiency of different peptides was compared, in terms of affinity constant and capacity. Such columns were used for purification of t-PA from a CHO cell supernatant. Again it was confirmed that even a very short peptide exhibited very high affinity constant although being immobilised without a spacer. Interestingly, longer peptides showed lower affinity constants, which might be the result of multipoint attachment. Because of the purification speed, high biological activity of the t-PA was preserved.

318 High molecular mass ligands The first report about immobilisation of high molecular weight ligand on methacrylate monolith dates back to 1992 when the immobilisation of heparin and collagen on epoxy groups was performed [99]. Using a heparin affinity column, a purification of very hydrophobic plasma proteins from plasma membranes was possible, while collagen monolith was used for separation of annexins. In both cases good recovery was obtained. In further work of the same group, application of heparin immobilised methacrylate monolith was extended to monitoring of isolation of antitrombine III and Factor IX [136]. Separation was in both cases completed in 6 min. Another interesting application was the immobilisation of annexin CBP 65/67 on epoxy groups present in methacrylate monolith [100]. Monolith was used for the purification of monospecific, polyclonal antibody, which was subsequently used for successful cloning and sequencing of cDNA of this protein. In 1998 there was a report about immobilisation of Concanavaline A on methacrylate monolith epoxy groups [137]. The characterisation of affinity monolith was performed with glucose oxidase. The affinity unit was used for purification of the enzyme dipeptidyl peptidase IV from rat liver. There are several reports about immobilisation of immunoglobulins for purification of Protein G on CIM epoxy monolithic columns. Kasper et al. immobilised human IgG for purification of Protein G from E. coli cell lysate [138]. The study of immunoaffinity properties revealed a high selectivity of separation method resulting in high product-purity. Due to negligible mass resistance and low pressure drop, very short purification times were possible. Developed affinity monolith was further incorporated in a flow injection analysis (FIA) system and applied for determination of Protein G content in E. coli cell lysate [139]. A linear calibration curve over several orders of magnitude was obtained. The analysis was very reproducible although being completed within 5 min. A more detailed study of different types of acidic eluents performed recently demonstrated multisite character of Protein G–IgG linkage resulting in high thermodynamic strength [140]. The study of the effect of experimental conditions on the properties of the affinity unit included, besides immobilised human IgG, also immobilised bovine serum albumin (BSA) and soybean trypsin inhibitor [141]. As test substrates, two artificial solutions with trypsin, blood serum containing antibodies against BSA and cell lysate with recombinant protein G were used. The effect of different ligand densities on immunoaffinity properties like dissociation constant and maximal binding capacity was evaluated with adsorption isotherm. Higher ligand density resulted in higher binding capacity and higher dissociation constant. The effect of flow rate was determined through measurement of recovery and it was found in all cases to be negligible. Finally, the effect of the temperature was investigated. Interestingly, it was found for two tested systems that the capacity increased in the range between 0 C and 20 C. A similar trend was also found for the capacity of proteins adsorbed on CIM anion-exchange monolithic

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column [142]. At an even higher temperature of 40 C, a substantial decrease in capacity was observed. Immunoglobulins represent one of the main purification targets in the pharmaceutical field. Therefore, it is not surprising that there are several reports also about immobilisation of the high molecular ligands for their isolation. In 1998 the immobilisation of Protein A and Protein G on epoxy methacrylate monolith was performed [90,137]. Affinity columns were used for the separation of monoclonal antibodies from mouse ascites and human plasma. Interestingly, in both the cases affinity monoliths were combined with weak anion-exchange monoliths to perform purification of IgG and separation of sample proteins in one step – the so called Conjoined Liquid Chromatography (CLC) approach. In the year 2000, a comparative study of the immobilisation of Protein A, Protein G and Protein L for isolation of immunoglobulins was performed [143]. All ligands were immobilised on CIM epoxy monolithic columns. High immobilised amount was achieved with Protein G, followed by Protein A and finally by Protein L. The capacity for IgG followed same order while the ligand utilisation depended on the immunoglobulin type. The isolation was, in all cases, completed within 1 min. Another study involved immobilisation of three different recombinant forms of Protein G, namely monofunctional IgG-binding, monofunctional SA-binding and bifunctional IgG/SA-binding Protein G [144]. They were compared with respect to their specific affinity to blood immunoglobulin G (IgG) and serum albumin (SA). One order of magnitude higher adsorption capacities for IgG in comparison to SA was found, both for monofunctional and bifunctional Protein G forms. However, the measured dissociation constants of affinity complexes seemed to be very close. The methods developed were also scaled-up using 8-ml CIM tube monolithic columns and applied for IgG isolation from crude biological sample. High purity IgG was obtained in extremely short time. Affinity columns described so far were applicable for the purification of a wide range of immunoglobulin classes. On the other hand, when a target antibody is to be isolated, a specific antigen can be used. Ostryanina et al. have described an elegant method for the simultaneous binding of several different antibodies during a single loading step [145]. The method is based on the immobilisation of different ligands, namely bradykinin, bovine serum albumin (BSA), succinylated bovine serum albumin (BSA-S) and a conjugate of bradykinin with bovine serum albumin (BSA-S-BK). Each ligand was immobilised on a separate CIM epoxy disk. As a sample, the pool of polyclonal antibodies obtained by immunisation of rabbits with the covalent conjugate (BK-BSA-S) was used. Such a pool contained both monospecific antibodies against each part of the conjugate used for immunisation and some so-called ‘‘crossreactive’’ antibodies that have epitopes for complementary binding to all parts of the complex antigen. All the four disks with immobilised ligands were placed into a single housing and a sample was loaded on the column. For elution, three CIM disks were taken out of the housing and elution was performed from each single CIM disk separately. In this way a fractionation of the serum was obtained. By changing the order of

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Fig. 28. 2D gel separation of plasma proteins. A represents original plasma sample, B plasma sample with removed 6 main proteins and C the same plasma 2-fold concentrated. First dimension is pH 3–10 non-linear; second dimension 14% SDS-PAGE. (Reprinted from [146].)

