review of literature hepatitis b is the most serious liver...

Review of Literature

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2.1 Hepatitis B disease

Hepatit is B is the most serious liver infection in the world and can

lead to liver failure, c irrhosis or cancer of the liver (Hepati tis B

Foundation). It is often characterised by jaundice, abdominal pain, l iver

enlargement and fever. According to the WHO, it is estimated that 400

mil l ion people worldwide are already chronically infec ted with hepatit is

B and th is disease leads to over 1 mill ion deaths per year. This disease

is transmitted by blood and body f luids, frequently during sexual

intercourse. Treatment is generally ineffective (Hepatit is B Foundation)

and thus prevention and vaccination remain the best a lternatives for

this disease.

Hepatit is B is caused by the hepatit is B v irus (HBV), an envelope

virus contain ing a partially double s tranded, circular DNA genome and

classif ied within the family hepadnav irus (Shepard et al. , 2006). The

virus interferes with the function of the liver in replicating hepatocytes

caus ing the immune sys tem to be activated leading to liver

inflammation.

The Hepati tis B v irus is a spherical partic le with a diameter

of 42 – 47 nm and circulates in the blood in concentrations as high

as 108 v irions per mL (Shepard et al., 2006). The partic les are

composed of an envelope, which consis ts of proteins known as HBs or

surface prote ins. These self-assembling units form non-infectious

spherical or tubular partic les (Robinson and Lutwick, 1976) containing


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the surface antigens (HBsAg) which are respons ible for elic it ing the

immune response. These HBsAg partic les contain only envelope

glycoproteins and host-derived lipids and outnumber v ir ions by 1000:1

to 10,000:1 (Ganem and Prince, 2004). The surface coat surrounds an

inner protein shell , composed of HBc proteins. Final ly, the v iral DNA

and enzyme DNA polymerase can be found in the inner most region of

the partic le.

2.2 Structural Components of HBV

Infection by HBV results in secretion of high t iters of an antigen

called Australia antigen in the sera (Dane et a l., 1970) on three types

of partic les: (1) pleomorphic spheres of approximately 20 nm diameter,

(2) f i laments of variable length and diameter of 20 nm, and (3)

spherical double shelled partic les of 42 nm that are referred to as Dane

partic les . The inner protein shell of the Dane partic le is called the core

partic le, which contains core antigen (HBcAg). The antigen present on

the outer shell of the Dane partic le is referred to as hepatit is B surface

antigen (HBsAg). The core antigen surrounds the v iral genome, a small

DNA molecule of unusual s truc ture. Another antigen, which is a

derivative of the core antigen and known as e antigen (HBeAg) is found

in various forms (Mackay et a l., 1981). Among the three antigens,

HBsAg was found to be protective antigen suitable for vaccine

development.


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2.3 HBV Structure and Pathogenicity

The Human HBV virus consists of a part ially double-stranded DNA

genome of 3.2 kb enclosed by envelope proteins (HBsAg). The genome is

packaged wi th a core protein (HBcAg) and a DNA polymerase. Fol lowing the

receptor-mediated entry into a hepatocyte, it enters in to the nucleus and i ts

DNA becomes integrated into the host genome. Prote in synthesis proceeds

f rom four open reading f rames (ORFs): the envelope proteins (large, middle

and major HBsAg) f rom the S gene, pre-S1, and pre-S2 gene sequences; the

e antigen (HBeAg) and HBcAg f rom the C gene and pre-C gene sequence;

the DNA polymerase pro tein f rom the P gene; and the transactivator X

protein f rom the X gene (Fig. 2.1). DNA replication proceeds v ia RNA

intermedia tes in the nucleus. The vi rus part icles are then assembled in the

cytoplasm and re leased by the hepatocyte (Pugh and Bassendine, 1990).

Al though the integration of the vi ral genome is not necessary for i ts

repli cation, i t allows the persistence of the v i ral genome in the cells of host

(Tiollais et a l ., 1985; Ganem and Varmus, 1987).

Fig. 2.1: Hepatitis B virus (HBV) genome organization

HBV is a member of the 'hepadnavirus' group, double-stranded DNA viruses, which repl ica te, unusual ly, by reverse transcription of an RNA in termediate, the pregenomic RNA. The f igure shows the physical map of the HBV


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genome. The genome is approximately 3200 nucleotides in length . HBV vi rions contain both DNA and RNA and some regions of the packaged genome can be single stranded, double stranded or even triple stranded. There are four def ined overlapp ing open reading f rames (ORFs) in the genome which resul t in the transcript ion and expression of the seven dif fe rent hepatit is B proteins through the use of varying in-f rame start codons. The four ORFs transcription are controlled by four promoter elements (preS1, preS2, core and X), and two enhancer elements (Enh I and Enh II). Source: J Viral Hepat 2004.

The core and po lymerase genes are essential for v i ral DNA replica tion,

and the envelope proteins are essential for envelopment of nucleocapsids.

HBx pro tein and HbeAg are expressed during natural infect ions and their

funct ion is not too c lear. HBx pro tein has been seen to be required for the

establ ishment of an infection in vivo (Chen et al . , 1993; Zoulim et a l ., 1994)

but is dispensable for v i rus replica tion in transfected ce lls (Blum et al . , 1992;

Yaginuma et al . , 1987).

2.4 The HBV replication cycle

Despite 40 years of HBV research no widely available cell l ines

permissive for HBV or any other member of the Hepadnav ir idae family

has been described. Studies on the repl ication cycle of

Hepadnavir idae , i.e., attachment, entry, genome replication,

transcription and expression of v iral genes, assembly, and budding

cannot be fully studied or are limited to small series of experiments

with primary permissive hepatocytes (Aldrich et a l., 1989; Gripon et al. ,

1993; Gripon et al., 1988; Ochiya et al., 1989; Tutt leman et a l., 1986).

Unfortunately, these primary hepatocytes remain permiss ive for only a

short t ime after being obtained from the intact l iver.


