Characterisation of chromatography adsorbents for

antibody bioprocessing

A thesis submitted to University College London for the degree of Master of

Philosophy

Bela Sheth

Department of Biochemical Engineering

University College London
2009
I, Bela Sheth, confirm that the work presented in this thesis is my own.
Where information has been derived from other sources, I confirm that this
has been indicated in the thesis.

Signature

Date

2
Dedicated to my husband, Michael

3
Acknowledgements

I would like to acknowledge the enormous contribution of my supervisor,

Daniel Bracewell, in helping to develop this project.

For inviting me to visit Millipore (UK), I wish to thank my industrial supervisor,

Jon Huddleston and the whole team at Durham.

For funding this project, I wish to acknowledge the EPSRC and Millipore
(UK).

Finally, I wish to thank my husband, Michael and my parents, Suresh and

Yashoda for their support.

4
 Abstract

Currently the purification of monoclonal antibodies for therapeutic purposes is

reliant on protein A affinity chromatography. The rapid growth of this class of
therapeutic and their high value makes the understanding of protein A
chromatography an important target. There is a range of commercially available
protein A chromatography media. The main differences between these media are
the support matrix type, the pore size, the particle size, the amount of ligand
attached to the matrix and the kind of protein A modification. The differences in
these factors give rise to differences in compressibility, chemical and physical
robustness, diffusion resistance and binding capacity of the adsorbents. The ideal
media would have high specificity, high mass transfer and binding capacity, low
non-specific adsorption and ligand leakage, incompressibility, resistance to alkaline
condition for sanitization, chemical stability and cost effectiveness. Current resins
offer a compromise, which balances what is achievable in respect of these features
giving rise to an array of different solutions. Measurement of these parameters is
often complex and agreed standards have yet to be determined. The objective of
this study is to further develop understanding of these measurements for the
assessment of the matrix performance.

This thesis employs a suite of techniques to characterise commercial and prototype

adsorbents. The adsorbents that will be looked at are MabSelect (GE Healthcare),
MabSelect Xtra (GE Healthcare), Prosep Ultra (Millipore), Protein A immobilised on
4CL Sepharose (GE Heatlhcare) in house and a prototype adsorbent with a Protein
A mimic ligand (Millipore). Both down-scaled techniques of fixed bed
chromatography, together with supporting analysis of equilibrium and dynamic
behaviours are used. The latter will adopt standard and novel ‘wet chemistry’
approaches together with the increasingly adopted techniques of laser scanning
confocal microscopy. Experiments are carried out using hIgG to study the static
capacity, adsorption equilibrium and dynamic capacity of adsorbents. Other
techniques will be used to study the kinetic uptake and desorption rates of
adsorbents in different conditions. A novel approach using confocal microscopy is
used to further understand the adsorption behaviour of individual beads of different
sizes.

5
The main results that were drawn from these techniques are that MabSelect Xtra
had the highest static capacity of 61.8mg/ml. It also showed the highest dynamic
capacity at 2 mins, 4 mins and 8 mins residence time (0.66cm Omnifit column, bed
height 6cm) when compared to other adsorbents. This is mainly due to the more
porous nature of the MabSelect Xtra beads, which increased the surface area
available for Protein A ligand immobilisation. From the adsorption equilibrium data
the Kd values ranged from 181nM to 36nM. Such low values are expected by
affinity adsorbents such as these. The uptake rate curves were similar for all the
adsorbents. Hence the difference in particle size, pore size, the type of ligand or the
material of the adsorbent itself did not have an effect on the uptake rate when
carried out in a batch mode. A similar behaviour was shown for the desorption
curves. The confocal analysis using a flow cell showed that all the adsorbents
showed a shrinking core effect except for the prototype where the hIgG didn’t
penetrate into the bead and was only attached to the surface of the bead. It was
found that the adsorption rate to the centre of each bead was linear. The different
particle sizes within any particular type of matrix and also across different matrix did
not result in different diffusion rates. From the adsorption curves produced it was
seen that smaller beads reached saturation much faster than larger beads at any
given time. This technique can have great benefits in understanding how individual
beads of different adsorbents behave in different circumstances.

6
 Table of Contents

AKNOWLEDGEMENTS 4

ABSTRACT 5

Chapter 1 - Introduction to characterisation of affinity adsorbents for

antibody bioprocessing

1.1 CHROMATOGRAPHY IN BIOPROCESSING 13

1.2 AFFINITY CHROMATOGRAPHY 16

1.3 TYPES OF MEDIA 17

1.3.1 Natural polymers 17

1.3.2 Synthetic polymers 17

1.3.3 Inorganic media 18

1.4 MODES OF CHROMATOGRAPHY 18

1.4.1 Elution 18

1.4.2 Frontal Analysis 18

1.4.3 Displacement 19

1.5 ANTIBODY 19

1.6 PROTEIN A AFFINITY CHROMATOGRAPHY 21

1.6.1 Protein A 21

1.6.2 Commercially available Protein A media 22

1.7 FACTORS EFFECTING PERFORMANCE OF ADSORBENTS 26

1.7.1 Pore size 26

1.7.2 Particle size 26

1.7.3 Support material 27

1.7.4 Protein A modification 27

7
1.7.5 Column Packing 28

1.8 PERFORMANCE MEASUREMENTS 29

1.8.1 Adsorption isotherms 29

1.8.2 Breakthrough curves (Dynamic Capacity) 31

1.8.3 Batch adsorption 33

1.8.4 Shallow beds 33

1.8.5 Desorption 33

1.8.6 Protein A leakage 34

1.8.7 Purity 34

1.8.8 Life Cycle Studies 34

1.9 CONFOCAL MICROSCOPY 35

1.10 CONCLUSION 36

1.11 TARGET OF THE THESIS 36

Chapter 2 – Performance measurement of affinity chromatography

adsorbents

2.1 ABSTRACT 38

2.2 INTRODUCTION 39

2.3 STATIC CAPACITY 39

2.3.1 Materials for determining the static capacity 39

2.3.2 Method for determining the static capacity 40

2.3.3 Results 41

2.4 ADSORPTION EQUILIBRIUM DATA USED TO DETERMINE

MAXIMUM CAPACITY AND DISSOCIATION CONSTANT 41

2.4.1 Materials for adsorption equilibrium experiments 41

2.4.2 Method for adsorption equilibrium experiments 42

2.4.3 Results 43

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2.5. DYNAMIC CAPACITY 48

2.5.1 Material for determining dynamic capacity 48

2.5.2 Method for determining dynamic capacity 49

2.5.3 Results 50

2.6 BATCH UPTAKE RATE 51

2.6.1. Method to determine uptake rate 51

2.6.2 Results 56

2.7 DESORPTION KINETICS 58

2.7.1 Method to analyse desorption rate 58

2.7.2 Results 59

2.8 DESORPTION AT 4OC 60

2.8.1 Method to analyse desorption kinetics at 4oC 60

2.8.2 Results 61

2.9 DISCUSSION 61

2.10 CONCLUSION 63

Chapter 3- Confocal analysis using flow cell

3.1 ABSTRACT 65

3.2 INTRODUCTION 66

3.3 MATERIALS AND METHOD 69

3.3.1. Materials 69

3.3.2 Equipment 69

3.4. METHOD 70

3.4.1 Sample preparation 70

3.4.2 Equipment setting 70

3.4.3 Analysis 72

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3.5 RESULTS AND DISCUSSION 74

3.5.1 Visual images 74

3.5.2 Diffusion rate 75

3.5.3 Adsorption rate 77

3.6 CONCLUSION 78

Chapter 4- Future Work

4.1 INTRODUCTION 80

4.2 LIGAND DENSITY 80

4.3 USE OF OPTICAL BIOSENSOR TECHNOLOGY 80

Chapter 5 - Thesis conclusion

5.1 SUMMARY OF THE THESIS 82

REFERENCES 84

APPENDIX 88

LIST OF FIGURES

Fig 1.1 Five major steps in the recovery of biomolecules 13

Fig 1.2 Projected annual production of monoclonal antibodies 15

Fig 1.3 Stages during the affinity chromatography process 17

Fig 1.4 Structure of IgG molecule 20

Fig 1.5 Schematic drawing of regions encoded by the gene for Staphylococcal

protein A 21

Fig 1.6 C-terminal cysteine favours oriented thioether coupling 27

Fig 1.7 A typical Van Deemter Plot 29

Fig 1.8 Types of adsorption isotherms 31

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Fig 1.9 Breakthrough curve 32

Fig 1.10 Flow cell set up used to view labelled individual beads under a

confocal microscope 35

Fig 2.1 Hanes Plot – MabSelect 44

Fig 2.2 Adsorption Equilibrium – MabSelect 44

Fig 2.3 Hanes Plot – MabSelect Xtra 45

Fig 2.4 Adsorption Equilibrium – MabSelect Xtra 45

Fig 2.5 Hanes Plot – Prosep Ultra 46

Fig 2.6 Adsorption Equilibrium – Prosep Ultra 46

Fig 2.7 Hanes Plot – CIGL 47

Fig 2.8 Adsorption Equilibrium – CIGL 47

Fig 2.9 Dynamic capacity determined at 2min, 4min and 8min residence time 50

Fig 2.10 Uptake rate equipment setup 51

Fig 2.11 Response time for the system. This equilibration time (response time) is

recorded at 4ml/min and 6ml/min flow rate which shows to be ~20-25sec 53

Fig 2.12 Uptake kinetic curves for two samples of CIGL 55

Fig 2.13 Uptake kinetic curves for two samples of CIGL where maximum capacity

at the end of adsorption for each sample equals to 1 55

Fig 2.14 Uptake kinetic curves for MabSelect, MabSelect Xtra, Prosep Ultra

and CIGL 56

Fig 2.15 Uptake kinetic curves for MabSelect, MabSelect Xtra, Prosep Ultra and

CIGL where maximum capacity at the end of adsorption for each media equals to 1 56

Fig 2.16 Uptake kinetic curves for MabSelect and MabSelect Xtra using higher

concentration of hIgG (3mg/ml) and an impeller speed of 145rpm 57

Fig 2.17 Normalised version of uptake kinetic curves for MabSelect and

MabSelect Xtra using higher concentration of hIgG (3mg/ml) and an impeller

speed of 145rpm 57

Fig 2.18 Desorption kinetic curves for two samples of CIGL 59

Fig 2.19 Desorption kinetic curves for MabSelect, MabSelect Xtra, Prosep Ultra and

CIGL 60

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Fig 2.20 Desorption carried out on CIGL and MabSelect Xtra at 4oC 61

Fig 3.1 Flow cell with a channel of diameter 0.1cm and a length of 1cm was used

to pack ~8µl of media, which could be placed under an inverted confocal microscope 70

Fig 3.2 Flow cell setting under confocal microscope. 2mg/ml hIgG was passed through

the channel at 0.1ml/min flowrate with a syringe pump 71

Fig 3.3 Image of beads seen under the microscope at 30 mins. The images of the

chosen beads are from the centre and of different sizes (50µm and 70µm) 72

Fig 3.4 Intensity profile across a small bead (~50µm) and a large bead (~70µm) at

A; 10mins, B; 20mins and C; 30 mins time intervals 73

Fig 3.5 Visual Images of Mabselect, MabSelect Xtra, Prototype and Protein A

immobilised on 4CL Sepharose media taken at 10, 20 and 30 mins in a flow cell 75

Fig 3.6 Adsorption rate for different size beads for two different samples of MabSelect,

1 sample of MabSelect Xtra and Protein A immobilised on 4CL Sepharose 76

Fig 3.7 Uptake curves for different size beads of MabSelect Xtra plotted from data

calculated from intensity profiles 78

LIST OF TABLES

Table 1.1 Approved monoclonal antibodies available in the market 14

Table 1.2 Commercially available native, recombinant and mimic Protein A affinity

chromatography media 22

Table 2.1 Static capacity for MabSelect, MabSelectXtra, Prosep Ultra and CIGL 41

Table 2.2 A summary of maximum capacity and dissociation constant determined

using Hanes Plot for MabSelect, MabSelect Xtra, Prosep Ultra and CIGL 48

Table 2.3 A summary of maximum capacity and dissociation constant determined

using Solver function in Excel for MabSelect, MabSelect Xtra, Prosep Ultra and CIGL 48

Table 2.4 Summary of dynamic capacity determined at 2min, 4min and 8min

residence time for MabSelect, MabSelect Xtra, Prosep Ultra and CIGL 50

Table 3.1 Diffusion rate for different size beads and for different media 77

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Chapter 1 - Introduction to characterisation of
affinity adsorbents for antibody bioprocessing

1.1 Chromatography in Bioprocessing

The centre focus of bioprocessing is using living cells to make desired products,
which is commonly carried out in a bioreactor. Downstream processing from this
reactor gives concentrated and purified products. These products can be
therapeutic proteins such as factor VIII and urokinase. It could also be diagnostic
enzymes, insulin, monoclonal antibodies, dehydrogenase, antibiotics, proteases or
amylases. In the recovery of biomolecules 5 stages can be distinguished. These
can be seen in the figure below (Krijgsman, 1995).

Cell disruption
Stabilisation
Pre-treatment Sterilisation
Pasteurisation
Flocculation

Filtration
Solid/Liquid
Separation Sedimentation
Centrifugation

Membranes
Precipitation
Concentration
Evaporation
Extraction
Freeze Concentration

Precipitation
Extraction
Purification
Diafiltration
Adsorption
Chromatography

Drying
Prilling
Formulation
Extrusion
Granulation
Tabletting

Fig 1.1 - Five major steps in the recovery of biomolecules

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Just focusing on the antibody based therapies, the current global antibody pipeline, which comprises of more than a hundred antibodies in
development, was ready to deliver 16 new products by 2008 (Swinnen et al., 2007). The table below shows some of the approved monoclonal
antibodies present in the market (Shukla et al., 2007).

Table 1.1 - Approved monoclonal antibodies available in the market

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The graph below shows the projected annual production of monoclonal antibodies
(Low et al., 2007).

18
Projected annual production (t)

16
14
12
10
8
6
4
2
0
2006 2007 2008 2009 2010
year

Fig 1.2 - Projected annual production of monoclonal antibodies. The numbers are in metric
tonne.

In response to increasing market demands and pressure for cost reduction, the
biotech industry has successfully increased bioreactor volumes up to 20,000 L
(Swinnen et al., 2007). However process bottleneck has always been downstream
and purification costs are outweighing cell culture costs.

