Comparative stability and analytical performance of anti-miroestrol recombinant antibody in different cassettes.

Immunoassays are efficient for the phytochemical analysis of various matrices. However, producing an appropriate recombinant antibody for small molecules is challenging, resulting in costly analyses. In this study, we aimed to develop recombinant fragment antigen-binding (Fab) antibodies against miroestrol, a potent phytoestrogen marker of Pueraria candollei. Two expression cassettes of Fab were established for the production of active Fab antibodies using SHuffle® T7 Escherichia coli cells. The orientation of variable fragment heavy chain (VH) and variable fragment light chain (VL) in the expression vector constructs influences the reactivity, stability, and binding specificity of the resultant Fab. Stability testing of antibodies demonstrated that Fab is a more stable form of recombinant antibody than a single-chain variable fragment (ScFv) antibody in all conditions. Based on the obtained Fab, the ELISA specifically detected miroestrol in the range of 39.06-625.00 ng/mL. The intra- and inter-assay precisions were 0.74-2.98% and 6.57-9.76%, respectively. The recovery of authentic miroestrol spiked into samples was 106.70-110.14%, and the limit of detection was 11.07 ng/mL. The results for P. candollei roots and products determined using our developed ELISA with Fab antibody and an ELISA with anti-miroestrol monoclonal antibody (mAb) were consistent (R2 = 0.9758). The developed ELISA can be applied for the quality control of miroestrol derived from P. candollei. Therefore, the appropriate expression platform of Fab resulted in the stable binding specificity of the recombinant antibody and was applicable for immunoassays.Key points• ELISAs with Fab has higher sensitivity than that with ScFv.• Fab is more stable than ScFv.• Fab-based ELISA can be used for miroestrol determination of Pueraria candollei.

Immunoaffinity separation of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham using fragment antigen-binding antibody produced via Escherichia coli.

INTRODUCTION: Miroestrol and deoxymiroestrol are potent phytoestrogens and are oestrogen markers of Pueraria candollei var. mirifica. However, purifying these compounds is difficult because they only exist in trace amounts. OBJECTIVES: Active fragment antigen-binding (Fab) antibodies were produced via Escherichia coli SHuffle® T7 and used to selectively separate these compounds. MATERIALS AND METHODS: Two immunoaffinity separation approaches were developed, namely the immunoaffinity column (IAC) and a cell-based method. Group-specific Fab antibodies against miroestrol and deoxymiroestrol (anti-MD Fab) were used as biological binding reagents for selective separation. RESULTS: The Fab-based IAC effectively separated miroestrol and deoxymiroestrol (0.65 and 2.24 µg per 2 mL of resin, respectively) from P. mirifica root extract. When P. mirifica extract was added to E. coli cultures during Fab expression via a cell-based method, the target compound accumulated in intracellular compartments and, thus, were separated from E. coli cells after the removal of other compounds. A yield of 1.07 µg of miroestrol per gram of cell pellet weight was obtained. Miroestrol and deoxymiroestrol were successfully purified from P. mirifica extract using anti-MD Fab via the IAC and an intracellular cell-based method. CONCLUSION: The proposed methods can simplify the miroestrol and deoxymiroestrol extraction process and provide a basis for applications utilising recombinant antibodies to separate target compounds.

A rapid ELISA for the detection of matrix metallopeptidase 9 using a recombinant Fab-type antibody.

Matrix metalloproteinase 9 (MMP9) contributes to several aspects of inflammation and cancer pathology, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant fragment antigen-binding (Fab)-type anti-MMP9 antibody in Escherichia coli with high purity within five days and confirmed the nanomolar order of antigen-binding efficiency of the recombinant Fab. Moreover, we optimized the experimental time for performing enzyme-linked immunosorbent assay (ELISA), and decreased the reaction time from the conventional 20.5 h to 3.5 h. The rapid and sensitive MMP9 detection system developed in this study can be applied to a range of applications, including the diagnosis of diseases with MMP9 overexpression including inflammatory and cancer-related diseases.

Improvement in the binding specificity of anti-isomiroestrol antibodies by expression as fragments under oxidizing conditions inside the SHuffle T7 E. coli cytoplasm.

