Ligands from Combinatorial Peptide Libraries for Virus Removal

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Abstract

Salm, Jeffrey Richard. Ligands from Combinatorial Peptide Libraries for Virus Removal.

(Under the direction of Dr. Ruben G. Carbonell and Dr. Dennis T. Brown)

Small peptides were investigated as affinity ligands for virus removal from human blood plasma (HBP). Sindbis virus (SV) was radiolabeled with 35S and screened against a solid phase peptide library. The screening identified 9 hexapeptides that were

synthesized on a TosoBioseparations Toyopearl 650M Amino Resin. The peptide sequences SGKPVA and IATDGG were found to remove approximately 3 logs and 2.4 logs of SV from buffer respectively and approximately 1.5 logs of SV from 50% (HBP). Toyopearl Amino bound only 0.6 logs of SV in both buffer and 50% HBP. Good agreement was seen between infectious quantification methods and quantification of the virus using radiation. Injections of several batches of SV showed variations between the batches.

A similar screening procedure was also applied to radiolabeled canine parvovirus (CPV). Screening against a solid phase library identified 25 leads through to be specific to CPV. Tests with a portion of these leads found less than 0.5 logs of CPV clearance in both buffer and 50% HBP. Electron microscopy of the CPV used in these experiments showed that large aggregates of virus were present in the radiolabeled virus. Infectious assays for CPV were also problematic and inconsistent.

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Ligands from Combinatorial Peptide Libraries

for Virus Removal

by

Jeffrey R. Salm

A dissertation submitted to the Graduate Faculty of North Carolina State University

In partial fulfillment of the requirements for the Degree of Doctor of Philosophy

Department of Chemical Engineering May, 2004

Approved By:

__________________________ __________________________ Dr. Peter K. Kilpatrick Dr. Jan Genzer

__________________________ __________________________ Dr. Dennis T. Brown Dr. Ruben G. Carbonell

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Dedication

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Biography

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Acknowledgements

I would like to thank the American Red Cross for the financial support for this work. Specifically David Hammond for always supporting the Bioseparations Group at NCSU.

I would like to thank the professors and researchers at North Carolina State University who helped me obtain my degree. Specifically, thank you to Dr. Raquel Hernandez for her expertise, patience, and belief in the abilities of a chemical engineer, Dr. Brown for his open door, his knowledge, and belief in this project, and Dr. Carbonell for his direction, his belief in me, and his ability to brighten the darkest periods of this research.

I would like to thank the members of the Bioseparations lab group, Honglue, Viterose and Guan, and the Biochemistry lab group Steevenson, Chris II, John, and Katie. Special thanks to Patrick and Patrick for their help and friendship, and to Chris for always having the time and the answers to help me.

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List of Tables

Table 2.1 SV Plaque Counts and Titers for Figure 1...28

Table 2.2 Criteria for Batches of SV...29

Table 2.3 Sequences Identified from Screening 35S-SV Against a Solid Phase Peptide Library ...34

Table 2.4 Sequences Identified from Screening 35S-SV Against a Solid Phase Peptide Library with a 1 M NaCl Wash...35

Table 2.5 Tertiary Screening of Resins Using 35_{S-labeled Sindbis Virus ...42}

Table 2.6 Log Reduction from Four Batches of SV ...45

Table 2.A.1 Secondary Screening of Resins Using 35S-labeled Sindbis Virus ...53

Table 3.1 Criteria for Well-Growth Batches of CPV...72

Table 3.2 Sequences Identified from Screening 35S-CPV Against a Solid Phase Peptide Library ...73

Table 3.3 Sequences Identified from Screening 35S-CPV Against a Solid Phase Peptide Library with a 1 M NaCl Wash...74

Table 3.4 Secondary Screening of Resins Using 35S-labeled Canine Parvovirus...77

Table 3.5 Secondary Screening of Resins with Varied Peptide Densities and Ligand Length Using 35S-labeled Canine Parvovirus...79

Table 3.6 Secondary Screening of TosoBioSep Resins Using 35S-labeled Canine Parvovirus ...80

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List of Figures

Figure 2.1 SV Plaques on BHK Cell Monolayers Observed, A. 10-8 Dilution, B. 10-7

Dilution, C. 10-6 Dilution...28

Figure 2.2 Effect of Radioactive Label on Stability of SV ...31

Figure 2.3 Effect of Radioactive Label on Stability of SV ...32

Figure 2.4 Effect of Specific Activity of Radio-labeled SV ...33

Figure 2.5 Loads of SV on SGKPVA at Varied Flow Rates ...37

Figure 2.6 Effect of Flow Rate on Log Removal ...38

Figure 2.7 Injections of Varied Quantities of SV on SGKPVA...39

Figure 2.8 Injections of SV in PBS on Several Resins ...40

Figure 2.9 Injections of SV in 50% HBP on Several Resins ...41

Figure 2.10 Comparison of Plaque Assay Quantification and Radiation Quantification for SV in PBS on SGKPVA ...43

Figure 2.11 Comparison of Plaque Assay Quantification and Radiation Quantification for SV in 50% HBP on SGKPVA ...44

Figure 2.12 Comparison of Chromatograms from Batches of SV on SGKPVA in PBS...46

Figure 2.13 Comparison of Chromatograms from Batches of SV on SGKPVA in 50% HBP.47 Figure 2.14 a, b, HBP Protein Adsorption to SGKPVA. A. Chromatograms of SV in PBS and HBP B. SDS-PAGE of Fractions from HBP Chromatogram...48

Figure 3.1 Negative Stain Electron Microscopy of CsCl Purified CPV ...68

Figure 3.2 Negative Stain Electron Microscopy of Purified CPV ...70

Figure 3.3 Scintillation Counts of CPV Dilutions...71

Figure 3.4 Comparison of CPV Monoclonal Antibody and Sequences from Primary Screening ...75

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Figure 3.6 Injections of Two Concentrations of CPV on RYNDWA...87

Figure 3.7 Injections of CPV on NIIVQR at Varied Flow Rates...83

Figure 3.8 Re-injection of Unbound Fraction on RYNDWA ...84

Figure 3.9 Re-injection of Unbound Fraction on Toyopearl Amino...85

Figure 3.10 Effect of Prefiltering CPV on GFYGAH Adsorption...86

Figure 3.11 Effect of Prefiltering CPV on RYNDWA Adsorption ...87

Figure 4.1 Log Clearance from 10 ml Injection of PPV in Varied HBP Concentrations on Amino Resin ...109

