As to prokaryotic systems for the production of antibodies see Ypatent Health

Definitions:

Chaotropic agent: refers to a compound that in a sutiable concentraiton is capable of chaing the spatila configuration or conformation of polypetpides through alterations at the surface so as to render the polypeptide soluble in an aqueous medium. The concentraiton of chaotropic agent will directly affect its strength and effectiveness. A strongly denaturing chaotropic solution contains a chaotropic agent in large concentrations which will unfold a polypeptide present in the solution effectively eliminating the prtoeins secondary structure. The unfolding will be relatively extensive, but reversible. Examples of chaotropic agents include guanidine hydrochloride, urea and hydroxides such as sodium or potassium hydroxide. 

Reducing agent: refers to a compound that in a suitable concentraiton in aqueous solution maintins free sulfydryl gorups so that the intra or intermolecular disulfide bonds are chemically disrupted. Examples of reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), beta-mercaptoethanol (BME), cystein, cysteamine, thioglycolate, glutathione and sodium borohydride. Pizarro (US 2008/0125580)

When the host cell is a Gram negative prokarytoic cell, newly synthesized protein is often isolated form the periplasmic space. Preferred Gram negative prokaryotic ccells used for periplasmic epxression are Escherichia coli strains. (Slough, US 15/538, 282, published as US 20170342105). Pizarro (US 2008/0125580)

Introduction:

Inclusion bodies are particles found in both the cytoplasmic and perioplasmic spaces of E. coli during high-level expression of heterologous protein. 

Inclusion bodies obtained by cytosolic microbial overexpression of a recombinant protein are large, spherical particles. Upon high-level expression, the inclusion bodies may span the entire diameter of an E. coli cell. Because of their refractile character, inclusion bodies can be observed directly in the living host cell by phase constrast microscopy. The term refractile body is therefore often used as a synoym for inclusion body. (Rudolkp “in vitro folding of inclusion body proteins” The FASEB J, 10(1)q, pp. 49-56, 2017). 

Inclusion body pellets consist of varying proportions of impurities (e.g., proteins, DNA and lipids) of host origin which may need to be purified prior to refolding the target protein. Purification of protein in the denautred state can be done as by RP-HPLC, gel filtration and ion exchange chromatography. (Mukhopadhyay, “inclusion Bodies and Purificaiton of Proteins in Biologically Active Forms” Advances in Biochemical Engineering/Biotechnology, 56, 1997).

The general strategy used to recover active protein from inclusion bodies involves three steps: inclusion body isolation and washing, solubiliation of the aggregated protein and refolding of the solubilized protein (Clark, “Protein refolding for industrial prcoesses” Current Opinion in Biotechnology, 2001, 12: 202-207). 

Isolation/Solubilization of Inclusion Bodies: 

Inclusion bodies must be separated from the host cells and solubilization of the inclusion bodies. There are many ways by which living organisms are disrupted to release intraceullar products. The outer membrane of E. coli si composed of lipoproteins, lipoolysaccharides, and proteins wehreas phospholipids and proteins are two components of the inner membrane. In addition, the periplasmic space contains peptidoglycan. These multilayer lipd protein glycan complexes contribute to the regidity of the E. coli cell wall and thus inclusion bodies produced in E. coli cannot be isolated satisfactrily by techniques like ultrasonication, osmatoic shock and eznymatic lysis. Although ultrasonic disintegration is the most widely used techniques, the disruption of bacterial cells is lower than 100%. The best technique for the disintegration of E. coli cells is high pressure homogeneization, although it suffers from the drawback of contamination of target protein with the cellular components.  (Mukhopadhyay, “inclusion Bodies and Purificaiton of Proteins in Biologically Active Forms” Advances in Biochemical Engineering/Biotechnology, 56, 1997). 

A variety of methods may be used to solubilize inclusion bodies; however, the choice of solubilizing agent can greatly impact the subsequent refolding step and the cost of the overall process. (Clark, “Protein refolding for industrial prcoesses” Current Opinion in Biotechnology, 2001, 12: 202-207).

Use of Chaotropic, Denaturing and Reducing agents:

After their isolation, inclusion bodies are commonly solubilized by high concentrations of chatropic agents such as guanidimium hydrochloride or urea. (Felipe, Microbial Cell Facotires, 2004, 3:11) 

–Guanidine+EDTA+DTE+Tris: 

Often functional heterologous proteins from E. coli or other bacteria are isolated form inclusion bodies and require solubiliation using strong denaturants. During the solubilization step, as is well known in the art, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is 0.1M Tris pH8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). (Dimitrov, US2010/0316641). 

Buchner (Anal Biochem, 205(2), 1992, pp. 263-70) discloses solubiliation of an inclusion body pellet in the presence of strong denaturants and reducing agents in a buffer consisting of 0.1 M Tris, pH 8, 6 M guanidine, 2 mM EDTA, 0.3M DTE. The solution is incubated for at least 2 h at room temeprature. 