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It is assumed that v irus entry and the host range of

Hepadnav ir idae is dependent on the N terminus of the large surface

antigen (Chouteau et al., 2001; Gripon et al., 2005; Ishikawa and

Ganem 1995; Lambert et a l., 1990; Verschoor et al., 2001). So far, the

intr insic HBV receptor has not been discovered, but from studies on

DHBV in primary duck hepatocytes it is assumed that around 104

receptor molecules per cell mediate the rapid binding, followed by a

slow uptake of the v irus to the cel l which can take up to 16 hours

(Hagelstein et a l., 1997; Kl ingmuller and Schaller, 1993; Kock et a l.,

1996; Pugh et al., 1995; Pugh and Summers, 1989; Rigg and Schaller,

1992). Following entry into the hepatocyte and uncoating, which may

proceed in parallel , the nuc leocapsid is transported into the cell’s

nucleus where the v iral nucleic acid is released. Release of the v iral

DNA and disintegration of the nuc leocaps id is assumed to take place at

the nuclear pore complex (Kann et a l., 1997; Rabe et a l., 2003).

In the infected hepatocyte the viral DNA is immediately

transformed into the covalently closed circular (ccc) DNA by cellular

enzymes. The cccDNA in turn is the template for transcription of v iral

genes, and acts chemically and structurally l ike an

episomal/extrachromosomal DNA and has a plasmid-like structure

(Bock et a l., 1994; Bock et a l., 2001; Newbold et a l., 1995). Congruent

with the fact that HBV infec ts hepatocytes, nearly all e lements

regulating v iral transcription have binding sties for l iver-specif ic

transcription factors (Courtois et al., 1987; Guo et al., 1993; Lopez-


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Cabrera et a l., 1990; Lopez-Cabrera et a l., 1991; Raney et a l., 1995;

Schaller and Fischer, 1991). Nevertheless, although a number of

factors and interactions regulating v iral transcription are known, the

exact mechanisms of HBV transcription remains unclear. However, v iral

transcription occurs in the nucleus, and both messenger and

pregenomic RNAs are transported into the cytoplasm where they are

translated or used as the template for the production of progeny

genome, respectively.

In the cytoplasm, the core protein which i tself can be

phosphorylated by several kinases forms the basis for the nucleocapid

and plays an ac tive role in binding and packaging of the pregenomic

RNA, recruitment of the v iral polymerase, and thus enables the RT

polymerase/RNA complex to init iate reverse transcription within the

newly forming nuc leocaps ids (Daub et al., 2002; Gerlich et a l., 1982;

Kann et al., 1993; Kau and Ting, 1998; Lan et al., 1999; Liao and Ou,

1995; Watts et al., 2002).

The three surface proteins of HBV have two major properties:

First, as transmembrane proteins they are anchored in the v iral

envelope and thus are located on the surface of the virus, being

respons ible for binding to the so-far-unknown v iral receptor. Second,

the three surface proteins are secreted as subviral partic les that do not

contain a functional nucleocapsid. The proteins di ffer in their N-

terminal sequences that are longer in case of the L and M protein. All


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proteins have in common the S domain, M addit ional ly has the preS2

domain, L has both the preS2 and in addition the preS1 domain (Figure

2). The surface proteins of mammalian Hepdnav ir idae have been shown

to be N- and O-glycosylated. These glycosylations have been shown to

be responsible for proper secretion of progeny v iral partic les and in

turn may represent novel targets for therapies with glycosylation

inhibi tors (Block et al., 1998; Block et al., 1994; Lu et al., 2003;

Schildgen et al., 2004; Schmitt et al., 1999; Schmitt et a l., 2004).

Moreover, the surface proteins have been demonstrated to be

activators of transcription by acting in trans (Caselman et a l., 1990;

Kekule et a l., 1990).

The v iral polymerase, s ingle most enzyme encoded by the

hepadnav iral genome, consists of three functional domains – the

terminal protein, the reverse transcriptase, and the RNaseH domain –

and a spacer domain that separates the terminal protein domain from

the polymerase domains. The terminal protein also serves as a primer

for the reverse transcription (Lanford et al., 1997; Wang and Seeger,

1992; Weber et al., 1994). Before or during formation of the cccDNA

the terminal prote in but also one of the redundant terminal repeats

present on the relaxed circular v iral genomic DNA that is released from

the nucleocapsid are removed and the cccDNA forms by not fully

understood mechanism, most probably dependent on cellular l igases

and maybe further enzymes. So far i t is assumed that cellular DNA


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repair mechanisms become active and convey the relaxed circular form

into the cccDNA (Seeger et al., 2007).

The cccDNA is the template also for the pre-genomic RNA

(pgRNA). This RNA is both the template for core and polymerase

protein trans lation but also the matr ix for the progeny genomes. The

pgRNA bears a secondary s truc ture - named e-structure - that is

present at both the 5’- and the 3’-ends. The e-hairpin loops at the 5’

end are f irst recognized by the v iral polymerase and act as the init ial

packaging signal (Bartenschlager and Schaller, 1992; Hirsch et al.,

1990; Huang and Summers , 1991). The synthesis of the DNA minus

strand, i .e. the intr ins ic reverse transcription, is then init iated by the

formation of a covalent bond between the tyrosine Y65 res idue of the

terminal protein domain and a desoxy-guanosine-monophosphate

(dGMP) (Lanford et a l., 1999; Wang and Seeger, 1992; Weber et al.,

1994; Zoulim and Seeger, 1994). The next few nucleotides following

this init ia l dGMP are complement to a small part of the e-structure. The

small terminal protein bound primer is subsequently translocated to the

3’ end v ia an unknown mechanism but remains covalently bound during

the whole t ime. Perhaps this process is a prerequisite for the correct

folding of the progeny genome within the newly forming nucleocapsid.

Finally, the minus strand is fully synthesized by the reverse

transcription reaction while the RNA is degraded by the RNaseH

activ ity of the enzyme. The fo llowing plus s trand synthesis is init iated

by an 18mer capped RNA ol igo that remains from the 5’ end of the


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pgRNA (Lien et a l., 1986; Loeb et a l., 1991). Nevertheless, it is

assumed that, whi le not actively replicating and even with conflict ing

data on its stability, there is ev idence that cccDNA may be stable in

infected hepatocytes, thus contr ibuting to chronic HBV infection,

leading to a need for long-term therapies to help eliminate the cccDNA

posit ive cells.

The final replication s tep, i .e., assembly and release of Dane

partic les , is not fully understood, although from one study on usage of

glycosylation inhib itors that at nontox ic doses suppress viremia in

WHV-infected woodchucks there is indirect evidence that assembly and

release occur v ia secretory pathways (Block et a l., 1998).