Figure 4.2 Log Clearance from 10 ml Injection of PPV in Varied HBP Concentrations on ARC30 Resin ...110

Figure 4.3 Log Clearance from 10 ml Injection of PPV in Varied HBP Concentrations on ARC49 ...111

Figure 4.4 Log Clearance from 10 ml Injection of PPV in Varied HBP Concentrations on ARC40 Resin ...112

Figure 4.5 Log Clearance from 10 ml Injection of PPV in Varied HBP Concentrations on FLLHPI ...113

Appendix B Figure 1 Electrophoretic Analysis of Ethidium Bromide Stained Agarose Gels of Template DNA...186

Appendix B Figure 2 Chromatograms of the Test PCR Mixtures and the Complete PCR Reactions...187

Appendix B Figure 3 Peak Fraction Analysis of the [α-32] P Labeled PCR Reaction ...188

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Chapter 1 Introduction and Overview

1.1. Introduction

1.1.1 Motivation

Biological products derived from either human or animal origins contain an

inherent risk of viral contamination. As a result, the United States Food and Drug

Administration has mandated that all biological products be treated with two virus

removal or inactivation steps. In response to federal regulations, manufacturers have

incorporating steps to determine the level of viral contamination and processes to remove

or inactivate viruses. These are referred to as viral validation and viral clearance

respectively.

Generally, viral clearance processes are one of the last steps in a production

process. This is a result of a lack of specificity demonstrated by most clearance

strategies. For example, membranes are commonly used for viral clearance as most

biologicals are substantially smaller than potential viral contaminants. However,

membranes are prone to fouling when challenged with complex feed streams such as cell

culture supernatant or human blood plasma. The cost of replacing or cleaning the

membranes generally relegates them to a late stage viral clearance process.

A viral clearance process that is specific to a virus would be of great value to the

biotechnology industry. For product streams such as human blood plasma that are

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viral clearance strategy that is used once in the beginning of a purification process instead

of multiple times for each product at the end of the process. A clearance strategy that is

virus specific could also target viruses such as human B-19 parvovirus that are not

adequately addressed by currently available clearance processes.

Affinity adsorption is a separation method that could meet the required specificity

needed for virus removal. However, this method has been largely ignored in viral

clearance. Current affinity ligands such as monoclonal antibodies are not suitable for

large scale processes because of the cost of producing the antibodies, the inability of such

ligands to withstand harsh solvent treatments, and the potential for ligand leakage. For an

affinity chromatography method to be industrially applicable, it must demonstrate

specificity to the target virus and overall cost efficiency. Small peptide affinity ligands

are inexpensive and robust which could make them ideal for a viral clearance method.

1.1.2 Overview

Several viral clearance strategies are currently used by the biotechnology industry

that are non-specific to virus particles. This work describes the development of a viral

clearance method that uses affinity peptides for virus removal from human blood plasma.

Combinatorial peptide libraries were screened to identify peptides specific to different

model viruses. After screening, peptides were tested for their ability to bind target

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and in the literature. Chapter 2 describes the identification and characterization of

affinity ligands specific sindbis virus. Chapter 3 details efforts to discover affinity

ligands for canine parvovirus. Chapter 4 focuses on affinity ligands specific to three

viruses from the parvovirus family. Overall conclusions and recommendations for future

work are summarized in Chapter 5. Appendix A has a review of the use of affinity

peptide ligands for protein purification. Appendix B is a manuscript detailing a

purification process for PCR products.

Introduction

1.1.3 Review of Viral Clearance

1.1.3.1 Virus Inactivation Techniques

Heat inactivation [4-7] and radiation inactivation [8, 9] are common inactivation

strategies that involve exposing the virus to harsh environmental conditions. These

methods balance the inactivation of the virus with maintaining product viability [3, 8, 10,

11]. For viruses like human immunodeficiency virus (HIV) and hepatitis B which are

denatured with treatment at 60ºC, heat inactivation strategies work extremely well

because the virus particles are significantly less stable than product proteins [4, 12].

Other viruses like human B-19 parvovirus, which is stable to 80ºC, require alternative

clearance strategies.

Photoinactivation [11, 13, 14] and solvent detergent inactivation [3, 15-17] rely

on the addition of denaturing chemical agents. Chemicals such as tri(n-butyl) phosphate

mixed with Triton X-100 or Tween 80 can easily destroy the lipid membrane of a virus

making it an excellent chemical inactivation strategy for enveloped viruses [18]. For

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concentration, virus specific chemicals such as Inactine™ have been shown to inactivate

viruses such as B-19 parvovirus and West Nile virus [19]. Naturally occurring molecules

such as the fatty acid caprylate have also been shown to result in significant viral

clearance [20]. Like heat inactivation, chemical inactivation methods can result in the

loss of the target molecule. Chemical inactivation methods have the added drawback of

requiring additional processing and validation steps to remove or neutralize potentially

toxic chemical additives [11, 15, 21, 22].

1.1.3.2 Virus Removal Techniques

Viral removal methods including filtration [22, 23], adsorption [24, 25],

precipitation [18], and chromatography [1, 4, 5, 22, 25-27] are also common industrial

viral clearance methods. Filtration methods use membranes to retain virus particles while

allowing protein products to pass through the membrane pores [23, 28]. Ultipor®

polyvinylidene fluoride membranes from Pall (East Hills, NY) have been shown to retain

a variety of virus particles including polio, Semliki Forest Virus, Herpes Simplex virus,

and HIV [28-30]. Viresolve NFP filters from Millipore (Billerica, MA) also have been

shown to clear viruses including porcine parvovirus, hepatitis A virus, murine

encephalomyocarditis virus, bovine viral diarrhoea virus, and bovine herpes virus [31].

Membrane removal systems are somewhat limited in the types of solutions they

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Adsorption to filters is also used to remove viral contaminants [24, 25]. These

techniques rely on the physiological properties such as charge or hydrophobicity of the

product and the potential contaminants. As a result, shifts in the feed stream composition

can result in dramatically different adsorptive properties and ineffective viral clearance

[24]. Precipitation can be used for viral clearance, but separating a solution into a liquid

and solid fraction usually generates a fraction with less virus and a fraction with more

virus [18, 32]. For a solution such a human blood plasma that will be separated into

many products, this result is of limited value.