This chapter mainly gives an introduction to chromatography focusing further on

affinity chromatography that is used to purify monoclonal antibodies. Protein A
chromatography is widely used to purify monoclonal antibodies mainly because of
its high selectivity. To meet the demands with the increasing bioreactor volumes
and cell culture expression levels an optimum use of expensive Protein A affinity
resin (€6000–€9000/L resin) can have significant benefits (Swinnen et al., 2007).

Despite Protein A affinity chromatography being a predominant mAb capture step in

bioprocessing it has several limitations. Firstly the high cost of the resins. Secondly
the differences in the dynamic binding capacity at various flow rates and in the
pressure-flow characteristics of various Protein A chromatographic media can result
in wide variations in throughput (Shukla et al., 2007). A Protein A step also adds
impurity in the process in the form of leached Protein A ligands. Another major
limitation is the inability to increase large-scale column diameter to beyond 2m

15
without encountering significant issues with flow distribution and packing (Shukla et
al., 2007). There is a ready market for the company that can produce a Protein A
resin with a dynamic binding capacity of >50 g/L at an even lower residence time
(Low et al., 2007). Some of the options that are currently being investigated include
higher ligand densities on their resins, ligand orientation/accessibility, particle size,
pore size and distribution, alternative protein A mimic synthetic ligands and more
stable support matrices with increased mass transfer.

1.2 Affinity Chromatography

Affinity chromatography involves the use of ligands that attach to the media and
that have binding affinity to specific molecules or a class of molecules.
Ligands can be bio-molecules, like protein ligands or can be synthetic molecules.
Both types of ligand tend to have good specificity. But protein ligands have the
disadvantage that they are expensive and mostly denature with the use of cleaning
solutions, whereas synthetic ligands are less expensive and more stable.
The most commonly used protein ligand in production is the protein A ligand, which
is specific to the IgG antibody (Section 1.6).
In affinity chromatography when the solution is introduced to the column the target
protein is adsorbed while allowing contaminants (other proteins, lipids,
carbohydrates, DNA, pigments, etc.) to pass through the column (Fig 1.3). The
adsorbent itself is normally packed in a chromatography column; though the
adsorption stage can be performed well by using the adsorbent as stirred slurry in
batch binding mode. The next stage after adsorption is the wash stage, in which the
adsorbent is washed to remove residual contaminants. The bound protein is then
eluted in a pure form. Elution is normally achieved by changing the buffer or salt
composition so that the protein can no longer interact with the immobilized ligand
and is released. Affinity chromatography can be performed in a fixed bed or a
fluidised bed.

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Fig 1.3 – Stages during the affinity chromatography process that includes adsorption, wash
and desorption

1.3 Types of media

The support to which the ligands can be attached to can be of different materials.

1.3.1 Natural polymers

Natural polymers can be of agarose, cellulose or dextran. Their most beneficial

point is that these materials have very low non-specific adsorption. This is because
the polymer chains are very hydrophilic due to the presence of many hydroxyl
groups and hence the proteins do not adhere to them.
Dextran and agarose have better flow properties than cellulose. Fibrous cellulose is
extremely hard to pack (Jaunbauer et al., 2005). Its usefulness is limited by its
fibrous and non-uniform character. The main disadvantage of dextran is its low
degree of porosity. Agarose beads on the other hand are extremely soft and
therefore cross-linked to increase the strength. However due to their loose structure
it allows ready penetration by macromolecules with molecular weights in the order
of several millions (Lowe et al., 1974). The uniform spherical shape of the particles
in particular gives good flow properties.

1.3.2 Synthetic polymers

The three synthetic polymers of importance are hydrophobic vinyl polymers,

polyacrylamide and polyvinylstyrene.

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All of these are relatively hydrophobic and hence needs to be modified by coating
the surface with a hydrophillic polymer to avoid low recovery (Jaunbauer et al.,
2005). An advantage of the synthetic polymer-based media is their resistance to
extreme chemical conditions, such as pH.
The main disadvantage with polystyrene adsorbents is their relatively low porosity.
Polyacrylamide beads on the other hand are superior to many polymeric supports
due to their polyethylene backbone, which increases chemical stability. They tend
to have a more uniform physical state and porosity, permitting the penetration of
macromolecules with molecular weights of upto 500,000 (Lowe et al., 1974).
Another advantage is that they posses many modifiable groups which enables the
covalent attachment of a variety of ligands.

1.3.3 Inorganic media

The commercially available inorganic media are made of ceramic, silicate or glass.
The silicate media are coated with several other materials. The most common glass
media used consists of irregularly shaped controlled porous glass (CPG) particles.
These adsorbents are rigid and can work at high flow rates. Due to their controlled
pore size they tend to generate sharp exclusion limits (Lowe et al., 1974).

1.4 Modes of Chromatography

1.4.1 Elution

Elution is carried out by first introducing a small sample of the mixture on to the
column. It is then eluted with a mobile phase, which has a lesser affinity to the
stationary phase than the sample components. The components then move along
the column depending on their relative affinity for the stationary phase but at a
slower rate than the eluent. Hence the components can be completely separated
with a zone of mobile phase between them. This mode of chromatography is
commonly used for analytical purposes (Braithwaite et al., 1985).

1.4.2 Frontal Analysis

Frontal analysis is executed by continuously adding the sample onto the column.
The component with the least affinity for the stationary phase will pass along the

18
column while the component with the greater affinity will get adsorbed to the
stationary phase. Eventually this component will pass along the column, when the
capacity limit of the stationary phase is exceeded. This mode of chromatography is
used to achieve breakthrough curves (Section 1.8.2).

1.4.3 Displacement

Displacement is carried out by first introducing the sample mixture onto the column.
Elution then occurs when a displacing solvent is passed through the column, which
has a greater affinity for the stationary phase than the sample components.

1.5 Antibody

Antibodies are protein molecules that play a crucial role in the immune system,
which defends the body against toxins that enter the bloodstream and counters the
disease threat posed by invading microbes and viruses. Antibodies are made in
white blood cells, B-lymphocytes. Each B-lymphocyte makes copies of its own
unique antibody, which are then displayed on its outer surface. Immunoglobin G is
the major antibody in serum. It has a Y-shaped structure composed of four protein
subunits which comprises of two identical light chains and two identical heavy
chains as shown in Fig 1.4.

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 N N N N

Fab Light Chain

S-S S-S

C C
S-S

Fc

Heavy Chain

C C

Fig 1.4 – Structure of IgG molecule (L. Stryer 1995)

Monoclonal antibodies can be used therapeutically, to protect against diseases;

they can also help to diagnose a wide variety of illnesses, and can detect the
presence of drugs, viral and bacterial products, and other unusual or abnormal
substances in the blood. Their specificity makes monoclonal antibody technology so
valuable. Given such a diversity of uses for these disease-fighting substances, their
production in pure quantities has long been the focus of scientific investigation. The
monoclonal antibody (mAb) market has grown rapidly in recent years, reaching
sales of $14bn in 2005, an increase of 36.5% from 2004 sales of $10.3bn.
Monoclonal antibodies are homogenous because they are synthesised by a
population of identical cells (a clone). Each such population is descended from a
single hybridoma cell formed by fusing an antibody producing cell with a tumour cell
that has the capacity for unlimited proliferation. Hence it possesses a structure that
can only bind to one epitopal group on one antigen. Polyclonal antibodies, in
contrast with monoclonal ones are products of many different populations of
antibodies-producing cells and hence differ somewhat in their precise specificity
and affinity for the antigen. Hence a polyclonal antibody mixture has typically most
or all of the antibodies acting against all epitopal groups for antigens on the
molecule.

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1.6 Protein A affinity chromatography

1.6.1 Protein A

Protein A affinity chromatography is widely applied in the commercial production of

IgG. Protein A, which has a molecular weight of 42,000, is a cell wall protein from
Staphlococcus aureas with affinity for the Fc region of IgG. The amino-terminal
region contains five highly homologous IgG-binding domains (Fig 1.5). All five IgG-
binding domains of SpA bind to IgG via the Fc region (Jansson et al., 1993).
Binding of the antibody is normally done at pH 7 and elution at pH 2-3 (Jungbauer
et al., 2005). The two binding domains at the C-terminal are non-immunoglobulin
binding regions and are thought to be responsible for the binding of Protein A to the
bacterial cell wall.

Fig 1.5 - Schematic drawing of regions encoded by the gene for Staphylococcal protein A. S
is the signal sequence. E, D, A, B and C are the immunoglobulin binding regions. Xr and Xc
are C-terminal located, non-immunoglobulin binding regions (Uhlen et al., 1984).

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1.6.2 Commercially available Protein A media

There are various commercially available protein A chromatography media. The main differences between these media are the support matrix
type, protein A ligand modification, the pore size and the particle size. The differences in these factors give rise to differences in compressibility,
chemical and physical robustness, diffusion resistance and binding capacity of the adsorbents (McCue et al., 2003). Products of different
manufacturers were researched to arrive at this table.

Adsorbent Manufacturer Matrix type Ligand type Particle/Pore size Static/dynamic Adsorbent special features
capacity
nProtein A Sepharose 4 GE Healthcare, UK Crosslinked Native protein A Particle size – Static capacity – 2 times total IgG capacity than Protein
Fast Flow agarose – 4% 90µm 35mg/ml A Sepharose CL-4B. Replaces former
Protein A Sepharose 4 Fast Flow
rProteinA Sepharose Fast GE Healthcare, UK Crosslinked Recombinant Protein A. Particle size – 60- Static Capacity – Much higher dynamic capacity.
Flow agarose – 4% C-terminal Cysteine 165µm 50mg/ml Thioether coupling providing single
attached to rProtein A point attachment of the protein A.
Oriented coupling enhances the binding
of IgG
rmp Protein A Sepharose GE Healthcare, UK Crosslinked Recombinant Protein A. Particle size – 60- Static capacity – Low ligand leakage. Increase chemical
Fast Flow agarose – 4% Multipoint ligand 165µm 35mg/ml stability due to multipoint ligand
attachment attachment
Protein A Sepharose CL- GE Healthcare, UK Crosslinked Native protein A Particle size – Static capacity – NG
4B agarose – 4% 90µm 20mg/ml

HiTrap rProtein AFF GE Healthcare, UK Crosslinked Recombinant Protein A Particle size – Static Capacity – Pre packed Column – 1ml and 5ml
agarose – 4% C-terminal Cysteine 90µm 50mg/ml
attached to rProtein A
Streamline rProtein A GE Healthcare, UK Crosslinked Recombinant Protein A. Particle size – 80- Static Capacity – Expanded bed adsorption. Capture IgG
agarose – 4%. C-terminal Cysteine 165µm 50mg/ml directly from unclarified feed stock.
Modified with inert attached to rProtein A
metal alloy core for
providing required
high density
MabSelect GE Healthcare, UK Crosslinked NG Particle size – Dynamic Large feed volumes – more than
agarose 85µm capacity – 10000L
30mg/ml

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 Adsorbent Manufacturer Matrix type Ligand type Particle/Pore size Static/dynamic Adsorbent special features
capacity
MabSelect Sure GE Healthcare, UK Crosslinked Alkali stabilised Protein Particle size – Dynamic Increase chemical stability during
agarose A 85µm capacity – sanitization
30mg/ml

MabSelect Xtra GE Healthcare, UK Crosslinked NG Particle size – Dynamic High dynamic capacity as adsorbent
agarose 75µm capacity – more porous than other MabSelect
40mg/ml family adsorbents

IPA-300 RepliGen Corp., Crosslinked NG NG NG NG

Waltham, MA, USA agarose

IPA - 400 RepliGen Corp., Crosslinked NG NG NG NG

Waltham, MA, USA agarose

IPA - 500 RepliGen Corp., Crosslinked NG Particle size – NG Modified to double binding capacity
Waltham, MA, USA agarose 90µm

Affi-Gel Protein A Gel Bio-Rad, Hercules, CA, USA Crosslinked Native protein A NG Static Capacity- NG
agarose 20mg/ml
Affi-Prep Protein A Bio-Rad, Hercules, CA, USA Polymeric – NG NG Static Capacity- Used for medium to high pressure
Macroporous 16-23 mg/ml
matrix
MAbsorbent A2P Affinity Chromatography Ltd. Crosslinked Synthetic ligand Particle size - Static Capacity – Use of Synthetic ligand – less
(Prometic Biosciences), agarose – 6% (aromatic triazine ~100µm 50mg/ml expensive than Protein A ligands. Only
Freeport, Isle of Man, UK derivative)- mimic used for humanized or human antibody.
recombinant and native
Protein A.
MAbsorbent A1P Affinity Chromatography Ltd. Crosslinked Synthetic ligand Particle size - Static Capacity – Use of Synthetic ligand – less
(Prometic Biosciences), agarose – 6% (aromatic triazine ~100µm 50mg/ml expensive than Protein A ligands. Only
Freeport, Isle of Man, UK derivative)- mimic used for murine antibody.
recombinant and native
Protein A.
Protein A Ceramic Pall corporation Polyacrylamide gel Recombinant Protein A Particle size - Dynamic Rigid. Less back pressure issues.
HyperD F 2200 Northern Boulevard in ceramic 50µm Capacity –
East Hills, USA macrobead 30mg/ml

23
 Adsorbent Manufacturer Matrix type Ligand type Particle/Pore size Static/dynamic Adsorbent special features
capacity
Poros A 50 High Capacity PerSeptive Biosystems, Polystyrene NG Particle size – NG Discrete classes of pores. Large
Framingham, MA, USA divenylbenzene 50µm ”through pores” allow convection flow
Pore size – 50 to through particles, quickly carrying
1000nm molecules to small ”diffusive pores”
inside. Used large scale for low
pressure application

Poros A 20 PerSeptive Biosystems, Polystyrene NG Particle size – Discrete classes of pores. Large
Framingham, MA, USA divenylbenzene 20µm ”through pores” allow convection flow
Pore size – 50 to through particles, quickly carrying
1000nm molecules to small ”diffusive pores”
inside. Used samller scale for high
resolution.
UltraLink Immobilized Pierce, Polymeric Native protein A Particle size–50 to Static Capacity – Rigid – Used for high pressure
Protein A Plus Rockford, IL, USA 80µm 30 mg/ml Higher capacity than UltraLink
Pore size-100nm Immobilized Protein A

UltraLink Immobilized Pierce, Polymeric Native protein A Particle size – 50 to Static Capacity – Rigid – Used for high pressure
Protein A Rockford, IL, USA 80µm 16 mg/ml
Pore size – 100nm
Agarose Protein A Pierce, Crosslinked NG NG Static Capacity – NG
Rockford, IL, USA agarose – 6% 12-19 mg/ml

Agarose Protein A Plus Crosslinked NG NG Static Capacity – Higher capacity than Agarose Protein A
Pierce, agarose – 6% 34 mg/ml
Rockford, IL, USA

Prosep-vA Ultra Miilipore Bioprocessing, Porous glass Native Protein A Particle size - Static Capacity – Rigid, incompressible, low back
Consett, UK ~100µm 56 mg/ml pressure and operate at high flow rates.
Pore size – 70nm
Prosep-vA High Capacity Miilipore Bioprocessing, Porous glass Native Protein A Particle size - Static Capacity – Rigid, incompressible, low back
Consett, UK ~100µm 40 mg/ml pressure and operate at high flow rates.
Pore size – 100nm
Protein A Cellthru 300 Sterogen Bioseparations, n.a. Agarose content – Recombinant Protein A Particle size – 200 Static capacity – Used for unclarified, viscous feed
200–300 n.a. 4% to 300µm 35mg/ml stream
Carlsbad, CA, USA Dynamic
capacity –
28mg/ml

24
 Adsorbent Manufacturer Matrix type Ligand type Particle/Pore size Static/dynamic Adsorbent special features
capacity
Protein A Ultraflow Sterogen Bioseparations, n.a. Agarose content – Recombinant Protein A Particle size – 40 to Static capacity – NG
200–300 n.a. 4% 160µm 50mg/ml
Carlsbad, CA, USA Dynamic
capacity –
40mg/ml

Table 1.2 – Commercially available native, recombinant and mimic Protein A affinity chromatography media

25
1.7 Factors effecting performance of adsorbents

The ideal features of a chromatographic media would be high specificity, high mass
transfer, high binding capacity, low non-specific adsorption, incompressibility,
resistance to alkaline condition for sanitization, chemically stability, low ligand
leakage and cost effectiveness (Jungbauer et al., 2005). There are various factors
that affect these features.

