Metabolomic characterization of monoclonal antibody-producing Chinese hamster lung (CHL)-YN cells in glucose-controlled serum-free fed-batch operation.

The fast-growing Chinese hamster lung (CHL)-YN cell line was recently developed for monoclonal antibody production. In this study, we applied a serum-free fed-batch cultivation process to immunoglobulin (Ig)G1-producing CHL-YN cells, which were then used to design a dynamic glucose supply system to stabilize the extracellular glucose concentration based on glucose consumption. Glucose consumption of the cultures rapidly oscillated following three phases of glutamine metabolism: consumption, production, and re-consumption. Use of the dynamic glucose supply prolonged the viability of the CHL-YN-IgG1 cell cultures and increased IgG1 production. Liquid chromatography with tandem mass spectrometry-based target metabolomics analysis of the extracellular metabolites during the first glutamine shift was conducted to search for depleted compounds. The results suggest that the levels of four amino acids, namely arginine, aspartate, methionine, and serine, were sharply decreased in CHL-YN cells during glutamine production. Supporting evidence from metabolic and gene expression analyses also suggest that CHL-YN cells acquired ornithine- and cystathionine-production abilities that differed from those in Chinese hamster ovary-K1 cells, potentially leading to proline and cysteine biosynthesis.

Production of Antibodies to Peptide Targets Using Hybridoma Technology.

Hybridoma technology is a well-established and indispensable tool for generating high-quality monoclonal antibodies and has become one of the most common methods for monoclonal antibody production. In this process, antibody-producing B cells are isolated from mice following immunization of mice with a specific immunogen and fused with an immortal myeloma cell line to form antibody-producing hybridoma cell lines. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents but have also emerged as the most rapidly expanding class of therapeutic biologicals. In spite of the development of new high-throughput monoclonal antibody generation technologies, hybridoma technology still is applied for antibody production due to its ability to preserve innate functions of immune cells and to preserve natural cognate antibody paring information. In this chapter, an overview of hybridoma technology and the laboratory procedures used for hybridoma production and antibody screening of peptide-specific antibodies are presented.

Recombinant Antibody-Producing Stable CHOK1 Pool Stability Study.

Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements.

CRISPR-Cas9 knockout screen informs efficient reduction of the Komagataella phaffii secretome.

BACKGROUND: The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality. RESULTS: We identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody. CONCLUSIONS: We demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.

Production of Secreted Antibody in Baculovirus Expression Vector System.

Monoclonal antibodies have widespread applications in disease treatment and antigen detection. They are traditionally produced using mammalian cell expression system, which is not able to satisfy the increasing demand of these proteins at large scale. Baculovirus expression vector system (BEVS) is an attractive alternative platform for the production of biologically active monoclonal antibodies. In this chapter, we demonstrate the production of an HIV-1 broadly neutralizing antibody b12 in BEVS. The processes including transfer vector construction, recombinant baculovirus generation, and antibody production and detection are described.

Dynamic optimization of an integrated cultivation-aggregation model for mAb production.

Due to their proteinaceous structure, monoclonal antibodies (mAbs) are susceptible to irreversible aggregation, with harmful consequences on drug efficacy and patient safety. To mitigate this risk in modern biopharmaceutical processes, it is critical to comply with current good manufacturing practices (cGMP) and pursue operating strategies minimizing irreversible aggregation whilst also maximizing mAb throughput. These conflicting objectives are targeted in this study by formulating and analyzing an integrated dynamic model accounting for both cultivation and aggregation of mAbs from a Chinese Hamster Ovary (CHO) cell line. Two manipulated dynamic variables are considered here in simulation studies: firstly temperature manipulation within a batch reactor, and secondly feed flow manipulation within a series of isothermal fed-batch reactors. Following this, dynamic optimization investigations have been conducted, firstly with the single objective of maximizing mAb throughput and secondly with multiple (two) objectives of maximizing mAb throughput while also minimizing irreversible aggregate content, simultaneously. The study provides key insight into tradeoffs of how simultaneous temperature and feed flowrate manipulation affects mAb throughput and aggregation inside bioreactors.

