Continuous recovery of bioproducts by adsorption

Continuous recovery of bioproducts

by adsorption

J.P. van der Wiel

r

|TR diss

Academisch Boeken Centrum

CONTINUOUS RECOVERY OF BIOPRODUCTS BY ADSORPTION

CONTINUOUS RECOVERY OF BIOPRODUCTS BY ADSORPTION

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Technische Universiteit Delft, op gezag van de Rector Magnificus, prof. drs. P.A. Schenck, in het openbaar te verdedigen ten overstaan van een commissie aangewezen door het College van Dekanen

op 11 april 1989 te 16.00 h door Johannis Pieter van der Wiel

geboren te Rotterdam, scheikundig ingenieur

Contents Contents I Summary II Samenvatting IV 1 Introduction 1 2 Adsorption of Proteins 8 3 Mass Transfer 34 4 Continuous Adsorption 59

5 Adsorption from Yeast Homogenate 78 6 Hydrodynamics of Fluidized beds 96

Symbols 116

Appendix 1 Adsorption of Antibiotics

Appendix 2 Staining Technique for Measuring Penetration of Protein in Adsorbents

Summary

Adsorption is often used in the recovery of products obtained from biotech-nological processes. This thesis describes the design of a continuous fluidized bed adsorption process for the recovery of proteins and antibiotics.

In the introduction (Chapter 1) the application of (countercurrent) fluidized bed adsorption processes for the recovery of bioproducts is discu­ ssed. For the design of these processes three sets of data are required:

- equilibrium parameters - mass transfer parameters and

- a hydrodynamic model of the equipment.

In Chapter 2 the equilibrium adsorption of proteins on various surfaces is discussed. The adsorption of Bovine Serum Albumin (BSA) on the anion ex­ changer QMA Spherosil has been studied as a function of pH and buffer concentration. It is concluded that electrostatic interactions play a dominant role in the adsorption process. These results are different from results obtained with other surfaces.

Many models are available for describing transport of proteins in adsorbents. A summary is given from which it is concluded that a simple pore diffusion model can be used to describe adsorption and desorption. This model has been applied successfully to describe the adsorption and desorp­ tion of BSA on QMA Spherosil (Chapter 3). A staining technique (Appendix 2) has been used to measure the penetration of protein in an adsorbent particle. This provides extra information about the mechanism of adsorption. Counter current adsorption of proteins has been studied using a multi stage fluidized bed. The hydrodynamic characteristics of a lab-scale contactor are discussed. Together with the equilibrium and mass transfer parameters the steady state performance can be predicted (Chapter 4). The effect of operat­ ing variables has been studied theoretically and experimentally.

It is often assumed that fluidized bed adsorption processes are attractive for treatment of turbid solutions (fermentation broth, cell homogenates).

Therefore continuous adsorption of BSA from a yeast homogenate has been studied also. The multi stage contactor has been operated successfully with cell homogenate. The steady state performance of the contactor can be described approximately with a relatively simple model.

For scale-up of the multi stage contactor some knowledge about the hydrodynamics is required. The expansion properties of a multi stage contac­

tor can be described with excisting correlations for open fluidized beds. The mixing of liquid is not affected by the diameter of the column. For this the compartment height should be chosen well above the height of the spouts coming from the perforations in the trays.

The main part of this thesis deals with adsorption of proteins. However continuous adsorption can also be used for recovery of antibiotics (Appendix 1). The adsorption of Penicillin V from synthetic solution and a fermenta­ tion broth has been studied. When using broth as feed only the equilibrium adsorption is affected. No effect on mass transfer has been observed. The multi stage contactor has been operated succesfully with synthetic solution and fermentation broth. The steady state performance can be predicted using data obtained from the batch experiments.

Samenvatting

Adsorptie wordt vaak gebruikt voor het opwerken van producten die langs biotechnologische weg zijn bereid. Het ontwerpen van een continu fluide bed adsorptie proces voor het opwerken van eiwitten en antibiotica wordt be­ sproken in dit proefschrift.

De inleiding (Hoofdstuk 1) behandelt de toepassing van (tegenstrooms) fluide bed adsorptie processen voor het schelden van bioproducten. Het ontwerp van deze processen vereist dat drie soorten gegevens beschikbaar zijn:

- evenwichts gegevens

- stofoverdrachts parameters en

- een stromings model voor het gebruikte apparaat

In Hoofdstuk 2 wordt de evenwichts adsorptie van eiwitten op verschillende oppervlakken besproken. De adsorptie van Runder Serum Albumine (BSA) op de anion wisselaar QMA Spherosil is bestudeerd als functie van zuurgraad en buffer-concentratie. De resultaten lijken er op te wijzen dat electros-tatische interacties een overheersende rol spelen in de adsorptie. Dit wijkt af van wat gewoonlijk gevonden wordt voor de adsorptie van eiwit.

Het massa transport van eiwitten in poreuze adsorbentia kan beschreven worden met een veelheid aan modellen. Een overzicht van deze modellen wordt gegeven waaruit geconcludeerd wordt dat een relatief eenvoudig model al een voldoende beschrijving geeft van het stoftransport. Dit eenvoudige model is toegepast voor het beschrijven van adsorptie en desorptie van BSA op QMA Spherosil (Hoofdstuk 3). Een kleur techniek is ontwikkeld (Appendix 2) die het mogelijk maakt de indringing van het eiwit in het deeltje te meten. Op deze wijze wordt meer informatie over het adsorptie proces verkregen.

Tegenstrooms adsorptie is bestudeerd in een meertraps fluide bed kolom. Gegevens over het stromings gedrag zijn bepaald voor een klein kolom. Tezamen met de eerder genoemde evenwichts- en stofoverdrachts-gegevens is het nu mogelijk de werking van het apparaat te voorspellen. De voorspelling komt goed overeen met de experimentele waarneming. De invloed van proces

parameters op de werking van het apparaat is zowel theoretisch als ex­ perimenteel bestudeerd (Hoofdstuk 4 ) .

De mogelijkheid om met fluide bed processen ruwe vloeistoffen (fermentatie vloeistof) te behandelen wordt vaak genoemd als een voordeel. Daarom is de opwerking van BSA uit een gemalen gist-suspensie bestudeerd. Het blijkt mogelijk om zo'n ruwe soep te behandelen in de meertraps fluide bed kolom. Het stationaire gedrag van de kolom kan voorspeld worden met een relatief eenvoudig model.

Het ontwerpen van grootschaliger apparatuur vereist kennis over de verander­ ing van stroming met groter wordende diameters. Het uitzetten van het fluide bed in de meertraps kolom blijkt te beschrijven met een relatie die is opgesteld voor een open fluide bed. Het menggedrag van de vloeistof wordt niet beïnvloed door de diameter van de kolom. Echter, dit geldt alleen als de schotelafstand beduidend groter is dan de hoogte van de vloeistof­ straaltjes die uit de schotelgaten komen (Hoofdstuk 6 ) .

Een belangrijk gedeelte van het proefschrift gaat over de adsorptie van eiwitten. Echter, continue adsorptie processen kunnen ook gebruikt worden voor het opwerken van antibiotica. De adsorptie van Penicilline V op een anionwisselaar is bestudeerd (Appendix 1). Zowel 'nette' oplossingen als fermentatie vloeistof zijn gebruikt als uitgangs materiaal. Wanneer gebruik gemaakt wordt van fermentatie vloeistof is alleen de evenwichts ligging duidelijk anders. De stofoverdrachts snelheid is niet beinvloed.

Beide uitgangsmaterialen zijn gebruikt voor het bedrijven van de meertraps contactor. Gebruik makend van de eerder gemeten gegevens blijkt het mogelijk het gedrag van de kolom te voorspellen.

Chapter 1 Introduction

The production and use of biological materials is becoming of increasing importance. This development has been triggered by the 'new biology' which offers a greatly improved understanding of what happens inside living cells. The developments are now also leading to an increased interest in the en­ gineering aspects of processes dealing with bioproducts. In recent studies it has been concluded that, among many others, the field of 'biotechnology' offers many challenges to chemical engineers. As one of the topics the developments of efficient separation processes for bioproducts has been mentioned [NRC 1988, Keller 1987].