1.7.1 Pore size

Smaller pores mean an increase in the surface area available for protein A
immobilisation. Previous studies have shown that Prosep vA-Ultra (pore size 70nm)
has a higher static capacity than Prosep vA-High capacity (pore size100nm) due to
the fact that it has got smaller pores (McCue et al., 2003). Smaller pores also
means a much stronger structure and hence the media being less compressible.
But the disadvantage would be an increase in the diffusion resistance. Hence the
time taken to reach the static capacity would be slower for adsorbents with smaller
pores when compared to adsorbents with larger pores (McCue et al., 2003). On the
other hand larger pores would mean less diffusion resistance but a decrease in
protein A immobilisation and a more compressible structure (Hahn et al., 2005).

1.7.2 Particle size

Smaller particle size would mean better mass transfer and better access to internal
bead volume. This is because pore diffusivity depends on the square of the particle
diameter (Hahn et al., 2003). But there would be a higher pressure drop across a
packed bed due to the smaller particle size. On the other hand larger particles
would mean lower pressure drop across a packed bed, but the mass transfer and
efficiency would be poorer. This was also shown in previous studies. Poros 50
(particle size 45-50Nm) showed a higher dynamic capacity than prototype Poros LP
(particle size 55-60Nm). But Poros LP had a lower pressure drop (higher
permeability) at higher flow rates when compared to Poros 50 (Fahrner et al.,
1999).

26
1.7.3 Support material

The material used for the matrix can affect the performance. The different kinds of
support materials used were given in Table 1.2. The main factor that is affected by
the support material is the compressibility of the media. Previous studies have
shown that agarose adsorbents are more compressible than porous glass
adsorbents (Stickel et al., 2001). Pressure-flow profiles of different adsorbents
showed that non-linear profiles are characteristics of compressible adsorbents.
Linear pressure - flow curves, on the other hand, as that of Prosep-rA adsorbent
are characteristics of incompressible adsorbents. Hence depending on the matrix
type adsorbents can be used at higher flow rates without being compressed.

1.7.4 Protein A modification

Ligands are always exposed to the harsh conditions of operational cycle especially
to the intermediate cleaning and sanitization procedures. The way the Protein A
ligand is modified makes a difference in the performance of the adsorbent. Many
companies tend to use the native form of Protein A. But many companies also tend
to use recombinant Protein A with specific modifications. Three examples are
described below.
Firstly, In the case of rProteinA Sepharose Fast Flow (GE Healthcare) the
recombinant protein A has been engineered to include a C-terminal cysteine. The
coupling conditions are controlled to favour a thioether coupling providing single
point attachment of the protein A (Fig 1.6). The oriented coupling enhances the
binding of IgG thus increasing the dynamic capacity of the adsorbent.

Fig 1.6 – C-terminal cysteine favours oriented thioether coupling (GE Healthcare)

Secondly, in the case of rmp Protein A Sepharose Fast Flow (GE Healthcare) a
recombinant protein A is used with a molecular weight of 44 600. It contains five
antigen-binding domains, which allow multiple attachment points to the Sepharose

27
Fast Flow support. The attachment is carried out through reductive animation,
which creates chemically stable amide bonds. This results in less ligand detaching
from the support during elution and cleaning, and hence less Protein A leakage.
Lastly, in the case of MabSelect Sure (GE Healthcare) the recombinant protein
used is modified such that the amino acids sensitive to alkali were identified and
substituted with more stable ones. This made the adsorbent more stable to alkali
and hence more stable during cleaning and sanitization.
There are also several Protein A mimics available in the market which are synthetic
ligands and hence less expensive than the native form. One example is the
MAbsorbent A2P by Prometic Biosciences.

1.7.5 Column Packing

To obtain an optimal separation, sharp symmetrical chromatographic peaks must

be obtained. A theoretical plate model is used to understand the processes going
on inside the column and understand the column efficiency. The plate model
supposes that the chromatographic column contains a large number of separate
layers, called theoretical plates. The efficiency of the column can be expressed in
terms of theoretical plates in a column N (the more plates the better), or by stating
the plate height, the Height Equivalent to a Theoretical Plate (HETP - the smaller
the better).

The HETP is calculated by the following formula: HETP = L / N where L is the

length of the column. A more realistic understanding of the processes at work inside
a column is using the Van Deemter Equation. The resulting band shape of a
chromatographic peak is affected by the rate of elution. It is also affected by the
different paths available to solute molecules as they travel between particles of
stationary phase. If we consider the various mechanisms, which contribute to band
broadening, we arrive at the Van Deemter equation for plate height (Deemter et al.,
1956);

HETP = A + B / u + C u

Here u is the average velocity of the mobile phase and A, B and C are factors which
contribute to band broadening. The constant A refers to eddy diffusion, B refers to
longitudinal diffusion and C refers to resistance to mass transfer. Below is a typical

28
Van Deemter Plot. Such plots are of considerable use in determining the optimum
mobile phase flow rate.

Fig 1.7 – A typical Van Deemter Plot (Deemter et al., 1956)

1.8 Performance measurements

There are various tests that can be carried out to evaluate the performance of the
adsorbents. It is generally not possible to reconcile all the ideal features and a
compromise would need to be made. It is then up to the manufacturer to decide
which criteria are more important in the manufacturing of the adsorbents.

1.8.1 Adsorption isotherms

When the antibody solution is in contact with the adsorbent an equilibrium is

achieved between the antibodies in the solution and the antibodies bound to the
adsorbent.

Any concave adsorbent isotherm is favourable which means strong adsorption

occurs even in dilute solution. Any convex adsorbent isotherm on the other hand is
said to be unfavourable. Fig 1.8 shows the different types of adsorption isotherms.

29
• Linear isotherm – Linear isotherms are not very common in bioseparation
(Belter et al., 1988) and are given by q*= KdC*, where q* is the amount of solute
adsorbed per amount of adsorbent, C* is the solute concentration in solution
and Kd is the equilibrium dissociation constant.

• Freundlich isotherm – This isotherm is normally used to describe adsorption of a

wide variety of antibiotics, steroids and hormones (Belter et al., 1988) and is
given by q*=KdC*n, where n is a constant.

• Langmuir isotherm – This isotherm is often used to describe adsorption data for
proteins. This is given by;

q* = (QmC*)/(Kd+C*)

Where Qm is the maximum amount of solute that can be adsorbed per amount
of adsorbent. The Kd value gives information on the selectivity of the affinity
system. Kd value of ~ 5 x10-7M is typical for a strong, highly selective affinity
system (Hahn et al., 2005).

The Langmuir isotherm is derived from kinetic mechanism. It is based on the

following hypotheses:

• The surface of the adsorbent is uniform; hence all the adsorption sites are
uniform.
• Adsorbed molecules do not interact.
• All adsorption occurs through the same mechanism.
• Only a monolayer of adsorbed molecules is formed. They do not deposit on
other adsorbed molecules.

30
 Freundlich

Langmuir

Adsorbed Concentration q* Linear

Unfavourable

Solution Concentration C*

Fig 1.8 - Types of adsorption isotherms, which are Freundlich, Langmuir, Linear and
Unfavourable

1.8.2 Breakthrough curves (Dynamic Capacity)

Breakthrough curves give an indication of when the column is completely saturated.

When this happens, the concentration of target protein going in equals to the
concentration of target protein coming out. It gives an indication to when to stop the
loading depending on how much bed remains unused and how much of the product
is lost (Fig 1.9). Excessive loading would give rise to loss of some product, whereas
insufficient loading would leave the column under utilized (Bailey et al., 1986).

31
 Unused column capacity

100%
Concentration

CBT % Breakthrough

Feed “wasted”
0
Feed Volume

Fig 1.9 – Breakthrough curve

The gradient of the curve gives an indication of how sepecific the binding is. For
example, steeper the curve the more specific the binding.
Breakthrough curves are normally used to measure the dynamic capacity of a
media. The column is loaded with protein solution at a specific concentration and
flow rate. The loading is stopped at a specific percentage breakthrough and the
protein is eluted to get the dynamic capacity.
Dynamic capacity can be increased by either decreasing the flow rate and hence
increasing the residence time in the column or by increasing the column height,
which also increases the residence time in the column (Hahn et al., 2003, Hahn et
al., 2005).
Increasing the flow rate normally decreases the dynamic capacity. This is mainly
due to the fact that using a faster flow rate decresaes the rate of mass transfer of
the protein to the interior adsorption sites of the matrix. In other words, less protein
would bind to the adsorbents. The extent of the decrease in the mass transfer rate
depends on the particle size and the pore size of the adsorbents. When comparing
different types of media, adsorbent that tend to show good mass transfer properties
will have a higher dynamic capacity at higher flow rates. However some media
which have poorer mass transfer properties might have a higher equilibrium binding
capacity overall and will tend to show a higher dynamic capacity at longer residence
time (Hahn et al., 2003).
In order to decrease the breakthrough time a load with higher concentration can be
used. The distribution of protein adsorbed in the resin is determined by the

32
intraparticle diffusion rate of the protein into the matrix, which depends on the
concentration gradient of the protein and the resin porosity (Pan et al., 2005). When
the feed concentration increases, it results in the solute shooting into the interior
adsorption sites of the matrix, which leads to the higher adsorption capacity at
larger initial feed concentration.

1.8.3 Batch adsorption

Batch adsorption mostly involves the mixing of the adsorbent with the protein
solution. Leaving it for an extended amount of time (24hours) would give an
indication of the static capacity of the adsorbent. Whereas drawing samples at
specific intervals and measuring the concentration would give the uptake kinetics
for the adsorbent. One of the accurate ways to carry this out would be to recirculate
the stream through a spectrometer (Chang et al., 1998, McCue et al., 2003). But,
again, this method causes a time delay and might not be suitable for media that
reaches its static capacity in the first few minutes of mixing. Hence other methods
like replacing the external loop with a dip probe can be considered (Tscheliessnig et
al., 2005). Confocal microscopy can also be very useful in understanding the kinetic
uptakes of the adsorbent (Section 1.9). Batch adsorption kinetics can give a very
good indication of the mass transfer properties and static capacity of adsorbents.

1.8.4 Shallow beds

This is commonly performed by using a micro-column with approximately 5–20 µl of

adsorbent (Lewus et al., 1998, Hahn et al., 2005). The IgG solution is passed
through the column at a specific flow rate. Desorption is then carried out to get the
mass of IgG adsorbed to the media. The kinetic uptake can then be constructed by
varying the contact time.

1.8.5 Desorption

There are a few papers available on adsorption uptake kinetics, but hardly any on
desorption of IgG when using Protein A ligand. The rate at which desorption takes
place for different adsorbents might give a lot of information about the media in
terms of mass transfer. Batch desorption kinetics can be performed in a similar way
to batch adsorption. When desorption is carried out in dynamic mode it gives an

33
idea of the amount of IgG that might still be bound to the adsorbent. Whereas the
peak of the eluent can show how concentrated the eluent might be. This is a factor
that needs to be taken into consideration as a very high concentration might lead to
aggregation of the antibody and hence it might be favourable to have broader
elution peaks.

1.8.6 Protein A leakage

In order to ensure product safety it is necessary to have a minimum amount of

protein A leakage in the eluent. Protein A leakage is mostly measured by using
Protein A ELISA to get the protein A concentration in the eluent (Hahn et al., 2006).

1.8.7 Purity

In order to understand the selectivity of the adsorbent, using just pure IgG solution
is not sufficient, adding impurities to the feed will give a better understanding. The
entire chromatographic cycle can be carried out by adding impurities to the protein
solution for example 2.5% Fetal calf serum (Hahn et al., 2003) or cell culture
supernatant (Hahn et al., 2006). The eluent can then be tested by SDS /Page to
measure its purity.

1.8.8 Life Cycle Studies

Having an indication of how many cycles an adsorbent can perform without it

affecting its performance can be very important for economic reasons. This can be
done by performing repetitive purification cycles, which includes loading, washing,
elution, regeneration and CIP steps. Factors like protein A leakage, host cell
protein in the eluent and dynamic capacity can then be studied. The type of protein
ligand used, has a great influence on the amount of protein A leakage. Previous
studies have shown that MabSelect Sure showed the least amount of protein A
leakage when compared to MabSelect Xtra and Prosep vA-Ultra due to its alkaline
stabilized protein A ligand (Hahn et al., 2006). The capacity can be studied by
measuring the IgG concentration in the eluent or also by using laser scanning
confocal microscopy, which can give an indication of adsorbent fouling.

