Biotechnol. Appl. Biochem. (1999) 30, 171–172 (Printed in Great Britain) 171

COMMENTConformation of engineered proteins

The characterization of any biopharmaceutical is a criticalstep in the overall manufacturing process [1,2] and is ofimportance to the concept of the ‘well-characterizedbiologic ’. In vitro determination of specific biological activityis, of course, a critical requirement for such characterization,but activity can be maintained when there are structuralchanges which could result, for example, in neoantigenicity.Thus independent assessment of conformation is also a keypart of the characterization process.

The ‘well-characterized biologic ’ concept is particularlyimportant for recombinant proteins, as illustrated by theproduction of the blood coagulation factor FactorVIII ineither baby-hamster kidney cells [3] or Chinese hamsterovary cells [4]. Although a ‘native ’ protein, structuralevaluation of fully active recombinant Factor VIII is critical toaddress the neoantigenicity issues raised above, as there is asignificant problem with the development of Factor VIIIinhibitors in patients with haemophilia A [5,6]. In this case,evaluation of the Factor VIII proteins suggests that thepolypeptide chain structure is identical with the nativeprotein, but that there are differences in protein glycosyl-ation [7,8] that do not appear to influence function.

The modification of natural structures through proteinengineering to produce new therapeutic proteins providesan additional and usually more daunting challenge. Here theprimary question is : can site-specific mutagenesis be used toprepare a modified protein without structural or con-formational change? This issue, of course, was importantduring the development of site-specific chemical modifi-cation of proteins, and advances in analytical technology[9,10] has permitted the sophisticated acquisition of struc-tural information ; likewise there are sophisticatedapproaches to the study of post-translational modificationsuch as glycosylation [11]. However, the above analyticaltechnologies, for the most part, address chemical structureand not conformation issues. Recent studies that addressthis issue include the work of Jones et al. [12], who have usedanalytical ultracentrifugation and EPR to examine the effectof protein engineering of human tissue factor on interactionwith a non-ionic surfactant, Nahri et al. [13], who have usedfar-UV CD spectroscopy (FUVCD) to study conformationalchanges during the reversible denaturation of recombinanthuman megakaryocyte growth and development factor, andTarelli et al. [14], who also employed FUVCD to comparethe structures of recombinant and native human albumin.NMR spectroscopy is also a powerful tool to study proteinconformation. Rajarathnam et al. [15] used it to examine the

effect of disulphide modification in interleukin-8, showingthat deletion of either disulphide by replacement of cysteineby alanine results in the loss of both structure and function.Interestingly, formation of disulphide analogues by usinghomocysteine, penicillamine or selenocysteine producednative structures, as judged by NMR spectroscopy, but therewere substantial differences in biological activity.

This issue of Biotechnology and Applied Biochemistrycontains an excellent paper by Professor John Brewer of theUniversity of Georgia, which addresses the determination ofthe conformation of engineered proteins. Brewer usesdifferential scanning calorimetry (DSC) to study the con-formation of engineered forms of yeast enolase and,specifically, the effect of ligand binding on the heat-de-naturation of the engineered forms of this protein. De-naturation of the protein is shown as ‘excess heat capacity ’.This is analogous to a DNA ‘melting ’ curve. With enolase,the binding of ligand is demonstrated to ‘stabilize ’ theprotein, as demonstrated by an increase in the temperatureat which ‘excess heat capacity ’ (Tmax) is observed. Thisapproach appears to us to add an important new method tothe arsenal of techniques for assessing the ‘conformation ’component of the ‘well-characterized biologic ’. The readeris directed toward this interesting paper for further details.

Roger L. LundbladDuarte, California

Ralph A. BradshawIrvine, California

2 August 1999

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