2.5 Hepatitis B Surface Antigen

The HBV genome, HBsAg encodes the S region, further consists of

two regions, preS (pre S1 and pre S2) and S, with an ini tiation codon in

each region. The three forms of HBsAg are, smal l (SHBs), middle

(MHBs), and large (LHBs). Each of the three proteins encoded by

preS/S ari ses from separate trans lation init iation at each of the three in-

frame s tart codons. The S region which encodes the smallest SHBs

exis ts in glycosylated (gp 27) and unglycosylated (p24) forms. The pre

S2/S domain encodes the middle MHBs consists of a hydrophilic N-

terminal extens ion of 55 aminoacids aris ing from translation of the pre

S2 region. It exis ts as mono (gp33) and diglycosylated (gp36) forms.

Production of LHBs is resulted with an addit ional 108 or 109


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aminoacids by the translation at the from the pre S1 region. LHBs

exis ts as glycosylated (gp42) and unglycosylated (p39) forms (Gerlich

and Bruss, 1992). SHBs are the major component in all three partic les

(Fig. 2.2).

Fig. 2.2: Structural organization of the HBV Antigen ORF-S that encodes the three forms of surface antigen proteins Source: International Journal of Medical Sciences 2005; 2:2-7

SHBs carry at least one neutra lizing epitope. Two other

determinants have been described for HBsAg (Gerlich and Bruss,

1992). One determinant has either d or y specif ic ity and the other has

either w or r specif ic ity. All combinat ions of determinants are found,

result ing in four subtypes of HBV: adw, adr, ayw, and ayr (Peterson et

al., 1984). HBV strains are further class if ied in to nine serotypes, ayw

1, 2,3,4, ayr, adw 2,4, adrq-and adrq+, on the basis of their antigenic

determinants and sub-determinants of their HBsAg (Courouce et a l.,

1976; Courouce-Pauty et a l., 1978). The occurrence of escape mutants

in many regions of the world with di fferent SHBs subtypes suggests

that subtypespecif ic immunity may not play an important role if SHBs is

the only vacc ine component. Intramuscular injec tion of serumor yeast-

derived recombinant HBsAg in healthy indiv iduals results in effective

immunizat ion and protection from v iral infection (Emini et a l., 1986).

The technical adv isory group of the World Health Organization (WHO)


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has recommended the addit ion of hepatit is B vaccine in the Expanded

Programme of Immunizat ion (EPI) in al l countr ies with moderate to high

endemicity of infection (WHO, 1988; Calandra et al., 1992).

2.6 Serological heterogeneity of hepatitis B surface antigen

The serological heterogeneity of hepatit is B surface antigen

(HBsAg) has long been established. All known serotypes of HBV

contain the common a determinant and one of each of the mutual ly

exc lusive determinants d/y and w/r (Le Bouv ier, 1971; Bancroft et al .,

1972). Additional serological specif ic it ies, originally designated as

subdeterminants of a and subsequently as subdeterminants of w, have

allowed the identif ication of four serotypes of ayw and two of adw

(Courouce et al., 1976). Thus, eight subtypes of HBsAg, aywl, ayw2,

ayw3, ayw4, ayr, adw2, adw4 and adr, have been serologically defined,

also designated as P1 to P8 (Courouce et al., 1976). The q determinant

was original ly found to be expressed on HBsAg of al l subtypes apart

from adw4 (Magnius et al., 1975). Subsequently, lack of q was also

demonstrated in some adr subtype-containing sera. Thus adr strains

can be defined as either adrq+ or adrq- (Courouce-Pauty et a l., 1978).

2.7 Pathogenesis of hepadnavirus infections

The transmiss ion of HBV and other members of the

Hepadnavir idae fami ly occur both vertical ly and horizontally v ia body

fluids . A maximum of 1010 to 1012 genome copies per ml serum or

body f luid can be found. In chronic infections, the v iremia is subjec t to


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natural f luc tuations of one log (Schi ldgen et al., 2006). The rate for

chronic ity, depending on the study, is >90% in neonates and

approximately 10-15% in adults. The r isk for trans fusion-acquired and

nosocomial infections in the past two decades decreased due to

optimized molecular diagnostics and more s tr ict hygiene and legal

regulations , although there is stil l a remarkable number of such

transmission caused by incautious behav ior of healthcare personal.

Once having entered the host, the Dane partic le reaches its major

target cell, the hepatocyte, the main site for repl ication and

persistence, as vir tually all hepadnav iruses display a pronounced and

distinct l iver tropism. Furthermore, some other cell types have been

shown to serve as non-hepatic reservoirs for mammalian

hepadnav iruses. Within the infected liver in immunocompetent hosts

there is a continued damage of infected hepatocytes by cytotoxic T

lymphocytes (CTLs) that leads to uninterrupted expression of collagen

fibres that in the wors t and untreated cases lead to liver cirrhosis (Liaw

et al., 2004; Mathew et a l., 1996; Maynard et al. , 2005;

Papatheodoridis et a l., 2005; Pinzani, 1995; Rizzetto et a l., 2005;

Rockey, 2005; Yoshida et a l., 2004).

It is worth note that there is s til l no evidence that HBV is cytotoxic

for the infec ted hepatocyte. In contrast to other v iruses that can infect

the liver l ike the herpes s implex v irus (HSV), HBV is unable to induce

cytopathic effects under normal infection condit ions (J ilbert et al.,


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1992; Kajino et al., 1994; Thimme et al., 2003; Wieland et al., 2004).

Liver damage (f ibrosis, c irrhosis, and probably hepatocellular

carcinoma) is believed to be induced by the ongoing immune reaction

and the steady s tate inflammation in the liver. Consequently, confirmed

by experimental data (Ando et a l., 1994; Guidotti et a l., 1994; Guidot ti

et al., 1999; Guidotti et a l., 1994b; Guidotti et a l., 1996; Guidott i et a l.,

2000; Guidotti et al., 1999b; Kakimi et al., 2001; Tsui et al., 1995), it is

generally assumed that massive CTL and NK T cell action result ing in

the kil l ing of infected hepatocytes is essential for elimination of the

infection. It is further assumed that in those cases in which chronic

infection evolves, the init ial cellular immune response is too weak and

thus not suffic ient to control the infec tion (Ganem et a l., 2004). It

remains unclear what mechanisms are responsible for the passage

from acute to chronic infection, thus this part of the v iral l ife cycle

remains a matter of speculation. As a matter of fac t it has been shown

that a suff ic ient Th1 response involving CD8 posit ive CTLs, natural

ki l ler T cells (NK T), cytokines (TNF-alpha, interferon gamma), and

cytokines like IL-12 and IL-15 and many others are involved in the

suff ic ient suppression of trans ient infections (Seeger et al., 2007).