the disks it was possible to separate quantitatively all types of antibodies. A similar approach was recently reported by Johnston et al. [146]. They used CIM column containing four disks, each having immobilised specific antibody against the selected plasma protein (fibrinogen, orosomucolid, a1-antitypsin and serotransferrin) for removal of those proteins for plasma with the already removed IgG and HSA before performing 2D electrophoresis. In this way a better detection of some otherwise ‘‘hidden’’ proteins present in traces in plasma, was possible (Fig. 28). Bioconversion Immobilised enzymes can be commonly used for two purposes: as bioreactors to obtain the product from the loaded substrate via an enzymatic reaction or as biosensors when a particular substrate is to be detected. In addition, due to the absence of diffusional limitation in the methacrylate monoliths, such systems can be used for investigation of real enzyme properties. In this section different applications of enzyme immobilisation on methacrylate monoliths are be presented. The first usage of methacrylate monoliths for bioconversion was performed in 1991 [147]. By immobilising carbonic anhydrase to epoxy groups the authors were able to investigate kinetic properties of the system. Surprisingly it was found that a higher flow rate resulted in higher enzymatic activity. This was possible because of negligible diffusional resistance present in such type of monoliths. Josic´ et al. reported about the immobilisation of invertase and trypsin on epoxy groups [137]. Both the enzymes were used as bioreactors. The efficiency of immobilised invertase was studied through the conversion of sucrose to glucose.

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A complete conversion was achieved with 5% sucrose solution regardless of the applied flow rate. Immobilised trypsin was tested with the digest of transferrin, ovalbumine and bovine serum albumin. The results were highly reproducible, indicating the possibility of applying such a unit for peptide mapping. A very interesting approach was reported by Berruex and Freitag [148]. They combined CIM disk with immobilised trypsin and CIM ion exchange disk constructing in this way a chromatographic bioreactor enabling continuous bioconversion (cleavage of HSA into peptides) and separation of formed peptides (Fig. 29). A detailed review about other chromatographic bioreactors can be found elsewhere [149]. Platonova et al. used CIM epoxy monoliths for immobilisation of polynucleotide phosphorylase from Thermus thermophilus and used it as a flow-through reactor [150]. They investigated its ability to synthesise polyriboadenylate from ADP and to carry out its reverse phosphorolysis. It was found that immobilisation reinforced differences in the specificity of the enzyme interaction with high- and low-molecular mass substrates. In contrast to other bioreactors using polynucleotide phosphorylase, no decrease either in synthase or in phoshorylase activity was observed during the continuous work of six months. Authors suggested that such units can be used in preparative-scale production of polyribonucleotides and nucleoside diphosphates. Vodopivec et al. performed an extensive study of various immobilised enzymes [137,151–153]. Glucose oxidase (GOX) was immobilised on different types of CIM columns bearing epoxy, aldehyde or amino groups. Several immobilisation

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methods were tested in order to optimise biologic activity. An immobilised enzyme was found to be stable over months and after several hundreds of injections [151]. CIM epoxy disks with the immobilised glucose oxidase were integrated as an enzyme reactor in a flow injection analysis (FIA) system and applied to on-line monitoring of glucose during cultivation of Saccharomyces cerevisiae and Aspergillus niger (Fig. 30). The developed CIM GOX disk–FIA system exhibited good signal reproducibility and satisfactory long-term stability with a linear response in the range of 10–200 mg/l [152]. The characterisation of the immobilised enzymes to CIM monolithic supports was extended to the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase, and lactate dehydrogenase [153]. The long-term stability, reproducibility, and linear response range of the immobilised enzyme reactors were investigated along with the determination of the kinetic behaviour of the enzymes immobilised on the CIM monoliths. The Michaelis–Menten constant Km and the turnover number K3 of the immobilised enzymes were found to be flow-unaffected. Furthermore, the Km values of the soluble and immobilised enzymes were found to be comparable, indicating negligible diffusional resistance. Another enzyme reactor (microreactor) was developed by immobilising a human recombinant acetylcholinesterase (hrAChE) on CIM EDA monolithic

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column previously activated with glutaraldehyde [154]. Although the enzyme retained only 3.0% of the initial activity, it was stable for over 60 days whereas the free enzyme lost over 80% of initial activity within one day. The effect of AChE inhibitors was evaluated by the simultaneous injection of each inhibitor with the substrate and the results were found to be in agreement with those derived by the conventional kinetic spectrophotometric method. In comparison with the previously developed AChE-based immobilised enzyme reactors, AChE monolithic microreactor showed advantages in terms of reduction of analysis time (2 min), lower aspecific matrix interactions and lower backpressure. An interesting enzyme system was immobilised by Podgornik et al. [155]. Two different isoforms of lignin peroxidase (LiP), namely LiP H2 and LiP H8 to CIM monoliths were immobilized on CIM epoxy monoliths. The characteristics of immobilised LiP were compared and the factors that influence their biologic activities were evaluated using flow-through experiments. Enzyme kinetics was determined via oxidation of veratryl alcohol into veratraldehyde (Vald). While VA oxidation rate increased with an increasing flow rate (upto 1.5 ml/min) for LiP H2, it was almost constant in a wide flow-rate range for LiP H8. This observation together with the stepwise deactivation of the enzyme by consecutive experiments was ascribed to the accumulation of the formed Vald inside the support. Calculated kinetic parameters showed 3–5 times higher Km value for VA for both tested isoforms in comparison with the free enzyme. Immobilised LiP H8 was used as a bioreactor for decolourisation of azo dye Mahogany. Bencˇina et al. introduced and tested a new immobilisation chemistry on CIM monolithic supports based on matrix activation with 1,10 -carbonyldiimidazole resulting in imidazole carbamate functionalities (commonly described in the literature as CDI activation) [156]. The new chemistry was compared with the immobilisation on epoxy groups by immobilising protein A, deoxyribonuclease I and trypsin, enzymes having large molecule substrates. Higher biologic activity was obtained with a new chemistry and shorter immobilisation time was required. In both the cases immobilised enzymes turned out to be stable over months. A detailed investigation of immobilised deoxyribonuclease I properties and usage as a bioreactor was also reported [157]. Columns with various levels of DNase activity were prepared varying immobilisation temperature, pH, time and characterised. The CIM DNase bioreactor was used for the elimination of DNA contaminants in RNA samples prior to reverse transcription followed by PCR. Further details about bioconversion on CIM and other monoliths can be found elsewhere [158]. Solid phase peptide synthesis So far, all the described applications were related to the separation or conversion of the sample. Due to the extreme chemical stability of CIM monolithic column, such a matrix can be an interesting support for solid state synthesis of various molecules. When specific affinity ligands are synthesised, usage of methacrylate