34
1.9 Confocal Microscopy

Dynamic capacity and kinetic adsorption have been studied by using packed
column or batch adsorption experiments as mentioned in section 1.8.2 and 1.8.3.
Confocal microscopy, on the other hand, can be used to analyse the adsorption of
IgG on a single particle.
In confocal microscopy the specimen is fluorescently labelled to be detected by the
confocal scanning microscope. The depth discriminating property of the confocal
microscopy can be used to carry out virtual optical sectioning of a specimen.
Adsorption and kinetic properties of the media can be studied by attaining images
of a single particle by incubating the adsorbent for different time intervals in
fluorescently labelled IgG solution (Ljunglof et al., 1996). In order to get images in a
more dynamic condition; flow cells (Fig 1.10) can be used (Dziennik et al., 2003).
Here the particles are packed behind a coverslip and the adsorbent is kept in place
with the help of a frit and the fluorescently labelled IgG feed solution is passed
through the adsorbent with the help of a pump.

Cell body
Adsorbent

Feed input

Fig 1.10 – Flow cell set up used to view labelled individual beads under a confocal
microscope

These images give an indication of where the IgG molecules tend to bind on a
single particle at different time intervals, whether on the surface or more in the
interior of the porous particles. A similar procedure can be used to get dynamic
desorption images.
Confocal microscopy can also be used to get an idea of adsorbent fouling by taking
samples of adsorbent from different chromatographic cycles and incubating them in
fluorescently labelled IgG and viewing the particles under a confocal microscope
(Siu et al., 2006, Thillaivinayagalingam et al., 2007). Confocal microscopy can also

35
be used to see where the ligand is attached to the adsorbent by fluorescently
labelling the ligands itself.

1.10 Conclusion

There are several factors to consider when designing a protein A affinity

chromatography adsorbent. Different elements would have an effect on different
performance measurements. Several methodologies tend to help manufacturers to
characterise these adsorbents. It is usually the case that not all the desired
characteristics of the adsorbents can be achieved and a trade off needs to be
made.

Selectivity of the adsorbent can be of importance as it can eliminate further

downstream processing steps, hence reducing costs. Higher binding capacity and
better mass transfer properties are important especially due to the fact that
feedstock expression levels are increasing and the affinity step needs to be able to
purify a large amount of feed. These factors are important especially to companies
where the affinity chromatography is a bottleneck in the bioprocess. On the other
hand, the production rate might be of great importance too. Some adsorbents might
have a higher dynamic capacity but can only perform at lower flow velocities due to
compressibility issues, whereas other adsorbents might have a lower dynamic
capacity but can withstand higher flow rates without getting compressed. Hence
more cycles would be required to process a given amount of antibodies, but as
each cycle is run quickly the overall production rate might increase. Then the factor
to consider would be whether the adsorbent’s performance is affected by the
increase in the number of cycles.

Hence by having a methodology that can be used to study various characteristics

and the performance of different adsorbents can help companies to design
adsorbents that would be beneficial to manufacturing companies, by taking the
economical factor into consideration too. The next chapter will look into some
methods that can be used to characterise affinity adsorbents.

1.11 Target of the thesis

This thesis will mainly focus on designing a set of techniques to understand the
performance of different affinity chromatography adsorbents. The different

36
adsorbents that are considered in this thesis are MabSelect (GE Healthcare),
MabSelect Xtra (GE Healthcare), Prosep Ultra (Millipore), Protein A immobilised on
4CL Sepharose (GE Heatlhcare) in house and a prototype adsorbent with a Protein
A mimic ligand (Millipore). These adsorbents are considered specifically as they
have different particle size, porosity, support material and the type of ligand. The
major methods used in this thesis looks at the static capacity, adsorption
equilibrium data, dynamic capacity, uptake kinetics and desorption kinetics of
different adsorbents. A more novel technology using confocal microscopy is also
designed using a flow cell, which gives a good indication of the visual
characteristics of an adsorbent, diffusion rate to the centre of the bead and the
adsorption rates at an individual bead level. It can be of great benefits to have a set
of methods that can be used to characterise adsorbents especially to manufactures
of novel adsorbents.

37
Chapter 2 – Performance measurement of
affinity chromatography adsorbents

2.1 Abstract

Affinity chromatography plays a big role in the purification of monoclonal antibodies.

To make the process more cost effective and to be able to handle the large
bioreactor volumes produced, manufacturers of chromatography media are looking
for robust methodologies to characterise the performance of these media. This
chapter looks at designing and analysing a range of different performance
measurement methods. Any new adsorbent in production can be subjected to these
methods to understand their characteristics and compare them with other
commercially available adsorbents.

Four different affinity adsorbents are looked at in this chapter, which are MabSelect
(GE Healthcare), MabSelect Xtra (GE Healthcare), Prosep Ultra (Millipore, Consett)
and a prototype adsorbent immobilised with a synthetic ligand (Protein A mimic). All
these four adsorbents have distinct qualities in terms of pore size, particle size,
ligand type and the material the adsorbent is made from.

In this chapter, 6 different methods were designed and analysed. The static
capacity experiment showed that MabSelect Xtra has the highest static capacity of
61.8mg/ml. The adsorption equilibrium data showed very low Kd values as
expected by such affinity adsorbents. MabSelect Xtra and Prosep Ultra showed
steeper equilibrium curves possibly indicating that these media have higher affinity
to hIgG at lower hIgG concentration. Dynamic capacity carried out in a 0.66cm
Omnifit column with a bed height of 6cm at 2, 4 and 8 min residence time showed
MabSelect Xtra to have the highest dynamic capacity of 37.1mg/ml at 2 min
residence time. By designing a method using the AKTATM Basic, studies of uptake
kinetics using 1mg/ml hIgG were carried out, which showed all the four media to
have very similar hIgG uptake rates. The same experiment was carried out using a
higher concentration of hIgG (3mg/ml) on MabSelect and MabSelect Xtra. The
uptake rate was much faster, however it was similar for both the adsorbents.
Similarly desorption rate was carried out on the four media at room temperature

38
and at 40C to slow the process down. Again all the four media showed similar
characteristics at both the temperatures.

2.2 Introduction

This chapter aims to study the performance of a few commercially available

Protein A media along with a prototype. These adsorbents are MabSelect (GE
Healthcare), MabSelect Xtra (GE Healthcare) and Prosep Ultra (Millipore, Consett).
MabSelect consists of highly cross-linked agarose beads with a mean particle size
of 85Nm. MabSelect Xtra also consists of cross-linked agarose beads but it has a
more open structure than that of MabSelect and hence greater porosity. The mean
particle size is 75Nm, smaller than MabSelect (Hahn et al., 2005). Prosep Ultra
consists of irregularly shaped porous glass particles of ~100Nm and a pore size of
70nm. The prototype consists of cross-linked agarose beads, but with a different
percentage of agarose used when compared with MabSelect and MabSelect Xtra.
The particle size is smaller with a mean of 51Nm and the ligand used is a chemical
ligand as opposed to Protein A ligand. This chapter gives an indication of a
methodology that can be used to characterise chromatographic adsorbents. It will
study the performance of different media, hence relating performance of the media
to the support matrix type, particle size and pore size.

2.3 Static capacity

2.3.1 Materials for determining the static capacity

Chromatography media

MabSelect, MabSelectXtra media were from GE Healthcare (UK) and Prosep Ultra
and a prototype media (CIGL) were from Millipore (Consett, UK).

Chemicals

Phosphate buffered saline (PBS) pH 7.4 was from Sigma-Aldrich (Gillingham,

Dorset), which is biotechnology performance certified (product – P5368). Human
polyclonal IgG was also from Sigma-Aldrich in the form of a 99% lyophilised powder
(product G4386).

39
2.3.2 Method for determining the static capacity

The static capacity for MabSelect, MabSelect Xtra, Propsep Ultra and the prototype
(CIGL) were determined by batch experiment. The method below needs to be
repeated for each adsorbent. 55 ml of hIgG with a concentration of 1 mg/ml of hIgG
in PBS pH 7.4 was made in a beaker and the solution was sterile filtered using
0.22Nm syringe filters (Sartorius). Three, 20ml falcon tubes were labelled A, B and
C and the fourth falcon tube was labelled as the adsorbent. 1 ml (2.3cm) of
adsorbent was measured in a 5ml extraction column (Bioconnections, Leeds) by
suction drying it with a syringe. The adsorbent was washed with 4CV of PBS. The
adsorbent was put into the falcon tube labelled as the adsorbent with the help of a
syringe. The extraction column was washed with 4ml PBS and the same 4ml was
added to the falcon tube with the adsorbent. The adsorbent falcon tube was placed
on a stirrer. When the beads were in uniform suspension, 500µL of the suspension
was taken using a 1ml pipette and added to each of the three 20ml falcon tubes
labelled A, B and C (hence making triplicates for each sample). 14ml of 1mg/ml
hIgG solution was added into each of the three falcon tubes, A, B and C. The three
falcon tubes were put on a rotator for 6 hours. It was taken off the rotator and the
beads were left to settle. Each sample was analysed in a spectrometer by
measuring the UV absorbance in a quartz cuvette at 280nm and the data was
recorded, using PBS as blank. The average of the 3 samples was used to calculate
the static capacity.

The static capacity Qs was calculated using the formula below:

Qs = {(1mg/ml)*14ml hIgG solution – [(Protein concentration in solution left after

run/measured feed concentration)*1mg/ml](14.5ml total volume)}/0.1ml beads

The concentration was measured using the formula below:

Concentration (mg/ml) = A (mAu)/1.38 (Ext.coeff)

40
2.3.3 Results

The table below shows the average static capacity of the three samples for each of
the adsorbents. Appendix A shows the static capacity for all the samples.

Adsorbent Static capacity (mg/ml)

MabSelect 57.6
MabSelect Xtra 61.8
Prosep Ultra 56.4
Prototype (CIGL) 59.3

Table 2.1 - Static capacity for MabSelect, MabSelectXtra, Prosep Ultra and CIGL.
MabSelect Xtra shows the highest static capacity of 61.8mg/ml. Average of 3 samples was
taken.

2.4 Adsorption equilibrium data used to determine maximum

capacity and dissociation constant

2.4.1 Materials for adsorption equilibrium experiments

Chromatography media

MabSelect, MabSelectXtra media were from GE Healthcare (UK) and Prosep Ultra
and a prototype media (CIGL) were from Millipore (Consett, UK).

Chemicals

Phosphate buffered saline (PBS) pH 7.4 was from Sigma-Aldrich (Gillingham,

Dorset), which is biotechnology performance certified (product – P5368). Human
polyclonal IgG was also from Sigma-Aldrich in the form of a 99% lyophilised powder
(product G4386).

41
2.4.2 Method for adsorption equilibrium experiments

Adsorption equilibrium data were determined by batch experiment for MabSelect,

MabSelect Xtra, Propsep Ultra and the prototype (CIGL). The method below needs
to be repeated for each adsorbent. 80ml of 0.5mg/ml of hIgG in PBS pH 7.4 was
made in a beaker and the solution was sterile filtered using 0.22Nm syringe filter
(Sartorius). Six, 20ml falcon tubes were labelled A, B, C, D, E and F and the
seventh 20ml falcon tube was labelled as the adsorbent.
1 ml (2.3cm) of adsorbent was measured in a 5ml extraction column
(Bioconnections, Leeds) by suction drying it with a syringe. The adsorbent was
washed with 4CV of PBS. The adsorbent was put into the falcon tube labelled as
the adsorbent with the help of a syringe. The extraction column was washed with
4ml PBS and the same 4ml was added to the falcon tube with the adsorbent. The
adsorbent falcon tube was placed on a stirrer. When the beads were in uniform
suspension, 500µL of the suspension was taken using a 1ml pipette and added to
each of the 20ml falcon tubes labelled A, B, C, D, E and F (hence making 6
samples). 4ml, 8ml, 10ml, 12ml, 16ml and 20ml of 0.5mg/ml hIgG solution was
added into falcon tubes labelled A, B, C, D, E and F respectively. The final volume
of liquid was made up to 20ml with PBS. The tubes were put on a rotator for 6
hours. It was taken off the rotator and the beads were left to settle. Each sample
was analysed in a spectrometer by measuring the UV absorbance in a quartz
cuvette at 280nm and the data was recorded, using PBS as blank.

The equilibrium concentration in bulk liquid (C*) was calculated using the formula
below:
C* mg/ml = Feed concentration * Protein concentration in solution left after
run/measured feed concentration

The equilibrium concentration on adsorbent (q*) was calculated using the formula
below:
q* mg/ml = (0.5mg/ml) * volume of hIgG solution – [(Feed concentration) * (Protein
concentration in solution left after run/measured feed concentration)](20.5ml total
volume)

This gave results for 0.1ml of media and hence the data was converted for 1ml of
media, which was then used to plot the graphs.

42
Kd (M) was calculated using the following formula:
Kd (M) = Kd (mg/ml)/ 150000 (Approximate RMM of IgG)

2.4.3 Results

Two different ways were used to determine the maximum capacity and the
dissociation constant. In the first way, the data collected from the experiment was
linearised and the linearised form of the Langmuir isotherm (Hanes plot) was used
to get the maximum capacity and the dissociation constant, where 1/slope gives the
maximum capacity and y intercept equals to Kd/Qmax.
In the second way the experimental data was fitted using Langmuir isotherm and
the maximum capacity and dissociation constant were determined using the Solver
function in Excel, without linearising the isotherm.

The data collected was for human polyclonal IgG, which included the IgG3 that
doesn’t bind to protein A (MabSelect, MabSelect Xtra, Prosep Ultra). Hence the
data was corrected to take this factor into account. To get the corrected values,
5.5% (Hahn et al., 2005) from the initial feed concentration was subtracted to give
the following results. This correction only applies to MabSelect, MabSelect Xtra and
Prosep Ultra as CIGL tends to bind IgG3.

43
 0.0050

0.0040
C*/q* (-)
0.0030

0.0020

0.0010

0.0000
0.000 0.050 0.100 0.150 0.200 0.250
C* (mg/ml)

Fig 2.1 – Hanes Plot – MabSelect. Linearised form of Langmuir isotherm for Mabselect.
1/slope gives the maximum capacity and y intercept equals to Kd/Qmax. The equation for the
2
line is y=0.01813x + 0.00047 with an R value of 0.99832. Here Qmax = 55.1 and Kd =
0.026mg/ml

60

50
q* (mg/ml)

40

30

20

10

0
0 0.1 0.2 0.3 0.4 0.5 0.6
C* (mg/ml)

Fig 2.2 – Adsorption Equilibrium – MabSelect. Experimental data for MabSelect fitted using
Langmuir isotherm and the maximum capacity and dissociation constant were determined
using the Solver function in Excel.

