For the purification of antibodies from inclusion bodies, see Protein Purification and Antibody Purification. 

Antibody fragments were usually manufactured in mammalian cells, but alternative production systems such as E. coli, Pichia yeasts and plants have also been used. These manufacturing alternative have been driven by the specific antibody product and the balance between scale, cost, speed and biological safety. Humphreys (WO2004/035792) discloses using E coli host cells for the manufacture of recombiantn antibodies where the E coli host cells have been genetically modified in order to change at least one physical property such as isoelectric point  of an E coli protein(w) which in the wild type copuries with the antibody.

Kolkman (US2009/0297535) discloses that nanobodies can be produced eitehr intracellularly (e.g., in the cytosol, in the periplasma or in inclusion bodies) and then isolated form the host cells and optionally further purified or can be produced extracellularly (e.g., in the meidum in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. Bacterial cells such as E. coli do not noramlly secrete proteins extracellularly, except for a few classes of proteins such as toxins and hemolysin, and secretroy production in E. coli refers to the translocation of proteins across the inner membane to the periplasmic space. Periplasmic production provides several advantages over cytosolic production. For example, the N-terminal amino acid sequence of the secreted product can be identical to the natural gene product after cleavage of the secretion signal sequence by a specific signal peptidase. Also, there appears to be much less protease activity in the periplasm than in the cytoplasm. In addition. protein purificaiton is simpler due to fewer contaminating protins in the periplasm. Another advnatage is that correct disulfie bonds may form becasue the periplasm provides a more oxidative environment than the cytoplasm. Proteins overexpressed in E. coli are often found in insoluble aggregates, so called inclusion bodies which may be located in the cytosol or in the periplasm. The recovery of biologically active proteins from these includion bodies requires a denaturation/refolding process. Many recombinant proteins, included therapetuci proteins, are recovered from inclusion bodies.

Sawasdaishi (US2006/0269977) also teaches the separation of a fragment antibody form a culture medium which is prepared by expression of a recombinant prokaryotic host cell encoding the fragment antibody and subsequent lysis or disuprtion of the host cell.

Bacterial expression systems have limitations in glycoprotein production due to the complete lack of the enzymatic machinery and compartmentalization required for mammalian type glycosylation, although engineering and functional transfer of the required glycosylation machinery into prokaryotes has been done. Gharderi, Biotechnolgy and Genetic Engineering Reviews 28, 147-176 (2012). 

Production in Gram Negative Bacteria (e.g., Escherichia coli)

Advantages, Disadvantages and Challenges

Much work on antibody fragment production has been focussed on E. coli. The advantage is its ability to produce proteins in relative large amounts. In addition, E. coli is easily accessible for genetic modifications, requires simple inexpensive media for rapid growth and can easily be cultured in fermentors permitting large scale production of proteins of interest. Several antibody fragments have been produced in functional form and expression of relevant gene segments also permit the production of the recombinant antibody fragments. The problem of stability has been tackled by generation of single-chain Fv (scFV) or disulfide stabilised Fv (dsFv) fragments. (Joosten, “The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi” Microbial Cell Factories 2003, 2 pp. 1-15).

A significant advance in the development of Ab engineering came about when it was demonstrated taht Ab fragments could be exported to the bacterial periplasm by the inclusion of an N-temrinal secretion signal sequence. Within the periplasmic space, Ab fragmetns are able to fold correctly, aided by the formation of intra-domain disulfide bonds made possible by the oxidative environment, and remain in a soluble form. The choice of bacterail signal sequence does not apepar to be of major importance and seeveral ahve been successfully used such as OmpA. Upon transport into the periplasmic space, signal peptides are cleaved leaving the correct N-terminal sequence. High-level expression of recobminant proteins in E. coli and subsequent export to the periplasm often result in the formation of insoluble aggregates. Such effects can be reduced by indcution and expression of Ab fragments at 20-25C. Significnat improvements in the yeilds of soluble recombinant Ab fragments have also been obtained by identifying and overexpressing E. coli periplasmic chaperones such as Skp and other proteins involved in modulating folding, such as the peptidylprolyl cis,trans-isomerase FkpA. Periplasmic aggregation can also be reduced by induction of cultures in growth medium supplemented with osmotic stress inducing concentraitons of non-metabolized compounds such as 1M sorbitol and 2.5 mM glycin betaine or 0.4 M sucrose. (Chariton “Expression and isolation of recombinant antibody fragments in E. coli” in Methods in Molecualr Biology, vol. 248: “Antibody Engineering: Methods and Protocols”, Humana Press )

A protein that does not require glycosylation is preferably expressed in prokaryotic expression systems employing prokaryotic cells such as gram-negative bacteria. For example, an antibody fragment such as Fab is preferably expressed in such systems. (Spitali, WO2012/013682) 

Expression of larger proteins, like Fc fusions, in E. coli can be a unique challenge becasue E. coli lack the chaperone proteins and other refolding machinery found in a eukaryotic expression system. The cytoplasms of E. coli is also a reducing environment, which is not favorable for the formuation of disulfide bonds. The Fc region of human IgG1 antibodies contains six disulfide bonds. Two disulfide bonds in the hinge region join two peptide chains frorm the homodeimeric molecule and there are two more disulfide bonds within each of the peptide chains.  (Hollander, US 14/766,848, published as US 10065987)