324 monoliths would be advantageous since there would be no need for cleavage of the ligand from the matrix and subsequent immobilisation to a new support. This option seems to be very attractive, since it was shown that ligands immobilised on the CIM methacrylate monoliths are well exposed and therefore the accessibility is very good as investigated in detail by Hanh et al. [130]. Because of the abovementioned advantages, it is not surprising that there are several articles dealing with solid state synthesis on methacrylate monoliths. The first publication appeared in 2000 by a Russian group which synthesised peptide bradykinin on the methacrylate beads as well as on the monolith [159]. To enhance the reactivity of the support, styrene was added to the basic chemistry. Protective ligand groups were successfully removed using trifluoromethylsulfonic acid without cleavage of the attached peptide. No unspecific adsorption was observed during the isolation of a target antibody. In 2002 two publications of the solid phase peptide synthesis on the CIM monoliths were published [160,161]. Peptide against human blood coagulation factor VIII was synthesised using Fmoc chemistry [160]. Original epoxy groups were converted into amino groups using ammonia or ethylenediamine. A similar density of the ligand was achieved as with immobilisation of the same ligand on epoxy groups. Successful purification of the Factor VIII was performed and no unspecific adsorption was observed with the ammonia functionalised support. In the second paper, CIM monoliths were used as a matrix for synthesis of the peptide library [161]. Small CIM disks were inserted in a 96-well plate and screening of the synthesised peptides was performed in a flow-through mode. Thus, besides the affinity of the synthesised ligands, their binding kinetics could also be investigated (Fig. 31). This information is very important to evaluate the applicability of the prepared peptide as an affinity ligand used in chromatography. Due to a flexibility of the developed manifold, the optimisation of purification conditions by changing running buffers, washing and elution conditions could be also performed. Affinity binding parameters of solid phase synthesised peptides were investigated by Vlakh et al. [162]. Several peptidyl groups complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesised using Fmoc-chemistry. The results have been compared with those established for CIM affinity sorbents obtained by the immobilisation of the same, but preliminarily synthesised on convenient resin, cleaved and purified ligands on the disks using one-step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude CHO cellular supernatant. In the second review dealing with the same system, emphasis was given on the preparation of the matrix before peptide synthesis [163]. Hydroxyl and amino groups were introduced on the matrix and two methods for introduction of b-alanine spacer were investigated as a starting point for F-moc chemistry. They demonstrated that the ligand density can be controlled carefully.

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Fig. 31. Mutational analysis of peptide against FVIII. (A) Detection of pdFVIII bound to peptides synthesised on a cellulose membrane. (B) Detection of pdFVIII bound to peptides synthesised on CIM minidisks in a flow through mode. In both cases bound FVIII was transferred to a nitrocellulose membrane before detection with MAb 038 directed against the light chain of FVIII and anti mouse peroxidase conjugate. The flow through of the minidisks was analyzed by a dot blot (C) with MAb 038 and anti mouse peroxidase conjugate and by VIII:CAg ELISA (D). (Reprinted from [161].)

Other details about solid-phase synthesis and combinatorial chemistry can be found elsewhere [164]. Conclusions Since their introduction in 1990, short-bed methacrylate monoliths have been successfully applied in many separation and purification methods. Recent developments proved that scale-up of such monoliths is feasible. Furthermore, new, non-invasive methods for the characterisation of each single monolith proved that full control over the production process and properties over time can be obtained, thus fulfilling all the demands related to cGMP. Because of their advantageous properties over the conventional chromatographic supports, especially the high flow-unaffected binding capacity for extremely large molecules, they were already implemented in the industrial process e.g., plasmid DNA purification. It can be anticipated that they will soon find more uses in processes like purification of antibodies and viruses. References 1. Peterson EA and Sober HA. Chromatography of Proteins. I. Cellulose Ion-exchange Adsorbers. J Am Chem Soc 1956;78:751. 2. Porath J and Flodin P. Gel filtration: a method for desalting and group separation. Nature 1959;183:1657.

