Bioprocess Development and Characterization of a 13C-Labeled Hybrid Bispecific Antibody Produced in Escherichia coli.

Monoclonal antibodies (mAbs) are highly efficacious therapeutics; however, due to their large, dynamic nature, structural perturbations and regional modifications are often difficult to study. Moreover, the homodimeric, symmetrical nature of mAbs makes it difficult to elucidate which heavy chain (HC)-light chain (LC) pairs are responsible for any structural changes, stability concerns, and/or site-specific modifications. Isotopic labeling is an attractive means for selectively incorporating atoms with known mass differences to enable identification/monitoring using techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR). However, the isotopic incorporation of atoms into proteins is typically incomplete. Here we present a strategy for incorporating 13C-labeling of half antibodies using an Escherichia coli fermentation system. Unlike previous attempts to generate isotopically labeled mAbs, we provide an industry-relevant, high cell density process that yielded >99% 13C-incorporation using 13C-glucose and 13C-celtone. The isotopic incorporation was performed on a half antibody designed with knob-into-hole technology to enable assembly with its native (naturally abundant) counterpart to generate a hybrid bispecific (BsAb) molecule. This work is intended to provide a framework for producing full-length antibodies, of which half are isotopically labeled, in order to study the individual HC-LC pairs.

Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli.

BACKGROUND: Escherichia coli (E. coli) is a promising host for production of recombinant proteins (including antibodies and antibody fragments) that don't require complex post-translational modifications such as glycosylation. During manufacturing-scale production of a one-armed antibody in E. coli (periplasmic production), variability in the degree of reduction of the antibody's disulfide bonds was observed. This resulted in variability in the free thiol content, a potential critical product quality attribute. This work was initiated to understand and prevent the variability in the total free thiol content during manufacturing. RESULTS: In this study, we found that the reduction in antibody's disulfide bonds was observed to occur during homogenization and the ensuing homogenate hold step where in the antibody is exposed to redox enzymes and small molecule reductants present in homogenate. Variability in the downstream processing time between the start of homogenization and end of the homogenate hold step resulted in variability in the degree of antibody disulfide bond reduction and free thiol content. The disulfide bond reduction in the homogenate is catalyzed by the enzyme disulfide bond isomerase C (DsbC) and is highly site-specific and occurred predominantly in the intra-chain disulfide bonds present in the Fc CH2 region. Our results also imply that lack of glycans in E. coli produced antibodies may facilitate DsbC accessibility to the disulfide bond in the Fc CH2 region, resulting in its reduction. CONCLUSIONS: During E. coli antibody manufacturing processes, downstream processing steps such as homogenization and subsequent processing of the homogenate can impact degree of disulfide bond reduction in the antibody and consequently product quality attributes such as total free thiol content. Duration of the homogenate hold step should be minimized as much as possible to prevent disulfide bond reduction and free thiol formation. Other approaches such as reducing homogenate temperature, adding flocculants prior to homogenization, using enzyme inhibitors, or modulating redox environments in the homogenate should be considered to prevent antibody disulfide bond reduction during homogenization and homogenate processing steps in E. coli antibody manufacturing processes.

Engineered sigma factors increase full-length antibody expression in Escherichia coli.

Escherichia coli (E. coli) is a promising platform for expression of full-length antibodies owing to its several advantages as a production host (fast growth, well characterized genetics, low manufacturing cost), however, low titers from shake flask (typically < 5 mg/L) has limited its use for production of research-grade material in antibody discovery programs. In this work, we used global transcriptional machinery engineering (gTME) with high throughput screening to increase the expression of full-length antibodies in E. coli. A library of E. coli mutants carrying mutations in the global sigma factor RpoD were generated and screened using the Bacterial Antibody Display (BAD) method for enhanced expression. RpoD mutants were isolated that resulted in full-length antibody titers of up to 130.7 ±â€¯6.6 mg/L of shake flask culture with chaperone co-expression. These results could be useful for production of several antibodies quickly in shake flasks for characterization (e.g. antigen binding, biological function) during the early discovery phase.

