Production Strategies for Antibody Fragment Therapeutics - Microbial systems such as E. Coli and yeasts are the most effective production systems for the production of antibody fragments. - BioPharm


Production Strategies for Antibody Fragment Therapeutics
Microbial systems such as E. Coli and yeasts are the most effective production systems for the production of antibody fragments.

BioPharm International Supplements


The term antibody fragments covers a large number of molecular structures derived from full-length antibodies. Some clarification of structural types is useful before discussing production aspects.

IgG Structure

Figure 1. An explanatory schematic of whole antibody domain structure
IgGs are the most abundant immunoglobulins in the blood and are commonly used as antibody therapeutics. They have a molecular weight of approximately 160 kDa comprising 12 domains of roughly equal size. They are made up of four separate peptide chains, two identical heavy (H) chain polypeptides and two identical light (L) chain polypeptides (Figure 1). The L and H chains are composed of two and four domains, respectively, with each domain having a similar β-barrel structure and containing one disulfide bridge. The H and L chains are linked together by a combination of disulfide and non-covalent bonds.4 Each chain contains both variable and constant domains. The variable domains of the H and L chain (VH and VL) contain the variable complementarity-determining regions (CDRs). These regions of extremely variable amino acid sequences are located at the N-terminal part of the antibody molecule. Together, VH and VL form the unique antigen-recognition site. The amino acid sequences of the remaining C-terminal domains are much less variable.

Fc Region

The Fc region, also referred to as the constant domain, is the nonantigen-binding part of the antibody molecule. Fc mediates several immunological functions, such as binding to receptors on target cells and triggering effector functions that eliminate the antigen. There are applications where the Fc-mediated effects are not required or are even desirable. The Fc fragment also is the site of glycosylation in IgGs.

Antigen-Binding Region

The unique antigen-binding site of an antibody consists of the H and L chain variable domains (VH and VL). Each domain contains four conserved framework regions and three CDR regions, which determine the specificity of the antibody. The VL and VH domains together form a binding site, which binds a specific antigen.

Antigen-Binding Fragments

Figure 2. Antibody fragment diversity
A wide variety of functional antigen-binding fragments has been constructed from whole antibody domains, some of which are illustrated in Figure 2 (see also reference 5). Selected fragment types are discussed in detail below.

Fab Fragments

Fab fragments (fragment antigen binding) are the antigen-binding domains of an antibody molecule, containing two amino acid chains composed of two domains, VH + CH1 and CL + VL. An interchain disulfide bond present at the C terminal of each constant domain links the CL and CH1 domains together, which gives a molecular weight of the heterodimer around 50 kDa.6 Lucentis and Cimzia are both examples of Fab antibody fragments.

Single-Chain Fv Fragments

A single-chain Fv fragment (scFv) is the smallest fragment (~30 kDa) that still contains the complete antigen-binding site (VH + VL) of a whole IgG antibody.7 Linker sequences joining the VH and VL domains in a single amino acid chain have been used to overcome the fact that native scFv fragments are unstable and tend to dissociate from one another.

Antibody Fab and scFv fragments, comprising both VH and VL domains, retain the specific monovalent antigen-binding affinity of the parent IgG while showing improved pharmokinetics for tissue penetration. However, these are not the smallest form of antibody fragments.

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