326 3. Porath J, Janson JC and Laas T. Agar derivatives for chromatography, electrophoresis and gel-bound enzymes: I. Desulphated and reduced cross-linked agar and agarose in spherical bead form. J Chromatogr 1971;60:179–184. 4. Kato Y, Nakamura K and Hashimoto T. Characterization of TSK-GEL DEAE-Toyopearl 650 Ion Exchanger. J Chromatogr 1982;245:193–211. 5. Chang SH, Gooding KM and Regnier FE. Use of oxiranes in the preparation of bonded phase suports. J Chromatogr 1976;120:321–333. 6. Lee W-C. Protein separation using non-porous sorbents. J Chromatogr B 1997;699:29–45. 7. Roudruges AE. Permeable packings and perfusion chromatography in protein separation. J Chromatogr B 1997;699:47–61. 8. Afeyan NB, Gordon NF, Mazsaroff I, Varady L, Fulton SP, Yang JB and Regnier FE. Flowthrough particles for the high-performance liquid chromatographic separation of biomolecules:perfusion chromatography. J Chromatogr 1990;519:1–29. 9. Horvat J, Boschetti E, Guerrier L and Cooke N. High-performance protein separations with novel strong ion exchangers. J Chromatogr A 1994;679:11–22. 10. Rodrigues AE, Lopes JC, Lu ZP, Loureiro JM and Dias MM. Importance of intraparticle convection in the performance of chromatographic processes. J Chromatogr 1992;590:93–100. 11. Roper DK and Lightfoot EN. Estimating plate heights in stacked-membrane chromatography by flow reversal. J Chromatogr A 1995;702:69–80. 12. Shiosaki A, Goto M and Hirose T. Frontal analysis of protein adsorption on a membrane adsorber. J Chromatogr A 1994;679:1–9. 13. Iberer G, Hahn R and Jungbauer A. Monoliths as stationary phases for separation of biopolymers: the fourth generation of chromatography sorbents. LC-GC 1999;17:998–1005. 14. Kubin M, Sˇpacˇek P and Chromecˇek R. Gel Permeation Chromatography on Porous Poly (Ethylene Glycol Methacrylate). Coll Czechosl Chem Commun 1967;32:3881–3887. 15. Ross WD and Jefferson RT. J Chrom Sci 1970;8:386. 16. Hjerten S, Liao J-L and Zhang R. High-performance liquid chromatography on continuous polymer beds. J Chromatogr 1989;473:273–275. 17. Tennikova TB, Belenkii BG and Sˇvec F. High-performance membrane chromatography. A novel method of protein separation. J Liq Chromatogr 1990;13:63–70. 18. Nakanishi K and Soga N. Phase separation in gelling silica-organic polymer solution: systems containing poly(sodium styrenesulfonate). J Am Ceram Soc 1991;74:2518–2530. 19. Minakuchi H, Nakanishi K, Soga N, Ishizuka N and Tanaka N. Octadecylsilylated porous silica rods as separation media for reversed-phase liquid chromatography. Anal Chem 1996; 68:3498–3501. 20. Fields SM. Silica xerogel as a continuous column support for high-performance liquid chromatography. Anal Chem 1996;68:2709–2712. 21. Mayr B, Tessadri R, Post E and Buchmeiser MR. Metathesis-based monolith: influence of polymerization conditions on the separation of biomolecules. Anal Chem 2001;73:4071–4078. 22. Chirica GS and Remcho VT. Novel monolithic columns with templated porosity. J Chromatogr A 2001;924:223–232. 23. Hahn R, Podgornik A, Merhar M, Schallaun E and Jungbauer A. Affinity monoliths generated by in situ polymerization of the ligand. Anal Chem 2001;73:5126–5132. 24. Martin del Valle E, Galan MA and Serrano Cerro RL. Use of ceramic monoliths as stationary phase in affinity chromatography. Biotechnol Prog 2003;19:921–927. 25. Sun X and Chai Z. Urea–formaldehyde resin monolith as a new packing material for affinity chromatography. J Chromatogr A 2002;943:209–218. 26. Liang C, Dai S and Guiochon G. A graphitized-carbon monolithic column. Anal Chem 2003;75:4904–4912. 27. Noel R, Sanderson A and Spark L. A monolithic ion-exhange material suitable for downstream processing of bioproducts. In: Cellulosics: Materials for Selective Separations and Other Technologies. Kennedy JF, Philips GO and Williams PA (eds), New York, E. Horwood, 1993, pp. 17–24.

327 28. Mercier A, Deleuze H and Mondain-Monval O. Preparation and functionalization of (vinyl)polystyrene polyHIPEÕ : Short routes to binding functional groups through a dimethylene spacer. React Funct Polymers 2000;46:67–79. 29. Krajnc P, Leber N, Sˇtefanec D, Kontrec S, Podgornik A. Preparation and characterisation of PolyHIPE methacrylate monoliths and their application as separation media. J Chromatogr A 2005;1065:69–73. 30. Gustavsson P-E and Larsson P-O. Continuous superporous agarose beds for chromatography and electrophoresis. J Chromatogr A 1999;832:29–39. 31. Arvidsson P, Plieva FM, Savina IN, Lozinsky VI, Fexby S, Bulow L, Galaev I and Mattiasson B. Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns. J Chromatogr A 2002;977:27–38. 32. Sˇvec F, Tennikova TB and Deyl Z. In: Monolithic material: Perparation Properties and Applications. Amsterdam, Elsevier, 2003, pp. 1–773. 33. Tennikova TB and Sˇvec F. High-performance membrane chromatography: highly efficient separation method for proteins in ion-exchange hydrophobic interaction and reversed-phase modes. J Chromatogr 1993;646:279–288. 34. Tanaka N, Motokawa M, Kobayashi H, Hosoya K and Ikegami T. Monolithic silica columns for capillary liquid chromatography. In: Monolithic Materials: Preparation Properties, and Applications. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 173–196. 35. Sˇvec F and Frechet JMJ. Molded rigid monolithic porous polymers: an inexpensive efficient, and versatile alternative to beads for the design of materials for numerous applications. Ind Eng Chem Res 1999;38:34–38. 36. Endres HN, Johnson JAC, Ross CA, Welp JK and Etzel MR. Evaluation of an ionexchange membrane for purification of plasmid DNA. Biotechnol Appl Biochem 2003;37:259–266. 37. Cabrera K, Lubda D, Eggenweiler H-M, Minakuchi H and Nakanishi K. A new monolithictype HPLC column for fast separaton. J High Resol Chromatogr 2000;23:93–99. 38. Zo¨chling A, Hahn R, Ahrer K, Urthaler J and Jungbauer A. Mass transfer characteristics of plasmids in monoliths. J Separation Sci 2004;27:819–827. 39. Mihelicˇ I, Koloini T, Podgornik A and Sˇtrancar A. Dynamic capacity studies of CIM (Convective Interaction Media)Õ monolithic columns. J High Resol Chromatogr 2000;23:39–43. 40. Yamamoto S. Molecular Recognition and Transporth Phenomena in Chromatography ol Large Biomolecules. Recovery of Biological Products IX, Banff, 14–19.9.2003, 91. 41. Sˇvec F and Tennikova TB. Historical Review. In: Monolithic Materials: Preparation Properties, and Applications. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 1–15. 42. Hahn R, Panzer M, Hansen E, Mollerup J and Jungbauer A. Mass transfer properties of monoliths. Separation Sci Technol 2002;37:1545–1565. 43. Sˇvec F and Fre´chet JMJ. Kinetic control of pore formation in macroporous polymers. The formation of ‘‘molded’’ porous materials with high flow characteristics for separations or catalysis. Chem Mater 1995;7:707–715. 44. Merhar M, Podgornik A, Barut M, Zˇigon M and Sˇtrancar A. Methacrylate monoliths prepared from various hydrophobic and hydrophilic monomers – structural and chromatographic characteristics. J Sep Sci 2003;26:322–330. 45. Okay O. Macroporous copolymer networks. Prog Polym Sci 2000;25:711–779. 46. Sˇtrancar A, Podgornik A, Barut M and Necina R. Short monolithic columns as stationary phases for biochromatography. In: Advances in Biochemical Engineering/Biotechnology; Modern Advances Chromatography. Vol. 76. Freitag R (ed), Heidelberg, Springer-Verlag, 2002, pp. 49–85. 47. Hahn R and Jungbauer A. Control method for integrity of continuous beds. J Chromatogr B 1995;908:181–186.