Generation of a Rat Monoclonal Antibody for Human PAICS, a de novo Purine Biosynthetic Enzyme.

Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a de novo purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial-mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the N-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation.

Expression of Multispecific Antibodies.

Bi- and multispecific antibody formats allow the development of new therapeutic strategies to address previously unmet medical needs. However, due to the increased complexity (e.g., the interface design and the presence of multiple binders), such molecules are generally more challenging to express and purify compared to standard monoclonal antibodies (mAbs). We describe here an optimized methodology to express and purify basic bispecific antibodies using the BEAT® interface. This interface allows to generate antibodies with very high levels of heterodimer product (reported titers exceed 10 g/L) and comes with a built-in purification strategy allowing removal of residual levels of undesired product-related impurities (e.g., homodimers and half molecules).

Quantitative proteomics reveals cellular responses to individual mAb expression and tunicamycin in CHO cells.

Chinese hamster ovary (CHO) cells are popular in the pharmaceutical industry for their ability to produce high concentrations of antibodies and their resemblance to human cells in terms of protein glycosylation patterns. Current data indicate the relevance of CHO cells in the biopharmaceutical industry, with a high number of product commendations and a significant market share for monoclonal antibodies. To enhance the production capabilities of CHO cells, a deep understanding of their cellular and molecular composition is crucial. Genome sequencing and proteomic analysis have provided valuable insights into the impact of the bioprocessing conditions, productivity, and product quality. In our investigation, we conducted a comparative analysis of proteomic profiles in high and low monoclonal antibody-producing cell lines and studied the impact of tunicamycin (TM)-induced endoplasmic reticulum (ER) stress. We examined the expression levels of different proteins including unfolded protein response (UPR) target genes by using label-free quantification techniques for protein abundance. Our results show the upregulation of proteins associated with protein folding mechanisms in low producer vs. high producer cell line suggesting a form of ER stress related to specific protein production. Further, Hspa9 and Dnaja3 are notable candidates activated by the mitochondria UPR and play important roles in protein folding processes in mitochondria. We identified significant upregulation of Nedd8 and Lgmn proteins in similar levels which may contribute to UPR stress. Interestingly, the downregulation of Hspa5/Bip and Pdia4 in response to tunicamycin treatment suggests a low-level UPR activation. KEY POINTS: • Proteome profiling of recombinant CHO cells under mild TM treatment. • Identified protein clusters are associated with the unfolded protein response (UPR). • The compared cell lines revealed noticeable disparities in protein expression levels.

[Preparation and assessment of a monoclonal antibody against mouse NLRP3].

Objective To prepare a monoclonal antibody (mAb) against mouse NOD-like receptor family pyrin domain-containing 3 (NLRP3) and assess its specificity. Methods A gene fragment encoding mouse NLRP3 exon3 (Ms-N3) was inserted into the vector p36-G3-throhFc to construct a recombinant plasmid named Ms-N3-throhFc. This plasmid was then transfected into HEK293F cells for eukaryotic expression. NLRP3-/- mice were immunized with Ms-N3 protein purified using a protein A chromatography column, and splenocytes from the immunized mice were fused with SP2/0 myeloma cells to generate hybridoma cells. Specific mAbs against murine NLRP3 from hybridoma cells were screened using ELISA and immunofluorescence assay(IFA). Results The Ms-N3-throhFc recombinant plasmid was successfully constructed and exhibited stable expression in HEK293F cells. Twelve hybridoma cell lines were initially screened using ELISA. IFA revealed that the mAb secreted by the 9-B8-3-2-C5 cell line specifically recognized the native form of mouse NLRP3 protein. The heavy and light chain subtypes of this mAb were identified as IgM and κ, respectively. Conclusion A monoclonal antibody against mouse NLRP3 has been successfully prepared.