For the recovery of bioproducts many techniques can be used. Among these adsorption is of major importance. Traditionally adsorption processes are carried out in packed bed columns. These columns are easy to construct and give sharp separations, but the liquid to be treated should be clear of suspended solids to avoid blocking of the column. With bioproducts this is often a problem: filtration of biomass is difficult while quite often a considerable amount of product is lost in the filter cake.

Therefore there is an interest in fluidized bed adsorption processes which allow the treatment of turbid liquids.

In an open fluidized bed (Figure 1) the particles are fluidized by the liquid to be treated and suspended solids can pass through.

The main problem here is the mixing of the solids, which reduces the sharp­ ness of the separation. Using several small fluidized beds in series,

instead of one large bed, the mixing of solids is restricted to one bed which improves the efficiency of the process as a whole.

A counter-current process is obtained when continuous flows of liquid and sorbent move in opposite directions and adsorption and desorption are in­ tegrated in a sequential closed loop process. This is schematically indicated in Figure 2.

WASTE

o o o

9 e

d

1

FEED

oo o

WASH

• •

ELUENT

o o o

o o o

• • »

PRODUCT

Figure 2 Principle of continuous adsorption

The open fluidized bed can be used for counter-current processing, but again mixing of the solid phase is a problem. This disadvantage can be overcome by staging the column using perforated plates: the multi stage fluidized bed column (Figure 1 ) . Although the solids are well mixed in each compartment, the large number of compartments in series gives an overall plug flow be­ haviour (also for the liquid).

For ion exchange the advantages of a continuous multi stage fluidized bed over a packed bed have been summarized before [Van der Meer 1984]. Here the main advantages are considered to be:

- the possibility to treat turbid solutions, thereby avoiding filtration - treatment of (relatively) large amounts of liquid at a low pressure drop - shorter residence time of products in the adsorbed state (cycle time);

this is especially advantageous when dealing with sensitive biological products

- lower adsorbent inventory

Disadvantages of counter-current processes are:

- continuous processing is difficult to combine with the typical batch production processes in 'biotechnology'

- the equipment is more complicated than a packed bed - attrition of resin occurs due to transport

- the design requires a chemical engineering type of approach with which many people in 'biotechnology' are not well acquainted.

Applications

Applications of batch and counter-current fluidized bed adsorption processes have been described for a variety of bioproducts. For some processes it is known that they are applied on an industrial scale; other applications have only been investigated on laboratory scale. A summary of published applica­ tions is given in Table 1.

The first application of a fluidized bed adsorption process has been for the recovery of antibiotics. Already in 1958 the use of a (series of) batch fluidized beds for the recovery of streptomycin has been patented [Bartels 1958]. This system has been used on a large scale and has probably been extended to a counter-current process. Later the use of a semi-continuous fluidized bed system for the recovery of the acidic antibiotic novobiocin has been described [Belter 1973]. Here also the scale-up of the process is described. A small scale column, equipped with internals, has been used for the adsorption of Cephalosporin C [Hicketier 1984] . The internals reduce the mixing of the solid phase thereby improving the performance of the column compared to that of an open fluidized bed.

A true counter-current adsorption process has been used for the recovery of various antibiotics (e.g. Kanamycin). The apparatus consists of a series of conically shaped vessels through which there is a continuous flow of both phases [Gel'perin 1972, Torganova 1975, Kluyeva 1975]. Although direct adsorption from unclarified solutions is often mentioned as an advantage of fluidized beds, few results have been reported for such an application [Van der Wiel 1987].

Table 1 Summary of fluidized bed adsorption processes

bioproduct

Streptomycin Novobiocin Cephalosporin C Bovine Serum Albumin Bovine Serum Albumin various

antibiotics Penicillin V whey protein

Bovine Serum Albumin

type of fluidized bed open open open open open multi-stage multi-stage multi-stage multi-stage mode of operation semi-cont. semi-cont. batch batch batch continuous continuous continuous continuous reference Bartels 1958 Belter 1973 Hicketier 1984 Burns 1985 Wells 1987 Gel'perin 1972, Torgovanova 1975 Van der Wiel 1987 Biscans 1984 Wesselingh 1987

While the fluidized bed adsorption processes have long been used for the recovery of antibiotics, it is only recently that there is an interest in continuous adsorption of proteins. The first study, published in 1984, is on adsorption of protein from sweet whey using a three-stage fluidized bed [Biscans 1984]. At this moment this process is being studied on a pilot-plant scale. Other studies on protein adsorption using fluidized beds are on a small scale, using Bovine Serum Albumin as a model protein. The use of an open fluidized bed [Wells 1987] and a magnetically stabilized fluidized bed [Burns 1985] has been described. A small scale multi-stage column has been

applied to adsorb Bovine Serum Albumin from synthetic solutions and yeast homogenate continuously [Wesselingh 1987].

Envisaged applications

From the examples it follows that continuous fluidized bed adsorption can be used for the recovery of bioproducts from crude solutions. The envisaged applications can be divided in two categories depending on the type of product.

If the product is extracellular (i.e. present in the liquid phase) con­ tinuous adsorption can be applied as a first step directly after the fermenter. Compared with conventional processes this means that a filtration step can be omitted at this stage. Before disposal of spent liquor the microorganisms have to be removed by filtration anyhow, but filtration is then less critical.

For an intracellular product the microorganisms are separated from the liquid and subsequently disrupted. The suspension obtained gives serious filtration problems and direct adsorption from the homogenate becomes attractive.

So far avoiding filtration has been mentioned as the main reason to use (continuous) fluidized bed adsorption. However the process is also attrac­ tive for concentration and purification.

Extracellular products are obtained in a solution containing many other components; the majority of the other components has different properties and the product is easily separated. In conventional processes for the recovery of antibiotics, adsorption is used successfully for purification directly from filtered broth. So it is expected that in the continuous process concentration and purification can also be achieved.

For intracellular products a completely different situation is encountered. Upon disruption , a wealth of components is released which have properties similar to the bioproduct of interest. If a non-selective adsorbent such as an ion exchanger is used, concentration and purification can only be achieved when adsorption and desorption conditions are chosen carefully. The use of more selective adsorbents will help to overcome these problems.

Organization of the thesis

The aim of the research has been to study the application of continuous adsorption for the recovery of bioproducts. To this purpose the design of a counter-current adsorption process has been studied. The information re­ quired for designing such a non-equilibrium process is given in Figure 3.

equilibrium experiments

batch sorption experiments . equilibrium

parameters batch model [

external mass transfer coefficient pore diffusion coefficient hydrodynamic model model for equipment external mass transfer coefficient prediction of performance

Figure 3 design of a Tion-equilibrium process, flow of data

Three sets of 'basic' data are required:

- equilibrium parameters - mass t r a n s f e r parameters

- hydrodynamic parameters —Q , . ^

I n t h i s t h e s i s a l l three categories w i l l be discussed, thereby providing information to enable the evaluation of the a p p l i c a b i l i t y of continuous adsorption to the recovery of bioproducts.

I n Chapter 2 the equilibrium adsorption of p r o t e i n on an anlon exchanger is discussed. I t has been attempted to determine the adsorption mechanism by studying the effect of external conditions.

In Chapter 3 the rate of mass transfer for adsorption of protein in a porous ion exchanger is discussed. The rate limiting mechanism is determined and a mathematical model is given. The model is verified experimentally by batch adsorption studies. In addition detailed measurements of the distribution of protein inside particles are used to verify the model.

In Chapter 4 counter-current adsorption using a multi stage fluidized bed contactor is discussed. A model describing the performance of the contactor is given. The data discussed in previous chapters are used to predict the performance.

In Chapter 5 various aspects of adsorption from disrupted microorganism are discussed. Basic data for a model system are studied and used for describing continuous adsorption.