Sensitive and specific analysis of isomiroestrol (Iso) is required for the quality control of Pueraria candollei, a herb used to treat menopausal disorders. The anti-isomiroestrol monoclonal antibody (Iso-mAb) exhibits cross-reactivity with miroestrol and deoxymiroestrol, which impacts the analytical results. Here, the active and soluble forms of the single-chain variable fragment (Iso-scFv) and fragment antigen-binding (Iso-Fab) against Iso were expressed using Escherichia coli SHuffle® T7 to alter the binding specificity. The Iso-scFv format exhibited a higher binding activity than the Iso-Fab format. The reactivity of Iso-scFv towards Iso was comparable with that of the parental Iso-mAb. Remarkably, the binding specificity of the scFv structure was improved and cross-reactivity against analogs was reduced from 13.3-21.0% to ˂ 1%. The structure of recombinant antibodies affects the binding characteristics. Therefore, the immunoassays should improve specificity; these findings can be useful in agricultural processes and for quality monitoring of P. candollei-related materials.

Expression of actively soluble antigen-binding fragment (Fab) antibody and GFP fused Fab in the cytoplasm of the engineered Escherichia coli.

The expression of recombinant antibody fragments in the cytoplasmic space of Escherichia coli and the refolding process for restoring the structure and activity of such antibodies are not efficient. Herein, fragment antigen-binding (Fab) antibodies against miroestrol and deoxymiroestrol (MD-Fab) and their fusions with a green fluorescent protein (GFP) were expressed. The reactive MD-Fabs were successfully expressed as soluble and active forms in the cytoplasm of the SHuffle® T7 E. coli strain. Regarding the construct of MD-Fab alone, VH-CH1 could associate VL-CL into Fab in the oxidizing cytoplasm of the E. coli strain, and no additional in vitro refolding was needed. In the case of the fusions with GFP, when the C-terminus of VH-CH1 was linked with the N-terminus of GFP, the MD-Fab binding reactivity was retained, but the fluorescent activity of GFP interfered. When the C-terminus of GFP was linked to the N-terminus of VL-CL, the binding activity of MD-Fab was not observed. The constructed MD-Fabs had higher specificity toward deoxymiroestrol than the parental monoclonal antibody clone 12G11. In conclusion, MD-Fabs could be expressed using SHuffle® T7 E. coli cells. This process could be considered an economical, productive, and effective method to produce antibody fragments for immunoassay techniques.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries.

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library.

A novel hypothesis for an alkaline phosphatase 'rescue' mechanism in the hepatic acute phase immune response.

The liver isoform of the enzyme alkaline phosphatase (AP) has been used classically as a serum biomarker for hepatic disease states such as hepatitis, steatosis, cirrhosis, drug-induced liver injury, and hepatocellular carcinoma. Recent studies have demonstrated a more general anti-inflammatory role for AP, as it is capable of dephosphorylating potentially deleterious molecules such as nucleotide phosphates, the pathogenic endotoxin lipopolysaccharide (LPS), and the contact clotting pathway activator polyphosphate (polyP), thereby reducing inflammation and coagulopathy systemically. Yet the mechanism underlying the observed increase in liver AP levels in circulation during inflammatory insults is largely unknown. This paper hypothesizes an immunological role for AP in the liver and the potential of this system for damping generalized inflammation along with a wide range of ancillary pathologies. Based on the provided framework, a mechanism is proposed in which AP undergoes transcytosis in hepatocytes from the canalicular membrane to the sinusoidal membrane during inflammation and the enzyme's expression is upregulated as a result. Through a tightly controlled, nucleotide-stimulated negative feedback process, AP is transported in this model as an immune complex with immunoglobulin G by the asialoglycoprotein receptor through the cell and secreted into the serum, likely using the receptor's State 1 pathway. The subsequent dephosphorylation of inflammatory stimuli by AP and uptake of the circulating immune complex by endothelial cells and macrophages may lead to decreased inflammation and coagulopathy while providing an early upstream signal for the induction of a number of anti-inflammatory gene products, including AP itself.

Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab.

Trastuzumab is a therapeutic monoclonal antibody developed to target human epidermal growth factor receptor 2 (HER2) present at higher levels in early cancers. Here we report the near complete resonance assignment of trastuzumab-scFab fragment backbone and the methyl groups of isoleucine, leucine and valine residues, as well as their stereo-assignments. The antibody fragment was produced using a single chain approach in Escherichia coli.

