Manufacture Of Recombinant Proteins Research Articles

Evolving Paradigms of Recombinant Protein Production in Pharmaceutical Industry: A Rigorous Review

Many beneficial proteins have limited natural availability, which often restricts their supply and thereby reduces their potential for therapeutic or industrial usage. The advent of recombinant DNA (rDNA) technology enables the utilization of different microbes as surrogate hosts to facilitate the production of these proteins. This microbial technology continues to evolve and integrate with modern innovations to develop more effective approaches for increasing the production of recombinant biopharmaceuticals. These strategies encompass fermentation technology, metabolic engineering, the deployment of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, synthetic biology, high-throughput devices for cloning, and process screening. This appraisal commences with a general overview regarding the manufacture of recombinant proteins by microbes and the production of biopharmaceuticals, their trends towards the development of biopharmaceuticals, and then discusses the approaches adopted for accomplishing this. The design of the upstream process, which also involves host selection, vector design, and promoter design, is a crucial component of production strategies. On the other hand, the downstream process focuses on extraction and purification techniques. Additionally, the review covers the most modern tools and resources, methods for overcoming low expression, the cost of producing biopharmaceuticals in microbes, and readily available recombinant protein products.

A Systematic Review of the Potential of Pichia pastoris (Komagataella phaffii) as an Alternative Host for Biologics Production.

The methylotrophic yeast Pichia pastoris is garnering interest as a chassis cell factory for the manufacture of recombinant proteins because it effectively satisfies the requirements of both laboratory and industrial set up. The optimisation of P. pastoris cultivation is still necessary due to strain- and product-specific problems such as promoter strength, methanol utilisation type, and culturing conditions to realize the high yields of heterologous protein(s) of interest. Techniques integrating genetic and process engineering have been used to overcome these problems. Insight into the Pichia as an expression system utilizing MUT pathway and the development of methanol free systems are highlighted in this systematic review. Recent developments in the improved production of proteins in P. pastoris by (i) diverse genetic engineering such as codon optimization and gene dosage; (ii) cultivating tactics including co-expression of chaperones; (iii) advances in the use of the 2A peptide system, and (iv) CRISPR/Cas technologies are widely discussed. We believe that by combining these strategies, P. pastoris will become a formidable platform for the production of high value therapeutic proteins.

A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: Yield improvements, bioreactor production and downstream processing

The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors for human applications. Despite its increased use, it is still considered in disadvantage in production aspects compared to cell lines such as the CHO cell line. We provide here a simple workflow for the rapid generation of stably transfected HEK293 cells expressing an engineered variant of the SARS-CoV-2 Receptor Binding Domain (RBD) carrying a coupling domain for linkage to VLPs through a bacterial transpeptidase-sortase (SrtA). To generate stable suspension cells expressing the RBD-SrtA, a single two plasmids transfection was performed, with hygromycin selection. The suspension HEK293 were grown in adherent conditions, with 20% FBS supplementation. These transfection conditions increased cell survival, allowing the selection of stable cell pools, which was otherwise not possible with standard procedures in suspension. Six pools were isolated, expanded and successfully re-adapted to suspension with a gradual increase of serum-free media and agitation. The complete process lasted four weeks. Stable expression with viability over 98% was verified for over two months in culture, with cell passages every 4–5 days. With process intensification, RBD-SrtA yields reached 6.4 μg/mL and 13.4 μg/mL in fed-batch and perfusion-like cultures, respectively. RBD-SrtA was further produced in fed-batch stirred tank 1L-bioreactors, reaching 10-fold higher yields than perfusion flasks. The trimeric antigen displayed the conformational structure and functionality expected. This work provides a series of steps for stable cell pool development using suspension HEK293 cells aimed at the scalable production of recombinant proteins.

Quantitative Risk Assessment of Product Disulfide Bond Reduction in A Recombinant Protein Manufacturing

Disulfide bond reduction occurred now and then during a recombinant protein manufacturing. Their reduction often led in the loss of batches. Various prevention measures were developed. Their implementation in a manufacturing inevitably increases its operation complexity and even might impact its product qualities and process performances. It is highly desirable to apply a reduction measure only if necessary and to apply no strong measure if a moderate one works. The reduction occurrence is determined both by the reduction sensitivity of a recombinant molecule and by the reduction power of harvested cell culture fluids. The reduction power of harvested cell culture fluids varies largely and depends on cells, lysis level, growth stage, and culture conditions. The method of the quantitative risk assessment of disulfide bond reduction was reported here. The quantitative assessment is realized by comparing the reduction sensitivity of a recombinant molecule with the reduction power of its harvested cell culture fluids.