Chromatography is another common method used for viral clearance [1, 4, 5, 22,

25-27]. Darling et al. reported a range of 1-6 log[10] removal of polio virus and murine

leukemia virus using a Protein A Sepharose resin, Q-Sepharose resin, and S-Sepharose

resin [1]. Adcock et al. used a combination of ion exchange chromatography, gel

filtration chromatography, and heat inactivation to remove both hepatitis A and B viruses

from a modified plasma stream [4, 5]. Biescas et al. reported a 3.8 log[10] removal of

HIV-1 and a 2.9 log[10] removal of Bovine Diarrhea virus from Cohn’s plasma fraction

IV1 using a heparin-agarose gel chromatography column [33]. A significant problem

with all of the chromatographic methods used to date is that they work by selectively

binding the product and allowing the virus to flow through. This method can actually

result in the concentration of the contaminating virus. For a solution such as human

blood plasma, virtually all of the molecules in solution have value. By separating a

specific protein from a solution while retaining the viral contaminant, the viral clearance

problem has not been answered, but saved for another step.

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The use of an affinity resin that targets the virus is a new viral clearance strategy.

A process that specifically targets the virus particle is advantageous for viral clearance as

it could treat a complex solution containing several products early in the production

process. Such a process would then serve as a viral clearance step for all of the products.

An addition advantage is that an affinity method should not result in product inactivation

or the need for the addition of chemicals that would require additional processing.

Affinity ligands such as monoclonal antibodies exist for many viruses, but are of

limited value from a viral clearance. Antibody columns are expensive to produce, and

are prone to ligand leakage and degradation. Other affinity ligands such as dyes or

metals generally lack the specificity that would be needed to bind a virus particle and

introduce potential toxicity issues if ligand leakage occurs.

Small peptide affinity ligands are reviewed in Appendix A of this thesis. Small

peptide affinity ligands offer many potential advantages for viral clearance. These

ligands are extremely robust allowing harsh solutions to be used for regeneration and

elution. Combinatorial screening techniques allow small peptide ligands to be identified

with specific viral interactions efficiently and with relatively low expense.

Several small peptides have been identified that are capable of inactivating

specific viruses in solution [34, 35]. Lutzke et al. reported that a hexameric peptide

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Chapter 2 Identification and Characterization of Peptide Ligands

from Combinatorial Peptide Libraries that Bind

Sindbis Virus

2.1. Introduction

2.1.1 Background

Protein therapeutics are an increasingly important sector of the health care and

biotechnology industry. Recombinant proteins have been developed from information

garnered from the human genome project and used to treat conditions ranging from

inflammation to cancer. Protein therapeutics derived directly from natural sources are

commonly used to treat a variety of conditions ranging from genetic disorders to blood

deficiencies.

With all protein therapeutics, there exists an inherent risk of viral contamination

[1-8]. Potential sources of contamination include the mammalian cells lines or animals

used to produce the proteins, materials derived from living sources such as blood plasma

or fetal calf serum, or contamination from workers involved in the production process.

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strategies include the use of heat and radiation to denature the virus particles [1, 2, 10,

11]. This strategy works well for several viruses such as Hepatitis A and B Viruses that

are not heat stable [1, 2]. More stable viruses, such as B19 parvovirus, denature only in

conditions that also result in significant protein degradation. Another common

inactivation strategy involves the addition of chemicals to the production process that

denature the virus [12]. Enveloped viruses such as HIV are commonly treated with

detergents such as tri(n-butyl) phosphate that destroy the lipid bilayer of the virus [13,

14]. Other viruses are treated with chemical agents, such as Inactine (Vitex Corp.) to

denature the viral proteins. One drawback of adding chemicals to a process is that they

must be removed due to their potential toxicity to patents [15, 16]. This results in

additional processing steps that can substantially add to the overall processing cost.

These chemicals can also modify or denature desirable target proteins or other

biomolecules in the mixture.

The other primary method of viral clearance used in the biotechnology industry is

virus removal. Removal strategies have the advantage that they generally do not result in

product inactivation. Ultra-porous and nano-porous membranes often composed of

polyvinylidene fluoride are used in many viral clearance processes as they are extremely

effective at removing viruses such as HIV that are significantly larger than other

molecules in the production stream [17-19]. For smaller virus particles such as B-19

parvovirus that are not easily captured by a membrane removal, adsorption methods such

as ion-exchange chromatography can be effective [20]. These methods are of limited

value as they lack viral specificity in complex feed streams such as cell culture

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Affinity adsorption has also been suggested as a method for viral clearance [21].

Antibodies have the ability to specifically target viruses in complex feed streams. In

principle, affinity adsorption offers a significant advantage over other removal strategies

because a single adsorption step could be used to remove viruses in the beginning of a

purification process that makes several products. However, purification and use of an

antibody specific to a virus particle is cost prohibitive. Antibody adsorption columns are

prone to ligand degradation and leakage resulting from washing conditions. Dye ligands

are more stable than antibodies allowing them to be treated with harsh elution conditions.

However, dye ligands lack the specificity needed for a viral clearance process and they

can be toxic if they leak from the column. For virus removal by adsorption, the ligand

must be relatively inexpensive and safe since affinity supports could be used for a very

limited number of batches.

An alternative approach to antibodies and dyes involves using small peptide (3-8

amino acids) affinity ligands. Small peptides can be synthetically produced making them

significantly cheaper than antibodies. Small peptides also are not likely to exhibit the

toxicity issues of antibodies or dyes should ligand leakage occur. Peptide identification is

the greatest challenge to using small molecule affinity ligands. Rational design

approaches cannot realistically be applied to viruses because of the complexities of the

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proteinase inhibitor [22], von Willibrand Factor [23], fibrinogen [24], and α lactalbumin

[25]. Appendix A contains an extended review of the literature on the use of small

peptides as ligands for affinity chromatography. Random library screening should

identify peptides that bind various sites on the virus surface.

The two major types of peptide libraries used in screening are synthetic libraries

and phage display libraries [26-29]. Phage libraries use bacteriophages with peptides

expressed on their surface. Peptides are produced by genetic manipulation of the phages

genome. The major advantages to phage libraries are that the phages can be propagated

through several rounds of screening to identify the strongest binders and that they can be

used to make large peptides. The disadvantages to phage libraries are that only natural

amino acids can be used, and that ligands identified on phages may not have the same

affinity properties when immobilized to a matrix suitable for a viral clearance step.

In synthetic libraries, peptides can be chemically synthesized onto a support

matrix such as a membrane or resin. One advantage of synthetic libraries is that they can

be synthesized on the matrix that will later be used in an industrial application. Synthetic

libraries also have the advantage of not being limited to naturally occurring amino acids.

One drawback to synthetic libraries is that the length of the peptide is limited to

approximately 8 amino acids by the physical size of the library and the cost associated

with sequencing the peptides.