Backbone and methyl side-chain resonance assignments of the Fab fragment of adalimumab.

Adalimumab is a therapeutic monoclonal antibody developed to target human TNF an important mediator of immune-mediated inflammatory diseases such as rheumatoid arthritis, amongst others. The 48 kDa Fab fragment of adalimumab was produced in Escherichia coli using a single chain approach to allow complete isotopic incorporation of deuterium, carbon-13 and nitrogen-15 along with the protonated isoleucine-d, valine and leucine methyl groups. Here we report the near complete resonance assignment of the polypeptide backbone and the methyl groups of isoleucine, leucine and valine residues.

Development of Anti-idiotypic Monoclonal Antibody Mimicking SARS-CoV-2 Receptor Binding Domain.

Using the hybridoma technique, we developed a panel of anti-idiotypic monoclonal antibodies (aId-mAb) that mimic The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Receptor-Binding Domain (RBD) molecule against Fragment antigen-binding (Fab) of anti-SARS-CoV-2 (S1, RBD) antibodies. Investigated the in vivo and in vitro effects of these aId-mAbs we developed and examined their antigenic mimicry abilities. Among these 12 antibodies, 6 aId-mAbs (designated FY1B4, FY2A6, H9F3, E6G7, FY7E11, and FY8H3) were selected for further characterization in a series of experiments. First, competitive receptor binding assay results confirmed that six aId-mAbs could specifically bind to the ACE2 receptor in target cells and block the interaction between the RBD molecule and the ACE receptor. Moreover, we examined the immunological activities of these aId-mAbs in female BALB/c and showed that E6G7, H7E11, and H8H3 aId-mAbs induce an antibody response by mimicking RBD and stimulating the immune system. It is considered that these three aId-mAbs will be evaluated as SARS-CoV-2 vaccine candidate molecules in future studies.

A new method for isolation and purification of fusion-competent inhibitory synaptic vesicles.

Synaptic vesicles specific to inhibitory GABA-releasing neurons are critical for regulating neuronal excitability. To study the specific molecular composition, architecture, and function of inhibitory synaptic vesicles, we have developed a new method to isolate and purify GABA synaptic vesicles from mouse brains. GABA synaptic vesicles were immunoisolated from mouse brain tissue using an engineered fragment antigen-binding region (Fab) against the vesicular GABA transporter (vGAT) and purified. Western blot analysis confirmed that the GABA synaptic vesicles were specifically enriched for vGAT and largely depleted of contaminants from other synaptic vesicle types, such as vesicular glutamate transporter (vGLUT1), and other cellular organelles. This degree of purity was achieved despite the relatively low abundance of vGAT vesicles compared to the total synaptic vesicle pool in mammalian brains. Cryo-electron microscopy images of these isolated GABA synaptic vesicles revealed intact morphology with circular shape and protruding proteinaceous densities. The GABA synaptic vesicles are functional, as assessed by a hybrid (ex vivo/in vitro) vesicle fusion assay, and they undergo synchronized fusion with synthetic plasma membrane mimic vesicles in response to Ca2+-triggering, but, as a negative control, not to Mg2+-triggering. Our immunoisolation method could also be applied to other types of vesicles.

Investigational thymic stromal lymphopoietin inhibitors for the treatment of asthma: a systematic review.

INTRODUCTION: Severe asthma patients often remain uncontrolled despite high-intensity therapies. Biological therapies targeting thymic stromal lymphopoietin (TSLP), a key player in asthma pathogenesis, have emerged as potential options. Currently, the only TSLP inhibitor approved for the treatment of severe asthma is the immunoglobulin G (IgG) 2λ anti-TSLP monoclonal antibody (mAb) tezepelumab. AREAS COVERED: This systematic review assesses the efficacy and safety of investigational TSLP inhibitors across different stages of development for asthma treatment. EXPERT OPINION: TSLP contributes to airway inflammation, making it a pivotal therapeutic target. Ecleralimab, an inhaled antibody fragment antigen binding, shows promising evidence in enhancing efficacy and reducing systemic adverse events. SAR443765, with its NANOBODY® formulation and bispecific inhibition of TSLP and IL-13, offers improved tissue penetration and efficacy. The mAB TQC2731 exhibits high in vitro bioactivity, and the strength of the mAb UPB-101 is to act against the TSLP receptor. Some studies include mild and moderate asthma patients, suggesting the potential for extending biological therapy to non-severe patients. This systematic review highlights the potential of TSLP inhibitors as valuable additions to asthma treatment, even in milder forms of the disease. Future research and cost-reduction efforts are needed to expanding access to these promising therapies.