Finally in Chapter 6 the hydrodynamic behaviour of fluidized beds and multi stage contactors of varying size is discussed.

The main part of this thesis deals with the adsorption of protein. The design of an adsorption process for the recovery of a small bioproduct, Penicillin V, is described in Appendix 1.

References

Bartels C R . , Kleiman G., Korzun N., Irish D.B. (1958) Chem. Eng. Prog. 54: 49-52

Belter P.A., Cunningham F.L., Chen J.W. (1973)

Biotechnol. Bioeng. 15: 533-549

Biscans B. (1984) PhD thesis University of Toulouse 1984 Burns M. (1985) Biotech. Progress 1: 95-103

Gel'perin N.I. et al (1972) Khim. Farm. Zhur. 6: 34-38 Keller G.E. (1987) A.I.Ch.E. Monograph Series no 17 vol 83 Kluyeva L.M. et al (1975) Khim. Farm. Zhur. 10: 95-97;

ibid (1974) 8: 37-40; 9: 30-32

NRC (1988), Frontiers in Chemical Engineering; Research Needs and Opportunities, National Research Council, National Academy Press, Washington 1988

Van der Meer A.P. (1984) PhD thesis Delft University of Technology 1984 Torgovanova T.V. et al (1975) Khim. Farm. Zhur. 9: 34-39

Wells C M . , Lyddiatt A., Patel K. (1987) in: Separations for Biotechnology,

M.S. Verrall, M.J. Hudson (eds) Ellis Horwood, Chicester, 1987 pp 217-224

Wesselingh J.A., Van der Wiel J.P. (1987) Proc. Fourth European Congress

Biotechnology, Elsevier, Amsterdam 1987 vol 2: 546

Van der Wiel J.P., Klinckhamers F.J.L., Wesselingh J.A. (1987) Proc. Fourth

Chapter 2 Adsorption of Proteins

Introduction

Proteins adsorb on a variety of surfaces. Sometimes these interactions are disadvantageous, for example in biomedical applications adsorption of proteins on plastics used in blood-contacting devices may cause clotting of blood [Vroman 1977]. On the other hand for the recovery of proteins adsorp­

tion on porous particles is an important purification process [Janson 1982]. The adsorption of proteins on well-defined solid surfaces has been studied extensively supplying data on the mechanism of adsorption. There are several reviews on protein adsorption [Norde 1986r, Andrade 1986, Macrltchie 1978]. The literature on solid-liquid chromatography of proteins describes interac­ tions with surfaces and porous particles, with emphasis on the practicality of a sorbent for a certain separation problem [Regnier 1982].

Table 1 adsorbents used in solid-liquid chromatography

type of

surface

hydrophoblc

uncharged

hydrophilic

p o s i t i v e

charged

negative

chromatographic

method

hydrophobic i n t e r a c t i o n

size exclusion

(no adsorption)

anion exchange

cation exchange

The adsorption process depends on the solid surface (adsorbent), the protein and environmental conditions (pH, temperature etc.).

The solid surfaces used for studying the mechanism of adsorption can be divided into several groups (Table 2). The adsorbents used for solid-liquid chromatography can be distinguished likewise (Table 1) but these materials often are less well-defined with respect to surface properties.

Table 2 Surfaces used in protein adsorption studies hydrophilic uncharged L hydrophobic -polyoxymethylene -cellophane -polycyanopropylmethyl siloxane -polyethyleneoxide -regenerated cellulose -cellulose -polystyrene -polyethylene -polyvinylchloride -silicone (polydlmethylsiloxane) -silicone coated glass -methylated silica Norde (1986) Dillman (1973) Cheng (1987) Cheng (1987) Bornzin (1982) Biltz (1913) Lensen (1984) Brash (1976,1978) Lensen (1984) Soderquist (1980),Lok (1983) Cheng (1987), Bornzin (1982) Mizutani (1981) van Dulm (1983) MacRitchie (1972), Koltisko (1986) positive charge positive/ negative charge charged negative charge -polystyrene latex -amine coated glass _^polypeptide -haematite (a-Fe203) -hydroxyapatite (Ca3(P04) 2) -polystyrene latex

(so;

)

-Agl -SiO„ -glass -polyethylene/SO, jpolyfluorcarbon/SO~ Koutsoukos (1982), van Dulm (1983) Koltisko (1986) Soderquist (1980) Norde (1986), Koutsoukos (1982), Biltz (1913) Hlady (1979) Norde (1978),Fair (1980) van Dulm(1981) Koutsoukos (1982), van Dulm (1983),Lensen(1984) Norde (1986),Cheng (1987) Koutsoukos (1982),Norde (1986) Bull (1956), MacRitchie (1972), Morrissey (1974), Lensen (1984), van Dulm

(1983), Norde (1986) Bull (1956), Absolom (1981),Brash (1976) Dillman (1974) Dillman (1974)

Adsorption of proteins is a broad subject. Therefore this chapter will be restricted to the adsorption of one protein: serum albumin. The objective of this chapter is to present some general principles for the adsorption of albumin (Human Serum Albumin, HSA and Bovine Serum Albumin, BSA) on various surfaces. Own experimental data for the adsorption of BSA on an anion ex-changer, QMA Spherosil , will be compared with these general principles.

Characteristic properties of serum albumin fPeters 19851

Serum albumin is a polypeptide with molecular weight of (67 1) *10 ; the molecule has the shape of a prolate ellipsoid with dimensions 4.2 * 14.1 run. The tertiary structure consists of three distinct domains and several sub-domains. Different isomers occur at various pH's, the 'normal' form occurring between pH 4.3 and 8.0. Reversible transitions to the 'fast' form or the 'basic' form occur at pH 4.3 and 8.0 respectively.

The charge on the molecule depends on pH, the net charge being zero at pH 4.9 (iso-electric point). It has been shown that the different domains have different net charges causing charge asymmetry.

Mechanism of adsorption

Research on the adsorption of proteins dates back to the beginning of this century when Landsteiner and Uhliz showed that various inorganic powders adsorb horse serum protein [Landsteiner 1906]. In 1913 Biltz and Steiner studied the adsorption of albumin on ferric oxide, kaolinite and cellulose and reported an increase in adsorbed amount with increasing liquid con­ centration and almost irreversible adsorption [Biltz 1913].

The concentration dependance of adsorption is given by the adsorption isotherm (Figure 1 ) . The adsorption isotherms can be characterized by the plateau-value concentration (capacity) and the initial slope (tendency to adsorb).

In most cases the adsorbed amount reaches a plateau-value at a moderate bulk

. 3

liquid concentration ( < 1 kg m ). These plateau-values often correspond to

.2

Direct measurements of the thickness of the adsorbed layer also point at side-on adsorption [Norde 1986r].

a*

Figure 1 Typical adsorption isotherms. 1 - high affinity isotherm, 2 affinity isotherm

low

In some cases the isotherm exhibits two plateaus indicating different ad­ sorption mechanisms at low and high surface coverage [Fair 1980, Soderquist 1980].

The adsorption isotherms usually show a very sharp upward slope in the low bulk concentration region of the isotherm ('high-affinity'), although isotherms having a finite initial slope are also encountered.

Ionic strength, pH and temperature are the main external parameters which influence the adsorption process. The effect of these will be described briefly, making a distinction between the initial part of the isotherm and

the plateau-values.

For the adsorption of albumin on various surfaces (Table 3) it has been observed that the plateau-value reaches a maximum in the iso-electrlc region of the protein, decreasing in the direction of lower and higher pH. Because of the similar curves for different surfaces it is thought that structural

rearrangements in the adsorbing molecules are the main driving force for adsorption.