Refolding Buffers; Conditions: 

To obtain native conformation, the polypeptide chain has to be refolded into correct secondary and tertiary structures, which are further stabilized by the formation of intramolecular disulfide bonds. In order to facilitate this, the concentration of denaturant can be reduced to a level at which intramolecular stabilizing forms (e.g., hydrogen bonding, hdrophobic interactions) exist, buffers containing oxidizing agents are applied and the concentration of denatured protein in the refolding buffer is maintained low to avoid intermolecular aggregation. Dialysis and diafiltration membrane of defined MW cut-off are used for the exchange of buffer. In refolding by single stage dilution, the refolding buffer is typically composed of 50 mmol/l tris-HCL containing different redox systems such as reduced/oxidzed glutathione or atmospheric oxygen. Air oxidaition is catalyzed by incorporation 01.-1.0 umoll Cu+2 ions in the refolding buffer. The formation of disulfide bonds in air oxidation has been found more effective in the presence of trace amounts of thiol agents (e.g., 2-mercaptoethanol, DTT or cystein). In vitro refolding efficiencies have been imporved by incorporating various additives in the refolding buffer such as amino acids (e.g., arginine) , sugars, neutral surfactants, and polymers. (Mukhopadhyay, “inclusion Bodies and Purificaiton of Proteins in Biologically Active Forms” Advances in Biochemical Engineering/Biotechnology, 56, 1997). 

The formation of incorrectly folded species and in particular aggregates is usually the casue of decreased renaturation yields. A efficient strategy to suppress aggregation is the inhibition of the intermolecular interactions leading to aggregation by the use of low molecular weight additives. The most commonly used low molecular weight additives are L-arginine, low concentraitons of denaturants such as urea and detergents. (Clark, “Protein refolding for industrial prcoesses” Current Opinion in Biotechnology, 2001, 12: 202-207).

Use of Arginine, EDTA, Oxidized Glutathione (GSSG):

Atkinson (US2009/0291428) discloses that for protein refolding, solubilized inclusion body protein was added dropwise over 36 h into refolding buffer of 400 mM L-arginine-HCL, 2 mM EDTA, 0.02 M ethanolamine, 0.5 mM oxidized glutathione and 5 mM reduced glutathione 

Buchner (Anal Biochem, 205(2), 1992, pp. 263-70) discloses renaturation by a rapid 100 fold dilution of the denatured and reduced protein into refolding buffer consisting of 0.1M Tris, pH 8.0, 0.5 M L-arginine, 8 mM GSSG, 2 mM EDTA. The concentration of inclusion body protein during refolding was 30 ug/ml. The samples were incubated at 10C. 

Dechavanne (Protein Expression and Purificaiton 75 (2011) 192-203) discloses that a wide range of chemical additive have been described to prevetn or reduce misfolding of proteins during the refolding process. L-Arginine is one of the most widely used additives for refolding proteins and at a concentration between 0.2-1M fequently increases the renaturation yield. PEG is another additive frequently used to improve correct structure formation by inhibiting aggregation. A low concentration of EDTA is frequently recommended to prevetn metal-catalyzed air oxidation of cysteines, which could result in wrong disulfide bridge formation. Addition of a mixture of reduced (RS-) and oxidized (RSSR) forms of low MW thiol reagents such as glutathione (GSH/GSSG), cysteine/cystine, cystamine-cysteamine and (DTTODTT) usually provies the appropriate redox potential to allow formation and reshuffling of correct disilfide bridges. 

An exemplary refolding/renaturation buffer is 0.1MTris, pH 8, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG) and 2 mM EDTA. (Dimitrov, US2010/0316641) 

Linke (US2013/0202626) discloses the refolding of CAT-8015 initiated by a 10 fold dilution of the clarified and concentrated inclusion body filtrate into refolding buffer (50 mM ethanolamine, 1 M arginine, 2 mM EDTA, 0.91 mM oxidized glutathione, pH 9.5). The refold solution was maintained at 2-8C for 48-72 hours with continous mixing. 

O’Connor, (US13/880424, published as US2013/0273607) discloses refolding of an anti-CD22 antibody fragment using a refolding buffer having about 2070 mM ethanolamine, about 0.5-.2M arginine, aobut 0.5-3mM EDTA and about 0.5-1.5 mMGSSG. Surprisingly reducing the GSSG concentration from 9.1 mM to 0.91 mMreduced glutathione adduct i the product, while not impacting the yield of the antibody fragment. The refolding buffer is integrated into a purificaiton scheme of first solubilzing a mixture having the recombinant protein and inclusion bodies, clarifying the recombinant protein from the mixture with one or more depth filters, recovering the calirifed protein, concentrating the clarified recombinnt protein and refolding the clarified recombinant protein in the protein refolding buffer.

–Solubilization Buffer – Refolding Buffer:

Gal (US 2009/0042248) discloses preparation of MASP fragmetns which includes solubilizaiton of the inclusion bodies and tranfering the solubilized prtoein which contains a solubilization agent such as GuHCl or urea, prferably in a buffer comprising 6 M GuHCl. fragmetns into a refolding buffer at pH higher than 7, perferably between 8.5 and 10.5. 

