Purity determination of adenovirus - UvA · Purity determination of adenoviruses 10 THEORY 2.1...

PURITY DETERMINATION

OF ADENOVIRUSES

Febri Annuryanti

Supervision:

Dr. Marta Germano Crucell Holland B.V.

Dr. Wim Th. Kok University of Amsterdam

Faculty of Science

University of Amsterdam

July 2013

MSc Chemistry

Analytical Sciences

Master Thesis

PURITY DETERMINATION OF

ADENOVIRUSES

by

Febri Annuryanti

July 2013

Supervisor:

Dr. Wim Th. Kok

Daily Supervisor :

Marta Germano, PhD

DEPARTMENT OF ANALYTICAL DEVELOPMENT

CRUCELL HOLLAND B.V.

Purity determination of adenoviruses

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ABSTRACT

Adenoviruses are potentially useful vectors for vaccination. Crucell

manufactures recombinant adenoviruses on a PER.C6® cell substrate. An extensive

purification is done during the production of recombinant adenoviruses in order to

obtain the pure final products. However, product-related impurities, such as virus

aggregates and virus incomplete particles, can be present in the final product and

may lead to adverse effects or lack of efficacy. Incomplete particles and complete

particles can be separated by cesium chloride (CsCl) density gradient

centrifugation. In addition, a qualitative method to determine the presence of

incomplete particles is available by differential centrifugal sedimentation (DCS).

The goal of this study was to obtain a suitable analytical method for

determining the purity of adenovirus based on the content of product-related

impurities. SEC-HPLC, IEX-HPLC, CE and RP-HPLC were used to distinguish between

the complete and incomplete particles. No separation was observed between the

complete and incomplete particles with SEC-HPLC, IEX-HPLC, and CE techniques.

These results indicated that the complete and incomplete particles have similar

hydrodynamic volume and charged surface. Only RP-HPLC was able to differentiate

between the complete and incomplete particles based on the protein profiles.

Protein 13 and protein 10 were chosen as markers of purity. Protein 13 is

associated with the encapsidation of the viral DNA and protein 10 is universally

present in all forms of adenovirus (both complete and incomplete particles). A

linear correlation was obtained by plotting the percentage of upper band from CsCl

gradient against the ratio of molar concentrations of protein 13 and protein 10.

The percentage of incomplete particles in an adenovirus reference material was

between 4% and 8%.

In conclusion, the purity of adenovirus (with respect to product-related

impurities) could be determined by RP-HPLC. The RP-HPLC method can potentially

replace the DCS method for determining the purity of the adenovirus.


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PREFACE

This research project is part of a Master in Chemistry. The research was carried out

at Crucell Holland B.V. for 8 months. The objective of the research is exploring new

approaches to determine purity of adenovirus and identifying the product-related

impurities, which are important parts of product characterization.

ACKNOWLEDGEMENTS

I would like to thank to my supervisor Marta Germano, Angel Huidobro and Ewoud

van Tricht (Crucell Holland B.V) and Wim Th. Kok (University of Amsterdam) for

their supervision.

Thanks to all my Crucell colleagues who trained and helped me during my

internship. I wish them big success with their life and career.

Last but not least, thanks to my family for supporting me during my study.


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ABBREVIATIONS

Ad : Adenovirus Ad35WT : Adenovirus type 35 Wild Type AEX : Anion Exchange Chromatography AUC : Analytical Ultracentrifugation BGE : Background electrolyte CE : Capillary Electrophoresis CEC : Capillary Electrochromatography CGE : Capillary Gel Electrophoresis CIEF : Capillary Isoelectric Focusing CITP : Capillary Isotacophoresis CsCl : Cesium Chloride CZE : Capillary Zone Electrophoresis d : Detector pathlength DCS : Differential Centrifugal Sedimentation DNA : Deoxyribonucleic acid DSP : Down Streaming Process EOF : Electroosmotic Flow f : Flow rate GMP : Good Manufacturing Practices HPLC : High Performance Liquid Chromatography ID : Internal diameter of capillary IEX : Ion Exchange Chromatography KDa : Kilo Dalton MDa : Mega Dalton MEKC : Micellar Electrokinetic Capillary Chromatography MS : Mass Spectrometry PDA : Photo Diode Array PVA : Polyvinyl alcohol q : Eluted quantity RfA : Peak area response factor Rfh : Peak height response factor RP-HPLC : Reversed Phase – High Performance Liquid Chromatography RSD : Relative standard deviation SEC : Size Exclusion Chromatography TFA : Trifluoroacetic Acid TRIS : Tris(hydroxymethyl)aminomethane UV / VIS : Ultraviolet / Visible


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TABLE OF CONTENTS

ABSTRACT ....................................................................................................................................... 3

PREFACE ......................................................................................................................................... 4

ACKNOWLEDGEMENTS ................................................................................................................... 4

ABBREVIATIONS ............................................................................................................................. 5

TABLE OF CONTENTS ....................................................................................................................... 6

INTRODUCTION .............................................................................................................................. 8

THEORY......................................................................................................................................... 10

2.1 ADENOVIRUS ......................................................................................................................... 10

2.2 METHOD FOR ANALYSIS OF ADENOVIRUS PURITY AND PRODUCT-RELATED IMPURITIES ........................ 12

2.2.1 UV absorbance .............................................................................................................. 12

2.2.2 Ultracentrifugation ........................................................................................................ 12

2.2.3 Differential Centrifugal Sedimentation (DCS) ................................................................ 13

2.2.4 High Performance Liquid Chromatography (HPLC) ....................................................... 14

2.2.4.1 Size Exclusion Chromatography (SEC-HPLC) ....................................................................... 15

2.2.4.2 Ion Exchange Chromatography (IEX-HPLC) ........................................................................ 16

2.2.4.3 Reversed Phase HPLC (RP-HPLC) ........................................................................................ 17

2.2.5 Capillary Electrophoresis (CE) ........................................................................................ 18

EXPERIMENTAL SECTION .............................................................................................................. 21

3.1 STANDARD AND SAMPLES ......................................................................................................... 21

3.2 DIFFERENTIAL CENTRIFUGAL SEDIMENTATION .............................................................................. 21

3.3 SIZE EXCLUSION CHROMATOGRAPHY .......................................................................................... 21

3.3.1 Materials and Methods ................................................................................................. 21

3.3.2 Preparation of Adenovirus Control Sample and CsCl Fractions ..................................... 21

3.4 ANION EXCHANGE CHROMATOGRAPHY ....................................................................................... 22



3.5 CAPILLARY ELECTROPHORESIS ................................................................................................... 22



3.6 ADENOVIRUS PURITY DETERMINATION USING REVERSED-PHASE HPLC ............................................ 22


3.6.2 Preparation of Adenovirus Control samples and CsCl Fractions .................................... 23

RESULTS AND DISCUSSION ............................................................................................................ 24