44
 0.003

0.0025

0.002
C*/q* (-)

0.0015

0.001

0.0005

0
0 0.05 0.1 0.15 0.2
C* (m g/m l)

Fig 2.3 – Hanes Plot – MabSelect Xtra. Linearised form of Langmuir isotherm for Mabselect
Xtra. 1/slope gives the maximum capacity and y intercept equals to Kd/Qmax. The equation
2
for the line is y=0.01554x + 0.00009 with an R value of 0.99975. Here Qmax = 64.4 and Kd =
0.006mg/ml

70
60
50
q* (mg/ml)

40
30
20
10
0
0 0.1 0.2 0.3 0.4 0.5 0.6
C* (mg/ml)

Fig 2.4 – Adsorption Equilibrium – MabSelect Xtra. Experimental data for MabSelect Xtra
fitted using Langmuir isotherm and the maximum capacity and dissociation constant were
determined using the Solver function in Excel

45
 0.004

C*/q* (-) 0.003

0.002

0.001

0
0 0.05 0.1 0.15 0.2
C* (mg/ml)

Fig 2.5 – Hanes Plot – Prosep Ultra. Linearised form of Langmuir isotherm for Prosep Ultra.
1/slope gives the maximum capacity and y intercept equals to Kd/Qmax. The equation for the
2
line is y=0.01630x + 0.00013 with an R value of 0.99927. Here Qmax = 61.3 and Kd =
0.008mg/ml

70
60
50
q* (mg/ml)

40
30
20
10
0
0 0.1 0.2 0.3 0.4 0.5 0.6
C* (mg/ml)

Fig 2.6 – Adsorption Equilibrium – Prosep Ultra. Experimental data for Prosep Ultra fitted
using Langmuir isotherm and the maximum capacity and dissociation constant were
determined using the Solver function in Excel

46
 0.05
0.04
C*/q* (-)
0.03
0.02
0.01
0
0 0.5 1 1.5 2 2.5
C* (mg/ml)

Fig 2.7 – Hanes Plot – CIGL. Linearised form of Langmuir isotherm for CIGL. 1/slope gives
the maximum capacity and y intercept equals to Kd/Qmax. The equation for the line is
2
y=0.0163x + 0.0039 with an R value of 0.9997. Here Qmax = 61.5 and Kd = 0.024mg/ml

70
60
50
q* (mg/ml)

40
30
20
10
0
0 0.1 0.2 0.3 0.4 0.5 0.6
C* (mg/ml)

Fig 2.8 – Adsorption Equilibrium – CIGL. Experimental data for CIGL fitted using Langmuir
isotherm and the maximum capacity and dissociation constant were determined using the
Solver function in Excel

47
Adsorbent Qmax (mg/ml) Kd (mg/ml) Kd (nM)
MabSelect 55.1 0.026 173

MabSelect Xtra 64.4 0.006 38.6

Prosep Ultra 61.3 0.008 53.2

CIGL 61.5 0.024 160

Table 2.2 - A summary of maximum capacity and dissociation constant determined using
Hanes Plot for MabSelect, MabSelect Xtra, Prosep Ultra and CIGL.

Adsorbent Qmax (mg/ml) Kd (mg/ml) Kd (nM)

MabSelect 55.9 0.027 181

MabSelect Xtra 63.4 0.005 35.8

Prosep Ultra 60 0.007 49.9

CIGL 61.3 0.023 156

Table 2.3 - A summary of maximum capacity and dissociation constant determined using
Solver function in Excel for MabSelect, MabSelect Xtra, Prosep Ultra and CIGL.

2.5. Dynamic capacity

2.5.1 Material for determining dynamic capacity

Chromatography media

MabSelect, MabSelectXtra media were from GE Healthcare (UK) and Prosep Ultra
and a prototype media (CIGL) were from Millipore (Consett, UK).

Chemicals

Phosphate buffered saline (PBS) pH 7.4 was from Sigma-Aldrich (Gillingham,

Dorset), which is biotechnology performance certified (product – P5368). Human
polyclonal IgG was also from Sigma-Aldrich in the form of a 99% lyophilised powder
(product G4386). Glycine was also from Sigma-Aldrich.

48
2.5.2 Method for determining dynamic capacity

The adsorbents were packed into a 0.66cm Omnifit column to a bed height of 6cm.
Frontal analysis runs were performed using AKTATM Basic and antibody
concentrations were monitored on a spectrometer by UV absorbance at 280nm.
The packed column was equilibrated with PBS pH 7.4. The column was loaded with
1mg/ml hIgG in PBS. The loading flow rate was varied between 1– 0.25 ml/min to
give residence times between 2-8mins respectively. The loading was stopped at
20% breakthrough to take into account 10% IgG3 (value decided internally by
Millipore, UK) that doesn’t bind to Protien A and hence give a 10% breakthrough
value (value decided internally by Millipore, UK). The column was washed with PBS
pH 7.4. The bound hIgG was eluted using 0.1M glycine pH 2 and collected. The
sample was analysed by measuring the UV absorbance on a spectrometer at
280nm to determine the hIgG concentration. The dynamic capacity of the
adsorbents was calculated.

Experimental data can be seen in Appendix A

49
2.5.3 Results

60

50
Dynamic Capacity (mg/ml)

40

30

MabSelect
20 MabSelect Xtra
Prosep Ultra
10 Prototype

0
0 2 4 6 8 10
Residence time (min)

Fig 2.9 – Dynamic capacity determined at 2min, 4min and 8min residence time for
MabSelect, MabSelect Xtra, Prosep Ultra and CIGL. 0.66cm Omnifit column with a bed
height of 6cm was used. Loading was stopped at 20% breakthrough.

Adsorbent Dynamic Binding Capacity (mg/ml)

2mins 4mins 8mins
MabSelect 30.1 32.3 36.4
MabSelect Xtra 37.1 40.4 41
Prosep Ultra 30.8 33.2 35
CIGL 36.4 42.9 47.9

Table 2.4 - Summary of dynamic capacity determined at 2min, 4min and 8min residence
time for MabSelect, MabSelect Xtra, Prosep Ultra and CIGL. An Omnifit column with a 6cm
bed height, appr. 2ml adsorbent was used.

CIGL shows an exceptionally high dynamic capacity at 8min residence time. This is
due to the fact that IgG3 tends to bind to CIGL and hence the loading should have
been stopped at 10% breakthrough instead of 20% for this media for 2min, 4min

50
and 8min residence time. Due to lack of time this experiment wasn’t repeated for
CIGL at 10% breakthrough.

2.6 Batch uptake rate

2.6.1. Method to determine uptake rate

In this experiment phosphate buffered saline (PBS) pH 7.4 was from Sigma-Aldrich
(Gillingham, Dorset), which is biotechnology performance certified (product –
P5368). Human polyclonal IgG was also from Sigma-Aldrich in the form of a 99%
lyophilised powder (product G4386).

Response time

Antibody uptake kinetics was determined by continuously recirculating a small

stream of hIgG solution through the AKTATM Basic. The hIgG solution is mixed with
the adsorbent in a vessel with the help of an impeller (Fig 2.10).

AKTATM Basic
UV detector
2
Pump A Rotator
1

Filter
IgG solution
+ media
Beaker

Tube 1 – Connected from filter to outlet of UV detector

TM
Tube 2 – Connected from inlet of UV detector to Pump A of AKTA Basic.
Tube 3 – Waste loop

Fig 2.10- Uptake rate equipment setup used to determine the uptake kinetics for different
TM
media. It consists of a stream of hIgG solution recirculating through the AKTA Basic from
a beaker consisting of homogenous mixture of hIgG and media solution.

51
At any point in time as a small stream of solution is leaving the system it is not in
contact with the adsorbent for a certain amount of period. This gives a difference in
the hIgG concentration between the stream of solution drawn and that in the
beaker. As a result it takes a certain amount of time before the system stabilises
and shows a constant UV 280nm reading. This in other words is the response time
of the system. In order to get the response time the following method was carried
out.

First the system was equilibrated with PBS. A solution of hIgG was prepared in a
250ml beaker. Stream of hIgG solution was drawn from the beaker through a filter
membrane by using AKTATM Basic. The point at which the flow was started equals
to time 0 in Fig 2.11. This was the start time, which was specified manually when
the flow was started. The time taken to give a constant UV 280nm reading equals to
the response time of the system. The aim is to keep the response time to a
minimum, as this would give more accurate results when carrying out the uptake
kinetic experiment. Two sets of experiments were carried out at a flow rate of
4ml/min and 6ml/min.

The response time was estimated from the graph below (Fig 2.11). Two flow rates
were used of 4ml/min and 6ml/min. The hIgG solution is detected quicker when
using a flow rate of 6ml/min but they both tend to show a response time of ~20-
25sec. Higher flow rates couldn’t be used as the media in the solution tend to stick
to the filter. Hence a flow rate of 4ml/min was used to carry out the experiment.

52
 1.20

Absorbance 280nm/Max absorbance (-)

1.00

0.80

0.60

0.40

0.20

0.00
0.00 0.10 0.20 0.30 0.40 0.50
Time (mins)

Flowrate - 4ml/min Flowrate - 6ml/min

Fig 2.11 – Response time here is defined as the amount of time before the system stabilises
and shows a constant UV 280nm reading. This equilibration time (response time) is
recorded at 4ml/min and 6ml/min flow rate which shows to be ~20-25sec.

Uptake Rate

Antibody uptake kinetics was determined by preparing 100ml of 1mg/ml of hIgG

solution in PBS. The solution was agitated with a marine impeller
(2cm diameter, 2 blades) at 145rpm. The hIgG concentration was monitored by
continuously recirculating a small stream of solution through the AKTATM Basic. The
response time was ~20-25sec at a flow rate of 4ml/min. 1ml of media was
measured with the help of an extraction column (Bioconnections Ltd, Leeds). This
was suction dried and added to the solution. Time 0 on the graph is when the
suction dried media was poured into the hIgG solution, which was specified
manually. The setup was left to run for 2-3 hours. This same procedure was
repeated by preparing 100ml of 3mg/ml of hIgG solution in PBS.

53
Characterisation of mixing and reproducibility of results

Two sets of experiments were carried out with CIGL to get reproducible results.
One of them was carried out at 145rpm and the other at 200rpm.The difference in
the mixing speed is not very large but the fact that reproducible results are achieved
can prove that the mixing is uniform and the uptake rate is not affected by the
change in mixing speed. Extremely high mixing speed could be used but this might
damage the adsorbent. A very high speed was not needed to be tested, as from the
graph below both the results produced are very similar, thus proving that a mixing
speed above 145rpm doesn’t affect the results. Below 125rpm the beads were not
uniformly mixed visually. Moving of the impeller position was needed occasionally
at 145rpm and 200rpm due to the beads settling down in certain areas of the
beaker. This experiment was carried out without the use of any baffles. All the
experiments were carried out at 145rpm, except for Prosep Ultra, which needed
mixing at 200rpm as visually the beads tend to settle down at 145rpm. In Fig 2.12
Qmax equals to the maximum capacity of the adsorbents achieved at the end of the
experiment (120mins).

54
 60

50

Capacity q (mg/ml)
40

30

20

10

0
0 20 40 60 80 100 120 140
Time (mins)

CIGL 1(145rpm) CIGL 2 (200rpm)

Fig 2.12 - Uptake kinetic curves for two samples of CIGL where the impeller speed of
145rpm and 200rpm was used to check mixing characterisation and reproducibility.

1.2

0.8
q/qm (-)

0.6

0.4

0.2

0
0 20 40 60 80 100 120 140
Time (mins)

CIGL 1 (145rpm) CIGL 2 (200rpm)

Fig 2.13 - Uptake kinetic curves for two samples of CIGL where maximum capacity at the
end of adsorption for each sample equals to 1. The two graphs showed to be reproducible.

55
2.6.2 Results

70

60
Capacity q (mg/ml)

50

40

30

20

10

0
0 50 100 150 200
Time (mins)
MabSelect MabSelect Xtra CIGL 1 Prosep ultra

Fig 2.14 – Uptake kinetic curves for MabSelect, MabSelect Xtra, Prosep Ultra and CIGL.
Impeller speed of 145 rpm was used except for Prosep Ultra where 200rpm was used.
Maximum capacity determined at 120 mins.

1.2

0.8
q/qm (-)

0.6

0.4

0.2

0
0 50 100 150 200

Time (min)

MabSelect MabSelect Xtra CIGL 1 Prosep Ultra

Fig 2.15 – Uptake kinetic curves for MabSelect, MabSelect Xtra, Prosep Ultra and CIGL
where maximum capacity at the end of adsorption for each media equals to 1. The uptake
rate shows to be very similar for all four media.

56
 70

60

50
Capacity q (mg/ml)

40

30

20

10

0
0 5 10 15 20 25 30
Time (mins)

MabSelect Xtra MabSelect

Fig 2.16 – Uptake kinetic curves for MabSelect and MabSelect Xtra using higher
concentration of hIgG (3mg/ml) and an impeller speed of 145rpm. The uptake rate shows to
be very similar for the two media.

1.20

1.00

0.80
q/qm (-)

0.60

0.40

0.20

0.00
0 5 10 15 20 25 30

Time (mins)

MabSelect Xtra MabSelect

Fig 2.17 – Normalised version of the uptake kinetic curves for MabSelect and MabSelect
Xtra using higher concentration of hIgG (3mg/ml) and an impeller speed of 145rpm. The
uptake rate shows to be very similar for the two media.

57
2.7 Desorption Kinetics

2.7.1 Method to analyse desorption rate

In this experiment phosphate buffered saline (PBS) pH 7.4 was from Sigma-Aldrich
(Gillingham, Dorset), which is biotechnology performance certified (product –
P5368). Glycine was also from Sigma-Aldrich.

After the completion of the uptake the media was left to settle. The media was
collected in an extraction column (Bioconnections Ltd, Leeds) and washed with
PBS. It was suspended in 1ml of PBS in the extraction column. It was then
transferred to 100ml of 0.1M glycine pH 2. Time 0 on the graph is when the
suspended media was poured into the glycine solution, which was specified
manually. The solution was agitated with a marine impeller at 145rpm. The hIGg
concentration was monitored by continuously recirculating a small stream of
solution through the AKTATM Basic. The setup was left to run until a steady
absorbance reading was achieved. The data was used to get the desorption
kinetics.