Evaluation of Virus-Free Manufacture of Recombinant Proteins Using CRISPR-Mediated Gene Disruption in Baculovirus-Infected Insect Cells.

The manufacture and downstream processing of virus-like particles (VLPs) using the baculovirus expression vector system (BEVS) is complicated by the presence of large concentrations of baculovirus particles, which are similar in size and density to VLPs, and consequently are difficult to separate. To reduce the burden of downstream processing, CRISPR-Cas9 technology was used to introduce insertion-deletion (indel) mutations within the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp64 open reading frame, which encodes the major envelope protein of AcMNPV. After comfirming the site-specific targeting of gp64 leading to reduced budded virus (BV) release, the gag gene of human immunodeficiency virus type 1 was expressed to produce Gag VLPs. This approach was effective for producing VLPs using the BEVS whilst simultaneously obstructing BV release.

Development and validation of LC–MS/MS method for quantification of protease inhibitor Pepstatin A to monitor its robust clearance in vaccine downstream process.

Pepstatin A reversibly inhibits aspartic acid proteases and minimizes the impact of protease-induced degradation in recombinant protein manufacturing process. Pepstatin A is considered as a process-related impurity and must be characterized and controlled during manufacturing. Herein we describe the development and validation of an LC-MS/MS method for the quantitation of pepstatin A to monitor its robust clearance in vaccine purification process. Analyte extraction from process intermediates was carried out using 10% acetonitrile/water extraction method. Acetyl-pepstatin was used as internal standard (IS). Pepstatin A and IS were resolved on a C18 column using 10 mM ammonium acetate in water and methanol/acetonitrile mobile phase system. A triple quadrupole mass spectrometer operating in the positive electrospray ionization mode with multiple reaction monitoring was used to detect Pepstatin A and IS transitions of m/z 686.5 to 229.3 and 644.5 to 229.3, respectively. The method was validated for specificity, linearity, accuracy, repeatability (precision), intermediate precision, and assay robustness. The assay was linear over the range of calibration standards 0.5–100 ng/mL. The Lower-limit-of-quantification (LLOQ) of the method was 0.50 ng/mL.

Determination of recombinant thaumatin II secreted by Pichia pastoris using reversed-phase high-performance liquid chromatography

The manufacturing of recombinant proteins is increasing phenomenally, and yeasts serve as one of the most popular platforms for the large-scale production of these industrially relevant products. Protein analysis is a traditional step for the control and optimisation of such bioprocesses and often necessitates the combination of laborious sample treatments and expensive instruments. A hassle-free and cost-effective approach is always preferred for routine analyses and instigates a need for replacing these laborious tasks. In this study, a fast, simple, and reliable RP-HPLC method for the determination of the upcoming sweetener in the market, the sweet protein thaumatin was established. There is currently no reference method available for the quantification of this protein in fermentation samples. Due to an increasing industrial relevance foreseen for this sweetener, Pichia pastoris was used as a host for its bioproduction. The developed analytical method does not require a pre-treatment step of the biological sample and enables an easy quantification using a conventionally available HPLC system. The method facilitates the identification and quantification of recombinant thaumatin II and was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines(ICH, 2021). The developed technique enables the monitoring of thaumatin levels in fermentations, thereby facilitating bioprocess control and optimisation.

Transient Expression of Glycosylated SARS-CoV-2 Antigens in Nicotiana benthamiana.

The current COVID-19 pandemic very dramatically shows that the world lacks preparedness for novel viral diseases. In addition to newly emerging viruses, many known pathogenic viruses such as influenza are constantly evolving, leading to frequent outbreaks with severe diseases and deaths. Hence, infectious viruses are a recurrent burden to our daily life, and powerful strategies to stop the spread of human pathogens and disease progression are of utmost importance. Transient plant-based protein expression is a technology that allows fast and highly flexible manufacturing of recombinant viral proteins and, thus, can contribute to infectious disease detection and prevention. This review highlights recent progress in the transient production of viral glycoproteins in N. benthamiana with a focus on SARS-CoV-2-derived viral antigens.