4.1 DIFFERENTIAL CENTRIFUGAL SEDIMENTATION (DCS) AND AUC ...................................................... 24


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4.2 SIZE EXCLUSION CHROMATOGRAPHY (SEC-HPLC) RESULT ............................................................. 25

4.3 ANION-EXCHANGE CHROMATOGRAPHY (AEX-HPLC) RESULT ........................................................ 29

4.4 CAPILLARY ELECTROPHORESIS (CE) RESULT ................................................................................. 30

4.4.1 Capillary 1 ...................................................................................................................... 32

4.4.2 Capillary 2 ...................................................................................................................... 34

4.4.3 Capillary 3 with dynamic coating .................................................................................. 34

4.5 REVERSED-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (RP-HPLC) ............................... 36

CONCLUSION ................................................................................................................................ 43

FUTURE PERSPECTIVE ................................................................................................................... 45

REFERENCES .................................................................................................................................. 46


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INTRODUCTION

Crucell is a global pharmaceutical company that focuses on research,

development, and production of viruses and antibodies used in vaccines against

infectious diseases worldwide. Several products in development are based on

AdVac® technology, in which rare adenoviruses, such as Ad35 and Ad26, are used

as vectors for recombinant vaccines. Adenoviral vectors have been widely

investigated for clinical applications due to their ability to penetrate into the cell

[1-3]. AdVac® vaccination vectors can be manufactured to high titers on a PER.C6®

cell substrate, which does not allow replication-competent adenovirus to form

during the production process. These vectors combine all the advantages of

adenoviral vectors commonly used for vaccination, such as high production yields,

strong immunogenicity, and excellent safety profile, together with accurate dose

control of the vector-based vaccine. Thus, AdVac®-based vaccines are expected to

deliver consistent results in clinical studies.

The adenovirus production process, including down-stream processing,

results in purified mature recombinant adenovirus. This product expectedly

contains a certain level of impurities, either process-related or product-related.

Product-related impurities are defined as the percentage of virus subpopulations

such as aggregates and immature or defective particles against intact virus. The

presence of such impurities could initiate host immune response leading to

adverse reactions. Moreover, the presence of immature particles can either

enhance or inhibit transduction efficiencies. To ensure the safety, efficacy, and

quality of the vaccine, advanced measurement methods are needed to monitor the

relative quantities of intact virus and product-related impurities [4].

Purity of an adenovirus can be determined by cesium chloride (CsCl) density

gradient centrifugation. In this method, the complete virus is separated from

incomplete particles based on their density [5]. This method is only a qualitative

preparative assay [6, 7]. Differential centrifugal sedimentation (DCS) is another

method to characterize particle size distribution by utilizing sucrose gradient


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solutions [8]. The adenoviruses will sediment differently in a sucrose gradient

according to their size shape and density. In addition, an analytical ultra

centrifugation (AUC) method to determine the purity of adenovirus has been

reported by Berkowitz and Philo [9, 10]. AUC separation is based on differences in

sedimentation velocity and has the advantage of being a quantitative assay. The

disadvantages of this method are its cost, low throughput and an experienced

operator is required to operate the AUC. Hence, the use of AUC as a routine assay

is limited.

Recent studies have shown that column chromatography is a most versatile

and powerful method for adenovirus purification and quantification [11, 12].

Column chromatography may utilize different columns to achieve the desired level

of purification. For example, ion exchange [11, 13, 14], size exclusion

chromatography [12], and affinity chromatography [15], are becoming useful tools

for virus purification [12].

Capillary electrophoresis (CE) is another method that can be used to

analyze viruses. Several groups have investigated human rhinoviruses (HRVs) using

capillary zone electrophoresis (CZE) [16-18]. Mann et al. has developed a method

to separate recombinant adenovirus type 5 in 25 mM sodium phosphate buffer pH

7.0 using a capillary coated with polyvinyl alcohol (PVA) [19].

The aim of this study was to find suitable analytical methods for assessing

the purity of adenovirus preparations. A suitable method is rapid, reliable, and

informative about the quantity and quality of the different populations of

adenovirus particles. In this research, we explored chromatographic methods, such

as size-exclusion chromatography (SEC), ion exchange chromatography (IEX) and

reversed-phase chromatography (RP-HPLC) to determine the purity of adenovirus.

Capillary electrophoresis was also used to in this study, as its short analysis time

and high-efficiency for separations of macromolecules with minimum sample

preparation and sample consumption may be an important advantage [20, 21].

Differential centrifugal sedimentation (DCS) and analytical ultracentrifugation

(AUC) were used as orthogonal techniques to the chromatographic methods.


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THEORY

2.1 Adenovirus

Adenovirus is a non-enveloped, isocahedral DNA virus that belongs to

adenoviridae family [2, 3]. It is composed of an outer capsid surrounding an inner

DNA core with a particle size of approximately 80 nm and a molecular mass of

about 150 MDa [22, 23]. The mass is determined by the protein content (87%) and

DNA content (13%) [9]. Adenovirus proteins can be subdivided in three major

groups: major capsid proteins, minor proteins, and the core proteins. Figure 1

provides a schematic diagram of an adenovirus [2].

Figure 1: Schematic overview of adenovirus. Adenovirus proteins are divided in three major groups: major capsid proteins [hexon (II), penton base (III), fibre (IV)], minor proteins, and core proteins. The virus DNA, indicated by black thick line, is associated with core proteins. (Ref. [2])

The major capsid proteins are the hexon, penton-base, and fiber [3]. There

are 240 homotrimetric hexons (pentamers of protein II) and 12 pentons that

consist of 12 penton bases (protein III), and 12 extended fibers (trimers of protein

IV) [2, 3, 23]. Penton bases and fibers are involved in mediating adenovirus

infection. Protein IIIa, VI, VIII, and IX are minor components which are also


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associated with the capsid and located near the premises of major capsid proteins.

Protein VI is involved in facilitating endosomal escape by disruption of the

endosomal membrane and the main function of protein VI is to facilitate nuclear

import of hexon proteins [23]. Mutant virus analysis suggests that protein VIII plays

a role in the capsid structural stability. The precursor of protein VIII is undetectable

in complete particles while it is present in empty capsids or incomplete particles.

Protein IX acts as capsid cement. In addition, protein IX also affects the DNA-

packaging capacity of human adenovirus and transcriptional activity of several

promoters [23]. Six proteins are situated in the virus core. Five of the components

(V, VII, Mu, IVa2, and terminal protein) are associated with the double stranded

DNA genome, while the 23KDa virus protease plays an important role in the

assembly of the virus [2].