Development of peptide affinity ligands for the purification of polyclonal and monoclonal Fabs from recombinant fluids.

Engineered multi-specific monoclonal antibodies (msAbs) and antibody fragments offer valuable therapeutic options against metabolic disorders, aggressive cancers, and viral infections. The advancement in molecular design and recombinant expression of these next-generation drugs, however, is not equaled by the progress in downstream bioprocess technology. The purification of msAbs and fragments requires affinity adsorbents with orthogonal biorecognition of different portions of the antibody structure, namely its Fc (fragment crystallizable) and Fab (fragment antigen-binding) regions or the CH1-3 and CL chains. Current adsorbents rely on protein ligands that, while featuring high binding capacity and selectivity, need harsh elution conditions and suffer from high cost, limited biochemical stability, and potential release of immunogenic fragments. Responding to these challenges, we undertook the de novo discovery of peptide ligands that target different regions of human Fab and enable product release under mild conditions. The ligands were discovered by screening a focused library of 12-mer peptides against a feedstock comprising human Fab and Chinese hamster ovary host cell proteins (CHO HCPs). The identified ligands were evaluated via binding studies as well as molecular docking simulations, returning excellent values of binding capacity (Qmax ∼ 20 mg of Fab per mL of resin) and dissociation constant (KD = 2.16·10-6 M). Selected ligand FRWNFHRNTFFP and commercial Protein L ligands were further characterized by measuring the dynamic binding capacity (DBC10%) at different residence times (RT) and performing the purification of polyclonal and monoclonal Fabs from CHO-K1 cell culture fluids. The peptide ligand featured DBC10% ∼ 6-16 mg/mL (RT of 2 min) and afforded values of yield (93-96%) and purity (89-96%) comparable to those provided by Protein L resins.

Impaired proliferation and migration of HUVEC and melanoma cells by human anti-FGF2 mAbs derived from a murine hybridoma by guided selection.

Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.

The guided selection humanization process enabled the production of 20 human mAbs anti-FGF2;Seven human anti-FGF2 mAbs showed binding to the rFGF2 antigen in the SPR binding assay;Two human anti-FGF2 mAbs inhibited the proliferation and migration of HUVEC and SK-Mel-28 cells and were predicted to contact the FGF2 at a similar patch of residues than the original mAb.

A trimeric immunoglobin G-binding domain outperforms recombinant protein G and protein L as a ligand for fragment antigen-binding purification.

Fragment antigen-binding (Fab) has several advantages in the treatment and diagnosis of some diseases. The lack of highly efficient affinity chromatography platform creates a purification bottleneck for the downstream processing of Fab-based products, which raises the urgent need for a novel immunoglobin G (IgG)-binding domain (IgBD) with both high affinity and broad specificity for Fab. SpGC3FabRR (designated CFab) was previously identified as a Fab-selective IgBD, which triggered our interest in evaluating the potential of CFab for Fab purification. However, we found that monomeric CFab showed weak Fab-binding. To increase its affinity, a self-trimerizing domain (tri) was fused to CFab to produce CFab-tri. It was found that CFab-tri existed as a trimer and showed promising binding to Fab derived from IgG of humans, rhesus monkeys, mice, rats, and rabbits. Affinity chromatography demonstrated that the recovery rates of Fab derived from IgG of humans, rats, mice, and rabbits by CFab-tri-HP column were 2- to 5-fold of those by protein G-HP column. Human Fab was effectively purified by both protein L- and CFab-tri-HP column. However, unlike CFab-tri-HP column, protein L-HP column was inefficient for purification of Fab derived from IgG of rats, mice, and rabbits. Notably, rat Fab spiked into the extract of Escherichia coli (E. coli) was effectively recovered by CFab-tri-HP column. These results indicate that CFab-tri outperforms protein G and protein L as a ligand for Fab purification, and CFab-tri-based affinity chromatography might be developed as a novel platform for Fab purification.

Fragment antigen binding domains (Fabs) as tools to study assembly-line polyketide synthases.