Development of a novel platform for recombinant protein production in Corynebacterium glutamicum on ethanol

Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion, but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield. Here, we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer. First, we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture. The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter. Next, through transcriptional engineering that alters transcription factor binding sites (TFBSs) and the first transcribed sequence, the truncated promoter PA256 with a dramatically higher transcription level was generated. When producing the superfolder green fluorescent protein (sfGFP) under 1% ethanol conditions, PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36. This superior expression mode was further validated using two secreted proteins, camelid antibody fragment (VHH) and endoxylanase (XynA). Furthermore, utilizing CRISPRi technology, ethanol utilization blocking strains were created, and PA256 was shown to be impaired in the phosphotransacetylase (PTA) knockdown strains, indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation. Finally, this platform was applied to produce the “de novo design” protein NEO-2/15, and by introducing the N-propeptide of CspB, NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation. To the best of our knowledge, this is the first report of NEO-2/15 secretory overexpression.

Statistical experimental designs to optimize the transient transfection of HEK 293T cells and determine a transfer criterion from adherent cells to larger-scale cell suspension cultures

The transient transfection of mammalian cells is a rapid and versatile platform for the manufacture of recombinant proteins, but industrial processes depend on reliable scalability and efficient conversion from adherent to suspension cell cultures. Here we describe the optimized transfection of HEK 293T cells in both culture formats. DMEM was the best transfection medium for adherent HEK 293T cells, so we determined the kinetics of linear polyethyleneimine (LPEI) polyplex formation with plasmid DNA (pDNA) and subsequent cellular uptake. Statistical experimental designs revealed optimal transfection efficiency using 0.7 pg pDNA and 4.5 pg LPEI per cell. We used the amount of pDNA and LPEI per cell as the transfer criterion for HEK 293T/17 SF cell suspension cultures in FreeStyle 293 medium and confirmed optimal transfection at 1.1 pg pDNA and 6.6 pg LPEI per cell. We observed a strong correlation between polyplex size, transfection efficiency and post-transfection cell viability. Suspension cell transfection could be scaled to a 100-mL working volume without loss of efficiency. We conclude that pg pDNA and pg LPEI per cell is a suitable transfer criterion allowing the optimization of transient transfection using statistical experimental designs, thus minimizing the amount of pDNA and LPEI used without sacrificing transfection efficiency.

Technoeconomic Modeling and Simulation for Plant-Based Manufacturing of Recombinant Proteins.

Technoeconomic modeling and simulation is a critical step in defining a manufacturing process for evaluation of commercial viability and to focus experimental process research and development efforts. Technoeconomic analysis (TEA) is increasingly demanded alongside scientific innovation by both public and private funding agencies to maximize efficiency of resource allocation. It is particularly important for plant-based manufacturing, and other nontraditional recombinant protein production platforms, to explicitly demonstrate the manufacturing potential and to identify critical technical and economic challenges through robust technoeconomic analysis. In addition, in silico process modeling and TEA of scaled biomanufacturing facilities allows rapid evaluation of the impacts of process and economic changes on capital expenditures (CAPEX, also sometimes referred to as total capital investment), operational expenditures (OPEX, also known as total manufacturing costs or total production costs), cost of goods sold (COGS, also known as unit production costs), and profitability metrics such as net present value (NPV) and discounted cash flow rate of return (DCROR, also known as internal rate of return or IRR). These models can also be used to assess environmental, health, and safety impact of a designed biomanufacturing facility to evaluate its sustainability and environmental-friendliness. Here we describe a general method for performing technoeconomic modeling and simulation for and environmental assessment of plant-based manufacturing of recombinant proteins.

Recombinant Protein Production in Plants: A Brief Overview of Strengths and Challenges.

The first recombinant proteins were produced in microbes and animal cells cultivated in bioreactors. These systems have become the standard for industrial-scale recombinant protein manufacturing. Later, the production of recombinant proteins was demonstrated in whole plants, which differ morphologically from cell-based systems and require completely different cultivation conditions. Over time, additional plant-based production platforms were established, including hairy roots and cell suspension cultures, which are more similar to conventional cell-based systems in terms of morphology, procedures, and equipment requirements. In this brief overview of the field, we explain why plant-based systems are becoming increasingly attractive for the production of valuable proteins with scientific and commercial applications, but also highlight the challenges that these systems must overcome to achieve more widespread industrial utilization. We discuss various laboratory protocols and approaches for the production of recombinant proteins in plants, as well as strategies to optimize yields, and the regulatory and legal framework.