A purified adenovirus preparation consists of monomer adenovirus (particle

size 77 nm) and aggregates [24]. The aggregates are reversible and high ionic

strength conditions can induce dissociation of the aggregates [4]. It was shown in

pH stability data of the adenovirus that adenovirus type 5 spontaneously

dissociated at pH lower than 5.8 [25]. A small increase in the electrophoretic

mobility of adenovirus type 5 was observed by increasing the pH between 5.8 and

8.3 and the mobility stabilizes between pH 8.3 and 9.0 [19]. Based on pH stability

data and the electrophoretic mobility experiment, the pI value of adenovirus type

5 is predicted around 5.8 [19]. Other serotypes of adenovirus may have different pI

value dependent on their peptide compositions [26].

Empty capsids of adenoviruses are detected after the purification of

recombinant adenovirus. These empty capsids differ from complete virus in density

and DNA content. Due to the absence or lack of DNA, empty capsids have lower

density (approximately 1.29-1.30 g/mL) than intact adenovirus (1.34 g/mL) [5, 6].

Empty capsids generally contain hexon, penton and fiber, while the core proteins V

and VII are missing. Other proteins that are not present on the complete virus may

be observed in empty capsids. Some of the proteins have been identified as the


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precursor proteins, pVI and pVIII, or scaffolding proteins L1 52/55K [5]. Protein

52/55K is required for the infectious virus assembly [27].

2.2 Method for Analysis of Adenovirus Purity and Product-Related Impurities

2.2.1 UV absorbance

The complete virus, empty capsids, and aggregation can be analyzed by UV

absorbance at 260, 280, and 320 nm. The ratio A260/A280 shows the relationship

between nucleic acid and protein. A pure adenovirus type 35 shows an A260/A280

ratio of 1.2-1.3. Lower ratios of A260/A280 are typical for protein, while higher ratios

of A260/A280 indicate that the adenovirus preparation could be contaminated with

non viral DNA [28]. The A320/A260 ratio reflects the presence of aggregates in the

purified virus preparation. A typical range of A320/A260 for purified virus is 0.22-0.27

and can rise rapidly to 0.3-0.7 as virus aggregation is initiated [5].

2.2.2 Ultracentrifugation

Ultracentrifugation is a purification method to separate macromolecules,

such as DNA and viruses, based on their partial specific volume (combination of

density, size and shape). Macromolecules are separated from contaminants by

ultracentrifugation in a density gradient. For viruses, salts such as Cs2O4 or CsCl are

used to form the density gradient [7].

For adenoviruses, a sample is applied to a step gradient of CsCl where the

density of the bottom and the upper layer of CsCl are 1.4 g/mL and 1.25 g/mL,

respectively. Subsequently, the density gradients containing the sample are

centrifuged at a certain speed for a period of time. As a consequence, the virus

separates from cellular debris and collects in a band between the two CsCl layers.

This collected band is then mixed with CsCl at 1.35 g/mL and centrifuged overnight.

As a result, the intact virus is separated from incomplete particles (Figure 2). The

CsCl layers are collected from the tubes and CsCl can be removed by dialysis. Intact

viruses will be in the lower band since they have a higher density compared to


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incomplete and empty particles, whereas incomplete particles are present in the

upper band. This method is easy to perform and yields high-purity virus

preparations [6].

Figure 2. Example of recombinant adenovirus particles density forms separated by preparative CsCl gradient centrifugation. Upper light blue band contains incomplete particles; lower thick band contains intact virus. (from Ref. [29])

2.2.3 Differential Centrifugal Sedimentation (DCS)

Differential Centrifugal Sedimentation (DCS) is a method to determine the

particle size and mass distribution based on sedimentation of particles. Stokes Law

can be used to determine the spherical particle size from measurement of the time

needed for particles to settle a known distance in a liquid of known density and

viscosity. Sedimentation can either be based on gravitational or centrifugal force

[8]. Gravitational force is used to measure large particles. For small particles, like

virus, centrifugal sedimentation is preferably used to determine the size

distribution (the high centrifugal force makes the sedimentation of small particles

faster than Brownian motion so the particles start to settle according to Stokes

Law).

For this study, differential sedimentation was used. Samples are placed on

the top of a clear fluid and subjected to a centrifugal acceleration to yield particle

sedimentation [30]. Initially, the detector reads at maximum intensity that

decreases when particles pass the detector beam. The signal reduction is

proportional to the particle concentration when the X-ray detector is used. For


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monochromatic light source, Mie theory light scattering is used to estimate the

particle concentration [31].

DCS instruments use a hollow, optically clear disc that is driven by a motor

(Figure 3). A short wavelength (400-500 nm) monochromatic light beam is used as

detector beam, as it gives good sensitivity for measuring particles smaller than 100

nm diameter [8]. The DCS operation is very simple, and multiple measurements

can be done in one day. Furthermore, this method can produce accurate and

reproducible result in a short time. However, DCS cannot be used for quantitative

assay of the incompletes particles because of the difficulty to know the exact

values of refractive index, shape and particle density of incomplete particles.

Moreover, the DCS software cannot be qualified for Good Manufacturing Practices

(GMP) purposes so this is difficult to implement in quality control laboratories [32].

Figure 3. A typical DCS design. (Left) Front view; (Right) Side view (Ref. [8])

2.2.4 High Performance Liquid Chromatography (HPLC)

HPLC is a separation method based on the partition of sample compounds

between a solid stationary and a liquid mobile phase under high pressure [33]. It

potentially gives rapid separation of different sample compounds with an

extraordinary peak resolution. The highest efficiencies can be achieved by the use

of very small particles of stationary phase and high pressure to maintain a high

flow rate [12, 33].


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Many HPLC methods are developed for analyzing adenoviruses, such as ion-

exchange chromatography [11, 13, 28, 34, 35], size exclusion chromatography [12],

and reversed-phase chromatography [36, 37]. The modes of chromatography that

were explored in this study for assessing the purity of viral particles are size

exclusion chromatography (SEC), reversed phase high-performance liquid

chromatography (RP-HPLC), and ion exchange chromatography (IEC).

2.2.4.1 Size Exclusion Chromatography (SEC-HPLC)

Size-exclusion chromatography (SEC), also known as gel-filtration

chromatography, is a technique for separating analytes based on their size or

hydrodynamic volume. This technique can be used to analyze biological samples

that consist of proteins with different molecular weights [12, 38]. The solid-phase

matrix consists of porous beads packed into a glass or steel column so the mobile

phase can diffuse to the volume inside the pores and outside the beads. High

porosity beads lead to a total liquid volume of > 95% for packed columns [38].

In SEC (Figure 4), the pore structure of the resin provides a molecular sieve;

smaller molecules penetrate into the pores of the beads and larger molecules

remain outside of the beads as they are unable to enter the pores [7, 38, 39]. The

order of elution can be predicted by the size of the molecules, i.e. large molecules

elute first from an SEC column (as they are excluded from the pores) followed by

small molecules [12, 38, 39].