Peptides have been identified that are specific to viruses [30, 31]. Lutzke et al.

reported that HCKFWW identified by screening a soluble combinatorial library bound

specifically to human immunodeficiency virus [32]. However, these are generally

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small peptides attached to a solid phase matrix for viral adsorption is a new idea. A viral

clearance process that uses hexameric ligands could have the needed specificity to

remove viruses in one step while balancing issues such as cost and toxicity.

In this work, a solid phase hexa-peptide library was synthesized on

TosohBiosciences Toyopearl AF Amino-650M resin. The library was screened with 35S

labeled Sindbis virus (SV) to identify affinity ligands capable of removing SV from

whole human blood plasma (HBP). While SV is not a known blood pathogen, it is a

good model for highly structured, enveloped RNA viruses like Hepatitis C since it is

relatively easy to work with and is fairly well characterized. Two ligands were identified

that had moderate affinity to SV in the presence of HBP.

2.1.2 SV Background

SV is a 60-70 nm diameter human pathogen from the Togaviridae family of

viruses in the alphavirus genera. A structure for SV has been determined to 11 Å

resolution [33] using known atomic level resolution structures of the virus proteins and

cryo-electron microscopy. The virus consists of a single RNA molecule, a protein

capsid, a lipid bilayer, and two surface glycoproteins designated E1 and E2.

The SV genome consists of a single positive sense RNA strand approximately 12

kb in length. The 5’ end of the genome codes for four non-structural proteins that are

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virion. The spikes are formed by three copies of a dimer consisting of the 56 kDa E1 and

the 51 kDa E2 proteins.

2.2 Experimental

2.2.1 Materials

The American Red Cross and Vitex Corporation donated the HBP used in this

research. The HBP was received in frozen 15 to 50 ml aliquots. HBP aliquots from each

delivery were thawed in a 37°C water bath for approximately 5 min. The HBP was then

pooled and mixed with a bench top vortex mixer on a low setting. The plasma was then

aliquoted in 0.5 ml fractions and flash frozen in liquid nitrogen. The vials were then

stored at –20°C until needed. Before using, aliquoted HBP was thawed at 37°C,

vortexed, and then passed through a 0.22µm filter.

All cell culture work was done using proper flaming and sterilization techniques

in a laminar flow hood. Cells were grown in a NapCo E Series Model 5300 Incubator

saturated with water and kept at 5% CO2 and 37°C unless otherwise noted. Cells were

grown in Minimum Eagle Medium (MEM) with Earl’s salts purchased from Invitrogen

(Carlsbad, CA). ‘Supplemented MEM’ was made by adding 50 µg/ml gentamicin sulfate

and 2 mM glutamine form Invitrogen (Carlsbad, CA), 10% v_/

v heat inactivated fetal calf

serum (FCS) from Hyclone (Logan, UT), and 29.5 g/L tryptose phosphate broth from

Becton Dickinson (Sparks, MD).

35_{S-cysteine and}35_{S-methionine were purchased from NEN Life Science Products}

(Boston MA). Phosphate Buffer Saline (PBS) and Phosphate Buffer Saline with 0.1 v/v

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Cells were observed using a Zeiss Phase Inversion Light Microscope from Carl

Zeiss Instruments (Dallas, TX). Cell confluence was estimated after observation of the

cell monolayer under the microscope. Cells were counted using a hemocytometer and the

light microscope.

Heat resistant strain SV was acquired from Elmer Pfefferkorn at Dartmouth

College (Hanover, NH). Stocks of this virus were produced at a titer of 109 plaque

forming units per ml (pfu/ml) and stored at -80ºC by Dr. Dennis Brown’s Lab. When SV

was needed, stock virus was thawed on ice and diluted as appropriate.

2.2.2 Virus Growth

2.2.2.1 Cell Passing Procedure

‘Cell passing’ involves releasing a confluent monolayer of cells from a culture

flask and reseeding the cells into new flasks. To ‘pass’ cells, a confluent monolayer was

initially washed with PBS to remove the culturing media. A solution containing 0.25%

trypsin was added so that the cells were covered and the monolayer was observed for

detachment of the cells from the flask. After detachment, an equal volume of

supplemented media was added to the flask to inactivate the trypsin. The media

containing the cells was transferred to a 15 ml centrifuge tube and spun at 1000 rpm in a

HN-S II International Equipment Benchtop Centrifuge (Vernon Hill, IL) to pellet the

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SV was grown using a procedure adapted from Hernandez et al. [34]. Briefly,

baby hamster kidney cells strain 21 (BHK) were provided by Peter Faulkner (Queens

University, Kingston, Ontario). The cells were passed in supplemented MEM at least

twice prior to infection. The cells were seeded into 75 cm2 culturing flasks from Corning

(Corning, NY) at a density of approximately 7 x 106 BHK cell/flask. The cells were

grown overnight in the incubator to approximately 75% confluence. The medium was

decanted from the flasks, and 5 ml of fresh supplemented MEM containing 4 µg/ml

Actinomycin D (Calbiochem, San Diego CA) were added to the flasks of cells. The

flasks were covered and put in the incubator. After 1 hour, the medium was poured from

the flasks and the cells were infected by adding 1 ml of infection solution. The infection

solution contained 3% v/v FCS and approximately 1 x 108 infectious SV particles. The

flasks were placed on a Bellco Glass Inc. plate rocker at room temperature. After 1 hour

of gentle rocking, 4 ml of supplemented MEM were added to each flask. The flasks were

then placed in the incubator for 3 hours and 45 minutes. After incubation, the medium

was poured from the cells and a Pasteur pipette was used to insure complete removal of

the supplemented MEM. Five ml of supplemented MEM without Cysteine or

Methionine (starvation medium) were added to the cells. The cells were then placed in

the incubator for 1 hour. The starvation medium was poured from the flasks, and 5 ml of

fresh starvation medium with 35S-cysteine and methionine were added to each flask. The

flasks were incubated overnight at 37°C. After a cytopathic effect caused by the virus

was observed under the microscope, the cell supernatant was pooled from all the infected

flasks and stored at 4°C.

(32)

The SV cell supernatant was twice purified over a k-tartaric acid density gradient.