Reprogramming of Chinese hamster ovary cells towards enhanced protein secretion

The deficient secretory phenotype of Chinese hamster ovary (CHO) cells is a major limitation for high-level production of biopharmaceuticals, particularly for those with complex molecular architectures and post-translational modifications. To improve CHO cell secretory capacity, we recently engineered CHO cell hosts to overexpress BLIMP1 (CHOB), in a cell engineering strategy that transformed the cellular machinery and led to significantly higher product yields and cell-specific productivities for different rproteins. Here, as a follow-up to our previous study, we developed new CHO cell hosts that co-overexpress BLIMP1 and XBP1s ( CHOBX ), two transcription factors that together drive the professional secretory function of antibody-producing plasma cells. We found that the CHOBX cells presented an improved performance over that of CHOB cells, with better product yields and cell-specific productivities for a recombinant IgG1 and a ‘difficult-to-express’ EPO-Fc fusion protein. These improvements in the CHOBX-derived cell lines resulted from a series of physiological and metabolic changes due to the synergetic co-expression of BLIMP1 and XBP1s. Firstly, cells presented an inhibited cell growth and arrested cell cycle in G1/G0 phase, features that were directly linked to BLIMP1 expression levels. Secondly, cells increased protein translation (both overall and recombinant protein), expanded the endoplasmic reticulum and improved their capacity to secrete protein more effectively. Lastly, cells showed a metabolic profile favouring energy production, with a pronounced lactate switch and increased consumption of amino acids. This study highlights the value of transcription factors for reprogramming CHO cells towards a desired phenotype, offering the potential to engineer cells with new functionalities for enhanced manufacturing of recombinant therapeutic proteins.

Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants

Transgenic plants offer advantages for the manufacture of recombinant proteins with terminalmannose residues on their glycan chains. So plants are chosen as source of pharmaceutical products and forthe development of alternative expression systems to produce recombinant lysosomal enzymes. In thepresent study the sequence of the natural cDNA encoding for the human lysosomal enzymeglucocerebrosidase (GCD) was modified to enhance its expression in soybean plants. The glucocerebrosidasegene signal peptide was substituted with that signal peptide for the Arabidopsis thaliana basic endochitinasegene to support the co-translational translocation into the endoplasmic reticulum (ER), and the storagevacuole. So, targeting signal from tobacco chitinase A, to facilitate GCD trafficking from the ER to thestorage vacuole, appropriate primers were designed containing both an ER and vacuolar targeting signals,(VTS). Those primers were used for PCR amplification of the human GBA gene (Hu-GBA) gene fromconstructed PGEM-GBA plasmid which was cloned in the plant expression vector pCAMBIA1304. Theresulted construct was transported in Agrobacterium tumefaciens strain LBA4404 and was used fortransformation of cotyledon explants. After 5-day of seedling, cotyledons were cut and used as explants.After infection and co-cultivation, hygromycin B was added in selection media as a selective agent for thetransformants cotyledons. The presence of the Hu-GBA transgene in the genomes of transgenic plants wasdetermined by polymerase chain reaction PCR as a band of size1587 bp. The GBA mRNA expression inmodified soybean was detected by qRT-PCR compared with control GBA mRNA.

At-CycD2 Enhances Accumulation of Above-Ground Biomass and Recombinant Proteins in Transgenic Nicotiana benthamiana Plants.

Transient expression in Nicotiana benthamiana holds great potential for recombinant protein manufacturing due to its advantages in terms of speed and yield compared to stably transformed plants. To continue improving the quantity of recombinant proteins the plant host will need to be modified at both plant and cellular levels. In attempt to increase leaf mass fraction, we transformed N. benthamiana with the At-CycD2 gene, a positive regulator of the cell cycle. Phenotypic characterization of the T1 progeny plants revealed their accelerated above-ground biomass accumulation and enhanced rate of leaf initiation. In comparison to non-transgenic control the best performing line At-CycD2-15 provided 143 and 140% higher leaf and stem biomass fractions, respectively. The leaf area enlargement of the At-CycD2-15 genotype was associated with the increase of epidermal cell number compensated by slightly reduced cell size. The production capacity of the At-CycD2-15 transgenic line was superior to that of the non-transgenic N. benthamiana. The accumulation of transiently expressed GFP and scFv-TM43-E10 proteins per unit biomass was increased by 138.5 and 156.7%, respectively, compared to the wild type. With these results we demonstrate the potential of cell cycle regulator gene At-CycD2 to modulate both plant phenotype and intracellular environment of N. benthamiana for enhanced recombinant protein yield.