Figure 4. Schematic overview of a size exclusion column. Larger particles will elute first followed by smaller particles (Ref. [40])


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There are various types of stationary phase for SEC. The choice of stationary

phase depends on the molecular weight and the solubility of the analytes in the

sample. Silica porous beads are suitable for protein separations whereas polymer

porous beads are designed for organic polymers, poly- and oligosaccharides, and

viruses. Zhang et al., have developed a method for assessing adenovirus type 5

purity using Bio-Sep-Sec-S3000 column. The method allowed to quantify the

adenovirus and to detect impurities [12].

2.2.4.2 Ion Exchange Chromatography (IEX-HPLC)

Ion exchange chromatography is a separation process based on differences

in net surface charge of the molecules. This technique is appropriate for analysis of

proteins, oligonucleotides, nucleic acids or other charged biomolecules. There are

four steps during the separation process in IEX. The first step is the equilibration of

the column to the appropriate pH and salt concentration. The second step is the

sample loading, by which the analyte of interest will bind to the column. The third

step is elution of the analytes from the column by changing the pH or increasing

the salt concentration. The last step is column regeneration. IEX can be classified

into anion exchange and cation exchange (Figure 5). In cation exchange, a

negatively charged group in the stationary phase is used to attract positively

charged molecules. Sulfonic acid, phenolic hydroxyl and carbonyl are commonly

used as cation exchangers. The former is known as strongly acidic cation exchanger

while the others are weakly acidic cation exchangers. In anion exchange, negatively

charged molecules are attracted into positively charged groups in the stationary

phase. Quartenary, aromatic and aliphatic amino groups are examples of anion

exchangers [41, 42]. Separation is achieved as differently charged molecules have

different degrees of interaction with the ion exchanger. The separation can be

optimized by varying the pH and/or ionic strength of the mobile phase, or by

eluting in a pH or salt gradient [42, 43].


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Figure 5. Schematic of Ion-exchange chromatography. (Left) Anion Exchange column; (Right) Cation Exchange column. (from Ref. [44])

Several publications have been published on the use of ion-exchange

chromatography for analysis of adenoviruses. Uno-Q column [28, 34] and Q

Sepharose XL column [13, 35] have been used for quantifying adenovirus type 5.

Bio-Monolith QA column can be use to separate intact adenovirus type 5 from

contaminants [11].

2.2.4.3 Reversed Phase HPLC (RP-HPLC)

In reversed phase mode, a non-polar compound is utilized as the stationary

phase and a polar solvent is used as mobile phase. The separation mechanism is

based on the partitioning (differential affinity) of the analytes between stationary

and mobile phases.

Stationary phases of reversed phase columns are traditionally made up of

silica modified with alkyl chains of a certain length. The most common alkyl chains

are C2, C4, C8, C18. Phenyl can also be used as aromatic side chain. Short alkyl chains

are suitable for the separation of more polar analytes whereas longer alkyl chains

are preferred for moderately polar and non-polar analytes separation [42, 45].

Elution in RP-HPLC can be isocratic or in a gradient condition. In isocratic

elution, the concentration of the mobile phase remains constant during the

analysis. In gradient elution, the concentration of the organic solvent is increased

over a period of time. The analytes elute in order of increasing molecular

hydrophobicity [42].


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Lehmberg et al., have successfully quantified adenovirus type 5 by RP-HPLC

through quantification of the structural proteins [36]. Takahashi and co-workers

were also able to quantify the empty capsids of adenovirus type 5 by RP-HPLC.

They used precursor protein VII as the marker for quantifying the empty capsids

[37].

2.2.5 Capillary Electrophoresis (CE)

Capillary electrophoresis (CE) is a very efficient separation technique

available for the analysis of both small and large molecules [46]. Capillary

electrophoresis is defined as the differential movement of charged particles under

the influence of an electric field [47, 48]. A typical CE system, which is illustrated in

Figure 6, consists of a high-voltage power supply, a sample introduction system,

two buffer reservoirs, two electrodes, a capillary, a detector and an output device.

Each side of the high power supply is connected to an electrode. These electrodes

help to induce an electric field to initiate the migration of the sample from the

anode to the cathode through the capillary [49]. The capillary is filled with the

desired buffer solution and the ends of capillary are dipped in vials containing the

same buffer solution [50]. As the separation in CE depends not only on the

electrophoretic mobility but also on the viscosity of the solution, a temperature

control device is needed to ensure reproducible results. Most CE instruments make

use of forced air and liquid to control the temperature.

Figure 6. The instrumental set-up of CE system


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Capillary electrophoresis can be operated in several modes. Examples of CE

modes are capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE),

micellar electrokinetic capillary chromatography (MEKC), capillary

electrochromatography (CEC), capillary isotacophoresis (CITP), and capillary

isoelectric focusing (CIEF). In this research, only CZE was used.

Capillary zone electrophoresis (CZE) or free solution capillary

electrophoresis is the most common form of CE. The separation of a mixture in a

solution is based on the differences in electrophoretic mobility and the

electroosmotic flow (EOF). CZE is suitable for separation of both anionic and

cationic analytes. Neutral molecules are not separated based on electrophoretic

mobility and will migrate together with the EOF [48].

Electrophoresis

Electrophoresis is the migration of sample ions in a solution under the

influence of an electric field. The rate of mobility is directly proportional to the

applied electric field; the greater the strength of electrical field, the faster the

mobility. Furthermore, the electrophoretic mobility depends on the charge of the

molecule, the solution viscosity and the molecule’s radius. If two ions have the

same size, the one with greater charge will have a greater mobility. For ions with

the same charge, the smaller particle has less friction and will migrate at a faster

rate [51].

Electroosmotic Flow (EOF)

A fundamental characteristic of CE operation is the electroosmotic flow

(EOF). EOF is the bulk flow of solution caused by the application of a voltage to an

electrolyte-filled capillary (Figure 7). The flow occurs when the buffer (pH greater

than 3) running through the capillary so the free silanol groups are deprotonated

[47, 49]. As the whole system must be electrically neutral, the buffer solution will

form a diffuse double-layer close to the wall. The inner layer is fixed, while the


20

outer layer is mobile. When a voltage is applied to the capillary, the mobile layer

will move toward the cathode and creating an EOF [47, 49].

Figure 7. The principle of electroosmosis (from Ref. [52])

The advantage of the EOF is the flat flow profile in the capillary. There is no

pressure drop within the capillary because the electric force of the flow is

uniformly distributed along the capillary. As a result, no peak broadening is caused

by the EOF [47, 49].


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EXPERIMENTAL SECTION

3.1 Standard and Samples

A specific adenovirus type was used as control sample. CsCl fractions (upper

and lower band), obtained by ultracentrifugation, were used as samples. The upper

band represents incomplete particles (impurities of adenovirus), while the lower

band consists of intact particles of adenovirus. All samples were thawed slowly at

room temperature prior to analysis.