Evaluation of various membranes at different fluxes to enable large-volume single-use perfusion bioreactors.

The growing demand for biological therapeutics has increased interest in large-volume perfusion bioreactors, but the operation and scalability of perfusion membranes remain a challenge. This study evaluates perfusion cell culture performance and monoclonal antibody (mAb) productivity at various membrane fluxes (1.5-5 LMH), utilizing polyvinylidene difluoride (PVDF), polyethersulfone (PES), or polysulfone (PS) membranes in tangential flow filtration mode. At low flux, culture with PVDF membrane maintained higher cell culture growth, permeate titer (1.06-1.34 g/L) and sieving coefficients (≥83%) but showed lower permeate volumetric throughput and higher transmembrane pressure (TMP) (>1.50 psi) in the later part of the run compared to cultures with PES and PS membrane. However, as permeate flux increased, the total mass of product decreased by around 30% for cultures with PVDF membrane, while it remained consistent with PES and PS membrane, and at the highest flux studied, PES membrane generated 12% more product than PVDF membrane. This highlights that membrane selection for large-volume perfusion bioreactors depends on the productivity and permeate flux required. Since operating large-volume perfusion bioreactors at low flux would require several cell retention devices and a complex setup, PVDF membranes are suitable for low-volume operations at low fluxes whereas PES membranes can be a desirable alternative for large-volume higher demand products at higher fluxes.

Rapid generation of human recombinant monoclonal antibodies from antibody-secreting cells using ferrofluid-based technology.

Monoclonal antibodies (mAbs) are one of the most important classes of biologics with high therapeutic and diagnostic value, but traditional methods for mAbs generation, such as hybridoma screening and phage display, have limitations, including low efficiency and loss of natural chain pairing. To overcome these challenges, novel single B cell antibody technologies have emerged, but they also have limitations such as in vitro differentiation of memory B cells and expensive cell sorters. In this study, we present a rapid and efficient workflow for obtaining human recombinant monoclonal antibodies directly from single antigen-specific antibody secreting cells (ASCs) in the peripheral blood of convalescent COVID-19 patients using ferrofluid technology. This process allows the identification and expression of recombinant antigen-specific mAbs in less than 10 days, using RT-PCR to generate linear Ig heavy and light chain gene expression cassettes, called "minigenes", for rapid expression of recombinant antibodies without cloning procedures. This approach has several advantages. First, it saves time and resources by eliminating the need for in vitro differentiation. It also allows individual antigen-specific ASCs to be screened for effector function prior to recombinant antibody cloning, enabling the selection of mAbs with desired characteristics and functional activity. In addition, the method allows comprehensive analysis of variable region repertoires in combination with functional assays to evaluate the specificity and function of the generated antigen-specific antibodies. Our approach, which rapidly generates recombinant monoclonal antibodies from single antigen-specific ASCs, could help to identify functional antibodies and deepen our understanding of antibody dynamics in the immune response through combined antibody repertoire sequence analysis and functional reactivity testing.

An important consideration when expressing mAbs in Escherichiacoli.

Monoclonal antibodies (mAbs) are a driving force in the biopharmaceutical industry. Therapeutic mAbs are usually produced in mammalian cells, but there has been a push towards the use of alternative production hosts, such as Escherichia coli. When the genes encoding for a mAb heavy and light chains are codon-optimized for E. coli expression, a truncated form of the heavy chain can form along with the full-length product. In this work, the role of codon optimization in the formation of a truncated product was investigated. This study used the amino acid sequences of several therapeutic mAbs and multiple optimization algorithms. It was found that several algorithms incorporate sequences that lead to a truncated product. Approaches to avoid this truncated form are discussed.

[Advances of monoclonal antibodies and analysis of marketed antibody drugs].