Strain engineering to reduce acetate accumulation during microaerobic growth conditions in Escherichia coli.

Microaerobic (oxygen limited) conditions are advantageous for several industrial applications since a majority of the carbon atoms can be directed for synthesis of desired products. Oxygen limited conditions, however, can result in high levels of undesirable by-products such as acetate, which subsequently can have an impact on biomass and product yields. The molecular mechanisms involved in acetate accumulation under oxygen limited conditions are not well understood. Our results indicate that a majority of the genetic modifications known to decrease acetate under aerobic conditions results in similar or even higher acetate under oxygen limitation. Deletion of arcA, whose gene product is a global transcriptional regulator, was the only modification among those evaluated that significantly decreased acetate under both transient and prolonged oxygen limitation. Transcriptome results indicate that the arcA deletion results in an increased expression of the operon involving acs and actP (whose gene products are involved in acetate assimilation and uptake respectively) and some genes in the TCA cycle, thereby promoting increased acetate assimilation. These results provide useful cues for strain design for improved manufacturing of biopharmaceuticals under oxygen limited conditions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:303-314, 2018.

Toward an era of utilizing methionine overproducing hosts for recombinant protein production in Escherichia coli.

Amino acid sequence variants, especially variants containing non-canonical amino acids such as norleucine and norvaline, are a concern during therapeutic protein production in microbial systems. Substitution of methionine residues with norleucine in recombinant proteins produced in Escherichia coli is well known. Continuous feeding of amino acids such as methionine is commonly used in E. coli fermentation processes to control incorporation of norleucine in the recombinant protein. There are several disadvantages associated with continuous feeding during a fermentation process. For example, a continuous feed increases the operational complexity and cost of a manufacturing process and results in dilution of culture medium which could result in lower cell densities and product yields. To overcome the limitations of existing approaches to prevent norleucine incorporation during E. coli fermentations, a new approach using an engineered host was developed that overproduces methionine in the cell to prevent norleucine incorporation without negatively impacting fermentation process performance and product yields. In this commentary, the results on using methionine overproducing hosts for recombinant protein production in E. coli and some "watch outs" when using these hosts for recombinant protein production are discussed.

Effect of individual Fc methionine oxidation on FcRn binding: Met252 oxidation impairs FcRn binding more profoundly than Met428 oxidation.

The long serum half-lives of mAbs are conferred by pH-dependent binding of IgG-Fc to the neonatal Fc receptor (FcRn). The Fc region of human IgG1 has three conserved methionine residues, Met252, Met358, and Met428. Recent studies showed oxidation of these Met residues impairs FcRn binding and consequently affects pharmacokinetics of therapeutic antibodies. However, the quantitative effect of individual Met oxidation on Fc-FcRn binding has not been addressed. This information is valuable for defining critical quality attributes. In the present study, two sets of homodimeric site-directed IgG1 mutations were generated to understand how individual Fc Met oxidation affects FcRn binding. The first approach used Met to Leu mutants to block site-specific Met oxidation. In the other approach, Met to Gln mutants were designed to mimic site-specific Met oxidation. Both mutagenesis approaches show that either Met252 or Met428 oxidation alone significantly impairs Fc-FcRn binding. Met252 oxidation has a more deleterious effect on FcRn binding than M428 oxidation, whereas Met428 oxidation has a bigger destabilization effect on the thermal stability. Our results also show that Met358 oxidation does not affect FcRn binding. In addition, our study suggests that Met to Gln mutation may serve as an important tool to understand Met oxidation.

Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli.

Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes.

Repurposing lipoic acid changes electron flow in two important metabolic pathways of Escherichia coli.

