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Fragmentation occurs primarily in the hinge region of antibodies.2 There are two mechanisms underlying fragmentation, direct hydro- lysis and beta-elimination of disulfi de bonds. The reaction rate of direct hydrolysis is strongly pH-dependent with showing acceleration above as well as below pH 6.3

Beta-elimination of disulfi de bonds

occurs at the hinge disulfi de bonds and primarily occurs at basic pH.4 Since most fragmentation reactions occur at the hinge region, which is not variable, there are limited options for sequence engineering to infl uence fragmentation rates. However, formulation optimization can keep fragmentation reactions at a minimum.

Deamidation is the chemical transformation of an amino acid with an amide side chain (asparagine or glutamine) into a negatively charged carboxylic acid side chain (aspartate or glutamate).5,6 determine the reaction rate are:

Factors which

• the amino acid itself (where asparagine is usually more critical than glutamine),

• the subsequent amino acid (N+1) where glycine in particular causes fast deamidation,

• to a lesser extend the previous amino acid (N-1),

• the local three-dimensional structure and • the local conformational fl exibility.7

The reaction requires a distinct geometry at which the nitrogen of the subsequent amino acid reacts with the carbonyl moiety of the amide group forming a cyclic intermediate (succinimide). When this intermediate is hydrolyzed the former amide group is transformed into a carboxylic acid group, either aspartate or iso-aspartate (Figure 1).

Isomerization is closely related to deamidation. Here the transformed residues are either aspartate or glutamate, again with aspartate being more critical due to the energetically favored fi ve-ring intermediate. Just as for deamidation the subsequent amino reacts with the carbonyl carbon and the same cyclic intermediates are formed - consequently the products of the hydrolysis of the cyclic intermediate are the same as for deamidation (Figure 1)

The consequence of the deamidation and isomerization reactions can be manifold. If the respective amino acid is involved in antigen binding substantial potency loss may result. But even if the transformation is neutral with respect to antigen binding it might be unfavorable for protein stability and accelerate other protein degradation pathways, e.g. by reducing the conformational stability.

Oxidation primarily aff ects the sulfur containing amino acids cysteine and methionine, but also aromatic amino acids like tryptophan. Free cysteines should generally be avoided since cysteine is the most reactive of all proteinogenic amino acids undergoing a variety of chemical reactions. For oxidation sensitive amino acids the probability of oxidation and hence the rate at which the reaction occurs correlates with solvent accessibility. This principle can be intuitively understood as solvent accessibility increases the probability of forming contacts with dissolved oxygen and catalysts. The degradation products for methionine are the respective sulfoxide and the sulfon. Tryptophan most frequently is transformed to Kynurenine which itself is reactive and can undergo a variety of subsequent reactions.

42 | | May/June 2016

Figure 1. Deamidation and aspartate isomerization: Asparagine and aspartate can react with the subsequent amino acid into a

cyclic intermediate (succinimide). When this cyclic intermediate is hydrolyzed, the ring opening can either result in the reaction back to aspartate or into Iso-aspartate.

The consequences of oxidative degradation can be the same as for deamidation and isomerization. Affi nity to the antigen can be lost and protein stability compromised. In addition, all chemical transformations create new epitopes which in principle could be immunogenic.8 Therefore, antibody optimization eff orts aim at detecting potential sites susceptible to accelerated chemical degradation and to engineer sites with an increased risk to sequences with more favorable properties.

Besides chemical degradation reactions, aggregation is the second dominant antibody degradation pathway (see Figure 2). Antibody aggregation, which denotes the formation of high molecular weight particles, is a problem very frequently encountered in antibody development.9,10

Aggregation is not a well-defi ned process leading

to a well-defi ned product. In fact, aggregation pathways can be highly diverse. One reason for accelerated aggregation is reduced conformational protein stability. Proteins exist in equilibria between folded, partially unfolded and unfolded states where the population of the individual states is determined by the folding free energy diff erences between the individual states. In less stable proteins the fraction of partially or completely unfolded protein is higher than in stable proteins. In these non-native states hydrophobic residues, which in the folded state are buried in the protein core, become exposed to the solvent and, when coming into contact with unfolded parts of other molecules, tend to form disordered aggregates.11

A second aggregation pathway, which has been shown to be relevant for antibodies, involves the formation of cross-beta-sheet fi brils.12

Certain amino acid sequences have a strong tendency

to align with their counterparts from other molecules in a highly ordered fashion forming fi bril structures similar to those known from protein deposition diseases like Alzheimer’s Disease, Parkinson’s and Huntington’s Disease.

Thirdly, self-association through hydrophobic or charged surface patches can lead to formation of oligomers. With increasing protein concentrations oligomer formation can promote the formation of

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