Pizarro (US 2008/0125580) discloses a method for recovering reoflded recombinant prteoins from cell culture that includs solubilizng the protein in a first buffered solution with pH greater than 9 such as 9-10 using a first chatropic agent and then refolding the folubilized protein in a second buffer between pH 9 and 11 with a second chatropic agent, two or mroe reducing agents and addition of air or oxygen for such time and under such conditions that refolding of the recombinant protein occurs. In one emobdiment, the frist and second buffers also incues arginine and the chaotropic agent is 1 M urea. 

Sinner (US 2011/0237509) disclsoes purifying HGH from E. coli by obtaining the hGH containing includsion bodies by lysing (Tris/salt lysis buffer) to release the inclusion bodies, centrifugation, suspending them in a Tris buffer and solubilziing the inclusion body fraction with 8 mM followed by refolding the hGH containing granules in the presence of a phospahte buffer by dialysis at at pH about 80 and temeprature about 2-10C and then ioslating a precipitate of the refolded hGH udner appropirate pH of about 6.8-7.00. 

—-Alkaline pH (solubilization) – Lower but alkaline pH (refolding) + Glutathione

Hollander, (US 14/766,848, published as US 10,065,987; see also US 16/050,417, published as US 2019/0077828) discloses a method of isolating an Fc protein having at least one disulfide bond such as an antibody or fibronectin from inclusion bodies (IBs) using an alkaline pH whithout using a significant amount of denaturing and/or reducing  agent. The method includes suspending a denatured protein in a suspension solution and then using a solubilization buffer having a pH in the range of 10.5-13 (Arginine can be used to buffer the pH in this range) to obtain a solubilized denatured proteins and then using a refold buffer having a pH in the range of 9-11 to obtain the refolding protein. In other words, the method using a strongly alkline pH, followed by incubation at reduced pH. The refold buffer may contain Arginine or another positively charged amino acid to maintin the pH in the range of 9-11. In some embodiments in which the protein to be refolded includes one or more disulfide bonds, the refold buffer may also include an oxidizing agent such as glutatione to facilitate the formation of disulfide bonds. Following the refolding the pHmay be reduced to a lower value such as 6-8. 

Gonzalez-Villasenor (WO 03/102013) discloses a method of solubilizing and recovering a target peptide such as an antibody from a host organisms by (i) disrupting the host cell to produce a lystate, recovering the lysate precipitate containing the polypeptide (iii) resuspending the lysate precipitate in a denaturant free, non-buffered solubilzation solution. In one embodiment, the alkaline solubilizaiton solutions has a pH of about 11.2. The inclusion bodies are solubilized by stirring gently at room temperature (between about 20-25C) between about 20-40 minutes. After 20-30 min of stirring at room temperature, the pH of the preparation drops to pH between about 9.5-10.2s the protein solubilizes and interacts with the OH-ions of the NaOH in the solubilizaiton solution. The preparation is then centrifuged for about 15 minutes and the sueprnatanst provies the target peptide with a final pH of between about 9.5-10.3 

–Use of glutathione during chromatography process

Slough, US 15/538, 282, published as US 20170342105) discloses purifiction of proteins such as antibodies, and particularly a Fab bound to PEG, where throughout the chromatography scheme the protein/antibody is miantained in the presence of a reducing agent such as glutathione, beta-mercaptoethanol, beta-mercaptoehylamine, dithiothreitol, tris (2-carboxyethyl)phosphine, and cysteine. 

 Arginine, CHES, EDTA, Urea: 

Pizarro (US2008/0125580) discloses isolating a recombinant protein from a prokaryotic cell culture, solubilzing in a first buffered solution which can comprise 1M Urea, 300 M arginine, 10 mM CHES, 5 mM EDTA, pH 11, pH greater than 9, having a first chaotropic agent, refolding the solubilized protein in a second buffered solution pH 9-11 comprising 1 M Urea, 15 mM cysteine, 2 mMDTT, 100 mM arginine, 10 mM CHES, 5 mM EDTA, pH 10, having a second chaotropic agent, two or more reducing agents and addition of air or oxygen for such time and under such conditions that refolding of the recombinant protein occurs, and recovering the refolded protein. 

CuCl2+H2O: 

Burnie (US2007/02021160 discloses that for refolding the inclusion body solution was made with refolding buffer and CuCl2, 2H2O, The preparation was incubated for 48 h at 2-8C under strong agitation. The inclusion bodies were filtered and then filter sterilised with a depth filter. 

Purification of Non-antibody Proteins from Inclusion Bodies

Human Insulin:

Hartman (US 6,001,604) discloses a prcoess for the production of recombinant human insulin by folding a proinsulin hybrid polypeptide where the refolding includes incubating the polypeptide at about 4-37C for a period fo about 1-30 hours at a pH of about 8.5-12.

Human Growth Hormone:

Patra (Protein Expression and Purificaiton 18, 182-192 (2000) discloses large scale purification of r-hGH from inclusion bodies by solubilization in 16 ml of 100 mM Tris buffer, pH 12.5, containing 2 M urea. The oslubilized r-hGH was diluted 5 times with Milli-Q water and the pH was broght down to 8.5 by adding 1 N HCL.