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

Antibody Fragment Production by E. coli Expression

To facilitate developers of antibody fragment-based therapeutics, Avecia Biologics has developed the pAVEway system, an expression system in E. coli.

The pAVEway system offers biopharmaceutical developers key development needs including the delivery of clinical material for clinical and nonclinical development; processes providing optimized CoGs for commercial manufacture; and the application of generic methods.

Production strain selection is a critical early target in process development. Although it is possible to change the production strain at any stage in clinical development, this can lead to repeated clinical trials, with subsequent additional costs in both time and money. These costs increase substantially for later changes. It is also important to select an appropriate strain to avoid problems later in development, including process development, fermentation scale-up, purification, and manufacturing. Many expression systems and strains used at laboratory scale are not suitable for large-scale fermentation.

Table 2. A typical pAVEway expression development study—achievable timeline to from gene to process.
The pAVEway system consists of a combination of expression vectors, selected host strains, and generic fermentation processes, which can be used to go from a product gene to a high yield fermentation process for proteins, including antibody fragments, within one month. A typical pAVEway expression development study is outlined in Table 2.

Expression Vector

Fig 3. DNA loop formation to achieve tight expression shut off
The pAVEway system is tightly controlled, which means that protein expression in the absence of induction is very low, allowing tight control of fermentation conditions. This tight control is achieved by optimizing the spacing between the operator sequences (as perfect palindromes) to allow DNA looping to take place (Figure 3). In addition, the rate of protein expression can be directly controlled by the concentration of the inducer (Figure 5). This is particularly important for the expression of soluble and secreted proteins, in which the best yields are obtained when the rate of expression can be tuned to the folding and secretion capacity of the cell. This effect on expression rate can optimize the folding and secretion of a number of different antibody fragment structures and other proteins.

Fig 4. Properties of pAVEway expression vectors. No leaky expression is observed, even after overnight incubation.
The basis for this tight expression control is the use of perfect palindromic operator sequences. The use of these sequences from T7 RNA polymerase-based promoters (as used in the pET system) has been extended to a range of E. coli RNA polymerase dependent promoters. These sequences have a much wider host range and avoid the use of the λDE3 lysogen that can give rise to lytic phage, which would present problems for multiproduct manufacturing facilities. Using these control elements in combination with strong promoters gives protein titers equivalent to those seen for T7-based systems in shake flasks. Additionally, the pAVEway system also produces low levels of product in the absence of induction (Figure 4). This reduced leakiness leads to superior stability of the strains and enables rationally designed fermentation protocols.

Fig 5. Properties of pAVEway expression vectors - the modulatable response of expression rate to the inducer concentration.
To confirm the usefulness of the pAVEway systems, the cloning and expression of a variety of antibody fragment types (single chain and Fab) have been demonstrated and then moved to fermentation (Table 3). Intracellular soluble and insoluble protein expression have been demonstrated and are fully scaleable. Note that the active secretion protocol is different from the intracellular protocol.

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