Before the gradients were run, the supernatant was spun at 3000-5000 rpm in a HN-S II

International Equipment Benchtop Centrifuge (Vernon Hill, IL) to pellet cell debris. The

supernatant was then poured into a fresh tube to isolate the virus from the cell debris. A

two step gradient (15% w/v and 35% w/v tartaric acid in PBS) was formed in Ultraclear

SW28 ultracentrifuge tubes from Beckmann with at least 5 ml of both the 15% and 35%

solutions. The cell supernatant was then layered on top of the gradient. The gradient was

spun for 16 hours at 4°C and 24,000 RPM in an Optima L-90K Beckmann Ultra

Centrifuge. After spinning, the virus band was visualized at the interface of the 15% and

35% layers. The bottom of the ultracentrifuge tube was pierced with a 20 gauge needle

and dripped. The band of virus was collected and stored at 4°C. A second linear gradient

(15% w/v to 35% w/v tartaric acid in PBS) was prepared in Ultraclear SW40

ultracentrifuge tubes from Beckmann with a total volume of at least 9 ml. The tartaric

acid in the collected band was diluted by adding an equal volume of PBS. The band was

then layered on top of the linear gradient. The linear gradient was spun for 3 hours at

4°C and 26,000 RPM in a Beckmann Ultra Centrifuge. After spinning, the virus band

was visualized and drip collected using the same technique applied to the step gradients.

The collected bands were combined and dialyzed to remove the tartaric acid against PBS

(33)

The infectivity of SV was assayed using an end point plaque assay adapted from

Knipfer et al. [35]. Briefly, ten fold serial dilutions of virus samples were prepared with

supplemented MEM containing 10 mM HEPES-HCl (Sigma) pH=7.4 in sterile glass test

tubes on ice. A 25 cm2 culturing flask from Corning (Corning, NY) was prepared for

each dilution by seeding with ~2 x 106 BHK cells. The flasks were grown overnight to

75% confluence in the incubator. The medium was poured from the flasks of cells, and

200 µl of a given virus dilution was pipetted onto a given flask. The virus was allowed to

adsorb to the monolayers for 1 hour at room temperature on a plate rocker. After viral

adsorption, the liquid in the flasks was removed using a Pasteur pipette. The cells were

overlaid with 1% w/v agarose from Sigma (St. Louis, MO) at 45°C in supplemented

MEM. The agarose was allowed to solidify before the flasks were returned to the

incubator. After 2 days, the flasks were overlaid with 1% w/v agarose in supplemented

MEM containing 60 µg/ml Neutral Red from Fisher (Pittsburgh, PA) at 45° C. The

flasks were kept in the dark and placed in the incubator. After 6 hours, the flasks were

viewed on a light box and the plaques on each monolayer were counted. Individual

circular plaques observed on the cell monolayer represent 1 pfu, which is equivalent to

one infectious virus particle that results in plaque formation.

2.2.3.2 Protein and Radiation Characterization

The amount of viral protein for pure virus samples was quantified using a

MicroBCA assay kit from Pierce. Virus samples were boiled for 5 minutes to denature

the virus before using the kit. The kit was capable of detecting protein concentrations

(34)

For virus samples containing plasma proteins, the virus was quantified using the

radiation incorporated into the virus. Samples smaller than one ml were put in 20 ml

glass scintillation vials with 15 ml of Ecolume® Scintillation Fluid from ICN (Costa

Mesa, CA). The vials were sealed and shaken to homogenize the virus sample and the

added fluid. After five minutes to insure the sample had dissolved into the scintillation

fluid, the vials were counted using a A2010 TriCarb Liquid Scintillation Counter from

Packard (Boston, MA). A standard curve relates the amount of radiation, the protein

from MicroBCA and the infectious plaque assay.

2.2.4 Library and Peptide Synthesis

The hexameric peptide library was synthesized on TosohBiosciences Toyopearl

AF Amino-650M resin (TosohBiosciences, Montgomerywille, PA) as has been done

previously in the literature [28]. The methacrylate-based resin has an average pore size

of 100 nm and a particle diameter of 65 µm. The surface area of the resin is 30 m2_/g

while the external surface area is 0.094 m2/g. The resin was selected as the base support

because of its mechanical and chemical stability. The synthesis was performed by

Peptides International (Lexington, KT) using the ‘divide, couple, recombine’ technique.

Eighteen of the twenty naturally occurring amino acids (excluding cysteine and

methionine) were used to produce the one bead, one peptide library. The final library

(35)

Individual peptide leads were synthesized on Toyopearl AF Amino-650M resin

by Peptides International in a similar fashion to the peptide library. The leads were

synthesized to a final density of 100 µmols per gram of resin. Unreacted amino groups

on the surface of the resin were acetylated while the amino terminus of the peptide and

any functional groups on the peptide were maintained. 1 gram of resin expanded to

approximately 4 ml when swollen in methanol for 1 hour.

2.2.5 Library Screening (Primary Screening)

A detection scheme similar to the technique described by Mondorf et al. was used

to identify peptides specific to SV [36]. For each experiment, 20 mg of dry library

(approximately 20,000 beads) was weighed out and put in a 1.8 ml microcentrifuge tube.

The beads were swollen in methanol for 1 hour and then washed in PBS buffer. The

beads were allowed to settle and excess PBS was taken from the library. The beads were

mixed with 1 ml of whole human plasma overnight on an orbital rotator. After mixing,

100 µl of a solution containing approximately 108 purified SV pfu/ml in PBS was added

to the plasma/library mix. The library was then tumbled on an orbital rotator for 1 hour.

After tumbling, the library was removed from the rotator, and the resin was allowed to

settle to the bottle of the tube. Excess plasma and virus was pipetted from the resin and

the amount of unbound virus was quantified using the scintillation counter. The library

was then resuspended in one ml of PBS and transferred to a 10 ml BioRad wash column.

The library was washed with PBS containing Tween 20 until radiation was not detected

by scintillation in the flow through of the column. In later primary screening, a second

wash step of 1 M NaCl in PBS was used to remove non-specific or loosely bound SV

(36)

After all the washes, the library was resuspended in 2 ml of PBS. The beads were

transferred to a 50 ml conical tube and 38 ml of 1% w/v low melt agarose at

approximately 40° C were mixed with the library so that the beads were evenly

distributed throughout the agar. Approximately 20 ml of agarose was poured evenly onto

two 8” x 10” sheets of GelBond (FMC, Rockland, ME) forming a monolayer of the

library. The agarose gel was allowed to air dry overnight before being exposed to β-max

radiographic film (Kodak) for 1-4 days. After developing the film, positive beads were

identified by aligning the gel and film. Each gel was exposed to film at least twice to

confirm the positive signals. Selected beads were excised from the gel under a

microscope using a scalpel. Individual beads were placed in water and heated to 70°C to

remove agarose and bound SV. The beads were put in fresh water, and the heated wash

process was repeated. Library beads were sequenced at Texas A&M University or at

Iowa State University by Edman degradation.