328 48. Kaltenbrunner O, Jungbauer A and Yamamoto S. Prediction of the preparative chromatography performance with a very small column. J Chromatogr A 1997;760:41–53. 49. Podgornik A, Barut M, Jaksˇ a S, Jancˇar J and Sˇtrancar A. Application of very short monolithic columns for separation of low and high molecular mass substances. J Liq Chrom & Rel Technol 2002;25:3099–3116. 50. Mihelicˇ I, PhD Thesis, University of Ljubljana, Ljubljana, 2002, 162. 51. Meyers JJ and Liapis AI. Network modeling of the intraparticle convection and diffusion of molecules in porous particles packed in a chromatographic column. J Chromatogr A 1998;827:197–213. 52. Meyers JJ and Liapis AI. Network modeling of the convective flow and diffusion of molecules adsorbing in monoliths and in porous particles packed in a chromatographic column. J Chromatogr A 1999;852:3–23. 53. Liapis AI, Meyers JJ and Crosser OK. Modeling and simulation of the dynamic behavior of monoliths. Effects of pore structure from pore network model analysis and comparison with columns packed with porous spherical particles. J Chromatogr A 1999;865:13–25. 54. Tallarek U, Leinweber FC and Seidel-Morgenstern A. Fluid dynamics in monolithic adsorbents: phenomenological approach to equivalent particle dimensions. Chem Eng Technol 2002;25:1177–1181. 55. Leinweber FC and Tallarek U. Chromatographic performance of monolithic and particulate stationary phases: hydrodynamics and adsorption capacity. J Chromatogr A 2003; 1006:207–228. 56. Vervoort N, Gzil P, Baron G and Desmet G. A novel correlation for the pressure drop in monolithic silica columns. Anal Chem 2003;75:843–850. 57. Vervoort N, Gzil P, Baron G and Desmet G. Model column structure for the analysis of the flow and band-broadening characteristics of silica monoliths. J Chromatogr A 2004;1030:177–186. 58. Hahn R and Jungbauer A. Peak broadening in protein chromatography with monoliths as very fast separations. Anal Chem 2000;72:4853–4858. 59. Mihelicˇ I, Nemec D, Podgornik A and Koloini T. Pressure drop in CIM disk monolithic columns. J Chromatogr A 2005;1065:59–67. 60. Happel J. Viscous flow in multiparticle systems: slow motion of fluids relative to beds of spherical particles. AIChE J 1958;4:197. 61. Tennikov MB, Gazdina NV, Tennikova TB and Sˇvec F. Effect of porous structure of macroporous polymer supports on resolution in high-performance membrane chromatography of proteins. J Chromatogr 1998;798:55–64. 62. www.biaseparations.com. 63. Moore RRM and Walters RR. Protein separations on reversed-phase high-performance liquid chromatography minicolumns. J Chromatogr 1984;317:119–128. 64. Snyder LR and Stadalius MA. In: High-Performance Liquid Chromatography Advances and Perspectives, Vol. 4. Horvath Cs (ed), Orlando, Academic Press, 1986, p. 195. 65. Snyder LR, Stadalius MA and Quarry MA. Gradient elution in reversed-phase HPLC separation of macromolecules. Anal Chem 1983;55:1412. 66. Sˇtrancar A, Koselj P, Schwinn H and Josic´ Dj. Application of compact porous disks for fast separations of biopolymers and in-process control in biotechnology. Anal Chem 1996;68:3483–3488. 67. Tennikova TB and Sˇvec F. Theoretical aspects of separation using short monolithic beds. In: Monolithic Materials: Preparation Properties, and Applications. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 351–372. 68. Barut M, Podgornik A, Merhar M and Sˇtrancar A. In: Monolithic Materials: Preparation Properties, and Applications F, Vol. 10. Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 51–76. 69. Podgornik A, Barut M and Sˇtrancar A. In: Encyclopedia of Chromatography. Cazes J (ed), New York, M Dekker, 2003, pp. 1–7.