The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions. Therefore, discovering tools that aid crystallization is crucial. In this review, the versatility of fragment-antigen binding domains (Fabs) as protein crystallization chaperones is discussed. Fabs have aided the crystallization of membrane-bound and soluble proteins as well as RNA. The ability to bind three Fabs onto a single protein target has demonstrated their potential for crystallization of challenging proteins. We describe a high-throughput workflow for identifying Fabs to aid the crystallization of a protein of interest (POI) by leveraging phage display technologies and differential scanning fluorimetry (DSF). This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases (PKSs), which harbor flexible domains and assume transient conformations. PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds. Despite years of research studying these megasynthases, their overall topology has remained elusive. One Fab in particular, 1B2, has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic (cryoEM) analyses of multiple modules from distinct assembly-line PKSs. Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM, thereby enabling the visualization of intact PKS modules at near-atomic (3-5 Å) resolution. The identification of PKS-binding Fabs can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.

De novo discovery of peptide-based affinity ligands for the fab fragment of human immunoglobulin G.

Antibody fragments and their engineered variants show true potential as next-generation therapeutics as they combine excellent targeting with superior biodistribution and blood clearance. Unlike full antibodies, however, antibody fragments do not yet have a standard platform purification process for large-scale production. Short peptide ligands are viable alternatives to protein ligands in affinity chromatography. In this work, an integrated computational and experimental scheme is described to de novo design 9-mer peptides that bind to Fab fragments. The first cohort of designed sequences was tested experimentally using human polyclonal Fab, and the top performing sequence was selected as a prototype for a subsequent round of ligand refinement in silico. The resulting peptides were conjugated to chromatographic resins and evaluated via equilibrium and dynamic binding studies using human Fab-κ and Fab-λ. The equilibrium studies returned values of binding capacities up to 32 mg of Fab per mL of resin with mild affinity (KD ∼ 10-5 M) that are conducive to high product capture and recovery. Dynamic studies returned values of product yield up to ∼90%. Preliminary purification studies provided purities of 83-93% and yields of 11-89%. These results lay the groundwork for future development of these ligands towards biomanufacturing translation.

Fragment antigen-binding (Fab) antibody-based lateral flow immunoassay for rapid and sensitive detection of potent phytoestrogen, deoxymiroestrol.

Pueraria candollei is an ingredient of Thai herbal medicine, dietary supplements, and cosmetics. The in vitro and in vivo studies of this plant supported anti-osteoporotic activity and used for hormone replacement therapy. Deoxymiroestrol shows the most potent phytoconstituent in tuberous root of P. candollei with estrogenic activity. The quality controls are important for good agricultural practice (GAP) and good manufacturing practice (GMP) of plant-derived raw materials. The rapid detection of lateral flow immunoassay (LFIA) using colloidal gold is simply method, easy visualize detection and produce less waste than conventional chromatographic detection. In this study, LFIA for qualitative detection of deoxymiroestrol using antigen-binding fragment antibody (Fab) was developed. The result showed that the developed LFIA displays specific detection of deoxymiroestrol. Cross reactivity of this method was analyzed with miroestrol, isomiroestrol and methylisomiroestrol which showed 39.97%, 7.71% and 5.72%, respectively. After optimal condition, limit of detection (LOD) for deoxymiroestrol is 250 ng/ml. Plant samples were applied to strip test compare with indirect competitive ELISA using polyclonal antibody to confirm the application of LFIA. The results of LFIA method were comparable with those from ELISA. This developed lateral flow immunoassay can apply to detect deoxymiroestrol for the rapid testing. The developed method can use for quality control in plant samples as deoxymiroestrol is biomarker compound in P. candollei.

Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains.

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb2 format where a set of mutated amino acid residues in the CH3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab CH1/CL domain pair with a pair of antigen-binding CH3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding CH3 domains. Such bispecific molecules were produced in a "Fab-like" format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding CH3 domains offer an alternative solution in positioning and valency of antigen binding sites.

Microbioreactor Cultivations of Fab-Producing Escherichia coli Reveal Genome-Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects.

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome-integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon-limited microbioreactor cultivations with yields of 0-7.4 mg Fab per gram of cell dry mass. Antigen-binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid-free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80-300% of plasmid-based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.