Table 3 Surfaces where maximum adsorption around the iso-electric region of protein has been observed

uncharged

hydrophillc -polyoxymethylene hydrophobic -methylated silica

Norde (1986) MacRitchie (1972), charged — positive charge positive/ negative charge _negative charge -polystyrene latex -haematite (a-Fe203) -hydroxyapatlte (Ca3(P04) 2) -polystyrene latex (S0~ ) -Agl

-sio

2 jj>olyethylene/SO, van Dulm (1983) Koutsoukos (1982) Norde (1986), Koutsoukos (1982) Hlady (1979) Norde (1978) Koutsoukos (1982), van Dulm (1983), Norde (1986) Koutsoukos (1982), Norde (1986) Bull (1956), MacRitchie (1972),Norde (1986) Dillman (1974)

It has been shown, by measuring the a-helix content after adsorption/desorption, that albumin loses a part of its helical structure [Norde 1986, Soderqulst 1980]. Results from H -titratlon, electrophoresis and micro-calorimetry [Norde 1978] also point in this direction. Using spectroscopie techniques it is possible to monitor the structure of molecules in the adsorbed state; this also indicates a change in structure upon adsorption [Norde 1986r, Hlady 1988]. The decreased plateau-values at higher and lower pH are attributed to a decrease in stability of the albumin molecule giving a 'flatter' or more expanded molecule on the surface.

The adsorption of albumin on positively charged polystyrene behaves somewhat differently : at high ionic strength the general trend seems to be followed but at low Ionic strength the plateau-value increases with increasing pH and reaches a maximum at a higher pH [Koutsoukos 1982, van Dulm 1983]. In this

case the ionic interactions between surface and protein seem to play a dominant role.

The variation of initial slope with pH depends on the type of surface. For uncharged surfaces the initial slope decreases with increasing pH due to increased repulsion between (charged) protein and the surface [Koutsoukos 1982, MacRitchie 1972]. The variation of initial slope for adsorption on charged surfaces can to a large extent be explained from the interaction between the charges on the protein and those on the surface. For positively charged surfaces this leads to high-affinity isotherms over the whole pH-range studied (pH 4-7) [Koutsoukos 1982], while for negatively charged surfaces a decrease in initial slope with increasing pH has been observed

[Bull 1956, MacRitchie 1972, Norde 1978, Koutsoukos 1982].

The amount and type of electrolyte present in solution also influences the adsorption. For the adsorption on hydrophobic surfaces it has been found that the adsorbed amount increases with increasing concentration of electrolyte, especially when little or no electrolyte is present [Mizutani 1981, Soderquist 1980].

When the surface is negatively charged, an increase in electrolyte does not change the adsorbed amount to a significant extent [Fair 1980, Koutsoukos 1982, Norde 1978, Soderquist 1980]. Here co-adsorption of ions in the ad­ sorbed layer plays an important role in the adsorption process, and the effect of the type of electrolyte on the adsorbed amount has been explained in terms of the tendency of small ions to co-adsorb [Norde 1978, van Dulm 1983].

For the adsorption on positively charged surfaces or surfaces with a pH dependant charge the effect of the electrolyte can be explained in terms of ionic interactions: when the charges of surface and albumin are opposite, the adsorbed amount decreases with increasing ionic strength and vice versa [Hlady 1979, Koutsoukos 1982, Soderquist 1980].

The effect of temperature has not been studied in detail [Norde 1986r]. For uncharged surfaces the plateau value increases with increasing temperature [Bornzin 1982] although a much smaller effect of temperature has also been observed [Dillman 1974]. For negatively charged surfaces the effect of temperature depends on pH. In the iso-electric region the plateau values and initial slopes at 5 and 22 °C are equal. An isotherm with a steeper initial

slope and a lower plateau is found at 37 °C. Outside the iso-electric region increasing temperature causes only an increase in initial slope [Norde 1978].

Sofar the adsorption of albumin on various surfaces seems to follow qualita­ tively what might be expected by intuition. However the adsorption process is much more complicated; a significant amount of data has been published which do not fit in the qualitative model.

The most important feature not yet understood is the reversibility of the adsorption process. If the adsorption process is reversible, then upon changing to conditions less favourable for adsorption, desorption of protein should occur. Often this is not observed. A distinction has been made be­ tween [Norde 1986] :

-desorption after dilution of the solution -desorption after changes in pH

-desorption after addition of low molecular weight substances -exchange against dissolved proteins.

In general it has been found that no desorption occurs after dilution. Partial desorption from some charged surfaces [Koltisko 1986, MacRitchie 1972, Soderquist 1980] and hydrophobic silicone [Soderquist 1980] has been observed. The desorption depends on the residence time of albumin in the adsorbed state: at longer times the rate of desorption is slower. The desorbed molecules lose a significant amount of the a-helix content

[Soderquist 1980].

For negative polystyrene particles additional adsorption is observed when changing the pH towards the iso-electric point [Norde 1978], however the reverse process does not take place. For the more hydrophilic hydroxy apatite, haematite and silica, reversibility towards pH changes is observed

[Hlady 1979,Koutsoukos 1982].

For hydrophobic surfaces addition of electrolytes does not lead to a change in adsorbed amount while for hydrophilic surfaces the adsorption is (partially) reversible towards changes in ionic strength. Using a small organic molecule like morpholine [Norde 1986] or surfactant [Lensen 1984] it is possible to desorb albumin from various surfaces.

Although often no desorption upon dilution is observed, the adsorbed protein is in dynamic equilibrium with protein in solution. Both the fraction of exchangeable protein and the rate of exchange increase with shear rate and concentration of solution [Brash 1978].

A model for the adsorption mechanism has been proposed which describes the adsorption as a three step process [Soderquist 1980]. At short residence times the protein molecule adsorbs reversibly (step 1) ; at longer times a molecule on the surface undergoes a structural transition to optimize the interaction with the surface (step 2). The altered molecule can desorb (step 3) but at a much slower rate than in the initial period.

This model predicts 'Langmuirian' adsorption at short residence times and irreversibility at longer times, which Is in agreement with many findings.

Solid-liquid chromatographv

Solid-liquid chromatography has found many applications both in the isola­ tion and purification of proteins from biological sources [Janson 1982] and In protein analysis, where high performance liquid chromatography (HPLC) is a widely used technique [Regnier 1982].

The adsorbents used consist of a support (carrier) to which the active groups (llgands) are attached. The support should show minimal interaction with proteins in order to restrict the adsorption to interactions between the active groups and the protein.

For isolation and purification supports made of uncharged polysaccharides like cellulose, dextran and agarose [Janson 1982] have been used; these supports show minimal protein adsorption. A common support for HPLC is porous silica; here chemical modification of the internal surface is neces­ sary to minimize the interaction between support and protein. In general the adsorbents are poorly defined with respect to surface properties; this hampers a fundamental understanding of the mechanism of adsorption on these materials. Only recently some attention has been paid to these adsorption processes in terms of equilibrium parameters [Chase 1985, Graham 1982, Huang 1987, Kopaciewicz 1987, Roy 1984, Tsou 1985, Yamamoto 1983].

The adsorption on ion exchangers seems to be determined mainly by ionic interactions [Chase 1984, Yamamoto 1983]. This is not surprising regarding the efforts to minimize the interactions between protein and support. It should be noted that the ion exchangers have a higher charge density than the charged materials described in Table 1. Indirect evidence for reversibility can be found in the modes of operation applied in HPLC. Chromatography at constant liquid composition shows that desorption upon dilution is possible, while chromatography with a gradient in either pH or ionic strength points at desorption upon changing environmental conditions. The simplest model used to describe the chromatographic behaviour of proteins assumes that the net-charge of a molecule is a good measure for the retention behaviour; but this model fails to predict retention on ion ex­ change columns at the iso-electric point of the protein [Kopaciewicz 1983] . Improvement was obtained with the 'stoichiometric displacement model' [Kopaciewicz 1983]. Here it is assumed that the protein, upon adsorption will displace multiple ions from the sorbent (surface) :

P + z Ï <]'/*_ P + z I (1)

with an equilibrium constant K expressed as :

K - t ' M n '

( 2 )

e q [ p ] . [ i ] z

Using this model the capacity factor k', a measure for retention, can be related to the concentration of displacing ions :

k' - - £ — (3)

[ I ]

where K is a protein specific constant. Recently this model has been criticized [Fraaije 1987].