Despite the fact that only antibodies against the s protein are

neutralizing and are the only markers of immunity it was hypothes ized

that transient infection is kept in check by gamma interferon and other

cytokines re leased by immune cells, leading in turn to a shutdown of

v iral replication (Pasquetto et al., 2002; Schulz et a l., 1999; Schulz et


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al., 1999b). However, this does not expla in why only in those patients

who c lear the v irus HBsAg antibodies are present. This is assumed to

be a continuous control of the infection as cccDNA can be found in

these patients decades later (Maynard et al., 2005; Werle-Lapostolle et

al., 2004), while this is not the case if the infection passes to a chronic

stage.

2.8 Brief history of treatment for the Hepatitis B disease

Hepatit is B vaccines have been available since the early 1980s.

The first vaccine (HEPTAVAX®), l icensed in 1981, was a subunit

vaccine isolated from the blood of hepatit is B infected donors (Prince,

1982). In hepatit is B patients suffer ing from acute infec tion and in

hepatit is B carr iers, 22nm partic les containing HBV surface antigen

subunits of the v irus are found expressed in copious amounts. These

partic les themselves are not infectious but are effective in s timulating a

strong immune response (Dekleva, 1999). Since these partic les are

derived from blood, extensive purif ication is cr it ical to ensure that the

f inal product exceeds 99% purity and is free from extraneous infectious

viruses.

There were many l imitations in the manufacture of plasma-derived

hepatit is B vaccines. Primarily, a constant l imitation exists in the

supply of human plasma. Due to the intensity in the manufacturing

process, to produce just one batch, more than 1 year is required

(Dekleva, 1999). During the 1980s there was also growing concerns


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about AIDS infection through blood derived products (Francis et al.,

1986) that led to give a pressure to develop a recombinant vaccine for

human use.

RECOMBIVAX HB® from Merck was the f irs t recombinant hepatit is

B VLP vaccine. Recombinant hepatit is B vacc ine, ENGERIX B® w as

produced by GlaxoSmithKline (GSK), which is also currently in the

market. In addit ion, there is now a vaccine for both hepatit is A

and B together, known as TWINRIX®, which is bivalent in

nature, containing antigenic components used in producing HAVRIX®

(Hepatit is A inactivated vaccine) and ENGERIX-B® (Hepatit is B,

recombinant vaccine), by GlaxoSmithKline (GSK).

2.9 Hepatitis B Surface Antigen (HBsAg) VLP vaccine

The firs t commercially manufac tured recombinant hepatit is B VLP

licensed in 1984 (Hil leman, 2001; Hil leman, 2003; McAleer et a l. , 1984;

Valenzuela et a l. , 1982) was cloned and expressed in S. cerev isiae

(McAleer et a l., 1984; Miyanohara et al. , 1983). The VLP is assembled

from up to 100 copies of the hepatit is B surface antigen monomer and

host-derived lipids forming highly immunogenic spherical partic les

measuring ~ 22nm in diameter (Fig.2.3a). The VLP is widely referred to

as the hepatit is B surface antigen (HBsAg) after its or igins . The

comparison of the HBsAg produced by recombinant yeast for HBsAg of

human blood plasma has been reported (Yamaguchi et a l. , 1998).


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In S. cerevisiae, by the insertion of the hydrophobic segments of

the HBsAg monomer into the endoplasmic reticulum (ER) is init iated by

the assembly of the recombinant HBsAg partic les (Zhou et al. , 2006)

which then bud into the lumen of the ER as 20-nm l ipoprotein partic les

using host-derv ied machinery and l ip ids (Eble et a l. , 1986). In humans,

the HBsAg maturation process will proceed through the Golgi and the

HBsAg partic les are subsequently secreted. In yeast cells however, due

to the lack of protein transport machinery (Biemans et al. , 1992), the

HBsAg partic les are permanently localised in the ER.

(a) (b)

Fig. 2.3: (a) Cross-section of an HBsAg particle (b) Interaction between a p24s protein monomer and the lipid bilayer on the

surface of the HBsAg particle Source: (a)Koist inen (1980); (b)Mahoney and Kane, 1999.

The recombinant HBsAg partic les are cys teine-rich and the

structural evolvement and maturation depends on the level of

disulphide formation. The formation of correct intra- and intermolecular

disulphide bonds are cr it ical as they determine the conformational

stabil ity and immunogenicity of HBsAg vacc ines (Guerrero et al. , 1988;


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Zhou et al. , 2006). HBsAg derived from human blood plasma are

already in the mature form but in recombinantly expressed HBsAg, the

disulphide bond formation and the cross-l inking process continues

during purif ication. Addit ional s teps of treatment with potassium

thiocyanate (KSCN) and storage at elevated temperatur es have been

included at the end of the purif ication chain to complete the maturation

process of recombinant HBsAg (Zhou et al. , 2006).

HBsAg characterisation studies have revealed that the partic le is

composed of ~ 75% protein and ~ 25% lipid by mass (Dreesman et a l. ,

1972; Gav ilanes et al., 1982). The lipid-protein interac tions are

respons ible for the formation of the desirable helical structures of the

proteins forming the epitopes (Gav ilanes et al. , 1990) (Fig. 2.3b).

Sterol accounts for 30% of the lipid constituent of the HBsAg partic le

while phospholipids account for the remaining 70%, of which

phosphatidylcholine (PC) is the most abundant. Negatively charged

phospholipids, phosphatidylserine (PS) and phosphatidylinositol (PI), in

particular, have been shown to have s ignif icant influence on HBsAg

activ ity (Gomez-Gutierrez et a l. , 1994). A ful l account of the role of

l ipids in maintaining the structure and antigenicity of HBsAg and the

effects of l ipid reconstitution is available elsewhere (Gav ilanes et al. ,

1990; Gomez-Gutierrez et a l., 1994).