3.2 Differential Centrifugal Sedimentation

DCS analysis of adenovirus was performed using a CPS instrument model

DC24000 equipped with a detector. 200 µL of each adenovirus control sample and

samples was injected into the DCS instrument. Samples were injected as received

(no sample treatment was required for this analysis).

3.3 Size Exclusion Chromatography

3.3.1 Materials and Methods

An Alliance 2695 HPLC system with a PDA detector was used for the SEC

experiment. Silica- and polymer-based columns were utilized as analytical columns.

To determine the optimal conditions for purity determination of adenovirus,

buffers with different pH values were used as mobile phase. The injection volume

was 50 µL.

3.3.2 Preparation of Adenovirus Control Sample and CsCl Fractions

A specific adenovirus was used as control sample and CsCl fractions (upper

and lower band) were used as samples. All samples were thawed slowly at room

temperature. Control sample and samples were transferred into HPLC vials and

placed in the autosampler for analysis.


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3.4 Anion Exchange Chromatography


Anion-exchange chromatography was performed on a quaternary column.

An Alliance 2695 HPLC system with a PDA detector was used. Chromatograms were

extracted from the PDA data. Buffers with gradient elution were used as mobile

phase. The injection volume was 100 µL.


Adenovirus control sample and CsCl fractions are prepared as in 3.3.2.

3.5 Capillary Electrophoresis


CE analysis was carried out using an Agilent CE (G7100A). The different

capillaries were used for the experiments. Buffers with different pH values were

used as BGE.


A specific adenovirus control sample and CsCl fractions were thawed slowly

at room temperature.

3.6 Adenovirus Purity Determination Using Reversed-Phase HPLC


The RP-HPLC experiments were performed on an Alliance 2695 HPLC

system equipped with a PDA detector. C4 column was used for separation. The

mobile phases were eluted with the gradient program.


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3.6.2 Preparation of Adenovirus Control samples and CsCl Fractions

The adenovirus control sample and the CsCl fractions were thawed slowly

at room temperature. The upper and lower CsCl fractions were mixed to have

certain amount of the incomplete particles.


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RESULTS AND DISCUSSION

4.1 Differential Centrifugal Sedimentation (DCS) and AUC

The adenovirus control sample and the CsCl fractions were analyzed by DCS

using CPS instrument. The result is shown in Figure 8. The adenovirus control

sample showed 2 peaks. The small peak represents incomplete particles, with an

estimated diameter approximately 65 nm, whereas the higher peak represents

complete particles with an estimated diameter of about 69 nm. The upper

(incomplete particles) and lower (complete particles) band of CsCl fractions

showed differences in particle diameter. The peaks of the upper band and lower

band were detected at approximately 65 nm and approximately 71 nm,

respectively, overlapping with the two peaks of the adenovirus control sample.

Figure 8. Result of DCS analysis (–) Adenovirus control sample; (−) lower band of CsCl fraction; (–) upper band of CsCl fraction.

DCS result was confirmed by AUC (Figure 9). The AUC results further

showed that there were complete particles in the upper band (see peaks between

600 and 700 s in the red trace in Figure 9).


25

Figure 9. AUC results: Sedimentation distribution of (–) adenovirus control sample, (–) upper band of CsCl fraction and (–) lower band of CsCl fraction.

The DCS and AUC results indicated that the incomplete/empty and

complete particles can be distinguished based on their density. This was an

expected result as these different types of particles can also be separated by CsCl

density gradient ultracentrifugation. The disadvantages of the DCS technique as a

method for analysis of product-related impurities are that it is not quantitative and

the instrument cannot be operated in a GMP laboratory. This is also a drawback of

the AUC technique. Although AUC does yield quantitative result, it requires an

experienced operator. Furthermore, the sample throughput of AUC is low

compared to HPLC. Therefore, the feasibility of HPLC methods for the purpose of

this analysis was explored.

4.2 Size Exclusion Chromatography (SEC-HPLC) Result

An alternative method to determine quantitavely the purity of adenovirus is

needed since AUC is not the method of choice, as explained above. Size exclusion

chromatography (SEC) was chosen as the first chromatography method for

determining the purity of adenovirus since adenoviruses are already purified using


26

membrane-based anion-exchange in Downstream Process (DSP). A different

chromatographic mode of separation, such as size exclusion, is expected to resolve

the impurities in adenovirus products better than an analytical method with the

same mode of separation as that used for purification. The adenovirus control

sample was initially analyzed on a column, which has a pore size of 200 nm and is

made from silica. The chromatograms of the adenovirus control sample in Figure

10 show that buffer pH 1 gives a good peak response of adenovirus. Fixing this pH,

the flow rate and column temperature were optimized. The result is shown in

Figure 11.

Figure 10. Chromatograms of adenovirus control samples at different pH values. buffer (–) pH 1;

(–) pH 2; (–) pH 3.


27

Figure 11. Chromatograms of adenovirus control samples in pH 1 (–) default condition; (–)

condition 3; (–) condition 2; (–) condition 3; (–) blank.

Figure 11 shows that default condition is the best separation conditions for

adenovirus. However, a blank peak eluted at the same retention time as the

adenovirus control sample, as observed in the chromatogram. The result was

unexpected since blank has particle size smaller than the adenovirus. The

unexpected result could be an indication that the column is damaged.

Furthermore, the repeatability of adenovirus peak height between injections was

not good (data not shown). We hypothesized that the poor repeatability was

caused by strong interaction between adenovirus and silica. This assumption was

reinforced by the publication of Mann et al., which indicated that adenovirus are

adsorbed at silica surfaces [19]. Since the silica column interacts with adenovirus

and resulted in poor reproducibility, a different SEC column was needed.

Polymer column with a pore size 100 nm was chosen for the next SEC

experiment. This column was used successfully for analysis of other viruses in-

house and it was expected that the column would also work for adenovirus. Based

on the method used in-house for analysis of other viruses, buffer pH 8.0 containing

salt was used as mobile phase. The purpose of adding salt is to minimize

hydrophobic interactions between the polymer matrixes and adenovirus which

could lead to low recovery and carry-over effects. The adenovirus eluted at 13 min


28

and was well separated from the blank peak (Figure 12). Six injections of

adenovirus control sample showed a good repeatability of peak area (RSD < 5%).

No carry over was observed in the blank (black trace in Figure 12).

Figure 12. SEC chromatogram of adenovirus control sample in polymer-based column. Adenovirus

eluted at 13 min while the blank peak eluted at 20 min. (–) Adenovirus; (–) Blank.