2.2.6. Binding Confirmation (Secondary Screening)

Secondary screening was used to verify the ability of leads identified during

primary screening to bind specifically to SV. Leads were tested using the secondary

screening method previously described by Bastek et al. [22] and Gurgel et al. [37]. The

leads were synthesized directly onto Toyopearl AF Amino-650M resin at a substitution

(37)

was added to the resin. The resin and the virus were incubated for 1 hr at 25°C on an

orbital rotator. After one hour, the virus was collected by centrifugation. The resins were

then washed sequentially for 1 hour each with PBS, PBS with Tween 20, 1 M NaCl, 3 M

NaCl, 2% v_/

v acetic acid, and 6 M guanidine hydrochloride. Each wash was collected and

quantified using the radiation and the scintillation counter.

2.2.7 Column Screening (Tertiary Screening)

Leads tested during secondary screening and untested leads identified during

primary screening were synthesized onto TosohBiosep Toyopearl 650M Amino resin by

Peptides International as detailed above. FractoGel ion exchange resins from E-Merck

Separations, Toyopearl amino resin, and acetylated Toyopearl amino resin were used as

controls. The resins were swollen in methanol for at least 1 hr and then washed with

water. The resins were wet packed into 5 x 0.4 cm Omega columns (Upchurch, Oak

Harbor, WA) and tested in a chromatographic format on a Waters 626 HPLC with a built

in UV detector. Samples were manually injected using a 100 µl or 1 ml sample loop.

The columns were maintained at 25° C by immersing the columns in a water bath.

For each injection, the column was equilibrated in PBS at pH 7.4. Samples were

prepared with 108 SV pfu/ml in PBS or 108 SV pfu/ml in 50% human blood plasma

(HBP) and PBS unless otherwise noted. PBS buffer was used to initially load the sample

at a flow rate of 0.05 ml/min unless otherwise noted. After 80 minutes, the buffer was

changed to 4 M Urea and 2.5 M KCl in PBS and the flow rate was changed to 0.2

ml/min. After 15 minutes, the buffer was switched back to PBS for 15 minutes and then

back to 4 M Urea and 2.5 M KCl in PBS for a second wash. After regenerated, the

(38)

Fractions for each sample were collected manually either in 1.8 ml

microcentrifuge tubes or in 20 ml glass scintillation vials. Samples collected in

microcentrifuge tubes were stored at 4 °C and tested for infectivity using the plaque assay

described above within 24 hours. The amount of radiation in the samples was then

quantified using the Scintillation Counter.

2.3 Results and Discussion

2.3.1 Preparation and Characterization of SV

Several factors can influence SV grown in BHK cells. The health of the cells,

fresh solutions, and a controlled cell growth environment all play a critical role in the

development of uniform virions [35, 38-40]. Early experiments found large variations in

the adsorption of a peptide lead to SV between different batches of virus. Several

experiments and observations were made that were found to influence the uniformity of

the SV grown.

One of the most important aspects of growing consistent batches of virus was

found to involve having healthy cells in the log phase of growth. Before infecting, cells

were passed at least twice and examined carefully under a phase inversion light

microscope at 40x. Cells that did not have several mitotic cells in the viewing field were

not used to culture virus. Cells were only infected if the cell density was approximately

(39)

product. Dialysis in PBS buffer was used to remove the k-tartaric acid from the purified

SV. The radioactivity of samples before and after dialysis was quantified using the

scintillation counter. Approximately half of the virus was lost during dialysis.

Three methods were used to quantify the virus once it had been purified. An

assay was used to quantify the number of infectious particles. Plaques are white circles

observed on stained monolayers of cells. Figure 1 shows plaques caused by SV observed

on a light box for three serial dilutions of the SV plaque assay. A plaque is the result of

infected cells that cannot absorb the Neutral Red dye applied to the monolayer.

Uninfected cells turn red as they metabolize the dye. Only one virus particle is needed to

infect a cell. As the virus multiplies in the cell, neighboring cells become infected in a

radial manner resulting in a plaque. Therefore, each plaque represents one virus particle

that was infectious, or one plaque forming unit (pfu). If enough virus particles are added

to the monolayer simultaneously infecting all of the cells, dye will not be adsorbed into

any of the cells.

A general linear behavior is observed through the dilutions shown in Figure 2.1.

By counting the plaques on each dilution, the titer of the virus can be calculated using the

equation _d

sv PC ml

pfu Titer

10

× =

= where PC is the plaque count, sv is the volume of sample

applied to the monolayer in ml, and d is the dilution of the sample being tested. For

Figure 1a, PC=4, d=-8, and sv=0.2 since 200 µl of sample was applied to the cells. The

(40)

A. B. C.

Figure 2.1 SV Plaques on BHK Cell Monolayers Observed, A. 10-8 Dilution, B. 10-7

Dilution, C. 10-6 Dilution. Serial dilutions of SV were prepared and applied to BHK cell

monolayers using the plaque assay procedure. Plaques were counted by visualization on a light box.

Table 2.1 shows plaque counts for each dilution shown in Figure 2.1 and the

resulting titers. The -7 and -8 dilutions result in titers that are almost identical, 2 x 109

and 1.6 x 109 respectively. Based on the -7 and -8 dilutions, the -6 dilution should have

approximately 400 plaques. However, the size of the flask used prevents this many

distinguishable plaques form forming on the monolayer. Virus particles from

neighboring plaques begin to merge resulting in smeared or non-circular plaques. As a

result of plaque crowding, only the -7 and -8 dilutions were used in determining the titer

of the virus in Figure 2.1 as approximately 2 x 109 SV pfu/ml. For a well-grown batch

of virus, the titer of the purified virus is expected to 108 to 109 pfu/ml.

(41)

A protein assay was used to detect the overall amount of viral protein for both

infectious and non-infectious purified virus. For well-grown virus, the concentration of

viral protein was approximately 5-30 µg/ml. Generally, batches of virus with less protein

also had lower titers while batches with more protein had higher titers. SV has a

molecular weight of 60 x 106, half of which is viral protein. Therefore, a well-grown

batch of virus has a particle to pfu ratio of approximately 10 to 1.

The radiation in the virus particle was also quantified using a scintillation counter

by the number of disintegrations per minute (dpm). Dilutions of radiolabeled canine

parvovirus demonstrated that radiation counts were linear within the range of 103_{to 10}5

dpm (Chapter 3). Past experience indicates that linearity in the dpm extends from at least

102 to 107 dpm [42]. For well-grown virus, there were approximately 5 x 106 to 1 x 108

dpm/ml.