The intr ins ic charac teristics of the HBsAg partic les , outlined

above, are cri tical considerations in ensuring product quality and


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potency. Like other VLPs, HBsAg partic les are large macromolecular

entit ies which are diff icult to characterise and are susceptible to

conformational and biological changes in d ifferent microenv ironments.

As prev iously d iscussed, the process of producing the vaccine itself

often defines the end product (Buckland, 2005) and regulatory demands

are str ingent considering that the vaccine products are adminis tered to

healthy indiv iduals. It is required that assurance of the identity and

composit ion of the adminis tered dosage is provided to meet the

demands of product safety and eff icacy. Hence, in this study, the

biological and phys iochemical aspects of the HBsAg sys tem are

carefully considered alongside the purif ication process development

efforts. Ul tra-scale down (USD) techniques are particular ly valuable

here in simulating industria l scale condit ions to allow the

characterisation of the biochemical and biophysical responses of the

HBsAg partic les to different processing env ironments .

2.10 Hepatitis B Surface Antigen (HBsAg) platform for novel

vaccines

In addit ion of their use as direct immunogens, VLPs have shown

enormous potential as platforms for the development of future

generation vaccines based on hybrid or chimeric VLP. VLPs are highly

eff ic ient in stimulating cellular and humoral responses as they prov ide

the spatial structure for the display of conformational epitopes that

mimic the native v iral structure leading to enhanced antibody

production (Grgacic and Anderson, 2006).


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The hepatit is B surface antigen (HBsAg), with the abil ity to sel f-

assemble with host-derived l ipids into empty non-infec tious VLPs

(Cheong et al. , 2009), is attractive as a delivery platform for foreign

epitopes. The eff ic iency of the HBsAg partic les in pr iming cellu lar and

humoral responses even in the absence of an adjuvant has been

reported (Boisgerault et al. , 2002). Chimeric vaccines for Hepatit is C

(Netter et al. , 2001), dengue (Bisht et al. , 2001) and HIV (Michel et al. ,

2007; Greco et al. , 2007) have been developed using this platform.

The HBsAg partic les are composed of a lipid b ilayer surrounded by

external hydrophobic loops which could be genetically engineered to

carry the heterologous antigens for delivery (Delpeyroux et al. , 1986;

Netter et al. , 2001; Phogat et a l., 2008). The ability of the hydrophobic

loops to accept peptides facilitates the insertion of selec tive epitopes

agains t diseases including HIV-1 and hepatit is C (Lee et al. , 1996;

Schlienger et al. , 1992; Boisgerault et al. , 2002).

The lipid-envelope structure of the HBsAg is an attractive attr ibute

as it allows the incorporation of foreign antigens requiring the support

from a lipid bilayer structure. This aspect is particular ly relevant in the

development of HIV vaccines where a lipid membrane env ironment is

necessary to orientate the antigenic epitopes for eff ic ient induction of

broadly neutral is ing antibodies (Grunder et a l., 2002). Preliminary

studies showed that the HIV epitopes could be appended to the C-

terminus of the HBsAg S-1 protein and the l ipid env ironment prov ided

the support necessary for effec tive antibody binding (Phogat et a l.,


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2008). Simi larly, the chimeric VLP for dengue v irus envelope protein

showed enhanced immunogenic ity when assembled on the HBsAg

platform (Bisht et al. , 2002).

HBsAg partic les have a hollow core with an encapsulation

space of 900-8000 nm3 (Reimann et al. , 2006) and access to the

inter ior is mediated by pores in the bilayer. The hollow structure of the

HBsAg partic les could also be employed for the encapsulation or

entrapment of antigenic proteins and peptides , oligonucleotides or

cytokines which would be exposed to the surface by v ir tue of the pores.

Early proof-of-concept s tudies for this HBsAg appl ication have been

demonstrated (Reimann et a l. , 2006).

2.11 Production and purification methods for HBsAg

2.11.1 HBsAg expression

The HBsAg partic les used as the VLP model in this study was

developed by Merck (West Point, PA, USA) us ing S. cerevisiae as the

host cell expression system. Detai ls of the plasmid and strain

construc tion are reported elsewhere (Carty et a l. , 1989; Hinnen et a l. ,

1978).

The crucial start ing point in the development of a VLP vaccine is

the design of a cell based system which is s table over many

generations and produces high t itres of the VLP product (Buckland,

2005). The choice of the expression system is particular ly important for

l ipid-envelope VLPs for the reason that the lip id constituents are


40

derived from the host cel l (Betenbaugh et al. , 1995; Buonaguro et al.,

2005). Although the production of HBsAg has also been achieved using

bacteria (Shu et al., 2006), transgenic plants (Kumar et al. , 2005) and

mammalian cel ls (Diminsky et a l. , 1997), S. cerev isiae remains a highly

attractive host. Large scale fermentation is wel l established, highly

reproducible and amenable by Yeast fermentation (Buckland, 2005).

Addit ional ly, yeast cells have the abili ty of performing complex

eukaryotic- like post-translational modif ications producing proteins

similar to those of the mammalian origin and a wealth of genetic

information is avai lable for this strain wh ich facilitates the engineering

of the desired cell metabolism and expression characteristics for the

VLP.

Fermentat ion condit ions for VLP production would depend on the

host express ion system chosen. The recombinant HBsAg used as the

VLP model in th is study is transcribed under the control of the GAL-10

promoter in S. cerevisiae. Hence, HBsAg expression is regulated by the

levels of glucose and galac tose in the cul ture media. Fermentation

conditions and glucose-galactose interactions in HBsAg expression

have been reported prev iously (Carty et a l. , 1987; Carty et al. , 1989).

For plasmid selection and maintenance, the Merck HBsAg strain also

carr ies a Leu+ gene in the same plasmid as the HBsAg gene. This

allows the use of a leucine-free basal media for fermentation which

would select for cells containing the plasmid with the Leu+ and HBsAg

genes. Fermentation for HBsAg production is typically carr ied out in a


41

batch process (Carty et al. , 1987) but the use of continuous

fermentation has also been reported (Fu et al. , 1995).