Unfortunately, injections of the upper and lower band of the CsCl gradient

showed similar retention times (Figure 13). These results were unexpected

because we had determined by DCS and AUC that the lower (complete particles)

and upper (incomplete particles) bands of CsCl fractions consisted of particles with

different particle sizes (based on their separation by density). It is possible that the

polymer-based column is not able to resolve the small particle size differences

between incomplete particles and complete particles of adenovirus.


29

Figure 13. SEC chromatograms of adenovirus in polymer-based column. (−) adenovirus control sample; (−) the lower band of CsCl fractions; (−) the upper band of CsCl fractions; (−) blank.

Even though the particles of the two CsCl fractions cannot be separated

using SEC-HPLC method, this technique can still be used for quantification of

adenovirus particles in purified samples.

4.3 Anion-Exchange Chromatography (AEX-HPLC) Result

Anion-exchange analytical column was explored to determine the purity of

adenovirus. 100µL samples were injected and eluted in an increasing salt gradient

with mobile phase B. Figure 14 shows the AEX chromatograms.


30

Figure 14. AEX-HPLC chromatograms of (−) adenovirus control sample; (−) upper band and (−) lower bands of CsCl gradient. The upper and lower bands of the CsCl gradient have similar retention times, while the retention time of control sample is slightly different, which is explained by the differences in inserted gene for these two types of samples.

The chromatograms clearly show that there is no significant difference in

retention time between the upper and lower bands of the CsCl gradient. The

complete and incomplete/empty particles eluted at 11.27 min and 11.30 min,

respectively. The adenovirus control sample eluted at 11.52 min. The retention

time difference between the control sample and the CsCl gradient fractions can be

explained by the different inserted genes in these adenovirus samples. The results

for the upper and lower bands indicated that the incomplete/empty and complete

adenovirus particles are similarly charged at the pH of the mobile phase used, so

they cannot be distinguished from each other by AEX-HPLC. An investigation on the

effect of mobile phase pH on the resolution between the upper and lower bands

may be useful for further development.

4.4 Capillary Electrophoresis (CE) Result

CE was chosen as an alternative method since SEC cannot resolve

incomplete/empty from complete adenovirus particles, and only a slight difference

in retention times was observed by AEX-HPLC. CE is expected to separate particles

based on their charge to mass ratios with resolution higher than AEX-HPLC.


31

In the first run, the experimental condition were according to Mann et al.

[19] with slight modifications. In addition, different capillaries, pH values and

molarities were evaluated to determine the optimum condition for adenovirus

separation. Prior to the experiments, a range of different voltages was applied to

the background electrolyte (BGE) to observe excessive Joule heating effect by

plotting the applied voltage and the measured current (Table 1).

Table 1. A range of different voltages was applied to the BGE for observation of Joule heating effect

BGE

Voltage

-5 -10 -15 -20 -25 -30

Current (µA)

BGE 1 9 19 30 43 59 79

BGE 2 30 65 NA NA NA NA

BGE 3 7 13 20 28 38 49

BGE 4 11 22 35 51 68 NA

BGE 5 7 13 20 28 37 48

Applied voltages that still showed linear correlation with the current were

chosen as applicable voltages in order to have good efficiency and resolution. The

maximum applicable voltage for each BGE is presented in Table 2. Separation was

done in reversed polarity as adenovirus is negatively charged at pH above 6.0. BGE

2 was not used for the experiment due to low applicable voltage that would result

in longer analysis time for adenovirus.

Table 2. Maximum applied voltage for BGE solutions

No Background Electrolyte (BGE)

solution

Maximum applied voltage

( - kV)

1 BGE 1 20

2 BGE 3 25

3 BGE 4 18

4 BGE 5 25


32

4.4.1 Capillary 1

The electropherograms of adenovirus and CsCl fractions in each BGE

obtained with capillary 1 are shown in Figure 15 to Figure 18. For all samples, the

adenovirus peak eluted in less than 5 min in all BGE. No differences in migration

times were observed between the CsCl fractions and between these fractions and

the adenovirus control sample.

Figure 15. Electropherograms of different adenovirus samples. Electrophoresis in BGE 1; 20 kV in reverse polarity. (−) upper band of CsCl gradient; (−) lower band of CsCl gradient (−) adenovirus control sample.

Figure 16. Electropherograms of different adenovirus samples. Electrophoresis in BGE 3; 18 kV in reverse polarity. (−) upper band of CsCl gradient; (−) lower band of CsCl gradient. (−) adenovirus control sample.


33

Figure 17. Electropherograms of different adenovirus samples. Electrophoresis in BGE 4; 25 kV in reverse polarity. (−) upper band of CsCl gradient; (−) lower band of CsCl gradient; (−) adenovirus control sample.

Figure 18. Electropherograms of different adenovirus samples. Electrophoresis in BGE 5; 25 kV in reverse polarity. (−) upper band of CsCl gradient; (−) lower band of CsCl gradient; (−) adenovirus control sample.


34

4.4.2 Capillary 2

The separation of complete and incomplete adenovirus particles in capillary

1 was unsuccessful. Therefore, another experiment was done using capillary 2. This

capillary was successfully used by Mann et al. to analyze recombinant adenovirus

type 5. The separation of adenovirus in capillary 2 was evaluated in BGE as used for

capillary 1. The maximum applicable voltages were the same as for capillary 1 and

no significant differences in migration times were observed for each BGE value.

The results of adenovirus analysis in capillaries 1 and 2 are summarized in Table 3.

The unknown peaks, which looked like electrodispersion phenomenon,

were observed in the adenovirus control sample and the CsCl fractions when BGE 3

and 4 was used as BGE in the capillaries 1 and 2. We attributed those peaks to pH

instability.

Table 3. Migration times of adenovirus in capillaries 1 and 2

BGE Applied

Voltage

(- kV)

Migration Times

(Minutes)

Resolution

complete vs

incomplete

Cap. 1 Cap. 2 Cap. 1 Cap. 2

BGE 1 20 3.2 3.3 No No

BGE 2 5 - - No No

BGE 3 25 2.1 2.2 No No

BGE 4 18 3.4 3.6 No No

BGE 5 25 2.5 2.2 No No

In conclusion, capillaries 1 and 2 were not able to resolve the CsCl fractions.

The adenovirus control sample and the CsCl fractions showed similar migration

times in both capillaries 1 and 2.

4.4.3 Capillary 3 with dynamic coating

A capillary 3 dynamically coated with buffer pH 6.0 and 7.1 was tested. The

different coating mode was expected to be able to separate complete and

incomplete particles. The electropherograms of adenovirus at pH 6.0 and pH 7.1

are shown in Figure 19 and Figure 20, respectively.


35

Figure 19. Electropherograms of different adenovirus samples with dynamic coating. BGE: buffer pH 6.0. (−) adenovirus control sample; (−) lower band of CsCl gradient; (−) upper band of CsCl gradient.