It has been found that under different conditions of pH and ionic strength the capacity factors for several proteins can be described with the 'stoichiometric displacement model' [Kopaciewicz 1983, Rounds 1984, Drager 1986].

The fit-parameter z is interpreted as the number of charges apparently

involved in the adsorption/desorption proces. It is smaller than the charge

of the protein; this was explained from the non-uniform charge distribution

on the protein molecule: due to steric limitations only a part of the ionic

groups on the surface of a protein can interact with the sorbent surface

[Kopaciewicz 1983]. This is supported by the fact that the retention be­

haviour of BSA on anion exchange columns was found to depend on the tertiary

structure of the molecule [Withka 1987]. It is also known that proteins need

only a small 'foot-hold' to attach to a surface [Andrade 1986].

Mathematical description

Sofar two different models for the adsorption of proteins have been discu­

ssed. For many purposes a mathematical description of the adsorption

isotherm is desirable. Then the Langmuir equation can be used:

Q = Qm a x K, C +C <4>

lang

O is the maximum adsorbed amount, while K. represents the

concentra-Tiiax lang tlon where the adsorbed amount equals 0.5 0

The Langmuir model assumes:

-reversible adsorption

-no change of adsorbed molecules

-no lateral interaction between adsorbed molecules

-one adsorption site per molecule

-all adsorption sites are equal

Experimental

Materials

Chemicals : Bovine Serum Albumin, fraction V is obtained from Boehringer

Mannheim Germany (Albumin > 98% ) . All other chemicals are analytical grade. The concentration of BSA is determined with a modification of the Folin-Lowry method [Schacterle 1973].

r A

Adsorbent : QMA Spherosil is obtained from Rhone Poulenc, Villeneuve la

Garenne,France.

The sorbent is prepared following the recommendation of the manufacturer [Rhone Poulenc 1984] . Before use the particles are slurried in the buffer to be used in the experiment. The buffer is removed by filtration (10 minutes) on a sintered glass filter (G2). In each case the amount of liquid inside the particles is measured by drying at 80 °C to constant weight.

Methods

Composition of coating : the composition of the coating on the inner surface

of the adsorbent is determined by elemental analysis [ Perkin Elmer elemen­ tal analyzer). Nitrogen is analyzed by the method of Kjeldahl [Steyermark 1961].

Ionic capacity : the ionic capacity of the sorbent is determined by packing

a weighed amount of particles in a glass column followed by equilibration with 0.1 N HCl. The column is flushed with demineralized water until the pH of the effluent is equal to that of the influent. Then the Cl is displaced with a 2.0 M KN0_ solution; the chloride concentration in the effluent is determined by titration with AgNO,.

The total chloride-content in the equilibrated particles is determined by the method of Schöniger [Steyermark 1961].

Pore size distribution : the pore size distribution of various particle size

fractions is determined by Hg porosimetry using a Carlo Erba Mercury Porosimeter.

Particle size distribution : several samples of particles are photographed

and the size of approximately 3000 particles is measured.

Isotherm measurements

.3

static adsorption : a 0.5-2 .10 kg sample of equilibrated particles is

.6 3

carefully weighed and suspended in 30-100 .10 m protein solution.

.3

Starting protein concentration varies from 0.2 to 10 kg m . All solutions contain 0.02% sodium azide. The suspension is either gently stirred in a capped beaker glass or placed in a capped tube with adjustable volume and rotated. With the stirring method it is difficult to keep the particles in suspension, because at the required speed the particles are crushed by the stirrer thereby exposing non-coated silica surface to the solution causing unwanted adsorption.

In the other case care should be taken not to include air bubbles in the tube because this will give rise to denaturation upon long exposure.

Various particle size ranges have been used (90-106, 106-125 and 125-150 micrometer); in all cases at least 2 days are required before equilibrium is fully established, under some conditions it even took about 3 days. The concentration in the liquid was measured several times during the experiment with at least 6 hours between sampling.

Frontal analysis : a weighed amount of particles is packed in a glass column (1 x 14 cm) and washed with buffer. The protein solution is fed to the

.8 3 .1

column at 3 .10 m s . The UV-absorbance (280 nm) of the effluent is recorded continuously using an on-line spectrophotometer (Pharmacia); in the steep part of the breakthrough curve samples of the effluent are taken and the protein concentration assayed.

All equilibrium experiments are carried out at room temperature ( 2 0 + 2 °C) unless noted otherwise.

QMA i

+ + + + + + + + + + + + ^

Results & discussion

Characterization

The chemical composition and physical properties of the adsorbent have been measured, results are summarized in Table 4. The results agree with those obtained previously [Dromard 1982].

The polymer coating takes about 10% of the dry weight of the particle. Assuming a homogeneous coating on the pore walls, the thickness of the layer can be calculated to be A nm, which points at a multi-layer of polymer. A schematic drawing of the adsorbent is given in Figure 2.

Table 4 Elemental composition of QMA Spherosil

elemental composition C H N ion exchange capacity total capacity

mean pore radius surface area mean particle size

5.6 8.6 0.4 0.1 0.4 660 25 190 mmole/g mmole/g mmole/g mmole Cl /g mmole Cl /g nm m /g 10 m

The amount of nitrogen corresponds to the the total amount of chloride; however the chloride content measured by displacement is significantly lower. The difference is probably caused by the inaccessibility of charged groups trapped in the polymer layer. Following this assumption, a surface

2

charge of 2.4 charged groups per nm for the polymer surface can be calculated. This is higher than the charge density of materials often used in

protein adsorption studies [Koutsoukos 1982, Norde 1978] and comparable but somewhat lower than the charge density of materials used for analytical chromatography [Alpert 1976].

The pore size distribution (Figure 3) indicates that all of the surface will be available for protein molecules. The smallest pores are about 5 times the size of an albumin molecule, which makes exclusion highly unlikely.

~o

> (1) U

o

CU

>

• .-I

JO

"3

100

50

0

r ^

10 10 "

10'

a

pore r a d i u s (nm)

Figure 3 Pore size distribution of QMA Spherosil from Hg porosimetry

Adsorption

Adsorption isotherms have been measured at different conditions of pH and ionic strength; some isotherms are given in Figures 4, 5 & 6.

The accuracy of the measurements was found to be 5%; the measurements at 10 mM buffer concentration however show a larger scatter. The different ex­ perimental methods used for equilibration give identical results within the experimental error.

In all cases studied Langmuir-like adsorption isotherms are obtained. At low buffer concentration and at high pH the isotherms show a 'high-affinity' character and the adsorbed amount reaches a maximum above about 1 g/1. In other cases the plateau is reached at much higher liquid concentrations (5 g/1 )•

250

.200

^ 1 5 0 ^

o- 8

50

0

200

an

no°c6

)

n

a

a

1 0 0

p

a a

D D A

a □

A A 5 AA AA &A AA AA

0

—i 1 r~

₂

₄

"6

C

(g/1)

C

(g/1)

Figure 4 Equilibrium curve for adsorption of BSA on QMA Spherosll acetate buffer pH 5, o 10 mM, 0 25 mM, A 50 mM

ar

250

,200

i

' 1 5 0

:

1 0 0 $

50

0

CJDO

o°

o°o

°

O o " O

CPr,

D D CD D D D D Q

j£& &*

D A A PA

a

A

^p

ik

il

2 4

C

( g / l )

C

( g / l )

Figure 5 Equilibrium curve for adsorption of BSA on QMA Spherosllr

250

o

D

A A

D

2 4

C

(g/D

=B= A &_

0.5

C

( g / l )

1.0

Figure 6 Equilibrium curve for adsorption of BSA on QMA Spherosil1

The plateau-values at different pH's and buffer concentrations are given in

2

Figure 7; the maximum adsorbed amount varies from 4 to 12 mg/ m (BET-surface). This is higher than expected for a mono-layer of side-on adsorbed

2

albumin molecules (2.5 mg/m [Norde 1978]). There is no evidence of multi­ layer adsorption, so it is assumed that adsorption takes place in the end-on

2 configuration ( 9 mg/m [Norde 1978]).