In recent years, there has been a frequent occurrence of various epidemics worldwide such as COVID-19, monkeypox, influenza, and others additionally, there has been an increase in the number of new patients diagnosed with various types of tumors. Traditional drugs have limited effectiveness against emerging infectious diseases, tumors, and autoimmune diseases. However, with the emergence of hybridoma technology, monoclonal antibodies have achieved extensive applications and antibody drugs are playing an important role in modern medicine. Monoclonal antibodies have undergone various development stages, starting from mouse-derived antibodies to human-mouse chimeric antibodies, humanized antibodies, and ultimately human antibodies. Throughout this process, their immunogenicity has gradually decreased, while their safety for human use steadily increased. Fully human antibodies are currently the safest form of antibody, because their sequences all come from human sources and they do not induce human anti-murine antibody reactions. With the advance of genetic engineering technology, flow cytometry coupled to single B cell gene amplification technology has made it easier to construct and screen for fully human monoclonal antibodies. The development of antibody drugs has provided new opportunities, and the market for monoclonal antibody drugs will further expand. This article reviews the research progress of monoclonal antibodies and presents information on the 163 monoclonal antibody drugs approved by the United States Food and Drug Administration (FDA) as of Oct 1st, 2023. The aim is to offer new insights for the development and production of monoclonal antibodies in China.

[Preparation of a mouse monoclonal antibody against the NS1 protein of respiratory syncytial virus].

The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody's application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli. The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.

[Prokaryotic expression of N protein of akabane disease virus and preparation of monoclonal antibody].

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

[Human tau N-terminal domain-specific monoclonal antibodies: screening and application in blood detection].

The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.

Recombinant monoclonal antibody production in yeasts: Challenges and considerations.

Monoclonal antibodies (mAbs) are laboratory-based engineered protein molecules with a monovalent affinity or multivalent avidity towards a specific target or antigen, which can mimic natural antibodies that are produced in the human immune systems to fight against detrimental pathogens. The recombinant mAb is one of the most effective classes of biopharmaceuticals produced in vitro by cloning and expressing synthetic antibody genes in a suitable host. Yeast is one of the potential hosts among others for the successful production of recombinant mAbs. However, there are very few yeast-derived mAbs that got the approval of the regulatory agencies for direct use for treatment purposes. Certain challenges encountered by yeasts for recombinant antibody productions need to be overcome and a few considerations related to antibody structure, host engineering, and culturing strategies should be followed for the improved production of mAbs in yeasts. In this review, the drawbacks related to the metabolic burden of the host, culturing conditions including induction mechanism and secretion efficiency, solubility and stability, downstream processing, and the pharmacokinetic behavior of the antibody are discussed, which will help in developing the yeast hosts for the efficient production of recombinant mAbs.

Development of a 10 g/L process for a difficult-to-express multispecific antibody format using a holistic process development approach.

Ichnos has developed a multi-specific antibody platform based on the BEAT® (Bispecific engagement by antibodies based on the T-cell receptor) interface. The increased complexity of the bi- and multi-specific formats generated with this platform makes these molecules difficult-to-express proteins compared to standard monoclonal antibodies (mAbs). This report describes how expression limitations of a bi-specific bi-paratopic BEAT antibody were improved in a holistic approach. An initial investigation allowed identification of a misbalance in the subunits composing the BEAT antibody as the potential root cause. This misbalance was then addressed by a signal peptide optimization, and the overall expression level was increased by the combination of two vector design elements on a single gene vector. Further improvements were made in the selection of cell populations and an upstream (USP) platform process was applied in combination with a cell culture temperature shift. This allowed titer levels of up to 6 g/L to be reached with these difficult-to-express proteins. Furthermore, a high-density seeding process was developed that allowed titers of around 11 g/L for the BEAT antibody, increasing the initial titer by a factor of 10. The approach was successfully applied to a tri-specific antibody with titer levels reaching 10 g/L. In summary, a platform process for difficult-to-express proteins was developed using molecular biology tools, cell line development, upstream process optimization and process intensification.

Alternative splicing for tuneable expression of protein subunits at desired ratios.

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.