Characterisation of mixing and reproducibility of results

Two sets of experiments were carried out with CIGL to get reproducible results.
One of them was carried out at 145rpm and the other at 200rpm for the same
reason as mentioned in section 2.4. From the graph below both the results
produced are very similar, thus proving that the mixing speed doesn’t affect the
results.

The end point of the experiment was taken as zero, hence assuming that the
amount of antibody released is equal to the amount that was bound to the
adsorbent initially. This was done because the transfer of the suspended adsorbent
from the extraction column to the elution buffer caused some loss of the media.

58
 1.2

0.8
q/qm (-)

0.6

0.4

0.2

0
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
Time (mins)

CIGl 1 - 145rpm CIGL 2 - 200rpm

Fig 2.18 – Desorption kinetic curves for two samples of CIGL where the impeller speed of
145rpm and 200rpm was used to check mixing characterisation and reproducibility

2.7.2 Results

Fig 2.19 shows the desorption curves for all the four media. From the graph it is
difficult to say if the slight differences are due to the type of adsorbents or due to
experimental inaccuracies.

59
 1.2

0.8
q/qm (-)
0.6

0.4

0.2

0
0.00 0.50 1.00 1.50 2.00
Time (mins)

CIGL 1 Prosep Ultra

MabSelect MabSelect Xtra
response time - 4ml/min (Control)

Fig 2.19 – Desorption kinetic curves for MabSelect, MabSelect Xtra, Prosep Ultra and CIGL.
Impeller speed of 145 rpm was used except for Prosep Ultra where 200rpm was used.
Desorption rate shows to be very similar for all four media.

2.8 Desorption at 4oC

2.8.1 Method to analyse desorption kinetics at 4oC

In this experiment phosphate buffered saline (PBS) pH 7.4 was from Sigma-Aldrich
(Gillingham, Dorset), which is biotechnology performance certified (product –
P5368). Glycine was also from Sigma-Aldrich.

After the completion of the uptake experiment the media was left to settle. The
media was collected in an extraction column (Bioconnections Ltd, Leeds) and
washed with PBS. It was suspended in 1ml of PBS in the extraction column. It was
then transferred to 100ml of 0.1M glycine pH 2. This glycine solution in a beaker
was placed with ice around it to maintain the temperature at 4oC, which was
measured by a temperature probe. Time 0 on the graph is when the suspended
media was poured into the glycine solution, which was specified manually. The
solution was agitated with a marine impeller at 145rpm. The hIGg concentration
was monitored by continuously recirculating a small stream of solution through the

60
AKTATM Basic. The setup was left to run until a steady absorbance reading was
achieved. The data was used to get the desorption kinetics.

2.8.2 Results

1.2

0.8

0.6
q/qm (-)

0.4

0.2

0
0 0.5 1 1.5 2
Time (mins)
CIGL 4o C Room temperature CIGL MabSelect Xtra 4 oC

o
Fig 2.20 – Desorption carried out on CIGL and MabSelect Xtra at 4 C to slow down the rate
o
of desorption. Similar results showed for the two media at 4 C. Impeller speed of 145 rpm
was used

2.9 Discussion

From the static capacity data MabSelect Xtra has the highest static capacity of
61.8mg/ml. This was expected due to the more porous nature of the bead, which
increased the surface area available for Protein A ligand immobilisation. MabSelect
and Prosep Ultra showed similar static capacity data as proposed by the
manufacturers. The prototype in fact showed relatively good static capacity of
59.2mg/ml.

61
The adsorption isotherm data from the Hanes Plot and by using the Solver function
from Excel were very similar. Both the methods to analyse the data seem to be
accurate. The Qmax values estimated from the adsorption isotherms were very
similar to that achieved from the static capacity experiments. The Kd values range
from 181nM to 36nM. Such low values are expected by affinity adsorbents such as
these. The adsorption isotherms were slightly steeper for MabSelect Xtra and
Prosep Ultra when compared to MabSelect and the prototype. This probably means
that MabSlect Xtra and Prosep Ultra have higher affinity to hIgG at lower hIgG
concentration. It must be noted that 5.5% was subtracted from the initial feed
concentration. This percentage is the estimated amount of IgG3 that doesn’t bind to
MabSelect, MabSelect Xtra and Prosep Ultra. To make this method further accurate
it is recommended to use monoclonal IgG without IgG3.

Results from the dynamic capacity experiment showed MabSelect Xtra to have a
greater dynamic capacity than the other three adsorbents. This is again due to the
more porous nature of the beads and also due to the smaller size of the beads. The
dynamic capacity for all four media increased with increase in the residence time.
However the increase in the dynamic capacity for MabSelect Xtra from 4min
residence time to 8min was much less when compared to other adsorbents. This
could be due to an increase in resistance due to stoichiometric reasons. It must be
noted here that the prototype showed exceptionally high dynamic capacity. This is
due to the fact that IgG3 binds to CIGL and hence the loading should have been
stopped at 10% breakthrough instead of 20%. Hence CIGL cannot be used in this
case for direct comparison.

A reliable system was designed to understand the uptake kinetics of different

adsorbents. The system produced reproducible results for CIGL, proving the
system to be accurate and reliable. From the results all the four adsorbents had
very similar uptake rates. Hence the difference in particle size, pore size, the type of
ligand or the material of the adsorbent itself did not have an effect on the uptake
rate when carried out in a batch mode. The same experiment was carried out on
MabSelect and MabSelect Xtra at a higher hIgG concentration. The time taken to
approach saturation was much faster as expected and hence the uptake rate was
much faster. This is due to the increase in diffusion rate due to a higher
concentration gradient.

62
Desorption experiments again showed reproducibility when carried out on two
samples of CIGL at two different impeller speeds. The actual desorption of hIgG
from the adsorbent was achieved very quickly within a few minutes. Once again the
desorption rate was very similar for all the four adsorbents.

In order to slow down the desorption process to get a closer look at any differences
between the adsorbents, the process was carried out at 4oC for CIGL and
MabSelect Xtra. This definitely slowed down the process when compared to the
desorption experiment carried out at room temperature. However the desoprtion
rate was again very similar for CIGL and MabSelect Xtra.

2.10 Conclusion

The methods designed and carried out in this chapter demonstrates different ways
to characterise adsorbents in different situations. In a batch mode the static
capacity gives the maximum amount of hIgG that can bind to different adsorbents.
The adsorption equilibrium curve gives a strong indication of the affinity of the
adsorbent towards hIgG and the predicted maximum capacity. The uptake kinetics
and desorption kinetics can help someone understand the rate at which different
adsorbents can bind and release hIgG in a batch mode at different hIgG
concentrations. The dynamic capacity gives an indication of how different
adsorbents would perform when packed in a column. Clearly it can be seen that
some methods show a clear distinction between different adsorbents whereas
some show very similar results. This provides a set of methods to compare novel
adsorbents against commercially available adsorbents and help manufactures
understand which performance factors of an adsorbent need further analysis.
Batch methods can be very easy to perform and can give useful information on
different adsorbent kinetics. The method is also less expensive than using a column
and less time consuming. There are no issues involved with column packing and
there is consistency in the data, as opposed to fixed bed methods where column
packing can play a major role in the kind of data generated. There are also no
minor technical issues such as air bubbles being introduced in the column. The
data can also be comparable with different batch methods, where as comparing
data generated from fixed bed experiments can be an issue, especially when
different bed heights and column diameters has been used. Batch method used in
this thesis to find kinetic adsorption data is even more reliable because the stream

63
of hIgG is being re-circulated and at no time are any samples been taken out of the
system for analysis.

The next chapter looks into another more novel technology that uses laser scanning
confocal microscopy. This method gives a better indication of the performance of
matrices at a single bead level and also gives a more visual characterisation of the
adsorbents.

64
Chapter 3 - Confocal analysis using flow cell

3.1 Abstract

The use of laser scanning confocal microscopy has proved to have several
advantages. Its is a non-destructive process where individual beads can be
analysed over time. Quantitative and qualitative analysis can then be carried out on
the images produced. This chapter looks into the use of a flow cell placed under a
confocal microscope where a channel of the flow cell is packed with different
matrices and a continuous flow of 2mg/ml hIgG is passed through the channel with
a flow rate of 0.1ml/min. The channels are 1cm in length and have a diameter of
0.1cm. It can pack ~8µl of media. The microscope is set up such that xy images of
beads are taken every two minutes through the centre of various sized beads.

The following chapter studies four different matrices, which are MabSelect,
MabSelect Xtra, Protein A immobilised on CL4 Sepahrose and a prototype media
(CIGL). From the images a good understanding of the binding process could be
derived visually for all the matrices. They all showed a shrinking core effect except
for the prototype where the hIgG didn’t penetrate into the bead and was only
attached to the surface of the bead. Further quantitative analysis was carried out on
these images. From the intensity profiles of the beads diffusion rates and uptake
curves for individual beads could be calculated. It was found that the adsorption
rate to the centre of each bead was linear. The different particle sizes within any
particular type of matrix and also across different matrix did not result in different
diffusion rates. The diffusion rates here were found to be approximately constant at
around 0.72 min-1. These matrices are designed to perform effectively at high
concentrations. It could be possible that the difference in the diffusion rate might be
visible at much lower hIgG concentrations. From the adsorption curves produced it
can clearly be seen that smaller beads reached saturation much faster than larger
beads. From this study we can conclude that this methodology can prove to be very
useful in understanding the visual binding effects of different matrices and also
understanding diffusion rates and adsorption rates at an individual bead level
especially in chromatography media where heterogeneity in bead size exists.

65
3.2 Introduction

Chromatography is all but ubiquitous in the purification of biological molecules. The

performance of chromatographic separations is usually evaluated using
breakthrough curves, adsorption isotherms or static binding capacities. However,
these methods do not provide a complete understanding of the process because of
the complexity involved in this type of operation. At the level of individual beads it is
difficult to infer from these macroscopic methods the spatial and temporaly nature
of the binding within the bead. This maybe of particular relevance if the beads are
heterogeneous in nature i.e. have significant size distribution, as is the case with
most chromatographic media used in process scale operations. Microtome
sectioning of beads has been used in the past to study the adsorption process
within chromatographic media. Beads were sectioned using a microtome, and
fluorescently labelled proteins then visualised (Subramanian et al., 1994). This has
obvious disadvantages; it is a destructive technique and the method requires
significant time to prepare samples for imaging, making many events difficult to
study in this manner. More recently, the use of laser scanning confocal microscopy
(CSLM) was introduced to circumvent these issues and due to its ability to show an
optical section through the depth of beads (Ljunglof et al., 1996). CLSM, which
allows quantitative measurement of the product and/or contaminants as they are
adsorbed on to the beads, is an invaluable tool for a direct assessment of binding
events at the bead level. The technique has been used to study the mechanism of
transport and adsorption of various protein molecules.

CLSM can also be used to calculate the uptake curve for different chromatography
matrices. By relating the relative fluorescence intensity obtained at different times to
the value at equilibrium, the degree of saturation versus time could be calculated.
This would then be used to calculate the solid phase concentration at different time
intervals (Ljunglof et al., 1998). CLSM has been used to assess matrix performance
over consecutive affinity runs (Thillaivinayagalingam et al., 2007) and to study
protein adsorption to chromatographic matrices (Protein A to IgG Sepharose 6 Fast
Flow) (Ljunglof et al., 1996). This method has also investigated the fouling in
chromatography and examined the effectiveness of CIP protocols (C. Siu et al.,
2006). CSLM has also been used in the past to understand the adsorption of
plasmid DNA to individual adsorbent particles (Ljunglof et al., 1999). The

66
mechanism of transport of the protein molecules into the pores of the adsorbent has
also been studied using this technique (Subramanian et al., 2005). The effect of pH
and conductivity during ion-exchange adsorption was evaluated using CLSM and
the mechanism of adsorption under varying conditions has been discussed in the
paper by Harinarayan et al. (2006).

Several experiments have been reported using a batch mode where the media and
the protein conjugated with dye have been stirred continuously and samples
withdrawn at regular intervals to be analysed under confocal microscope. The main
drawback of using finite bath method of adsorption is that the same bead is not
evaluated over a period of time, instead different beads are examined at different
times. Kasche et al. (2003) used a technique where the beads were fixed in the
wedge between the microscopic slide and a coverslide. The solution containing the
adsorbate was then flowed through these beads and the adsorption process
examined. A further improvement to this method is the use of flow cell by Schroeder
et al. (2006) and micro-column by Hubbuch et al. (2002). IEX adsorption process
has been carried out, by imaging particles under confocal microscopy using a flow
cell (Dziennik et al., 2003). This has the biggest advantage as contacting and
imaging are carried out simultaneously as opposed to sequentially hence removing
the time lag. Secondly the same bead can be evaluated over a certain period of
time which gives a more realistic picture compared to batch mode studies. The rate
of adsorption and diffusion coefficient of various protein molecules were studied by
this technique and compared with the conventional method of measurements. This
direct method of measurement of diffusion coefficient was further used to model the
adsorption process to a greater accuracy.

The increased use of monoclonal antibodies (MAbs) in medicine has generated a

greater interest in the efficient and economical purification strategies. The selective
binding of IgG to protein A is used to capture them directly by affinity
chromatography principles. A variety of adsorbents with protein A as ligands are
available commercially for the purification of IgG. The performances of 15 different
adsorbents were compared by Hant et al. (2003) in terms of dynamic binding
capacities and rate constants. Porous agarose based media were found to exhibit
higher dynamic binding capacity However, the higher mass transfer with non-
agarose based media noticed was attributed to the smaller particle diameter of
these adsorbents. These adsorbents were compared by indirect measurements and
not by CLSM. These indirect measurements do not allow the study of effect of

67
particle size of the adsorbents, inherent in the commercial adsorbents, while CLSM
can be very useful in this context.

