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ABSTRACT 
The Characterization of Immobilized Papain and Pepsin on to a Chitosan 
Support and Their Use in the Fragmentation and Separation of 
Immunoglobulin G on a Synthetic Affinity Column 
 
Miranda Faryal Williams 
A number of supports, both synthetic and naturally occurring have been 
reported in the literature for the immobilization of enzymes as well as for 
use as affinity matrices for the purification of immunoglobulins and 
albumin. However, there are advantages and limitations associated with 
these supports. To date the degree of selectivity of the affinity gels 
towards the various classes of immunoglobulins or their fragments have 
not been fully determined. 
The present study reports on the characterization of pepsin and papain 
covalently immobilized on to a chitosan support and the subsequent use 
of these immobilized enzymes to digest sheep IgG into its corresponding 
fragments F(ab)’2, Fab and Fc. These fragments were then applied to an 
Avid AL TM, affinity column to effect separation.  
The proteolytic enzymes pepsin and papain were first covalently 
immobilized on to a succinylated chitosan support through amide bond 
formation with carbodiimide which aided in the facile coupling of these 
enzymes. Optimal conditions of temperature and pH as well as loading 
capacity and storage stability were investigated for these two immobilized 
enzyme systems.  
The results of these initial studies showed that there was essentially no 
shift in the pH optimum for the immobilized enzymes when compared to 
the free enzymes. The pH optimum for immobilized papain ranged 
between 6.5 - 7.0 and between 1.7 - 2.2 for immobilized pepsin. In 
addition, an increase in temperature optimum was observed for both 
immobilized preparations (immobilized papain ranged between 50- 70o C, 
immobilized pepsin ranged between 45 – 50o C) and hence offers a wider 
operational temperature working range than those of the corresponding 
free enzyme. 
The results also showed that the loading at which maximum coupling 
efficiency is observed for immobilized pepsin and papain was 
approximately 30.6 and 27 mg/ g gel respectively. For both enzymes the 
coupling efficiency was found to be relatively low and to decrease as the 
amount of protein bound increased. In addition, storage stability studies 
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showed that immobilized papain retained at least 30% of its initial 
activity at 4o C for 4 months, while immobilized pepsin retained 35% of 
its initial activity over three months.  
Digestions of immunoglobulin G using both free and immobilized pepsin 
and papain at pH 4.0 and 6.5 respectively and incubation times up to 72 
h were investigated. Products of IgG digestion were analysed by non – 
reducing PAGE electrophoresis. Immobilized pepsin was found to be 
effective in digesting IgG yielding the bivalent 84-97 kDa F(ab’)2 and 
36kDa Fc fragment. Immobilized papain was effective in yielding both the 
bivalent F(ab’)2 and univalent  Fab 45- 55 kDa and Fc fragment. However, 
the times taken to effect digestion were relatively long. Avid AL was found 
to bind to all three fragments F(ab’)2, Fab and Fc in addition to intact 
IgG.  
Finally, a method is described for the affinity chromatographic 
purification of immunoglobulin M utilizing the affinity support Avid ALTM. 
The results show that Avid AL is capable of binding small quantities of 
IgM under similar conditions used in the purification of IgG (0.1 M 
NaH2PO4, 1.5 M NaCl, pH 7.4).  
Keywords: aza-arenophilic, chitosan, fragmentation, immobilization,  
immobilized enzymes, immunoglobulins, succinylated chitosan.