An issue for cons ideration when developing yeast fermentat ion

media is that yeast cel ls are susceptible to catabolite repression, a

condition where cel l growth is hindered when excess sugars such as

glucose are present (Gancedo, 1998). Oura (1974) developed a fully

defined media for yeast fermentation us ing glucose that has minimal

catabol ite repression effects. Yau (2005), in her MEng research project

in Biochemical Engineering (UCL), proposed a media for HBsAg

production which has the combined advantages of the Oura (1974) and

the Fu et a l. (1995) media. This media was shown to be successful for

the fermentation of wild type S. cerev isiae cells but was not tes ted for

the recombinant HBsAg s train due to t ime constraints. Joyce et al.

(1998) reported on a recombinant S. cerev isiae fermentation media

which is semi-defined for the production of the human pappilomav irus

(HPV) vaccine. It is known that the S. cerev isiae host expression

system for this vaccine is similar to that for hepatit is B and hence the

media may be employed for HBsAg production.

In this research, the recombinant S. cerevisiae for HBsAg was

donated by Merck (West Point, PA, USA) in the form of a master seed

stock. Fermentation is not a key element in th is projec t however it is

crucial to produce suff ic ient feed material for subsequent purif ication

studies which were representative of material in an industr ial process.


42

There is l imited information in the publ ic domain on fermentat ion media

for HBsAg production us ing the Merck recombinant S. cerev isiae strain.

To minimise t ime and research effort in generating feed stock for

purif ication studies, prior knowledge from the Yau (2005) and Joyce et

al. (1998) studies were used as the basis for fermentation

development.

2.11.2 Recovery and purification of HBsAg

For recovery of the intracellular HBsAg product, the yeast cells

following fermentat ion are harvested, concentrated and washed with

buffer for the removal of media components and anti foam. This is

usual ly accomplished us ing centr ifugation or microfiltration and the

cells are stored as cell pas te at -70 oC (Sitr in and Kubek, 1992).

Product recovery is performed by suspending a batch of frozen

cells in a buffer containing phenylmethylsulfonyl f luoride which is a

protease inhibitor (Dekleva, 1999), and the suspension is subjected to

cell disruption by homogenisation. Following cell breakage, the crude

extract is treated with a phosphate buffer containing a detergent,

usual ly Triton X-100 (Wampler et a l. , 1985). This is a c rit ical step in the

recovery of HBsAg as the detergent facil itates the liberation of the

HBsAg from tightly associated endoplasmic reticulum (ER) membrane

components (Dekleva, 1999). Removal of cell debris can be performed

by centri fugation (Wampler et a l. , 1985) or microfiltration (Deklava,

1999). Res idual detergent is removed by recirculation through


43

polystyrene XAD-4 beads and the product stream can be ultrafiltered

through a 100-kDa membrane to clear small molecular weight

contaminants and for product concentration.

Purif ication is typically accomplished by chromatography-based

operations (Dekleva, 1999; Wampler et a l. , 1985; Belew et a l. , 1999)

although the use of prec ipitation and density ultracentr ifugation has

been reported (Deml et al. , 1999). The HBsAg product is init ially

purif ied v ia adsorption and elution from colloidal s il ica (Aerosil)

(Wampler et a l. , 1985). Condit ions for this process are reported

elsewhere (Pillot et al. , 1976; Sitr in and Kubek, 1992). Final pol ishing

is performed using hydrophobic interaction chromatography using butyl

agarose (Dekleva, 1999).

As highlighted by Zhou et a l. (2006), the HBsAg produced in S.

cerevisiae is not in the fully d isulphide-bonded form as found in blood-

derived HBsAg. The maturation process for recombinant HBsAg

progresses through the purif ication process. To facili ta te the format ion

of disulphide cross-linkages in the mature form, the HBsAg partic les

are treated with th iocyanate and incubated at an elevated temperature

of 37 oC prior to product formulat ion (Zhou et a l. , 2006). The product is

f inally adjuvanted by co-prec ipitation with a luminium hydroxide

(Dekleva, 1999).


44

In the complex mult istage operations involved in HBsAg production

and purif ication, product yie ld is d ictated by the eff ic iency of the

detergent-mediated HBsAg liberation s tep while the purif ication

performance of membrane fi ltration and chromatographic operations

are influenced by the level of contaminants such as yeast proteins and

lipids . This thesis investigates the development of an improved primary

purif ication strategy to achieve higher HBsAg recovery and a cleaner

output to reduce the burden on downstream operations.

2.12 Pichia pastoris, a novel expression system

As a eukaryot ic organism, Pichia pastoris has many of the

advantages of higher eukaryotic express ion systems especial ly pos t-

translational modif ications such as protein process ing, protein folding

and protein secretion into the medium, along with the later facil itates of

easy puri f ication (Hollenberg and Gellissen, 1997). while being

manipulated as easy as Escherichia coli or S. cerevis iae. It is fas ter,

easier and less expens ive to use when compared to other expression

systems such as baculov irus or mammalian system, and generally

gives higher expression level. As yeast, it shares the advantages of

molecular and genetic manipulations with Saccharomyces, and i t has

added advantage of higher levels of expression. These features along

with easy maintenance, easy scale-up, and inexpensive growth

requirements make P. pastoris as a very useful protein expression

system. The process can be scaled up to a level of expression, which is

10-100 times higher than that of E. coli (Clare et a l., 1991; Faber et a l.,


45

1995; Vozza et al., 1996). In many instances, heterologous proteins

were produced at much higher level when compared to their respective

productiv it ies in the tradit ional S. cerevisiae host. Their growth to high

cell density prov ides a means to produce large quantit ies of antigen

economically (Romanos et a l., 1992). Since 1984, over 300

heterologous proteins have been well expressed in P. pastoris (Cregg

et al., 2000; Cereghino et al., 2000). P. pastoris has gained more

popularity because of several factors such as alcohol oxidase I (AOX

I), which is one of the strongest and most regulated promoters, its

ability to integrate expression plasmids in its own genome in one or

more specif ic s ites, its ability to culture s trains in high density

fermenters; and its ready availabili ty as a kit ( Inv itrogen, USA).