Figure 20. Electropherograms of different adenovirus samples with dynamic coating. BGE: buffer pH 7.1. (−) adenovirus control sample; (−) lower band of CsCl gradient; (−) upper band of CsCl gradient.

The peaks of adenovirus control sample and CsCl gradient fractions were

split at pH 6.0, and only the adenovirus control sample showed a peak at pH 7.1.

The CE results using capillary 3 were inconclusive due to a broken electrode.

None of CE experiments led to satisfactory results because the

incomplete/empty particles could not be distinguished from the complete particles


36

under the tested conditions. It seems that the charge to mass ratio of adenovirus in

these different types of particles is only slightly different. As a result, a different

technique is needed to determine the concentration of incomplete particles. The

CE method, in which a capillary 1 with BGE 1 and BGE 5 was used, may still be used

for quantification of adenovirus particles.

4.5 Reversed-Phase High Performance Liquid Chromatography (RP-HPLC)

An RP-HPLC method has been developed for stability and purity testing of

adenovirus control sample but this method has only been used for qualitative

purposes. The CsCl fractions containing incomplete/empty particles and complete

particles have not yet been analyzed by this method. The conditions of RP-HPLC

disrupt the adenovirus particles and allow for the separation of the individual

adenovirus proteins. Representative chromatograms of the upper and lower bands

of CsCl gradient are presented in Figure 21.

Figure 21. RP-HPLC chromatogram overlay of: (−) upper band and (−) lower band of CsCl gradient. The main differences between the chromatograms are marked with arrows.

As it can be seen in Figure 21, differences were observed between the

chromatograms of the upper and lower bands. The peak areas of some proteins

(protein 1, 5, 7 and 8) were decreased in the upper band whereas other proteins

(protein 3, 9, 12, and 13) were increased in the upper band, compared to the lower

band of the CsCl gradient. Proteins 2, 6 and 10 had the same peak areas in the


37

upper and lower bands. In addition, two unknown peaks were observed in the

upper band of the CsCl gradient. The decreased peak area of the DNA-associated

proteins in the upper band is understood, as this band consists of

empty/incomplete particles. The absence of protein 9 in the lower band is

expected as this protein is not supposed to be present in complete adenovirus

particles. Interestingly, the protein 13 was also mainly present in the fraction

corresponding to empty/incomplete particles. Protein 13 is related with the

infectious virus assembly and it is also required for the encapsidation of the

genome. Predominance of the protein 13 in the upper band indicated that this

fraction contained virus particles which are still being formed or for which genome

encapsidation is ongoing, in agreement with the expectation that this fraction

contains mainly empty/incomplete adenovirus particles [53].

In order to determine if there is a linear relationship between the relative

peak area of the protein 13 and the percentage of empty/incomplete particles,

different ratios of the upper and lower band were mixed as described in section

3.6.2. The final virus particle concentrations of the mixed samples are to be

considered as estimations. The concentration of empty/incomplete particles can

be calculated according to Vellekamp et al. [5], but this calculation still needs to be

validated before it can be considered reliable.

The AUC data were used to estimate the percentage of complete particles

that were present in the upper band of CsCl gradient. Based on the AUC relative

peak areas, there were 17 % of complete particles in the upper band. The

percentages of empty/incomplete particles in the mixed samples were corrected

by this factor. It is stressed that these percentages remain estimations, as the

quantitative ability of the AUC method has not yet been demonstrated. Linear

correlations for each protein were observed by plotting the percentage of

incomplete/empty particles versus peak area. The RP-HPLC chromatograms are

shown in Figure 22.


38

Figure 22. RP-HPLC chromatograms of samples with varying concentrations of empty/incomplete particles obtained by mixing bands of the CsCl gradient.

For this report, protein 13 was chosen as marker of empty/incomplete

particles because it is only expected to be present in particles that are being

assembled or filled with viral DNA. Table 4 shows that the peaks assigned to

proteins 2, 6 and 10 were the same for all the mixed fractions. The % RSD value of

protein 10 peak area for duplicate analysis of the same sample is comparable to

the % RSD calculated for all the mixed samples, indicating that the relative

concentration of this protein is the same for all types of virus particles (empty,

incomplete and complete). It is expected for protein 10 to be present in equal

concentrations in all types of adenovirus. Therefore, protein 10 was chosen as

internal control peak. The areas of the other peaks in the chromatogram can then

be reported relative to peak 10 in order to eliminate possible result artifact due to

variations in sample concentration (VP/mL).


39

Table 4. The peak areas and RSD value of protein 2, 6 and 10 in all mixed fractions of incomplete particles.

Protein n=2 % of empty/ incomplete particles RSD

(%) 0% 21% 42% 63% 83%

2 Peak Area 20829 20569 19327 19565 17280 7.19

RSD (%) 1.4 0.3 1.3 0.1 2.1

6 Peak Area 27820 27900 27160 27110 26005 2.79

RSD (%) 0.7 0.9 0.9 1.0 0.0

10 Peak Area 25710 26160 25560 26372 25646 1.37

RSD (%) 0.9 0.8 1.5 1.3 1.1

Quantitation of Proteins Using HPLC-Detector Response

The molar concentration of proteins were calculated based on papers from

Eberlein and Lehmberg [36, 54]. According to Eberlein [54], the detector response

factors from HPLC could be used to measure the protein concentration of

adenovirus. The peak area, peak height, flow rate, extinction coefficient and

molecular weight data were needed for the calculations of protein concentration.

The extinction coefficients and molecular weights of proteins 10 and 13 were

obtained from ProtParam tool, ExPASy [55].

The detector responses were calculated in two steps as follows:

A. Peak height response factor from extinction coefficient (Rfh)

The response factor for the peak height (Rfh) was calculated as in Equation 1.

Rfh = absorbance (at 1 AU) x extinction coefficient Equation 1

The theoretical extinction coefficients for proteins 10 and 13 were 1.142 and

0.846, respectively. The absorbances of proteins 10 and 13 at 1 AU and the

responses factor for the peak height (Rfh) in the different mixed samples are listed

in Table 5 and Table 6.


40

Table 5. Absorbances of proteins 10 and 13

% of empty/incomplete particles Absorbance at 1 AU

Protein 10 Protein 13

0% 892 926.3

21% 908 984.3

42% 924 1006

62% 938.5 985

83% 934 1018.2

Table 6. Response factors for peak height (Rfh) of proteins 10 and 13

% of empty/incomplete particles Rfh (mV)

Protein 10 Protein 13

0% 1018.6 783.6

21% 1036.9 832.7

42% 1055.2 851.1

62% 1071.8 833.3

83% 1066.6 861.4

B. Peak area response factor (RfA) from extinction coefficient

The peak area response factor (RfA) could be obtained from the extinction

coefficient by integrating the peak height (Rfh) over its duration (i.e calculating the

peak area) [Equation 2]. The duration is determined by the flow rate ‘f’ (ml/60s).