Still, many therapeutic proteins in the native form are non-glycosylated. Non-glycosylated protein has shown distinct therapeutic advantages over its native glycosylated form. The majority of the expressed heterologous proteins accumulate in the cytoplasm of E. coli due to the absence of a proper secretory mechanism. Purification of these proteins into therapaeutic grade is tricky. Attempts have been made to secrete these proteins into the periplasm with the help of a signal peptide of a membrane or secretory protein. So far, such attempts have met with only limited sucess due to the complex nature of the heterologous proteins and their secretion mechanism. Therefore, many therapeutic proteins are still produced as inclusion bodies expressed in the cytoplasm. Because of their high concentration and aggregated form, inclusion bodies can be isolated from the cellular proteins in highly purified form. Inclusion bodes are aggregated, extremely dense structures of protein, products mostly in the cytoplasm of E. coli and can be as large as a normal bacterial cell. The large inclusions are visible under an optical microscope as particles reflecting light, and termed “refractile bodies”. They are morphologically different from any other intracellular strutures found in the cytoplasm of prokaryotes. However, the problem remains for an efficient route for the production of biologically active proteins the inclusion bodies. Most of the eukaryotic proteins expressed in E coli require correct disulfide paring to become biologcially active molecules. Oxidative folding of these proteins in the cytosol of E. coli is prevented due to the very high reducing environment (GSH/GSSG = 100-40). On the other hand, periplasm extends a congenial environment for folding due to its non-reducing nature and the presence of high energy protein disulfide reagents (DsbA) and prolyl-peptdyl cis-trans isomerase (PPI). Disulfide bond formation with concomitant folding complicates the in vitro regeneration of native proteins form their reduced and denatured state. (Mukhopadhyay, “inclusion Bodies and Purification of Proteins in Biologically Active Forms” Advances in Biochemical Engineering/Biotechnology, 56, 1997).  

Bacterial cells such as strains of E. coli do not secrete proteins extracellulalry, except for a few classes of proteins such as toxins and ehmolysin, and secretory production in E. coli refers to the translocation of proteins across the inner membrane to the periplasmic space. Periplasmic production provides several advantages over cytosolic production. For example, the N-terminal amino acid sequence of the secreted product can be identical to the nature gene product after cleavage of the secretion signal sequence by a specific signal peptidase. Aslo, there appears to be much less protease activity in the periplasm than in the cytoplasm. In addition, protein purification is simpler due to fewer contaiminating proteins in the periplasm. Another advantage is that correct disulfide bonds may form becasue the periplasm provides a more oxidative environment than the cytoplasm. Proteins overexpressed in E. coli are often found in insoluble aggregates, called inclusion bodies which may be located in the cytosol or in the periplasma; the recovery of biologically active proteins from these inclusion bodies requires a denaturation/refolding process. (Saunders (WO/2009/068627).  

Production in Gram Positive Bacteria

Gram-positive bacteria directly secrete proteins into the medium due to the lack of an outer membrane which could facilitate production of antibody fragments. The Gram-positive bacteria Bacillus brevis, Bacillus subtilis and Bacillus megaterium ahve already been successfully used for the produciton of different antibody fragments. In addition, B. megaterium does not produce alklaine proteases and provides high stabiliyt of plasmid vectors during growth allowing stable transgene expression during long term culutivation in bioreactors. Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Bacillus subtilis: can offer greater scale-up advantages compared with yeasts. Becasue of its faster growth rates and shorter fermentation cycles, using B. subtillis as a microbal host can lead to higher productivity. For instance, the organism can acheive a doubling time of as littel as 20 minutes under an optimal growth temperature of 30-35 C. Those conditions enable tye typical B. subtillis fermentation cycle to be completed in about 48 hours, whereas teh fermentaiton cycle of Saccharomyces crevisiae is much longer at about 180 hours. B. subtilis occurs naturally in soil and has evolved to secrete a complex suite of enzyems that allow it to access nutrients in surrounding organic matter, compete with other organisms. Gram-engative hosts such as Escherichia coli produce endotoxins and otehr impurities that must be removed during downstream processing. 

B. subtilis plays many roles in industrial applications becasue of its natural ability to serete enzymes of commerical interest, including amylases, xylanases, and proteases. Amylases release simple sugards from starch, one of the most abundant naturally occurring polymers. The inherent secretory mechanisms of B. subtilis make it well suited to the production of various enzymes needed to establish green alternatives, inclduing enzymes that enable the breakdown of abundant lignocellulosic biomass into simple, fermentable sugars. These sugars then can be converted into biofuels, including ethanl and butanol, through fermentation. 

Production of Antibody Fragments in Prokaryotics

Today, scFv (single chain fragment variable) and Fab are the most widely sued antibody fragments which are produced in prokaryottes. Other antibody formats ahve been produced in prokarytoic and eukaryotic cells, for example, disulfide-bond stabilized scFv (ds-ScFv), single chain Fab gragments (scFab) combining scFv and Fab properties as well as di- and multimeric antibody formts like dia-, tria-, o tetra-bodies or minibodies comprising different formats consisting of scFvs linked to oligomerization domains like immiunoglobulin CH3 domain. Frenzel, “Expression of recombinant antibodies” Frontiers in immunology volume 4, July 2013)

Genetic Optimization for Enhanced Protein Production:

Protein expression levels are influenced by several factors, including the activity of promoters used to regualte gene transcription, the strenght of the ribosome binding site, and the number of recombinant gene copies. The choice of a secretion signal peptide is also important for effective production in for example B. subtilis.