From the three characterization methods, a list of criteria was developed defining

SV that would be used in secondary and tertiary screening experiments. These criteria

are summarized in Table 2.2 and show the relationship between virus protein, infectivity,

and specific activity. This range of viral properties is consistent with SV described

elsewhere [41, 42].

Table 2.2 Criteria for Batches of SV. Several batches of SV were grown, and a list of

criteria was developed that established batches of virus that could be used in experiments.

Desired Characteristics of SV Batches

Infectivity Range 108 to 109 pfu/ml

Virus Protein Range 5-30 µg/ml

Radiation Range 5 x 106 to 1 x 108 dpm/ml

Particle to pfu Ratio 10:1

(42)

Viral incorporation of the 35S labeled cysteine (Cys) and methionine (Met) was

initially thought to play a role in the stability and uniformity of SV. Batches of SV were

grown with Cys, Met or both Cys and Met 35S label. Virus for each labeling condition

was purified and titered on a daily basis. Several experiments are shown in Figures 2-4.

In Figure 2.2, all the SV batches that were radioactively labeled have a starting

titer of approximately 1 x 109 pfu. After three days of storing the virus at 4°C in PBS pH

7.4, the titers of the all of the viruses dropped to 1 x 107 pfu or less. Virus grown,

purified and stored in the same manner that was not radiolabled had a titer that varied

between 1 x 108_{pfu/ml on day 3 to 1 x 10}10_{pfu/ml on day 6. Figure 2.3 shows a repeat}

of the same experiment. In this case, the virus titer was 1 x 108 pfu for both labeled and

unlabeled virus. For all virus batches, the titer remained constant for 10 days. Figure 2.4

shows additional batches of SV that were grown with specific activities of 5 x 106

DPM/ml and 5 x 105 DPM/ml. The initial titer of the high specific activity virus and the

low activity virus were 1 x 1010_{pfu and 1 x 10}9_{pfu respectively. After ten days, both}

batches had lost one log of titer when compared to the stock SVHR. Therefore, in this

run, the amount of 35S incorporation was not thought to have an effect on virus stability

and variability.

Several repetitions of this experiment failed to produce consistent titer results

(43)

Effect of Time on Radiolabeled SV

1E+05 1E+06 1E+07 1E+08 1E+09 1E+10 1E+11

1 2 3 4 5 6 7 8 9 10

Age of Virus (days)

Ti

te

r

Cys Labeled Cys/Met Labeled Met Labeled Unlabeled SV

SVHR (Control Virus)

Figure 2.2 Effect of Radioactive Label on Stability of SV. Several batches of SV

(44)

1.E+05 1.E+06 1.E+07 1.E+08 1.E+09 1.E+10 1.E+11

1 2 3 4 5 6 7 8 9 10

Ti

te

r

Cys Labeled Met Labeled Unlabeled SV

SVHR (Control Virus)

Figure 2.3 Effect of Radioactive Label on Stability of SV. Several batches of SV

(45)

1.E+05 1.E+06 1.E+07 1.E+08 1.E+09 1.E+10 1.E+11

1 2 3 4 5 6 7 8 9 10

Ti

te

r

High Low SV SVHR

Figure 2.4 Effect of Specific Activity of Radio-labeled SV. Batches of SV were

grown and labeled with Cys and Met. The batches were purified and stored in polypropylene tubes. The specific activity of the labeled batches differed by

approximately 1 order of magnitude. ‘High’ was SV with a specific activity of 4 x 106 CPM/ml. ‘Low’ was SV with a specific activity of approximately 5 x 106 CPM/ml. SV is virus that was grown and purified that was not radioactively labeled. SVHR is stock virus that normally titers at 1 x 109.

2.3.2. Lead Identification (Primary Screening)

Two different rounds of primary screening were performed to identify potential

binders of SV. In the first round of screening, the library was contacted with the virus in

HBP. The library was washed with saline buffer and plated out. Approximately 0.18

grams of the 18 g library was screened, representing roughly 340,000 sequences out of

the possible 34 million sequences. Five leads were identified and are summarized in

Table 2.3. Two of the five sequences ended in the same four amino acids (VIFLVR and

(46)

of the sequences contained a positively charged amino acid (Arg or Lys) in addition to

the terminal amine at the end of the sequence. The remaining amino acids appeared to be

randomly distributed.

Table 2.3 Sequences Identified from Screening 35S-SV Against a Solid Phase

Peptide Library. A solid phase peptide library was screened in whole human blood

plasma and 7 log10 pfu SV at 25°C for 1 hour. After washing, the library was exposed to

radiographic film and leads that bound SV were identified. Five sequences were isolated and sequenced using Edman chemistry. The sequences are listed from the amino to carboxy terminus.

Sequence Short Hand

Ala Tyr Phe Leu Val Arg AYFLVR

Phe Arg Ser Pro Asn His FRSPNH

Val Ile Phe Leu Val Arg VIFLVR

Asn Ile Ile Val Gln Arg NIIVQR

Lys Leu Tyr His Lys Ala KLYHKA

Characterization of these leads was done using secondary and tertiary screening.

None of the identified leads were found to consistently bind at least 2 log10 SV pfu/ml in

saline. A second round of screening was performed to try and identify additional leads

that might have greater adsorption to SV. An additional wash step of 1 M NaCl in PBS

buffer was added after contacting the library with the virus to try and avoid identification

of weak binders. Approximately 0.09 g of the 18 gram library was screened, representing

roughly 170,000 sequences out of the possible 34 million sequences. Four leads were

identified and are summarized in Table 2.4. No true consensus sequence was observed,

(47)

glutamic acid or aspartic acid. While the majority of the sequences from the first round

of screening had a net positive charge, the sequences of the second round of primary

screening were generally more neutral.

Table 2.4 Sequences Identified from Screening 35S-SV Against a Solid Phase

Peptide Library with a 1 M NaCl Wash. A solid phase peptide library was screened in

whole human blood plasma and 7 log10 pfu SV at 25°C for 1 hour. The library was

washed extensively with 1 M NaCl in PBS before exposing to radiographic film. Four sequences were isolated and sequenced using Edman chemistry. The sequences are listed from the amino to carboxy terminus.