200

3

x

6

at

150-

100-50

%.

P—

1 1 1 1 1 1 1 1 1 1 t i i

* *

if

_/

/

c r '

/ / l ^Q, N

.--o.

. A -\ \ \ A

_\

•--\-A

o S

0 5.0 6.0

pH

7.0 8.0

Figure 7 Maximum amount of BSA adsorbed on QMA Spherosil

Effect of buffer concentration

The effect of buffer concentration on the adsorption is studied by measuring the adsorption at three different concentrations : 10, 25 and 50 mM.

As can be seen in Figures 4-6, upon increasing the buffer concentration adsorption decreases; the effect being smaller with increasing pH.

The initial parts of the isotherms are also given in Figures 4, 5 & 6; the initial slope decreases with increasing buffer concentration at pH 5 and pH 6 while at pH 7 no significant difference can be observed due to the high-affinity character of the isotherms.

These results point at ionic interactions between the protein and the sur­ face as the governing factor in the adsorption process. This was also concluded for the adsorption of HSA on positively charged polystyrene par­ ticles [Koutsoukos 1982].

Effect of pH

The influence of pH on the adsorption process is studied between pH 4 and pH 8. In this pH region there are no isomer transitions for albumin; below pH 4.3 and above pH 8.0 other albumin isomers occur which may have different adsorption characteristics [Janatova 1974, Peters 1985].

The interpretation of the effect of pH is somewhat complicated because of the use of different, weak electrolytes: for pH < 5 acetate is used, while for higher pH's phosphate is used.

Some experiments have been carried out at pH 5 and pH 6 to study the in­ fluence of the type of anion used. With a 10 mM phosphate solution pH 5 the maximum adsorbed amount is about 20% lower than the plateau-value from the isotherm measured with acetate as anion. For a 25 mM solution no significant difference between phosphate and acetate could be observed. At pH 6 the results obtained with acetate and phosphate solutions are also similar. These results are in agreement with the behaviour of the electrolytes used : at pH 6 both acetate and phosphate are completely dissociated while at pH 5 only phosphate is completely dissociated. It can be concluded that the results obtained with different anions are comparable. For 10 mM solution a small effect of the type of anion should be taken into account.

A pronounced effect of pH below and around the iso-electric point of the protein can be observed. Upon increasing the pH the adsorption increases sharply and reaches a maximum around pH 6. Upon further increasing the pH, Q either remains constant (50 mM) or decreases (10 & 25 mM).

Tnax

The initial slope increases with increasing pH for 25 and 50 mM solutions. With 10 mM solution all isotherms are of the high-affinity type and no difference in initial slopes can be observed.

The variation of Q with pH is similar to the results obtained for a _max

r

silica based DEAE anion exchanger [Roy 1986]. Also for the adsorption of albumin on various polymeric surfaces (Table 3) a similar effect of pH on the adsorbed amount is found. However, there maximum adsorption usually

takes place at a lower pH (about pH 5). One of the materials mentioned in Table 3, positively charged polystyrene latex, resembles the coating used in QMA Spherosil . The results obtained for the adsorption of albumin on positively charged polystyrene latex at 10 and 100 mM KNO, correspond with the results reported here. For 10 mM solution maximum adsorption is found to occur around pH 6; for a higher concentration of anion maximum adsorption seems to occur at a lower pH but the variation with pH then is very small [Koutsoukos 1982]. Subsequent to these results it has been shown that at pH 7.4 for different amounts of anion the adsorption is similar [van Dulm 1983]. This is in excellent agreement with the results for QMA Spherosil It is interesting to note that for the adsorption on positively charged polystyrene latex an 'end-on' configuration is suggested [Koutsoukos 1982]. Different explanations for the effect of pH on the adsorption have been given. In general it is assumed that adsorption is to a large extent governed by structural alterations in the albumin molecule [Norde 1986r]. For a DEAE anion exchanger, Roy [1986] assumes that the increase in capacity at low pH is caused by the increase in (negative) charge on the protein. The decrease at a higher pH is thought to be caused either by deprotonation of DEAE-groups or repulsion by (negatively charged) silanol groups.

Koutsoukos [1982] also assumes electrostatic interactions to'play a decisive role in the adsorption on a positively charged surface. In addition a small effect of conformational stability of the albumin molecule is incorporated, mainly to explain the decrease in adsorption at higher pH.

Adsorption mechanism

Many aspects of the adsorption of BSA on QMA Spherosil can be explained by assuming that the adsorption occurs via an ion exchange mechanism. This is schematically indicated in Figure 8.

The protein does not adsorb at a low pH when there are few negative charges on the molecule. Upon increasing the pH the number of negative charges increases and the affinity of protein for the surface increases. This can be seen from te sharp increase in adsorption in the iso-electric region and the increase in initial slope upon increasing the pH. The effect of buffer concentration is interpreted as a competition between anions : at low pH, because of the low charge on the protein, small anions can influence the

adsorption to a great extent by competing for the same sites. At higher pH values, due to the larger number of negative charges on the protein, the binding of protein to the surface is stronger. Therefore the effect of small

ions will be limited here.

+ + + + + + + ♦ + + + + + +

pH )

i l

c o n c e n t r a t i o n )

O W Q Q V / Q Q

tJJJcJJJ^

' ******** *******

* * * * * * * * *******

Figure 8 Proposed mechanism for the adsorption of BSA on QMA Spherosil

The ion exchange mechanism does not explain the decrease in capacity upon decreasing the pH away from the maximum. As discussed previously several explanations can be given, but sofar there is no experimental evidence to support one of the possibilities.

It is known that there is a non uniform distribution of charge on the al­ bumin molecule [Peters 1985]. If ionic interactions play an important role in the adsorption process, this might stimulate an end-on orientation upon adsorption.

Effect of temperature

The adsorption at 50 mM phosphate pH 6 was studied at 10 and 22 °C. The results are shown in Figure 9. There is no significant effect of lowering the temperature on the adsorption process. These results are in agreement with previous observations for charged surfaces [Norde 1978] but not in agreement with the proposed ion exchange mechanism.

150

f—H

JMlOO

ar

50

8:

'.t

o

—i—i—r~'V' '

1.0

c

(g/D

~ i — i — i — i — i — e ' T1 T — r

-2.0

Figure 9 Effect of temperature on the adsorption of BSA 50 mM phosphate pH 6, 10 °C, H 22 °C

Desorption of adsorbed albumin

If the adsorption is governed by electrostatic interactions the adsorption should be reversible. This has been tested by measuring the amount of protein desorbed upon putting the loaded adsorbent in a fresh solution. The results of a desorption experiment are given in Table 5.

Here particles are loaded at 10 and 25 mM pH 6 and desorbed at various conditions. As can be seen from Table 5 desorption of albumin takes place. When the adsorption conditions are also used for desorption, the final situation corresponds with the adsorption isotherm. When the conditions are changed, the final situation approaches the situation according to the adsorption isotherm. However the capacities after desorption are systemati­ cally higher than predicted by the isotherm.

Table 5 Desorption of BSA from loaded QMA Spherosil . Adsorption in 10 mM and 25 mM solution pH 6, final capacity after adsorption 164 g/1 and 136 g/1 respectively. adsorption 10 mM pH 6 desorption solution 10 mM pH 5 25 mM pH 5 50 mM pH 5 10 mM pH 6 10 mM pH 6 10 mM pH 6 25 mM pH 6 25 mM pH 6 25 mM pH 6 50 mM pH 6 50 mM pH 6 50 mM pH 6 10 mM pH 7 25 mM pH 7 50 mM pH 7 final cone.

(g/D

0.35 0.66 0.73 0.024 0.14 0.33 0.53 1.24 1.83 0.87 1.37 2.04 0.20 0.54 1.21 Q exp.

(g/D

153 115 79 152 157 160 136 139 141 106 107 111 154 131 107 Q isotherm

(g/D

145-160 92 40 160 160 160 110-125 125-140 132 80-95 90-105 100-110 105-132 90 100-110 adsorption 25 mM pH 6 final cone.