The porosity of the adsorbent beads depends on the matrix type and the size of the
beads. The transport of protein molecules through the beads depends on the
porosity and the size of the adsorbate. The effect of bead size on the adsorption
process has not been studied using CLSM. However, using the results from various
literatures, Kasche et al. (2003) deduce that the concentration of the adsorbed
molecules in the first 10-30 % of the radius of the adsorbent increases for both
macroporous and polymeric network adsorbents (radius of adsorbent in the range
of 20-100 micrometers). Higher percentage values apply for smaller sized
adsorbent, which indicates that the penetration of the product is higher for smaller
sized particles. A significant scatter in the uptake profile of lysozyme (on to
Sepharose SP) by CLSM studies (direct measurement) was observed when
comparing with indirect measurement (fluid phase concentration measurement)
(Ljunglof et al., 1998). This was proposed to be the difference in the particle size of
the beads, which differed, by 5 micrometer even after a visual selection of similar
sized beads.

This chapter will be looking at hIgG adsorption on different affinity matrix using
CSLM and a flow cell. The adsorption profile has been used to understand the
difference in the adsorption of hIgG to beads of different sizes that belongs to the
same matrix.

68
3.3 Materials and Method

3.3.1. Materials

Chromatography media

MabSelect and MabSelectXtra media were from GE Healthcare (UK). The

prototype media was from Millipore (Consett, UK). The media where Protein A is
immobilised on 4CL Sepharose (GE Healthcare, UK) was produced in-house at
Millipore.

Chemicals

Chemical for buffer preparation was from Sigma-Aldrich (Gillingham, Dorset).

Phosphate buffered saline pH 7.4 is biotechnology performance certified (product –
P5368). Sodium carbonate was also from Sigma-Aldrich. Human polyclonal IgG
was also from Sigma-Aldrich in the form of a 99% lyophilised powder (product
G4386). Cy3 fluorescent dye was purchased from GE Healthcare (UK). Sephadex
G-50 used to remove unconjugated Cy3 dye was purchased from Sigma-Aldrich
(Gillingham, Dorset).

3.3.2 Equipment

Leica inverted confocal microscope was set up. An objective of 20X was used.
Image size was set at 512x512 pixels. The beads were excited at 568nm and
emitted between 573-643nm. Leica software was used for analysis.

A flow cell manufactured in-house at University College London was used to pack
the chromatography beads (Figure 3.1). It is a pyrex block with four channels drilled
through it. The channels are 1cm in length and have a diameter of 0.1cm. It can
pack ~8µl of media. Frits were placed just outside the channel to keep the beads in
place. A coverslip is attached on the face of the block with the help of an adhesive
(Araldite) to seal the channel.

69
 Channel packed with
media

Fig 3.1- Flow cell with a channel of diameter 0.1cm and a length of 1cm was used to pack
~8µl of media, which could be placed under an inverted confocal microscope.

3.4. Method

3.4.1 Sample preparation

1ml of hIgG (5mg/ml) was made in 0.1M sodium carbonate (pH 9.3 at room
temperature) which was fluorescently, labelled with Cy3. The hIgG solution was
added to a vial of fluorophore (1 vial contains 0.2-0.3mg of dye) and was left to
incubate for 40 min at room temperature with occasional inversion. 1 vial of Cy3
dyes 1mg of hIgG.
The sample was passed through a gel filtration column to remove unconjugated
Cy3 dye. Column XD 16 (GE Healthcare) was packed with Sephadex G-50 under
gravity. The labelled protein, the first band to elute was collected by visual
inspection. The sample was then made to 10ml with PBS. The concentration was
made to 2mg/ml by adding 15mg of hIgG. The dilution of labelled protein with
unlabelled protein is necessary to avoid total internal filtering of the confocal laser
light by the fluorescent dye.

3.4.2 Equipment setting

The flow cell was packed with the chromatography media. A syringe pump was
connected to the flow cell with the help of tubings and HPLC connectors. The media
was washed with 0.5ml of PBS at a flow rate of 0.1ml/min.

70
The flow cell was placed under the inverted confocal microscope. The Z series and
the stage of the microscope were adjusted such that visually images of a few beads
could be taken from the centre and an area with different size beads could be
selected. The labelled hIgG solution was passed through the column with a syringe
pump at 0.1ml/min (Figure 3.2). The microscope was programmed such that xy
images were taken every 2mins for 40mins. The fluorescent intensity across the
beads at every time interval could then be studied using the Leica software (Figure
3.3).

Flow
cell body
HPLC Adsorbent
connector Frit Feed in

Waste Cover slip

Syringe pump
Objective

Fig 3.2 - Flow cell setting under confocal microscope. 2mg/ml hIgG was passed through the
channel at 0.1ml/min flowrate with a syringe pump.

71
Fig 3.3 - Image of beads seen under the microscope at 30 mins. The images of the chosen
beads are from the centre and of different sizes (50µm and 70µm). The line across the bead
helps produce the intensity profile across that section as seen in figure 3.4

3.4.3 Analysis

The intensity profile across each bead for each time interval is produced by the
Leica software. The distance penetrated by the labelled hIgG to the centre of the
bead for each time interval can be estimated from the profile by choosing a certain
intensity baseline (Figure 3.4). This was chosen in the following way. The average
maximum intensity was calculated from the intensity value shown on the outside
surface of the bead. This was subtracted from the intensity reading of the
background (where there are no beads). 10% of this value was added to the
background intensity and this was taken as the baseline.

72
 100

80

Intensity (arbitary units)

60

40

20

0
0 10 20 30 40
Distance from edge to centre of bead (µm)

100 Edge of bead

80
Intensity (arbitary units)

60

40 Centre of bead
Base line

20

0
0 10 20 30 40
Distance from edge to centre of bead (µm)

73
 100
Small bead Large bead

80

Intensity (arbitary units)

60

40

20

0
0 10 20 30 40
Distance from edge to centre of bead (µm)

Fig 3.4 - Intensity profile across a small bead (~50µm) and a large bead (~70µm) at A;
10mins, B; 20mins and C; 30 mins time intervals. The distance travelled by hIgG to the
centre of the bead with time can be estimated from the profile

3.5 Results and Discussion

3.5.1 Visual images

The images obtained using confocal studies at the end of 10, 20 and 30 minutes for
the various adsorbent media is presented in Figure 3.5. Only one bead for each
matrix is selected and shown here. These specific beads are chosen such that the
images of these sections are taken from the centre of the bead. MabSelect and
MabSelect Xtra tend to show a typical shrinking core effect. Whereas for the
prototype it can be seen that hIgG doesn’t tend to penetrate inside the bead and
most of it just binds to the outer surface of the bead. The adsorbent where Protein
A is immobilised on 4CL Sepharose also shows a shrinking core effect, but the
areas where the beads are in contact with each other doesn’t tend to allow hIgG to
penetrate the inside of the bead until at a later stage in time. Hence this method
gives a clear visual characterisation of hIgG binding to different chromatography
matrices (Figure 3.5).

74
 Adsorbent Time (mins)
10 20 30

MabSelectTM
Average Particle
size - 85>m
4% agarose

MabSelect XtraTM
Average Particle
size - 75>m
4% agarose
ProteinA on 4CL
Sepharose
Average Particle
size - 100>m
4% agarose
Prototype
Average Particle
size - 60>m
6% agarose

Fig 3.5 - Visual Images of Mabselect, MabSelect Xtra, Prototype (Protein A Mimic ligand)
and Protein A immobilised on 4CL Sepharose media taken at 10, 20 and 30 mins in a flow
cell. 2mg/ml hIgG was passed through the channel at 0.1ml/min flowrate.

3.5.2 Diffusion rate

Analysis of data obtained from the intensity profile graphs is used to obtain the
diffusion rates shown in Figure 3.6. The analysis was carried out on 2 samples of
MabSelect, 1 sample of MabSelect Xtra and Protein A immobilised on 4CL
Sepharose. The experiment was carried on different days and using separately
prepared labelled protein samples each time. Hence the slight difference in packing
of the column, microscope specifications and labelled protein sample concentration
could contribute to the difference in the diffusion rate for different matrices. From
Figure 3.6 it can also be clearly shown that the adsorption rate to the centre of each

75
bead is linear. The results of diffusion rates obtained using confocal studies for
various matrices and difference particle sizes within the same matrix is presented in
Table 3.1. At lower feed concentrations (0.1-0.5 mg/ml) there is a dependency
between affinity and diffusivity (Hahn et al., 2005). But at higher concentration
(3mg/ml) all the media with different particle size had similar isotherm, thus affinity
is not dependent on diffusivity at this concentration. This means particle size
difference which could lead to higher diffusivity is not really beneficial in terms of
higher mass transfer if the feed concentration is increased beyond a certain level.

MabSelect Sample 1 beads

30 MabSelect Sample 2 beads

Distance from edge of bead (µm/min)

MabSelect Xtra beads

25
PrnA on 4CL Sepharose beads
20

15

10

0
0 5 10 15 20 25 30 35 40
Time (mins)

Fig 3.6 - Adsorption rate for different size beads for two different samples of MabSelect, 1
sample of MabSelect Xtra and Protein A immobilised on 4CL Sepharose

76
 -1
Chromatography media Bead size Gradient (min )
(µm)
MabSelect Xtra 75 0.74
Manufacture size - 75µm 70 0.72
66 0.74
55 0.73
53 0.74
MabSelect Sample 1 103 0.72
Manufacture size - 85µm 89 0.73
86 0.67
MabSelect Sample 2 72 0.71
82 0.77
ProteinA on 4CL 124 0.66
Sepharose 107 0.68
Manufacture size - 100µm

2
Table 3.1 - Diffusion rate for different size beads and for different media (R value of 0.977)

In the current study, the diffusion rates as seen from Table 3.1 seem to be same
among different bead sizes and also bead types. MabSelect and MabSelect Xtra
had similar diffusion rates. The different particle sizes within any particular type of
matrix and also across different matrix did not result in different diffusion rates. The
diffusion rates were found to be approximately constant at around 0.72 min-1.

3.5.3 Adsorption rate

By integration of intensity profiles the average volumetric intensity of the beads was
calculated. From this data uptake curves for different size beads of MabSelect Xtra
was plotted (Figure 3.7). It can clearly be seen that smaller beads reach saturation
much faster than larger beads indicating a higher adsorption rate as the proportion
of the bead that has absorbed the hIgG is higher for smaller beads than larger bead
at a given time.

77
 80 Bead size
Bead size- 53µm
- 53um
Bead size- 66µm
Bead size - 66um
Bead size- 75µm
Bead size - 75um
Intensity (arbitary units) 60

40

20

0
0 10 20 30 40
Time (mins)

Fig 3.7 - Uptake curves for different size beads of MabSelect Xtra plotted from data
calculated from intensity profiles

The adsorption rate as seen from Figure 3.7 is higher for smaller sized bead
compared to larger size bead. Perhaps constant diffusion rate means the front
reaches same length of the pore at any particular time for different sized beads.
From our studies we have demonstrated that particle size of the media needs to be
considered even if diffusion rates are similar. Modelling of chromatography
operations to predict the performance assumes a single particle size. The accuracy
of the modelling can be improved by considering the heterogeneity of the media.
The adsorption performance of individual beads can be measured quantitatively by
confocal microscopy thus allowing creating an accurate model.

3.6 Conclusion

Although using confocal microscopy to study adsorbents is a complex procedure it

can give very detailed information on different adsorbents. The methods looked at
in chapter 2 were very informative, however it did not give information on individual
beads. The uptake rate kinetics in chapter 2 showed similar uptake rates for all the

78
adsorbents, however using confocal microscopy showed a difference in the uptake
rates of different sized beads. Similarly in Chapter 2 the static capacity of the
prototype (CIGL) adsorbent was higher than MabSelect and Prosep Ultra, however
after carrying out the confocal experiment the visual images showed that hIgG only
tends to bind to the surface of the CIGL beads. This probably implies that the
prototype has excellent stoichiometric properties and needs further analysis. Hence
the use of confocal microscopy and the flow cell creates further questions on the
characteristics of different adsorbents, which helps make the analysis more robust.

79
Chapter 4 - Future Work

4.1 Introduction

So far this thesis looked into a few experimental methods that have proved to be
very accurate and reliable such as static capacity, adsorption isotherms, dynamic
capacity and uptake kinetics. These methods gave a good indication of the
performance of the different kind of matrices. This thesis also looked into a more
novel approach of using confocal microscopy using a flow cell to get a further
understanding of the different kind of matrices. However there are various other
areas that needs to be explored further. This chapter looks at some of these areas
where further work is necessary to achieve a toolbox of methods that can be used
to understand and evaluate the performance of different matrices.

4.2 Ligand density

It can be of great importance to understand how much protein A ligand is present in

1ml of media. Certain media might show a high static capacity but have relatively
low protein A ligand density and the high static capacity might be due to another
factor. In order to understand if this is the case, and compare adsorbents with each
other a robust technique to calculate the ligand density can be very useful.

One such method that can be carried out and tested further uses BCA
(Bicinchoninic acid). A standard curve is produced using different concentrations of
Protein A and adding BCA to it and measuring the OD values at 562nm. Similarly
BCA can be added to a known mass of adsorbent and the concentration of Protein
A can be calculated using the standard curve produced.

4.3 Use of Optical Biosensor Technology

SPR (surface plasmon resonance) technology enables real-time detection and

monitoring of biomolecular events and provides quantitative information. SPR
arises when light is reflected under certain conditions from a conducting film at the
interface between 2 media of different refractive index. Here the ligand is
immobilised on the sensor surface, or indirectly via an immobilised capturing

80
molecule and the solution with the desired product to be captured (e.g. IgG) is
passed over it. The data derived can be used to understand how specific the
binding is between two molecules. It can also be used to understand the kinetics
(rate of association and dissociation) and affinity. The data tends to give information
on the thickness, density and mass of the desired product attached, which can help
understand stoichiometry information.