Transformat ion of P. pastoris can easily be carried out either by

electroporation method or spheroplasting method, using linearized

recombinant plasmids and eff ic ienc ies are usually in several orders of

magnitude below those for other yeasts . Pretreatment of P. pastor is

with 0.1 M l i th ium acetate (LiAc) and 10 mM dithiothreito l (DTT) before

electroporation increased transformation eff ic iency approximately 150-

fold (Wu and Letchworth, 2004).

2.13 Detergent-mediated HBsAg liberation

The HBsAg product remains permanently localised on the yeast

endoplasmic reticulum (ER) fo llowing protein translation as transport

through the secretion pathway is blocked (Biemans et al. , 1992). A

detergent is required to facil i ta te the l iberation of HBsAg from tightly


46

associated ER membrane components (Dekleva, 1999). Typically Triton

X-100, a non-ionic detergent, is employed for this purpose although

alternative detergents such as polysorbate 20 and 80 (PS 20 and PS

80) and variants of Triton such as Tri ton X-101, CHAPS (3-[(3-

Cholamidopropyl)dimethylammonio]-1-propanesulfonate) and glycol

ether solvents have been reported for s imilar appl ications (Wijnendaele

et a l. , 1987; Kniskern and Hagopian, 1997; Allen et al ., 2007). The

key feature to a detergent’s function is in its amphipathic structure

comprising a hydrophil ic “head” region and a hydrophobic “tai l” region

which can assoc iate with the hydrophobic surfaces of proteins.

Detergents fac ilitate the recovery of membrane proteins by disrupting

the bipolar l ipid membrane of cells and by forming protein-detergent

complexes which are soluble. Of the detergents commonly employed

for the recovery of l ipid-envelope VLPs, Triton and polysorbate, both

fall under the non-inonic detergent class . Their difference lies in the

polymer type and length of the hydrophobic “tails”. Triton detergents

are made from ethyleneglycoether polymers and have shorter

hydrocarbon chains and higher CMC values than polysorbate

detergents which contain oxyethylene polymers (Cal igur, 2008).

Detergents with lower CMC values are more stable however the

interaction between the detergent and the protein is also stronger

which could be an issue for subsequent detergent removal efforts.

CHAPS in contrast is a zwitter ionic detergent which may have the

added benefit of being more effective for disrupting protein-protein

bonds in reduc ing aggregation due to its ionic nature although hav ing a


47

net neutral charge. In general, anionic or cationic detergents are less

preferred for biological applications as their charge charac teris tics

could modify protein s tructure leading to increased risks of protein

denaturation. Specif ically for HBsAg liberation, there are several

hypotheses as to how the detergent functions, mechanistically. Chi et

al (1994) proposed that detergents weaken the hydrophobic

interactions that ex ist between the VLP and the membrane

components. According to Sitr in and Kubek (1992), detergents facilitate

the disassociation of the HBsAg from the ER by promot ing the

separation of the yeast ER membrane from other unwanted cellular

debris. A study by Smith and co-workers suggests that detergent may

also promote VLP vesicle formation by inward budding of the ER

membrane (Smith et a l. , 2002). From the above, it appears that high

detergent concentrations would favour HBsAg liberation based on

reaction kinetics. However, to be taken also into cons ideration, is the

physiochemical nature of the HBsAg partic les which are 75% protein

and 25% lipid by mass (Gav ilanes et al. , 1982). There has been a lack

of consensus as to the optimal concentration of detergent for this

purpose. Other groups have reported that excess detergent could lead

to HBsAg partic le delipidation (Skelly et al. , 1981; Howard et a l. , 1982;

Sanchez et a l. , 1983) which in turn could result in a loss of antigenic

activ ity. This is unsurpris ing given that the lipid moiety has signif icant

roles in the maintenance of the structural and antigenic properties of

the protein components of the HBsAg (Gav ilanes et al., 1990).


48

The amount of detergent could also have indirect implications on

the contamination prof ile of the result ing process stream. Large

amounts of l ipid are produced by yeast cells over extended periods of

cell culture (Wijnendaele et al., 1987) which in the case of HBsAg

production is necessary to achieve the desired product t itres. The

majority of these lipids are assoc iated with membranous organelles

which also contain some proportion of proteins for spec if ic metabol ic

functions . During the detergent step, it is l ikely that the membrane

associated lipids and proteins are co-released alongside the HBsAg

product and the extent of co-l iberation is also a function of detergent

concentration.

Based on the factors h ighlighted above, it is cr it ical to identi fy a

suitable detergent and its optimal range of operating concentrations for

effective recovery of HBsAg, preservation of its antigenic properties

and minimal co-release of contaminating host l ipids and proteins .

2.14 Precipitation for partial purification

Precipitation is a tradit ional protein purif ication method and is

examined here as a means of achiev ing partial purif ication and

enrichment of HBsAg prior to h igher resolution chromatographic

operations. Polyethylene glycol (PEG) and ammonium sulphate are the

most widely cited precipitating agents for VLPs and are assessed for

the primary purif ication of HBsAg based on the degree of contaminat ion

reduction and product yield.


49

Polyethylene glycol (PEG) is attractive as a prec ipitating agent

owing to its low potential for protein denaturation even when present at

high concentrations (Ingham et a l. , 1990; Tsoka et a l. , 2000) and its

high fractionation eff ic iency when puri fying large size proteins such as

VLPs (Juckes et al., 1971). The appl ication of PEG precipitation for

HBsAg puri fication is not entirely new. Puri f ication of the f irst

generation hepati tis B vaccine based on HBsAg from plasma of carr iers

relied on mult iple PEG precipitation “cuts”. Since the recombinant

HBsAg examined here is not ident ical to the human form due to

differences in d isulphide c ross-linking, these recombinant partic les may

have different solubility behav iours in the presence of PEG. There is

also a difference in the role of the PEG prec ipitation step. In the 1970s

process for human HBsAg, PEG precipitation was the purif ication

workhorse and mult iple “cuts” were necessary to achieve the desired

f inal purity, often at the expense of s ignif icant product loss. As a

primary purif ication process for future-generation vaccines, its role

would be to reduce the contamination burden and process volume

entering downstream operations in a single “cut” with minimal product

loss . For PEG precipitation as a primary puri fication step product yield

is cr it ical and this rel ies on the effectiveness of precipitate recovery

from the liquor. Although PEG has a low intrinsic viscosity (Polson et

al. , 1964), concentrations above 10% w/v PEG may have substantial

effect on the v iscosi ty of the process stream. The ef fect of this on the


50

performance of the centr ifugation or f i l tration step for the precipitate

recovery would need to be evaluated.