In bacteria, cysteines of cytoplasmic proteins, including the essential enzyme ribonucleotide reductase (RNR), are maintained in the reduced state by the thioredoxin and glutathione/glutaredoxin pathways. An Escherichia coli mutant lacking both glutathione reductase and thioredoxin reductase cannot grow because RNR is disulfide bonded and nonfunctional. Here we report that suppressor mutations in the lpdA gene, which encodes the oxidative enzyme lipoamide dehydrogenase required for tricarboxylic acid (TCA) cycle functioning, restore growth to this redox-defective mutant. The suppressor mutations reduce LpdA activity, causing the accumulation of dihydrolipoamide, the reduced protein-bound form of lipoic acid. Dihydrolipoamide can then provide electrons for the reactivation of RNR through reduction of glutaredoxins. Dihydrolipoamide is oxidized in the process, restoring function to the TCA cycle. Thus, two electron transfer pathways are rewired to meet both oxidative and reductive needs of the cell: dihydrolipoamide functionally replaces glutathione, and the glutaredoxins replace LpdA. Both lipoic acid and glutaredoxins act in the reverse manner from their normal cellular functions. Bioinformatic analysis suggests that such activities may also function in other bacteria.

Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways.

The first and highly conserved step in glutathione (GSH) biosynthesis is formation of γ-glutamyl cysteine by the enzyme glutamate-cysteine ligase (GshA). However, bioinformatic analysis revealed that many prokaryotic species that encode GSH-dependent proteins lack the gene for this enzyme. To understand how bacteria cope without gshA, we isolated Escherichia coli ΔgshA multigenic suppressors that accumulated physiological levels of GSH. Mutations in both proB and proA, the first two genes in L-proline biosynthesis, provided a new pathway for γ-glutamyl cysteine formation via the selective interception of ProB-bound γ-glutamyl phosphate by amino acid thiols, likely through an S-to-N acyl shift mechanism. Bioinformatic analysis suggested that the L-proline biosynthetic pathway may have a second role in γ-glutamyl cysteine formation in prokaryotes. Also, we showed that this mechanism could be exploited to generate cytoplasmic redox buffers bioorthogonal to GSH.

Functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways.

In Escherichia coli, the glutathione/glutaredoxin and thioredoxin pathways are essential for the reduction of cytoplasmic protein disulfide bonds, including those formed in the essential enzyme ribonucleotide reductase during its action on substrates. Double mutants lacking thioredoxin reductase (trxB) and glutathione reductase (gor) or glutathione biosynthesis (gshA) cannot grow. Growth of Deltagor DeltatrxB strains is restored by a mutant (ahpC*) of the peroxiredoxin AhpC, converting it to a disulfide reductase that generates reduced glutathione. Here, we show that ahpC* also restores growth to a DeltagshB DeltatrxB strain, which lacks glutathione and accumulates only its precursor gamma-glutamylcysteine (gamma-GC). It suppresses this strain by allowing accumulation of reduced gamma-GC, which can substitute for glutathione. Surprisingly, new ahpC suppressor mutations arose in a DeltagshA DeltatrxB strain lacking both glutathione and gamma-GC, a strain that ahpC* does not suppress. Some of these mutant AhpC proteins channel electrons into the disulfide-reducing pathways via either the thioredoxins or the glutaredoxins without, evidently, the intermediary of glutathione. Our results provide insights into the physiological functioning of the glutathione pathway and reveal surprising plasticity of a peroxidase because different mutant versions of AhpC can channel electrons into the disulfide-reducing pathways by at least four distinct routes. Despite the reductase activity of mutant AhpCs, these various suppressor strains exhibit an oxidizing cytoplasm and accumulate correctly folded disulfide-bonded proteins in their cytoplasm. Proteins most effectively oxidized vary between strains, potentially providing useful tools for expressing different disulfide-bonded proteins.

The many faces of glutathione in bacteria.

Glutathione is one of the most abundant thiols present in cyanobacteria and proteobacteria, and in all mitochondria or chloroplast-bearing eukaryotes. In bacteria, in addition to its key role in maintaining the proper oxidation state of protein thiols, glutathione also serves a key function in protecting the cell from the action of low pH, chlorine compounds, and oxidative and osmotic stresses. Moreover, glutathione has emerged as a posttranslational regulator of protein function under conditions of oxidative stress, by the direct modification of proteins via glutathionylation. This review summarizes the biosynthesis and function of glutathione in bacteria from physiological and biotechnological standpoints.