81
Chapter 5 – Thesis Conclusion

5.1 Summary of the thesis

The work in this thesis was focused on designing reliable methods that can be used
to characterise different kinds of adsorbents. The batch methods used to analyse
different properties of adsorbents such as static capacity, adsorption equilibrium
data and kinetic uptake and desorption rate are easy to perform, can be reproduced
easily, can be performed at a low cost and there are no issues related to packing
columns. The batch uptake kinetic method was well designed and was accurate as
a small stream of hIgG solution was analysed continuously and at no point were
any samples taken out of the system for analysis. The results showed that
MabSelect Xtra had the highest static capacity. All the adsorbents had very low Kd
values as expected by affinity adsorbents such as these. The use of Hanes Plot
and the Solver function in Excel to calculate the Kd and Qmax values showed to be
accurate methods. All the media showed similar kinetic and desorption rates.
Dynamic capacity gives further information on how adsorbents would perform when
packed in a column. A small 0.66cm Omnifit column with a bed height of only 6cm
was used to minimise the time of experiment and cost. Data was collected at 2min,
4min and 8 mins residence time. The results showed MabSelect Xtra to have the
highest dynamic capacity. The relatively high static and dynamic capacity for
MabSelect Xtra is due to the more porous nature of the beads, which increased the
surface area available for Protein A ligand immobilisation and also due to the
smaller size of the beads.
Another more novel method using a flow cell, labelled hIgG and a laser scanning
confocal microscopy was designed to the see the adsorption properties of different
media at individual bead level. A channel of the flow cell was packed with different
media (~8µl) and placed under the microscope. Continuous flow of 2mg/ml hIgG
was passed through the channel with a flow rate of 0.1ml/min. This method uses a
minute amount of media, has shown to be accurate for the experiments carried out
in this thesis, gives information on individual beads visually and also gives uptake
rates of individual beads of different sizes of the same media. It was found that the
adsorption rate to the centre of the bead for all the matrices was linear and smaller
beads reached saturation much faster than larger beads at any given time. Visually
all the beads showed the shrinking core effect except for the prototype media. With
this method, images of the beads can be seen continuously with time and again no
samples need to be taken out of the system for analyses.

82
Manufacturers of affinity adsorbents need to take into account several factors while
designing and producing a specific matrix. Some of these factors are purity
(selectivity), capacity, recovery, binding and elution, alkali resistance, adsorbents
scale-up, stability, ligand loss (leakage) and safety (toxicity). Many manufactures
are showing increasing interest in designing synthetic ligands for affinity
chromatography. Synthetic ligands are less expensive than biological ligands,
synthesis of these ligands are simpler and they are more resistant to sterilisation.
There is a need to design methods that can be used to analyse the above
performance characteristics of affinity adsorbents. Some of these methods can be
used to analyse caustic stability of adsorbents, dynamic capacity using MAb
feedstock, pressure flow characteristics and life cycle studies. All these methods
put together can form a toolbox of methods to characterise different adsorbents.

83
References

Bailey. J. E, Ollis. D. F, Biochemical Engineering Fundamentals, 2nd ed., McGraw-

Hill (1986), 761

Belter. P. A, Cussler. E. L, Hu. W, Bioseperations- Downstream Processing for

Biotechnology, John Wiley and Sons (1988), 147-151

Braithwaite. A, Smith. F. J, Chromatographic Methods, 4th ed., Chapman and Hall

(1985) 8-9

Chang. C, Lenhoff. A. M, Comparison of protein adsorption isotherms and uptake

rates in preparative cation-exchange materials, Journal of Chromatography A, 827
(1998) 281–293

Dziennik. S. R, Belcher. E. B, Barker. G. A, DeBergalis. M. J, Fernandez. S. E,

Lenhoff. A. M, Nondiffusive mechanisms enhance protein uptake rates in ion
exchange particles, Proceedings of the National Academy of Sciences of the United
States of America, 100 (2003) 420-425

Fahrner. R. L, Whitney. D. H, Vanderlaan. M, Blank. G. S, Performance comparison

of Protein A affinity-chromatography sorbents for purifying recombinant monoclonal
antibodies, Biotechnology and Applied Biochemistry, 30 (1999) 121–128

Hahn. R, Bauerhansl. P, Shimahara. K, Wizniewski. C, Tscheliessnig. A,

Jungbauer. A, Comparison of protein A affinity sorbents II. Mass transfer properties,
Journal of Chromatography A, 1093 (2005) 98–110

Hahn. R, Schlegel. R, Jungbauer. A, Comparison of protein A affinity sorbents,

Journal of Chromatography B, 790 (2003) 35–51

Hahn. R, Shimahara. K, Steindl. F, Jungbauer. A, Comparison of protein A affinity

sorbents III. Life time study, Journal of Chromatography A, 1102 (2006) 224–231

84
Harinarayan. C, Mueller. J, Ljunglof. A, Fahrner. R, Van Alstine. J, Van Reis. R, An
exclusion mechanism in ion exchange chromatography, Biotechnology
Bioengineering, 95 (2006) 775-787

Hubbuch. J, Linden. T, Knieps. E, Thommes. J and Kula. M. R, Dynamic of protein

uptake within the adsorbent particle during packed bed chromatography,
Biotechnology and Bioengineering, 80 (2002) 359-368

Jansson. B, Niss. U, Nilsson. R, Sandberg. B, Nilsson. B, The interaction between

different domains of staphylococcal protein A and human IgG, IgA, IgM and Fab,
Molecular Immunology, 30 (1993) 1279-1285

Jungbauer. A, Chromatographic media for bioseparation, Journal of

Chromatography A, 1065 (2005) 3–12

Kasche. V, De Boer. M, Lazo. C, Gad. M, Direct observation of intraparticle

equilibration and the rate-limiting step in adsorption of protein in chromatographic
adsorbents with confocal laser scanning microscopy, Journal of Chromatography B,
790 (2003) 115-129

Krijgsman. J, Product Recovery in Bioprocess Technology, Butterworth-

Heinemann, (1995) 3-9

Lewus. R. K, Altan. F. H, Carta. G, Protein Adsorption and Desorption on Gel-Filled

Rigid Particles for Ion Exchange, Industrial and Engineering Chemistry Research,
37 (1998) 1079-1087

Ljunglof. A, Bergvall. P, Bhikhabhai. R, Hjorth. R, Direct visualisation of plasmid

DNA in individual chromatography adsorbent particles by confocal scanning laser
microscopy, Journal of Chromatography A, 844 (1999) 129–135

Ljunglof. A, Hjorth. R, Confocal microscopy as a tool for studying protein adsorption

to chromatographic matrices, Journal of Chromatography A, 743 (1996) 75-83

Ljunglof. A, Thommes. J, Visualising intraparticle protein transport in porous

adsorbents by confocal microscopy, Journal of Chromatography A, 813 (1998) 387-
395

85
Low. D, O’Leary. R, Pujar. N. S, Future of antibody purification, Journal of
Chromatography B, 848 (2007) 48–63

Lowe. C. R, Dean. P. D. G, Affinity Chromatography, John Wiley & Sons (1974),

14-17

McCue. J. T, Kemp. G, Low. D, Quinones-GarcZa. I, Evaluation of protein-A

chromatography media, Journal of Chromatography A, 989 (2003) 139–153

Pan. B. C, Meng. F. W, Chen. X. Q, Pan. B. J, Li. X. T, Zhang. W. M, Zhang. X,

Chen. J. L, Zhang. Q. X, Sun. Y, Application of an effective method in predicting
breakthrough curves of fixed-bed adsorption onto resin adsorbent, Journal of
Hazardous Materials, B124 (2005) 74–80

Shukla. A. A, Hubbard. B, Tressel. T, Guhan. S, Low. D, Downstream processing of

monoclonal antibodies - Application of platform approaches, Journal of
Chromatography B, 848 (2007) 28–39

Siu. C, Boushaba. R, Topoyassakul. V, Graham. A, Choudhary. S, Moss. G,

Titchener-Hooker. N. J, Visualising fouling of a chromatographic matrix using
confocal scanning laser microscopy, Biotechnology and Bioengineering 95 (2006)
714-723

Stickel. J. J, Fotopoulos. A, Pressure-flow relationships for packed beds of

compressible chromatography media at laboratory and production scale,
Biotechnology Progress, 17 (2001) 744-751

Stryer. L, Biochemistry, 4th ed., W.H. Freeman and Company (1995), 361-370

Subramanian. A, Hommerding. J, The use of confocal laser scanning microscopy to

study the transport of biomolecules in a macroporous support, Journal of
Chromatography A, 818 (2005) 89-97

Subramanian. A, Van Cott. K. E , Milbrath. D. S, Velander. W. H, Role of local

antibody density effects on immunosorbent efficiency, Journal of Chromatography
A, 672 (1994) 11

86
Swinnen. K, Krul. A, Van Goidsenhoven. I, Van Tichelt. N, Roosen. A, Van Houdt.
K, Performance comparison of protein A affinity resins for the purification of
monoclonal antibodies, Journal of Chromatography B, 848 (2007) 97–107

Thillaivinayagalingam. P, O’Donovan. K, Newcombeb. A. R, Keshavarz-Moore. E,

Characterisation of an industrial affinity process used in the manufacturing of
digoxin-specific polyclonal Fab fragments, Journal of Chromatography B, 848
(2007) 88–96

Tscheliessnig. A, Hahn. R, Jungbauer. A, In situ determination of adsorption

kinetics of proteins in a finite bath, Journal of Chromatography A, 1069 (2005) 23–
30

Uhlen. M, Guss. B, Nilsson. B, Gatenbäck. S, Philipson. L, Lindberg. M, Complete

sequence of the Staphylococcal gene encoding Protein A, Journal of Biological
Chemistry, 259 (1984) 695–1702

Van Deemter. J. J, Zuiderweg. F. J, Klinkenberg. A, Longitudinal diffusion and

resistance to mass transfer as causes of non ideality in chromatography, Chemical
Engineering Science, (1956) 271-289

87
Appendix
Appendix A - Static Capacity

MabSelect

Feed Concentration (mg/ml) 0.878

OD reading Concentration (mg/ml) Static capacity (mg/ml)
0.692 0.501 57.2
0.693 0.502 57.1
0.682 0.494 58.4
Average Static Capacity (mg/ml) 57.6

MabSelect Xtra

Feed Concentration (mg/ml) 0.946

OD reading Concentration (mg/ml) Static capacity (mg/ml)
0.7 0.507 62.3
0.706 0.512 61.6
0.705 0.511 61.7
Average Static Capacity (mg/ml) 61.8

Prosep Ultra

Feed Concentration (mg/ml) 0.896

OD reading Concentration (mg/ml) Static capacity (mg/ml)
0.71 0.5145 56.7
0.711 0.5152 56.6
0.718 0.5203 55.8
Average Static Capacity (mg/ml) 56.4

CIGL

Feed Concentration (mg/ml) 0.986

OD reading Concentration (mg/ml) Static capacity (mg/ml)
0.757 0.549 59.3
0.757 0.549 59.3
0.758 0.549 59.2
Average Static Capacity (mg/ml) 59.3

88
Appendix B – Dynamic Capacity

MabSelect

2mins residence time, 1ml/min flowrate

Plates/meter = 1674
HETP = 0.05972
As = 1.51

Feed concentration = 0.989 with OD reading of

273mAu
20% breakthrough is at 54.6mAu
concentration = 9.4mg/ml
Volume = 6.4ml
mass = 60.2mg
Dynamic capacity = 30.1mg/ml

4mins residence time, 0.5ml/min flowrate

Plates/meter = 1548
HETP = 0.05988
As = 1.71

Feed concentration = 0.989 with OD reading of

273mAu
20% breakthrough is at 54.6mAu
concentration = 9.93mg/ml
Volume = 6.5ml
mass = 64.5mg
Dynamic capacity = 32.3mg/ml

8mins residence time, 0.25ml/min flowrate

Plates/meter = 1520
HETP = 0.05980
As = 1.7

Feed concentration = 1.123 with OD reading of

310mAu
20% breakthrough is at 62mAu
concentration = 10.1mg/ml
Volume = 7.2ml
mass = 72.7mg
Dynamic capacity = 36.4mg/ml

MabSelect Xtra

2mins residence time, 1ml/min flowrate

Plates/meter = 2381
HETP = 0.04200
As = 0.84

Feed concentration = 1.025 with OD reading of

283mAu

89
20% breakthrough is at 56.6mAu
concentration = 13.5mg/ml
Volume = 5.5ml
mass = 74.25mg
Dynamic capacity = 37.1mg/ml

4mins residence time, 0.5ml/min flowrate

Plates/meter = 2167
HETP = 0.04628
As = 0.84

Feed concentration = 1.105 with OD reading of

305mAu
20% breakthrough is at 61mAu
concentration = 13.93mg/ml
Volume = 5.8ml
mass = 80.79mg
Dynamic capacity = 40.4mg/ml

8mins residence time, 0.25ml/min flowrate

Plates/meter = 2081
HETP = 0.04805
As = 0.85

Feed concentration = 1.08 with OD reading of

298.6mAu
20% breakthrough is at 59.72mAu
concentration = 12.06mg/ml
Volume = 6.8ml
mass = 82mg
Dynamic capacity = 41mg/ml

Prosep Ultra

2mins residence time, 1ml/min flowrate

Plates/meter = 1146
HETP = 0.08729
As = 1.86

Feed concentration = 1.01 with OD reading of 280mAu

20% breakthrough is at 56mAu
concentration = 8.8mg/ml
Volume = 7ml
mass = 61.6mg
Dynamic capacity = 30.8mg/ml

4mins residence time, 0.5ml/min flowrate

Plates/meter = 1051
HETP = 0.09514
As = 2.27

90
Feed concentration = 1.01 with OD reading of 280mAu
20% breakthrough is at 56mAu
concentration = 9.35mg/ml
Volume = 7.1ml
mass = 66.4mg
Dynamic capacity = 33.2mg/ml

8mins residence time, 0.25ml/min flowrate

Plates/meter = 1072
HETP = 0.09332
As = 2.44

Feed concentration = 1.01 with OD reading of 280mAu

20% breakthrough is at 56mAu
concentration = 10.77mg/ml
Volume = 6.5ml
mass = 70mg
Dynamic capacity = 35mg/ml

CIGL

2mins residence time, 1ml/min flowrate

Plates/meter = 2041
HETP = 0.04901
As = 1.6

Feed concentration = 1.07 with OD reading of 295mAu

20% breakthrough is at 59mAu
concentration = 8.67mg/ml
Volume = 8.4ml
mass = 72.8mg
Dynamic capacity = 36.4mg/ml

4mins residence time, 0.5ml/min flowrate

Plates/meter = 2148
HETP = 0.04656
As = 1.02

Feed concentration = 1.07 with OD reading of 295mAu

20% breakthrough is at 59mAu
concentration = 11mg/ml
Volume = 7.8ml
mass = 85.8mg
Dynamic capacity = 42.9mg/ml

8mins residence time, 0.25ml/min flowrate

Plates/meter = 1992
HETP = 0.05019
As = 1.66

Feed concentration = 1.07 with OD reading of 295mAu

91
20% breakthrough is at 59mAu
concentration = 13.68mg/ml
Volume = 7ml
mass = 95.77mg
Dynamic capacity = 47.9mg/ml

92