The use of ammonium sulphate, another popular precipitating

agent, is also investigated. It is generally known that the ammonium

sulphate salt is less selec tive compared to PEG which results in larger

quantit ies (> 40% w/v) of the salt being required for protein

precipitation (Wijnendaele et al., 1987) which could create waste

disposal issues. Lipid solubility, in contras t, is more sensit ive to

ammonium sulphate and lower concentrations of the salt are typically

suff ic ient for their precipitation.

Bracewell et a l., (2008) demonstrated an inverse purif ication

strategy us ing ammonium sulphate precip itation. In the study, l ipid

contaminants were precipitated out of solution whilst protein products

are reta ined in the soluble phase. This strategy would be more suitable

for large-scale VLP purif ication owing to the lower levels of ammonium

sulphate consumption which would pose less waste disposal issues.

2.15 Selective product release for primary purification

For expression of recombinant proteins in S. cerev isiae, there are

three main regions in the cell where the product of interest is l ikely to

be located: (1) the space between the cytoplasmic membrane and the

outermost mannan-protein layer of the cell wall , (2) in the cytosol, or

(3) embedded in subcellular organelles or partic les (Huang et al. ,


51

1991). It is within the third category that the production of recombinant

l ipid-envelope VLPs such as the HBsAg falls . The natural local isation

of the product of interest in a specif ic region of the cell can provide a

powerful means of recovering the product separately from bulk

contaminants originating from the host cell. This approach has been

demonstrated prev iously in the recovery of proteins from the

periplasmic space of E.coli cel ls using osmot ic shock or enzymes

(French et al. , 1996; Witholt et al. , 1976) and from specif ic regions in

yeast using detergent and enzymes (Chi et a l. , 1994; Huang et al. ,

1991; Asenjo et al. , 1993). Using these methods, several-fold product

enrichment was reported with product yields of up to 90%. As

highlighted prev ious ly, HBsAg partic les in yeast are permanently

local ised on the endoplasmic reticulum (ER) because transport through

the secretion pathway is blocked (Biemans et al. , 1992). It is for this

reason that a detergent is typically added to the cel l lysate following

homogenisation to release the HBsAg from the assoc iated ER

components. The ability to separate ER partic les containing the HBsAg

product from bulk cel l contaminants prior to the addit ion of detergent

would be invaluable as this would allow the HBsAg product to be

recovered later and in a cleaner process stream. The endoplasmic

reticulum which is slightly hydrophobic (Chi et al. , 1994) and denser

than the cell cytosol would be sedimented together with solid debris

partic les when centrifuged whils t bulk cytosolic materia l would be in the

supernatant. By discarding the supernatant fraction and treating only

the sol ids fraction with detergent, HBsAg would be released into a


52

process stream with s ignif icantly reduced host l ipid and protein

contaminants. The eff ic iency of this process would rely on the relative

partit ioning of the HBsAg product and host contaminants across the

cytosol and the membranous components.

It is possible to enhance further the selective recovery process by

f ine-tuning the cel l disruption condit ions. Characterisation of the size

distr ibution of yeast debris generated as a function of the

homogenisation condit ions revealed that the release of intracellular

proteins is often accompanied by fragmentation and micronisation of

the cell wall (Siddiqi et a l., 1995). Condit ions of the homogenisation

step could have a major influence on the eff ic iency of the selective

recovery process at three different levels: (1) the ex tent of cell

disruption would influence the accessibil ity of detergent to the ER

during the HBsAg liberation step and this would impact product yield,

(2) the population size of the ER components following homogenisation

would influence the effec tiveness of their recovery by sedimentation

during the init ia l centr ifugation s tep and this would also impact product

yie ld, and (3) the partic le size dis tribution of membranous debris would

influence the rate of co-ex trac tion of contaminating lipids and proteins

during the detergent step and this would impact the level of purif ication

achieved. Thus, a trade-off study on the impact of homogenisation

conditions would be warranted in developing any approach to selective

product release.


53

2.16 Scalability of selective product release

In process development, USD techniques are invaluable as they

are capable of predicting industrial scale performance of

biopharmaceutical protein downstream process ing in a rapid fashion

using as lit t le as mil l i l itres of material (Titchener-Hooker et a l. , 2008).

Identif ication of process challenges and predic tion of performance of

large scale protein precipitation operations using ultra scale-down

techniques has been demonstrated (Boychyn et a l. , 2000). Hence, the

focus here was on the scale-up of a selec tive product release method.

The selec tive product release methodology investigated in this

work relies on a twin centrifugation operation. The role of the f irst

centr ifugation step is to allow the elimination of bulk cytosolic

contaminants whi lst retaining the solids fraction containing the HBsAg

product. The second centr ifugation step which is performed following

detergent treatment recovers the HBsAg product in the supernatant

whilst solid contaminants are discarded. Different centr ifuge designs

such as the tubular bowl, multichamber bowl and disc-s tack centr ifuge

have been rev iewed (Salte et al., 2006; Boychyn et a l. , 2004). The

CARR PowerfugeTM centr ifuge, which is a tubular bowl variant, is most

suited for inclusion in the selective product release process sequence

due to its good solids compaction and high c lar if ication features . The

level of dewatering and clar if ication attained would dictate the

selec tiv ity and product yield from this approach.


54

The Sigma concept (Ambler, 1959) allows the comparison and

prediction of centr ifuge performance across scales and centr ifuge

designs by the introduction of correc tion factors which account for

differences in f low pat terns. USD models have been developed from the

Sigma theory (Tustian et a l., 2008; Boychyn et a l. , 2004; Boychyn et

al. , 2000; Salte et a l. , 2006) and are useful here to predict the

clar if ication performance of an industr ial CARR Powerfuge T M operating

at di fferent feed flow rates and rotational speeds. Hav ing determined

the optimal operating condit ions of the CARR Powerfuge T M centr ifuge

for the selective recovery process, a pilot-scale validation s tudy can be

performed to verify that the purif ication potentials of any methodology

are not compromised upon scale-up.

review of literature hepatitis b is the most serious liver...

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