Sequence Short Hand

Ile Ala Thr Asp Gly Gly IATDGG

Tyr Glu Trp Lys Trp Gly YEWKWG

Glu Trp Val Pro Thr Ile EWVPTI

Ser Gly Lys Pro Val Ala SGKPVA

One drawback to the primary screening method used in this work was the choice

of base resin. Toyopearl Amino resin has an average pore diameter of 100 nm. The

particle diameter of SV is 70 nm, so little if any pore penetration was expected in the

screening. This greatly reduces the surface area that is available to the virus to bind

during the screening process which could potentially result in missed leads. The impact

of the reduced surface area was somewhat nullified by the choice of 35S labeling. 35S has

a specific activity of 42,707 Ci/g compared to 4.46 Ci/g for 14C which was used in

previous work with proteins. A higher specific activity should allow smaller quantities of

SV to be detected, though the exact detection limits were not determined.

2.3.3 SV Binding to Resins (Secondary Screening)

Secondary screening was performed on several resins and the results are found in

Appendix 2.A.1. Secondary screening was unsuccessful because approximately 85% of

the virus added to the resin adsorbed to the reaction vessel holding the resin. Several

(48)

tertiary screening methods since the column system was found to adsorb less than 10% of

the virus.

Since a secondary screening system was not identified, equilibrium isotherm

experiments could not easily be performed. As a result, the resin capacity and

equilibrium-binding coefficient were not determined.

Experiments were performed that varied the flow rate through the column and the

concentration loaded onto the column. Figure 2.5 shows several chromatograms

generated by injections of SV in 50% HBP onto a 0.6 ml column of SGKPVA with flow

rates ranging from 0.05 ml/min to 0.8 ml/min. As the flow rate through the column was

reduced, the amount of SV found in the unbound fraction decreased. A flow rate of 0.05

ml was used for remaining injections to ensure adequate binding time in the column. A

second injection performed at 0.05 ml/ml demonstrated that the column was being

(49)

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Column Volumes

Fr

ac

ti

on of Tot

al

S

V

0.05 ml/min 0.1 ml/min 0.2 ml/min 0.8 ml/min 0.05 ml/min (2)

Figure 2.5 Loads of SV on SGKPVA at Varied Flow Rates. A column of SGKPVA

was challenged with SV at flow rates ranging from 0.05 ml/min to 0.8 ml/min. The flow through and eluted fractions were collected and quantified using radioactivity and the scintillation counter.

Figure 2.6 shows a plot of the log removal versus the flow rate through the

column. The log removal was calculated using the amount of radiation added to the

system and the amount of radiation recovered in the unbound fraction. For this batch of

virus, a maximum log reduction of 1.2 pfu was obtained at a flow rate of 0.05 ml/min.

As the flow rate was increased to 0.8 ml/min, the log reduction decreased to 0.36 pfu or

approximately the same log reduction found for the control resin. Since the flow rate

directly effects the residence time in the column, Figure 6 suggests that increasing the

(50)

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Flow Rate (ml/min)

Log Removal

Figure 2.6 Effect of Flow Rate on Log Removal. The log removal for injections of

SV on SGKPVA is plotted versus the flow rate.

Figure 2.7 shows three different injections of SV with the total amount of viral

load ranging from 2.03 µg (~5 x 108 pfu) to 0.46 µg (~1 x 107 pfu). Figure 2.7 shows

that increasing the viral load results in increasing corresponding eluted fraction peaks.

The areas of these peaks are roughly proportional to the amount of SV initially loaded.

For the largest injection load tested, SV bound in a monolayer to the resin would cover a

surface area of 3.4 x 10-4_m2_{. In the 0.6 ml Omega columns used in these experiments,}

the external surface area of the resin is 1.2 x 10-2 m2. Therefore, for the amount of SV

(51)

0 200000 400000 600000 800000 1000000 1200000 1400000 1600000

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Column Volumes

DPM/ml

2.03 µg 1.06 µg

0.46 µg

Figure 2.7 Injections of Varied Quantities of SV on SGKPVA. A column of

SGKPVA was challenged with varied concentrations of SV. The flow through and eluted fractions were collected and quantified using radioactivity.

2.3.4 Clearance of SV from Plasma (Tertiary Screening)

Tertiary screening was performed to determine the selectivity of leads identified

for SV in the presence of 50% HBP. Toyopearl Amino resin, acetylated Toyopearl

Amino resin, two ion exchange resins from E-Merck separations and a peptide ligand

identified for Canine parvovirus were used as controls.

Representative chromatograms for several resins are shown in Figure 2.8 and 2.9.

The log clearances for these chromatograms along with clearances for other resins that

were tested are summarized in Table 2.5. The clearance was calculated using the amount

of radiation loaded onto each column and the amount of radiation found in the unbound

fraction. Figures 2.8 and 2.9 show the virus loaded onto the column was not completely

(52)

0.00 0.05 0.10 0.15 0.20 0.25 0.30

0 1 2 3 4 5

Column Volumes

Fraction of Total SV

Actylated Amino Fractogel NIIVQR IATDGG SGKPVA

Figure 2.8 Injections of SV in PBS on Several Resins. Several resins were

(53)

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

0 1 2 3 4 5

Column Volumes

Fraction of Total SV

Acyetylated Amino Fractogel NIIVQR SV 1 SKKPVA

Figure 2.9 Injections of SV in 50% HBP on Several Resins. Several resins were

challenged with injections of SV in 50% HBP. Fractions were collected and quantified using radiation.

Table 2.5 summarizes the best log clearances achieved of SV from PBS and 50%

HBP. Resins with a hexapeptide were able to clear between 1.4 and 3.0 logs. IATDGG

and SGKPVA cleared the most SV with 2.4 and 3.0 logs respectively. Positively and

negatively charged ion exchange resins from E-Merck Separations achieved 2.1 and 1.3

logs of SV clearance respectively. Toyopearl amino resin and acetylated amino resin

bound 0.6 and 0.1 logs of SV respectively.

In general, resins that made use of a ligand bound more SV than resins without a

ligand. The positive charge on the amino resin also allowed more SV to be adsorbed than

an uncharged resin. For both the amino resin and the FractoGel resins, the interaction

with SV may be a result of ionic interactions with charged patches on the surface of the

Ligands from Combinatorial Peptide Libraries for Virus Removal

Abstract

Ligands from Combinatorial Peptide Libraries

for Virus Removal

Jeffrey R. Salm

Dedication

Biography

Acknowledgements

Table of Contents

List of Tables

List of Figures

Chapter 1

Introduction and Overview

Chapter 2

Identification and Characterization of Peptide Ligands

from Combinatorial Peptide Libraries that Bind

Sindbis Virus

Column Volumes

DPM/ml