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MICROBIOLOGY


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Developability Predictions for Antibody Engineering and Risk Mitigation


Daniel Seeliger1* , Timothy D. Fenn2 , and Anne R. Karow-Zwick3


1Discovery Research, Lead Identification and Optimization Support, Boehringer Ingelheim, Biberach, Germany 2Discovery Research, Immune Modulation and Biologics Discovery, Boehringer Ingelheim, Ridgefield, CT, USA 3Global Bioprocess and Pharmaceutical Development, Boehringer Ingelheim, Biberach, Germany *Corresponding author: daniel.seeliger@boehringer-ingelheim.com


Introduction


Biologics, in particular monoclonal anti- bodies, have revolutionized medicine in the last two decades and nowadays represent the standard of care for a variety of diseases, most notably in oncology and autoimmune disorders. This fact is reflected by the market share biologics have obtained in 2015, in which seven of the top ten selling drugs belong to this class.


Biologics differ substantially from classical small molecule drugs not only by their size (the molecular weight of an antibody is about three hundred fold higher than a typical small molecule drug) but also in the way they are discovered, and even more importantly, how they are manufactured and administered. In most cases antibodies are generated through immunization of mammalian species (e.g. mouse or rabbit) with the desired antigen, produced in cell cultures and administered either i.v. or s.c. since molecules of this size are generally not bioavailable after oral application.


Secondly, the function of antibodies is tightly coupled to the integrity of their three- dimensional structure, which is sensitive to heat,


40 | | May/June 2016


interfacial stress and pH conditions to which small molecule drugs are typically resistant. Hence, manufacturing of antibodies -which involves production in living cells, separation of the antibody from the remainders of the cells, purification, filling and other manufacturing steps which essentially denote stress for proteins - represent sub-stantial challenges for development. The suitability of antibodies to be manufactured in large-scale, which is commonly referred to as developability, therefore is not always a given.


Since antibodies are a product of an evolutionary process as part of the adaptive immune response their sequences vary substantially. The driving force for the in vivo selection and maturation is biological function and not suitability for large-scale manufacturing and long-term storage. Hence, antibodies derived from immunized animals do not necessarily fulfill the requirements to be developed into drugs. The same holds true for antibodies derived from in-vitro technologies like phage-display, where the selection cri- terion is typically based solely on binding.


The careful selection of suitable candidates and the rational engineering of antibody sequences for improved developability while maintaining biological function are therefore


intensive fields of research. Central to this effort is the understanding of the molecular mechanisms of antibody degradation and the ability to establish relationships between the sequence and undesired properties of antibodies to guide protein engineering.


Antibody Degradation Pathways


Antibodies can undergo various types of degradation which can be roughly divided into chemical degradation and protein aggregation.1


Chemical degradation denotes


the chemical transformation of the antibody. The most frequent chemical degradation reactions are:





fragmentation, which denotes the formation of antibody fragments through hydrolysis


• deamidation, the chemical transformation of an amide to a carboxylic acid


• isomerization, conversion from aspartate into iso-aspartate


• oxidation, reaction of an amino acid with oxygen


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