329 70. Tennikova TB and Freitag R. An introduction to monolithic disks as stationary phases for high performance biochromatography (review). J High Resol Chromatogr 2000;23:27–38. 71. Tennikova TB and Freitag R. High-performance membrance chromatography of proteins, in analytical and preparative separation of biomolecules. In: Analytical and Preparative Separation Methods of Biomolecules,. Vol. 10. Aboul-Ehnen HY (ed), New York, Marcel Dekker, 1999, pp. 255–300. 72. Josic´ Dj and Sˇtrancar A. Application of membranes and porous units for the separation of biopolymers. Ind Eng Chem Res 1999;38:333–342. 73. Mihelicˇ I, Krajnc M, Koloini T and Podgornik A. Kinetic model of a methacrylate-based monolith polymerization. Ind eng chem res 2001;40:3495–3501. 74. Peters EC, Sˇvec F and Fre´chet JMJ. The preparation of large diameter ‘‘molded’’ porous polymer monoliths and the control of pore structure homogeneity. Chem Mater 1997; 9:1898–1902. 75. Mihelicˇ I, Koloini T and Podgornik A. Temperature distribution effects during polymerization of methacrylate-based monoliths. J Appl Polym Sci 2003;87:2326–2334. 76. Podgornik A, Barut M, Mihelicˇ I and Sˇtrancar A. In: Monolithic Materials: Preparation Properties, and Applications, Vol. 10. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 77–102. 77. Podgornik A, Barut M, Sˇtrancar A, Josic´ Dj and Koloini T. Construction of large-volume monolithic columns. Anal Chem 2000;72:5693–5699. 78. Podgornik A, Barut M, Sˇtrancar A and Josic´ Dj. Chromatographic device. US 6,736,973; 18.05.2004. 79. Podgornik A, Jancˇar J, Merhar M, Kozamernik S, Glover D, Cˇucˇek K, Barut M and Sˇtrancar A. Large scale methacrylate monolithic columns: design and properties. J Biochem Biophys Methods 2004;60:179–189. 80. Bentrop D and Engelhardt H. Chromatographic characterization of ion exchangers for highperformance liquid chromatography of proteins : I. chromatographic determination of loading capacity for low- and high-molecular mass anions. J Chromatogr 1991;556:363–372. 81. Helfferich, FG. Ion Exchange, New York, McGraw-Hill, 1962. 82. Mihelicˇ I, Podgornik A and Koloini T. Temperature influence on the dynamic binding capacity of a monolithic ion-exchange column. J Chromatogr A 2003;987:159–168. 83. Lendero N, Brne P, Vidicˇ J, Podgornik A and Sˇtrancar, A. A fast, simple and non-destructive method for determining the amount of ion exchange groups on resins. J Chromatogr A 2005;1065:29–38. 84. Kang X and Frey DD. Chromatofocusing using micropellicular column packings with computer-aided design of the elution buffer composition. Anal Chem 2002;74:1038–1045. 85. Jungbauer A and Hahn R. Large-scale separations. In: Monolithic Materials: Preparation Properties, and Applications. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 561–599. 86. Yamamoto S. Plate height determination for gradient elution chromatography of proteins. Biotechnol Bioeng 1995;48:444–451. 87. Yamamoto S, Nomura M and Sano Y. Resolution of proteins in linear gradient elution ionexchange and hydrophobic interaction chromatography. J Chromatogr 1987;409:101–110. 88. Dubinina NI, Kurenbin OI and Tennikova TB. Peculiarities of gradient ion-exchange highperformance liquid chromatography of proteins. J Chromatogr 1996;753:217–225. 89. Milavec Zˇmak P, Podgornik H, Jancˇar J, Podgornik A and Sˇtrancar A. Transfer of gradient chromatographic methods on cim monolithic columns. J Chromatogr A 2003;1006:195–205. 90. Sˇtrancar A, Barut M, Podgornik A, Koselj P, Josic´ Dj and Buchacher A. Convective Interaction Media: polymer-based supports for fast separation of biomolecules. LC-GC 1998;11:660–669. 91. Vodopivec M, Podgornik A, Berovicˇ M and Sˇtrancar A. Application of convective interaction media (CIMÕ ) disk monolithic columns for fast separation and monitoring of organic acids. JCS 2000;38:489–495.

330 92. Podgornik H, Stegu M, Podgornik A and Perdih A. Isolation and characterisation of Mn(III) tartrate from phanerochaete chrysosporium culture broth. FEMS Microb Lett 2001;10029:1–5. 93. Lethanh H and Lendl B. Sequential injection Fourier transform infrared spectroscopy for the simultaneous determination of organic acids and sugars in soft drinks employing automated solid phase extraction. Anal Chim Acta 2000;422:63–69. 94. Svete P, Milacˇicˇ R, Mitrovicˇ B and Pihlar B. Potential for the speciation of Zn using fast protein liquid chromatography (FPLC) and convective interaction media (CIMÕ ) fast monolithic chromatography with FAAS and elctrospray (ES)-MS-MS detection. Analyst 2001;126:1346–1354. 95. Sˇcˇancˇar J and Milacˇicˇ R. A novel approach for speciation of airborne chromium by convective-interaction media fast-monolithic chromatography with electrothermal atomicabsorption spectrometric detection. Analyst 2002;127:629–633. 96. Nesterenko PN and Rybalko MA. The use of a continuous flow gradient for the separation of inorganic anions on a monolithic disk. 2240 FM Mendeleev Commun 2004;17:1–2. 97. Vlakh EG, Platonova GA, Vlasov GP, Kasper C, Tappe A, Kretzmer G and Tennikova TB. In vitro comparison of complementary interactions between synthetic linear/branched oligo/ poly-L-lysines and tissue plasminogen activator by means of high-performance monolithicdisk affinity chromatography. J Chromatogr A 2003;992:109–119. 98. Ren D, Penner NA, Slentz BE, Inerowicz HD, Rybalko M and Regnier FE. Contributions of commercial sorbents to the selectivity in immobilized metal affinity chromatography with Cu(II). J Chromatogr A 2004;1031:87–92. 99. Josic´ Dj, Reusch J, Lo¨ster K, Baum O and Reutter W. High-performance membrane chromatography of serum and plasma membrane proteins. J Chromatogr A 1992;590:59–76. 100. Josic´ Dj, Lim Y-P, Sˇtrancar A and Reutter W. Application of high-performance membrane chromatography for separation of annexins from the plasma membranes of liver and isolation of monospecific polyclonal antibodies. J Chromatogr B 1994;662:217–226. 101. Sˇtrancar A, Barut M, Podgornik A, Koselj P, Schwinn H, Raspor P and Josic´ Dj. Application of compact porous tubes for preparative isolation of clotting factor VIII from human plasma. J Chromatogr A 1997;760:117–123. 102. Branovic´ K, Buchacher A, Barut M, Sˇtrancar A and Josic´ Dj. Application of monoliths for downstream processing of clotting factor IX. J Chromatogr A 2000;903:21–32. 103. Branovic´ K, Buchacher A, Barut M, Sˇtrancar A and Josic´ Dj. Application of semi-industrial monolithic columns for downstream processing of clotting factor IX. J Chromatogr B 2003;790:175–182. 104. Podgornik H, Podgornik A and Perdih A. A method of fast separation of lignin peroxidases using convective interaction media disks. Anal Biochem 1999;272:43–47. 105. Barut M, Podgornik A, Podgornik H, Sˇtrancar A, Josic´ Dj and Mac Farlane J. Fast separation of biomolecules using polymer-based monolithic supports. Am Biotechnol Lab 1999;17:48–51. 106. Podgornik H and Podgornik A. Separation of manganese peroxidase isoenzymes on strong anion-exchange monolithic column using pH–salt gradient. J Chromatogr B 2004;799:343–347. 107. Podgornik H, Podgornik A, Milavec P and Perdih A. The Effect Of agitation and nitrogen concentration on lignin peroxidase(LiP) isoform composition during fermentation of Phanerochaete chrysosporium. J biotechnol 2001;88:173–176. 108. Cepeljnik T, Zorec M, Viktor-Nekrep F and Marinsˇ ek-Logar R. Isolation of Endoxylanases from Anaerobic Bacterium butyrivibrio SP. Strain MZ5 is possible by anion exchange chromatography on CIMÕ DEAE-8 monolithic column. Acta Chim Slov 2002; 49:401–408. 109. Isobe K and Wakao N. Thermostable NAD+-dependent Õ -specific secondary alcohol dehydrogenase from cholesterol-utilizing Burkholderia sp. AIU 652. J Biosci and Bioeng 2003;96:387–393.