RfA = Rfh x f -1 Equation 2

From the value of RfA, the eluted quantity (q) is calculated as in Equation 3:

q = Peak area x RfA-1 x d-1 Equation 3

where q = eluted quantity (mg), RfA = area response factor for peak area at f =0.2

ml/min, d = detector cell path length (1 cm). The molar concentration is calculated

by dividing the eluted quantity (mg/mL) by the molecular weight of each protein.

The measured peak area response factors (RfA), the injected quantities and

the molar concentrations are listed in Table 7.


41

Table 7. Peak area response factors, injected quantities and molar concentrations of protein 10 and 13 in the different mixed samples of upper and lower bands from the CsCl gradient.

% of empty/incomplete

particles

RfA (mV s mg-1

cm-1

) q (mg/mL) Molar conc. (mM)

Protein 10

Protein 13

Protein 10

Protein 13

Protein 10

Protein 13

0% 305580 235080 0.8 0.1 1.3E-05 2.7E-06

21% 311070 249810 0.8 0.6 1.3E-05 1.3E-05

42% 316560 255330 0.8 1.1 1.3E-05 2.6E-05

62% 321540 249990 0.8 1.7 1.3E-05 3.9E-05

83% 319980 258420 0.8 2.1 1.3E-05 4.7E-05

The correlation between the estimated percentage of incomplete/empty

particles and molar concentration of protein 13 (normalized by dividing it by the

molar concentration of protein 10) is shown in Table 8 and Figure 23.

Table 8. The correlation between the upper band and

the molar concentration ratio of protein 13 and protein 10

% of empty/incomplete particles

Molar conc. ratio of p13/p10

0% 0.2

21% 0.1

42% 2.0

62% 3.0

83% 3.6

Figure 23. Plot of the molar concentration ratio of protein 13 vs protein 10, against the estimated percentage of empty/incomplete particles. The solid line represents the linear regression fit.

y = 4,2454x + 0,185 R² = 0,9955

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

0% 20% 40% 60% 80% 100%

Mo

lar

con

c. r

atio

of

p1

3/p

10

Estimated % of empty or incomplete particles


42

The percentage of empty/incomplete particles in adenovirus control

samples were determined by calculating the molar concentration ratio of protein

13 vs protein 10 in adenovirus control sample and comparing it to the calibration

curve in Figure 23. The percentages of incomplete particles in the samples of

adenovirus control sample analyzed in this study were between 4.8% and 8.1%.

The percentage of empty/incomplete particles in adenovirus control sample

obtained from the calibration curve were two times lower than if it is calculated

based on AUC result (16%). There were two main reasons for the significant

differences in the results of the two methods. First, the AUC results of adenovirus

particles were determined at difference wavelength. This difference in wavelength

could affect the results due to the absorbance or the light scattering effects of the

adenovirus particles. Second, the AUC results show that there are two different

peaks assigned as empty/incomplete particles. One of the peaks may still contain

DNA whereas the other does not have DNA at all. On the other hand, the

percentages of incomplete/empty particles in adenovirus control sample by RP-

HPLC were determined based on the protein profiles.

In conclusion, the RP-HPLC method with the calculation as described could

be used to calculate the percentage of empty/incomplete particles in adenovirus

samples.


43

CONCLUSION

Several methods were explored in this study to determine the purity of

adenovirus (quantification of product related-impurities). SEC-HPLC, AEX HPLC and

CE methods did not lead to satisfactory separation of the upper and lower bands of

CsCl gradient. With SEC-HPLC, the retention times of both bands of CsCl gradient

were the same, whereas with AEX-HPLC only slightly different retention times were

observed between the upper and lower bands of CsCl gradients. CE, which is

expected to have high separation efficiency, was also explored to separate the

upper and lower bands of CsCl gradients. Different capillaries and background

electrolyte (BGE) were tried for the experiments, but none of the experiments

yielded satisfactory separation of upper and lower bands. Since none of the

methods could distinguish between upper and lower bands of the CsCl gradient,

we concluded that complete and incomplete/empty adenovirus particles have

similar hydrodynamic volumes and surface charges.

Another technique, RP-HPLC, was explored for distinguishing between the

CsCl fractions. In this method, we obtain the adenovirus particles’ protein profiles

after particle disassembly in an appropriate mobile phase. We observed different

protein profiles for each of the CsCl fractions. Most of the proteins in the profiles

were present in both fractions. We focused our research on protein 13 and protein

10. Protein 13 is associated with adenovirus assembly and encapsidation of the

viral DNA. A large relative concentration of protein 13 in the upper band revealed

that the adenovirus particles in this band lacked or had less DNA. Equal

concentrations of protein 10 in the two CsCl fractions indicated that this protein is

present in the same stoichiometry in complete and incomplete/empty adenovirus

particles.

A calibration curve was used to determine the purity of an adenovirus

control sample (% of product-related impurities). The calibration curve was made

by plotting the molar concentration ratios of protein 13 vs protein 10 against the

percentage of empty/incomplete particles in “calibration samples”. The calibration


44

samples were prepared by mixing defined volumes of the upper and lower bands

of the CsCl gradient. A good linear correlation was observed between the

estimated percentage of empty/incomplete particles and the molar concentration

ratio of protein 13 vs protein 10. The molar concentration ratio of protein 13 vs

protein 10 of adenovirus control sample indicated that the adenovirus control

sample samples tested contained less than 10% of incomplete particles (estimation

based on the calibration curve described above). The percentage of

empty/incomplete particles in adenovirus control sample determined by AUC

method was significantly different from the RP-HPLC (two times higher). This result

might be caused by the different wavelength and the different measurement

principles that were used for the two determinations.

In summary, RP-HPLC is a promising tool to determine the purity of

adenovirus, in terms of presence of product-related impurities (empty/incomplete

particles). The method can be used to replace the current DCS method that is used

for the same purpose in our laboratories. The percentage of empty/incomplete

particles in adenovirus-based vaccines can be very useful to estimate the vaccine

potency.


45

FUTURE PERSPECTIVE

It was concluded that RP-HPLC is the method of choice for determining the

percentage of empty/incomplete particles in adenovirus preparations. Currently,

the analysis time of RP-HPLC is more than two hours. The method should be

optimized and validated to obtain a fast and robust analysis. Mass spectrometry

(MS) analysis is needed to confirm the peak assignments in the RP-HPLC

chromatograms. In addition, the correlation between molar concentration of the

protein 13 (normalized to the concentration of protein 10) and the percentage of

empty/incomplete particles needs to be accurately established as the results

reported here are only estimations based on preliminary results (of both AUC and

RP-HPLC).

Although CE and HPSEC were not fit-for-purpose to determine the purity of

adenovirus preparations, these methods can be further developed to determine

virus particles concentrations.


46

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