(g/D

0.15 0.72 1.55 0.007 0.35 0.42 1.11 3.55 0.006 0.46 1.44 Q exp.

(g/D

131 115 80 136 125 94 103 115 136 121 98 Q isotherm

(g/D

130-145 90 55 130-160 115-125 65-80 95 105 105-135 90 100-110 Mathematical description

For calculation purposes a mathematical description of some of the adsorp­ tion isotherms is required. Therefore the Langmuir model has been fitted to some of the adsorption isotherms obtained. The fitted parameters are sum­ marized in Table 6 and the optimized curves are given in Figures 4,5 & 6. In general the fit is reasonable, the initial parts of the isotherms which are of interest for further studies, are described well. For the isotherms at pH 7 no conclusion about the fit can be reached because the scatter in the experimental results is too large.

Table 6 Values of parameters obtained by fitting of Langmuir equation to experimental data. c o n d i t i o n 10 mM 25 mM 50 mM pH 5 Tnax

(g/D

186 111 86 l a n g

(g/D

0.035 0.053 0.699 pH 6 Q max ( g / D 182 131 88 l a n g

(g/D

0.063 0.012 0 . 0 4 3 pH 7 Tnax

(g/D

120 120 100 lang

(g/D

0.002 0.018 0 . 0 H Concluding remarks

Some aspects of the adsorption of BSA on QMA Spherosil have been studied and are discussed in this chapter. Although this study is by no means com­ plete, some conclusions can be reached. From the amount of BSA adsorbed it follows that adsorption in the 'end-on' configuration is likely. From the variation of the adsorption with pH and buffer concentration it is concluded that the adsorption is, to a large extent, governed by electrostatic interactions. The finding that the adsorption is reversible is in line with this assumption. One aspect of the adsorption, the decreasing adsorption upon changing the pH away from the maximum, cannot be explained from the proposed mechanism. Probably a change in structure upon adsorption is in­ volved, as is often assumed for the adsorption of albumin.

The experimental data for a different silica based anion exchanger agree quite well with the data presented here. It might be that some of the aspects encountered are typical for these silica based adsorbents.

The general trends observed for the adsorption of albumin on polymeric surfaces are similar to the trends observed with the system studied here. However there are significant differences, of which the (ir)reversibility is

the mechanism of adsorption on porous adsorbents is completely different from the mechanism encountered with polymeric surfaces.

References

Absolom D.R., Michaeli I., van Os C.J. (1981) Electrophoresis 2 : 273-278 Alpert A.J., Regnier F.E. (1979) J. Chromatogr. 185: 375-392

Andrade J.D. (1986) Principles of protein adsorption, in: Surface and Interfacial Aspects of Biomedical Polymers, Andrade J.D. (ed.), Plenum Press, New York, 1986 vol2: 1-80

Biltz W., Steiner H. (1910) Blochem. Z. 23 : 27-44

Bornzin G.A., Miller I.F. (1982) J. Colloid Interface Sci. 86 : 539-558 Brash J.L., Davidson V.J. (1976) Thrombosis Research 9 : 249-259

Brash J.L., Samak Q.M. (1978) J. Colloid Interface Sci. 65 : 495-504 Bull H.B. (1956) Biochim. Biophys. Acta 19 : 464-471

Chase H.A. (1984) in: Ion Exchange Technology, Naden D., Streat M. (eds.), Ellis Horwood Ltd, Chicester 1984 pp 400-406

Chase H.A. (1985) Factors Important in the design of fixed bed adsorption processes for the purification of proteins, in: Discovery and Isolation of Microbial Products, Verrall M.S. (ed.), Ellis Horwood Ltd, Chicester 1985 Chapter 9

Cheng Y.L., Darst A..Robertson C.R. (1987) J. Colloid Interface Sci. 118 : 212-223

Dillman W.J., Miller I.F. (1973) J. Colloid Interface Sci. 44 : 221-241 Drager R.R., Regnier F.E. (1986) J. Chromatogr.359: 147-156

Dromard A., Ramanadin (1980) U.S. patent 4,327,191

Fair B.D. Jamieson A.M. (1980) J. Colloid Interface Sci. 77 :525-534 Fraaije J.G.E.M. (1987) Interfacial Thermodynamics and Electrochemistry of Protein Partitioning in Two-Phase Systems, PhD Thesis Agricultural University Wageningen

Graham E.E., FookC.F. (1982) AIChEJ. 28: 245-250 Hlady V., Andrade J. (1988) Colloids Surf. 32: 359-369

Hlady V., Furedi-Milhofer H. (1979) J. Colloid Interface Sci. 69 : 460-468 Huang J.-X., Horvath C. (1987) J. Chromatogr. 406: 285-294

Janatova J. (1974) J. Med. 5: 149-216

Janson J.-C, Hedman P. (1982) Advances Biochemical Engineering, Fiechter A. (ed.), Springer Verlag, Berlin vol 25: 43-99

Koltisko B., Walton A. (1986) Chromatographic analysis of protein adsorp­ tion, in: Surface and Interfacial Aspects of Biomedical Polymers, Andrade J.D. (ed.), Plenum Press, New York, 1986 vol2: 217-239

Kopaclewicz W., Fulton S., Lee S.Y. (1987) J. Chromatogr. 409: 111-124 Kopaciewicz W., Rounds M.A., Regnier F.E. (1983) J. Chromatogr. 266: 3-21 Koutsoukos P.G.,Mumme-Young C.A.,Norde W., Lyklema J. (1982) Colloids and Surfaces 5 : 93 - 104

Landsteiner K., Uhliz R. (1906) Centr. Bakt. Parensitenz., Abt. Orig. 40 : 265-270

Lensen H. (1985) Concurrerende adsorptie van plasma eiwitten aan vast/vloeistof grensvlakken, PhD Thesis Twente University

Lok B.K., Cheng Y.-L., Robertson C.R. (1983) J. Colloid Interface Sci. 91 : 104-116

MacRitchle F. (1978) Adv. Protein Chem. 32 : 283-326 Mizutani T. (1981) J. Colloid Interface Sci. 82 :162-166

Morrissey B.W., Stromberg R.R. (1974) J. Colloid Interface Sci. 46 Norde W. Norde W., Norde W., Norde W., Norde W., Norde W., Norde W., Norde W., Sci. 112: Peters jr Regnier F

(1986r) Adv. Colloid Interface Sci. 25 : 267 Lyklema J. (1978) J. Colloid Interface Sci. Lyklema J. Lyklema J. Lyklema J. Lyklema J. Lyklema J. MacRitchie F. 447-456 T. (1985) E. (1982)

(1978) J. Colloid Interface Sci. (1978) J. Colloid Interface Sci. (1978) J. Colloid Interface Sci. (1978) J. Colloid Interface Sci. (1979) J. Colloid Interface Sci. Nowicka G., Lyklema J. (1986) ace Sci. 46 : 152-164 - 340 66 66 66 66 66 71 J. 277 - 284 285 - 294 266 - 276 257 - 265 295 - 302 350 - 366 Colloid Interface Rhone Poulenc (1984)

Adv. Protein Chem. 37: 161-245 Anal. Biochem. 126: 1-7

product leaflet QMA Spherosil

Rounds M.A., Regnier F.E. (1984) J. Chromatogr. 283: 37-44 Roy A.K., Burgum A., Roy S. (1984) J. Chromatogr. Sci. 22: 84-86

Soderquist M.E., Walton A.G. (1980) J. Colloid Interface Sci. 75 : 386-397

Schacterle G.R., Pollack R.L. (1973) Anal. Biochem. 51: 654-655

Steyermark A. Quantitative Organic Microanalysis, Academie Press, New York,

337

AIChE J. 31: 1959-1966

Colloid Interface Sci. 91 : 248-255

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Vroman L., Leonard E.F. Withka J., Moncuse P.