331 110. 111.

112. 113.

114.

115. 116. 117.

118. 119.

120. 121.

122. 123.

124.

125. 126.

127. 128.

129.

130.

Luksˇ a J, Menart V, Milicˇicˇ S, Kus B, Gaberc-Porekar V and Josic´ Dj. Purification of human necrosis factor by membrane chromatography. J Chromatogr A 1994;661:161–168. Bavec A, Podgornik A and Zorko M. Purification of GTPgammaS binding proteins from membranes of porcine brain using convective interaction media (CIM) supports. Acta Chim Slov 2000;47:371–379. Hall T, Wood D and Smith C. Preparative and analytical chromatography of pegylated myelopoietin using monolithic media. J Chromatogr A 2004;1041:87–93. Branovic´ K, Lattner G, Barut M, Sˇtrancar A, Josic´ Dj and Buchacher A. Very fast analysis of impurities in immunoglobulin concentrates using conjoint liquid chromatography on short monolithic disks. J Immunol Methods 2002;271:47–58. Josic´ Dj. Separation of petides and peptides. In: Monolithic Materials: Preparation Properties, and Applications. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 389–415. Podgornik A, Barut M, Jancˇar J and Sˇtrancar A. Isocratic separations on thin glycidyl methacrylate-ethylenedimethacrylate monoliths. J Chromatogr A 1999;848:51–60. Podgornik A, Barut M, Jancˇar J, Sˇtrancar A and Tennikova T. High-performance membrane chromatography of small molecules. Anal Chem 1999;71:2986–2991. Merhar M, Podgornik A, Barut M, Jaksˇ a S, Zˇigon M and Sˇtrancar A. High performance reversed-phase liquid chromatography using novel (CIMÕ ) RP-SDVB monolithic supports. J Liq Chrom & Rel Technol 2001;24:2429–2443. Giovannini R, Freitag R and Tennikova TB. High performance membrane chromatography of plasmid DNA. Anal Chem 1998;70:3348–3355. Branovic´ K, Forcˇic´ D, Ivancˇic´ J, Sˇtrancar A, Barut M, Kosutic´-Gulija T, Zgorelec R and Mazuran R. Application of short monolithic columns for fast purification of plasmid DNA. J Chromatogr B 2004;801:331–337. Bencˇina M, Podgornik A and Sˇtrancar A. Characterization of methacrylate monoliths for purification of DNA molecules. J Sep Sci 2004;27:801–810. Jerman S, Podgornik A, Cankar K, Cˇadezˇ N, Skrt M, Zˇel J and Raspor P. Detection of processed genetically modified food using CIMÕ monolithic columns for DNA isolation. J Chromatogr A 2005;1065:107–113. Sˇtrancar A. Monolithic supports for chromatography. Genet Eng News 2003;19:50–51. Huber CG and Oberacher H. Nucleic acid analysis. In: Monolithic Materials: Preparation Properties, and Applications. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 417–456. Branovic´ K, Forcˇic´ D, Ivancˇic´ J, Sˇtrancar A, Barut M, Kosutic´-Gulija T, Zgorelec R and Mazuran R. Application of short monolithic columns for improved detection of viruses. J Virol Methods 2003;110:163–171. Kramberger P, Petrovicˇ N, Sˇtrancar A and Ravnikar M. Concentration of plant viruses using monolithic chromatographic supports. J Virol Methods 2004;120:51–57. Josic´ Dj, Buchacher A and Jungbauer A. Monoliths as stationary phases for separation of proteins and polynucleotides and enzymatic conversion. J Chromatogr B 2001;752:191–205. Josic´ Dj and Buchacher A. Application of monoliths as supports for affinity chromatography and fast enzymatic conversion. J Biochem Biophys Methods 2001;49:153–174. Platonova GA and Tennikova TB. Immunoaffinity assays. In: Monolithic Materials: Preparation Properties, and Applications. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 601–622. Platonova GA, Pankova GA, Il’lina IYe, Vlasov GP and Tennikova TB. Quantitative fast fractionation of a pool of polyclonal antibodies by immunoaffinity membrane chromatography. J Chromatogr A 1999;852:129–140. Hahn R, Amatschek K, Schallaun E, Necina R, Josic´ Dj and Jungbauer A. Performance of affinity chromatography with peptide ligands: influence of spacer matrix composition and immobilization chemistry. IJBC 2000;5:175–185.

332 131.

132. 133. 134.

135.

136. 137.

138.

139. 140.

141.

142. 143.

144.

145.

146.

147.

148.

149.