Retrospective assessment of clonal origin of cell lines.

Cell lines used for the manufacture of recombinant proteins are expected to arise from a single cell as a control strategy to limit variability and ensure consistent protein production. Health authorities require a minimum of two rounds of limiting dilution cloning or its equivalent to meet the requirement of single cell origin. However, many legacy cell lines may not have been generated with process meeting this criteria potentially impeding the path to commercialization. A general monoclonality assessment strategy was developed based on using the site of plasmid integration for a cell's identity. By comparing the identities of subclones from a master cell bank (MCB) to each other and that of the MCB, a probability of monoclonality was established. Two technologies were used for cell identity, Southern blot and a PCR assay based on plasmid-genome junction sequences identified by splinkerette PCR. Southern blot analysis revealed that subclones may have banding patterns that differ from each other and yet indicate monoclonal origin. Splinkerette PCR identifies cellular sequence flanking the point(s) of plasmid integration. The two assays together provide complimentary data for cell identity that enables proper monoclonality assessment and establishes that the three legacy cell lines investigated are all of clonal origin.

Transfection of miR-31* boosts oxidative phosphorylation metabolism in the mitochondria and enhances recombinant protein production in Chinese hamster ovary cells

MicroRNAs are increasingly being used to enhance relevant pathways of interest during CHO cell line development and to optimise biopharmaceutical production processes. Previous studies have demonstrated that genetic manipulation of microRNAs has led to the development of highly productive phenotypes by increasing cell density through modifying the cell cycle, extending the culture lifespan by delaying apoptotic mechanisms, or improving the energetic flux by targeting mitochondrial metabolism. Re-programming mitochondrial metabolism has arisen as a potential area of interest due to the potential to decrease the Warburg effect and increase cell specific productivity with significant impact on the manufacture of recombinant therapeutic proteins. In this study, we have demonstrated a role for miR-31* to enhance specific productivity in CHO cells by boosting oxidative phosphorylation in the mitochondria. A detailed analysis of the mitochondrial metabolism revealed that miR-31* transfection increases basal oxygen consumption and spare respiratory capacity that leads to an increase in ATP production. Additionally, a proteomic analysis unveiled a number of potential targets involved in fatty acid metabolism and the TCA cycle, both implicated in mitochondrial metabolism. This data demonstrates a potential role for miR-31* to reprogramme the mitochondrial energetic metabolism and increase recombinant protein production in CHO cells.

Sample Preparation of Secreted Mammalian Host Cell Proteins and Their Characterization by Two-Dimensional Electrophoresis and Western Blotting.

The manufacturing and purification of therapeutic recombinant proteins expressed by cultivated mammalian cells into the cell culture medium leaves the potential for contamination by host cell proteins (HCPs). Validation and quality control testing of any therapeutic protein needs to include a test to show that HCP contamination is at a minimal level. The presence of residual mammalian HCPs during purification and in the final drug product is typically determined using enzyme linked immunosorbent assay (ELISA), which is regarded as the gold standard. The complexity and heterogeneity of HCPs, which include proteins with significant differences in molecular weight (MW), isoelectric point (pI) and hydrophobicity, poses a challenge to detection and quantitation. Two-dimensional gel electrophoresis (2-DE) is one of the most powerful technologies for studying complex protein profiles and is a valuable analytical method in biologics manufacturing. In the purification process, it is very important to know the nature and composition of HCPs, and this information can be used in a rational process design in order to minimize HCPs from the product. Additionally, 2-DE in combination with western blotting can support ELISA development and quality control for the comprehensive immunochemical detection of HCPs by estimating the recognition capacity of the polyclonal serum used in those assays. Here, we present a standardized 2-DE western blotting protocol which takes into account the latest developments in sample preparation of HCPs, protein electrophoresis, protein transfer, immunostaining, and imaging.

Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells

Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/55E1 cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100−150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

BackgroundInclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model.ResultsIBs were isolated from E. coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol® also demonstrated a substantial molecular heterogeneity. Hybridization with 32P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA.ConclusionsThe results presented in this study indicate that the nucleic acids might be intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory. Further studies are needed to ascertain this notion.