398: 175-202

Yamamoto S., Nakanishi Bioeng. 25: 1465-1483

(1977) Ann. N.Y. Acad. Sci. 283: 65

, Baziotis A., Maskiewicz R. (1987) J. Chromatogr. Matsuno R., Kamikubo T. (1983) Biotechnol.

Chapter 3 Mass transfer

Introduction

In the preceding chapter equilibrium aspects of the adsorption of BSA on the anion exchanger QMA Spherosil have been discussed. For the design of adsorption equipment information about the rate of adsorption is also required. In this chapter the rate limiting steps in the adsorption process are discussed and a (general) model describing the transport in a porous adsorbent will be presented. So far, few systematic studies of protein adsorption rates have been published. The different models used in describ­ ing the transport are discussed and transport coefficients for various systems are compared.

Own experimental results for the rate of adsorption of BSA on QMA Spherosil will be presented and compared with published data.

Theory of mass transfer

In the uptake of a solute by an adsorbent usually four different transport mechanisms are distinguished:

-Film transport: mass transfer between the bulk liquid and the external

surface of the particle. The flux is described by:

Nf " kl <Cbulk-Cint> ( X ) -Pore diffusion: transport through the liquid contained in the pores of

the particle. Usually Ficks law is assumed to describe the transport:

N - - D -jj£- ( 2 )

pore p dv v '

-Adsorption kinetics: interaction of adsorbate with the adsorption

G(c,q) = kfl c (q^ - q) - kd q ( 3)

.3 .1

Where G(c,q) is given in mol (or kg) m s

-Surface diffusion: transport by diffusion of adsorbate in the adsorbed state:

N . - - D - I

3

" <

4

>

surf s 3r

A model taking into account all four mechanisms has been presented recently [Do 1987]. Usually such an extensive model is not necessary. Three dimen-sionless groups can be used to establish the relative importance of the various mechanisms: k r _. 1 film transpport , ,-. D pore diffusion P s "« surface diffusion . ,. D c0 pore diffusion 2 , G(Co,0) r . . ,■* " rate of adsorption v D C0 pore diffusion

With the Langmuir model: G(Co,0) - k C0 q

Recently a 'general' model has been proposed for (biospecific) adsorption of proteins [Arve 1987]; this is one of the special cases of the model presented by Do & Rice [Do 1987].

An a priori estimate of the contribution of the various transport mechanisms can be made using published coefficients for BSA. Typical values are indi­ cated in Table 1.

With these values estimates of the dimensionless constants (equation 5 - 7 )

2

can be obtained: both Bi and if> are much larger than 1, while A - 1.

This indicates that for the adsorption of BSA the adsorption kinetics will not be rate limiting. This conclusion is also valid for many other systems. Frequently limitation by adsorption kinetics has been incorporated in the

mass transfer models [Arve 1987, Hossain 1985, Do 1984] although with the parameters used, it can be shown that this limitation can be neglected. Only in a few cases there are indications that the actual adsorption reaction might play a role [Walters 1982, Sportsman 1984].

Table 1 Typical values for adsorption of BSA

rate coefficients -6 .1 k. 10 m s 1 .13 2 . 1 D 10 m s S .12 .11 2 D 10 - 10 m s P .2 3 .1 .1 k 10 m kg s

a

°

(a) (b) .1 (c) (d) characteristic properties of adsorbent .4 r 10 m P £ 0.5 P .3 q^ 100 kg m CQ 1 kg m" a Geankoplis.1983; Snowdon 1967 b Burghardt 1981

c Arve 1987; Hossain 1986; Arnold 1985 d Beissinger 1982; Cheng 1987; Do 1985

Although Bi » 1 film transport has to be taken into account because this will always be rate limiting in the initial period [Do 1985, Arve 1987]. The transport in a spherical particle for simultaneous pore and surface diffusion, assuming local equilibrium inside the pores, is given by:

. 3 0 . 3 c 1 3 . 2 ._ „ 3a . 3c

( e

p

+

3? > 3T °

J- 3r

[ r (D

p

+ D

s «Ü > "3r

( 8) The boundary conditions are given by:

-0 J f - 0 ;

( 9)

( no transport through the centre of the particle ) , and 3c

r=R

[D |f + D ] .

_{1 s 3c pJ 3r} - k.(C. .. -C. )

( no accumulation at the external surface )

It has been shown that the combined diffusion can be described with pore diffusion alone, lumping D together with D into an effective pore diffu­ sion coefficient. D ,.,. [Neretnieks 1976a] . The'contribution of pore and

p.eff *

surface diffusion can then be obtained from the variation of D „ with the p.eff

concentration ratio C0/q [Neretnieks 1976b],

With the assumption of only pore diffusion as the rate limiting step, the mass transfer can be described by:

c £p + ac > at 2 r r

a_

dr . 2 3c [ r

1Ï

(11) With and boundary conditions:

r=0 |f - 0

r=R - D _„. p P.eff ; dc dr r-=r l- kn(C. ., -C. ) v bulk m ty (12) (13) with t-0 c - q - 0 (14)

as initial condition if adsorption on an initially clean sorbent is studied. This set of equations, describing the mass transfer in a single particle, should be coupled to the mass balance of the reactor to be studied. For a stirred tank the mass balance is:

8 C.

bulk

a t k. A (C, ., - C. J 1 bulk int (15)

A special limiting case of the pore diffusion model, usually called the 'shrinking core' model, can be derived when the equilibrium can be described by a rectangular isotherm (which is often associated with irreversible adsorption). Then inside the particle two regions can be distinguished: a saturated outer zone and an unsaturated inner core (Figure 1). All molecules entering the particle are adsorbed at the boundary.

SATURATED

ZONE

d r c d t Cb u l k Tnax r (R - r ) . r c p e c

L

k

i

RP p p

Figure 1 Partly saturated adsorbent

Assuming a quasi steady state for diffusion, the movement of the boundary is given by:

(16)

Analytical solutions giving the time necessary to reach a certain bulk concentration have been presented [McKay 1984, Do 1984, Arnold 1985], but these equations are complicated and numerical solution of the differential equation is easier.

Desorptlon

Desorptlon of proteins can take place by changing pH or ionic strength of the solution. If low molecular weight substances are used for desorption it can be assumed that their distribution inside the adsorbent is fast compared to the diffusion of proteins. If the conditions for desorption are chosen in a proper way, it can also be assumed that all adsorbed material is released into the intraparticle liquid instantaneously. Then the overall rate of desorption is governed by pore diffusion and film transport.

For pore diffusion as rate limiting step the desorption into a finite bath, initially free of solute, is described by [Crank 1975]:

2 c_ e0 c i J_I \ - D t q t , „ ba (a+1) . pore n , .,_. 1 - £ ^ V"2 exPt c— 5 1 (17> a> n-1 9 + 9a + q a R n p with a ^ (18) £ P 3 <n

q the non-zero roots of tan q = =• (19) 3 + a q

S Qo

«O L + £ S p

(20)

Here it is assumed that at equilibrium the concentration of pore liquid and bulk liquid are identical.

Although desorption has been studied less frequently, different models for the rate of desorption have been proposed. It has been assumed that the overall rate of desorption is limited by desorption kinetics [Arnold 1985], internal mass transfer [Katoh 1978] or a combination of both mechanisms [Arve 1988].

Application of the models

Both the general pore diffusion model and the shrinking core model have been applied to describe the uptake of proteins by various adsorbents. A summary is given in Table 2.

The diffusion coefficients used in the pore diffusion models will be discu­ ssed in a subsequent paragraph.

Besides the diffusion models other models have also been used to describe mass transfer. The Linear Driving Force assumption has been used by various authors (Table 2) to describe batch and packed bed adsorption processes. While this model is not too bad for packed bed processes, serious errors can arise when this model is applied to adsorption from a finite bath [Hills 1986].

Finally the use of a pseudo kinetic model (analogous to the Thomas model [Thomas 1944]) should be mentioned. It has been applied succesfully to various adsorption systems. The parameters used in this model lump equi­ librium effects together with mass transfer resistances [Chase 1984].