Amatschek K, Necina R, Hahn R, Schallaun E, Schwinn H, Josic´ Dj and Jungbauer A. Affinity chromatography of human blood coagulation factor VIII on monoliths with peptides from a combinatorial library. J High Resol Chromatogr 2000;23:47–58. Pflegerl K, Hahn R, Berger E and Jungbauer A. Mutational analysis of a blood coagulation factor VIII-binding peptide. J Pept Res 2002;59:174–182. Hahn R, Podgornik A, Merhar M, Schallaun E and Jungbauer A. Affinity monoliths generated by in situ polymerization of the ligand. Anal Chem 2001;73:5126–5132. Hahn R, Pflegerl K, Berger E and Jungbauer A. Directed immobilization of peptide ligands to accessible pore sites by conjugation with a placeholder molecule. Anal Chem 2003; 75:543–548. Schuster M, Wasserbauer E, Neubauer A and Jungbauer A. High speed immuno-affinity chromatography on supports with gigapores and porous glass. Bioseparation 2001; 9:259–268. Josic´ Dj, Bal F and Schwinn H. Isolation of plasma proteins from the clotting cascade by heparin affinity chromatography. J Chromatogr A 1993;632:1–10. Josic´ Dj, Schwinn H, Sˇtrancar A, Podgornik A, Barut M, Lim Y.-P and Vodopivec M. Use of compact, porous units with immobilized ligands with high molecular masses in affinity chromatography and enzymatic conversion of substrates with high and low molecular masses. J Chromatogr A 1998;803:61–71. Kasper C, Meringova L, Freitag R and Tennikova TB. Fast isolation of protein receptors from streptococci g by means of macroporous affinity discs. J Chromatogr A 1998;798:65–73. Kasper C, Hagedorn J, Freitag R and Tennikova TB. High performance flow injection analysis by means of membrane chromatography approach. J Biotechnol 1999;66:3–12. Meringova LF, Leontjeva GF, Gupalova TV, Tennikova TB and Totolian AA. Isolation and investigation of recombinant IgG-binding receptor of group G Streptococci using of macroporous disks. Biotechnol (Rus) 2000;4:45–52. Ostryanina ND, Il’ina OV and Tennikova TB. Effect of experimental conditions on strong biocomplementary pairing in high performance monolithic disk affinity chromatography (HPMDAC). J Chromatogr B 2002;770:35–43. Mihelicˇ I, Podgornik A and Koloini T. Temperature influence on the dynamic binding capacity of a monolithic ion-exchange column. J Chromatogr A 2003;987:159–168. Berruex LG, Freitag R and Tennikova TB. Comparison of antibody binding to immobilized group specific affinity ligands in high performance monolith affinity Chromatography. J Pharm Biomed Anal 2000;24:95–104. Gupalova TV, Lojkina OV, Palagnuk VG, Totolian AA and Tennikova TB. Quantitative investigation of affinity properties of different recombinant forms of Protein G by means of high-performance monolith chromatography. J Chromatogr A 2002;949:185–193. Ostryanina ND, Vlasov GP and Tennikova TB. Multifunctional fractionation of polyclonal antibodies by immunoaffinity high-performance monolithic disk chromatography. J Chromatogr A 2002;949:163–171. Johnston, ML, Banas J, Laiberte, N, Stochaj, WR, Corbo, J, Nagel J, Adamec, J, and Meys, M. Sample Preparation for Proteomic Analysis by Removal of Abundant Proteins from Blood Plasma and Serum. Poster presented at the Annual meeting for the Association for Biological Resource Facilites, Denver, February, 2003. Abou-Rebyeh H, Ko¨rber F, Schubert-Rehberg K, Reusch J and Josic´ Dj. Carrier membrane as a stationary phase for affinity chromatography and kinetic studies of membrane-bound enzymes. J Chromatogr 1991;566:341–350. Berruex LG and Freitag R. Affinity-based interactions on disks for fast analysis, isolation and conversion of biomolecules. In: Methods for affinity-based separations of enzymes and proteins. Gupta MN (ed), Basel, Birkhauser, 2002, pp. 83–113. Podgornik A and Tennikova TB. Advances in biochemical engineering/biotechnology. Chromatographic reactors based on biological activity. In: Modern Advances in Chromatography, Vol. 76. Freitag R (ed), Heidelberg, Springer-Verlag, 2002, pp. 165–210.

333 150.

151.

152.

153. 154.

155. 156. 157.

158.

159.

160.

161. 162.

163. 164.

Platonova GA, Surzhik MA, Tennikova TB, Vlasov GP and Timkovskii AL. The catalysis of polriboadenylate synthesis and phosphoroolysis by polynucleotide phosphorylase immobilized on a new type of carrier. Russ Bioorg Chem (English) 1999;25:166–171. Podgornik A, Vodopivec M, Podgornik H, Barut M and Sˇtrancar A. Immobilisation and characteristics of glucose oxidase immobilised on convective interaction media (CIM) disks. In: Stability and stabilization of biocatalysts: proceedings of and International Symposium, Progress in biotechnology, Vol. 15. Ballesteros A, Plou FJ, Iborra JL and Halling PJ (eds), Amsterdam, Elsevier, 1998, pp. 541–546. Vodopivec M, Berovicˇ M, Jancˇar J, Podgornik A and Sˇtrancar A. Application of convective interaction media disks with immobilised glucose oxidase for on-line glucose measurements. Anal Chim Acta 2000;407:105–110. Vodopivec M, Podgornik A, Berovicˇ M and Sˇtrancar A. Characterization of CIM monoliths as enzyme reactors. J Chromatogr B 2003;795:105–113. Bartolini M, Cavrini V and Andrisano V. Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies. J Chromatogr A 2004;1031:27–34. Podgornik H and Podgornik A. Characteristics of LiP immobilized to CIM monolithic supports. Enz Microb Technol 2002;31:855–861. Bencˇina K, Podgornik A and Sˇtrancar A. Comparison of immobilization on epoxy and 1, 1carbonyldiimidazole activated CIM monoliths. J Sep Sci 2004;27:811–818. Bencˇina M, Bencˇina K, Sˇtrancar A and Podgornik A. Immobilization of deoxyribonuclease via epoxy groups of methacrylate monoliths: use of deoxyribonuclease bioreactor in reverse transcription-PCR. J Chromatogr A 2005;1065:83–91. Jungbauer A and Hahn R. Catalysts and enzyme reactors. In: Monolithic Materials: Preparation Properties, and Applications. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 699–724. Korol’kov VI, Platonova GA, Azanova VV, Tennikova TB and Vlasov GP. In situ preparation of peptidylated polymers as ready-to-use adsorbents for rapid immunoaffinity chromatography. LIPS 2000;5:1–9. Pflegerl K, Podgornik A, Berger E and Jungbauer A. Direct synthesis of peptides on convective interaction media monolithic columns for affinity chromatography. J Comb Chem 2002;4:33–37. Pflegerl K, Podgornik A, Berger E and Jungbauer A. Screening for peptide affinity ligands on CIMÕ monoliths. Biotechnol Bioeng 2002;79:733–740. Vlakh E, Ostryanina N, Jungbauer A and Tennikova TB. Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA). J Biotechnol 2004;107:275–284. Vlakh E, Novikov A, Vlasov G and Tennikova TB. Solid phase peptide synthesis on epoxy– bearing methacrylate monoliths. J Peptide Sci 2004;10:710–730. Jungbauer A and Pflegerl K. Solid phase synthesis and auxiliaries for combinatorial chemistry. In: Monolithic materials: preparation, properties, and applications. Sˇvec F, Tennikova TB and Deyl Z (eds), Amsterdam, Elsevier, 2003, pp. 725–741.