[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.

[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.

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.

A nanowell platform to identify, sort and expand high antibody-producing cells.

Increased use of therapeutic monoclonal antibodies and the relatively high manufacturing costs fuel the need for more efficient production methods. Here we introduce a novel, fast, robust, and safe isolation platform for screening and isolating antibody-producing cell lines using a nanowell chip and an innovative single-cell isolation method. An anti-Her2 antibody producing CHO cell pool was used as a model. The platform; (1) Assures the single-cell origin of the production clone, (2) Detects the antibody production of individual cells and (3) Isolates and expands the individual cells based on their antibody production. Using the nanowell platform we demonstrated an 1.8-4.5 increase in anti-Her2 production by CHO cells that were screened and isolated with the nanowell platform compared to CHO cells that were not screened. This increase was also shown in Fed-Batch cultures where selected high production clones showed titers of 19-100 mg/L on harvest day, while the low producer cells did not show any detectable anti-Her2 IgG production. The screening of thousands of single cells is performed under sterile conditions and the individual cells were cultured in buffers and reagents without animal components. The time required from seeding a single cell and measuring the antibody production to fully expanded clones with increased Her-2 production was 4-6 weeks.

Generation and functional evaluation of novel monoclonal antibodies targeting glycosylated human stem cell factor.

Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07 ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses. KEY POINTS: • Soluble glycosylated human SCF exerted improved proliferative activity on UT-7 cells. • Three mAbs with high specificity targeting glycosylated human SCF were obtained. • mAbs applications comprise sandwich ELISA, western blot, and immunofluorescence assays.

Development of monoclonal antibodies against prM of Dengue virus 4 / Desenvolvimento de anticorpos monoclonais para prM de Dengue vírus 4

Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

Reproducibility and flexibility of monoclonal antibody production with Nicotiana benthamiana.

The ongoing SARS-CoV-2 coronavirus pandemic of 2020-2021 underscores the need for manufacturing platforms that can rapidly produce monoclonal antibody (mAb) therapies. As reported here, a platform based on Nicotiana benthamiana produced mAb therapeutics with high batch-to-batch reproducibility and flexibility, enabling production of 19 different mAbs of sufficient purity and safety for clinical application(s). With a single manufacturing run, impurities were effectively removed for a representative mAb product (the ZMapp component c4G7). Our results show for the first time the reproducibility of the platform for production of multiple batches of clinical-grade mAb, manufactured under current Good Manufacturing Practices, from Nicotiana benthamiana. The flexibility of the system was confirmed by the results of release testing of 19 different mAbs generated with the platform. The process from plant infection to product can be completed within 10 days. Therefore, with a constant supply of plants, response to the outbreak of an infectious disease could be initiated within a matter of weeks. Thus, these data demonstrated that this platform represents a reproducible, flexible system for rapid production of mAb therapeutics to support clinical development.

Preparation and characterization of a high-affinity monoclonal antibody against nerve growth factor.

Nerve growth factor (NGF) is produced and released in injured tissues or chronic pain tissues caused by other diseases. Studies have shown that monoclonal antibodies targeting NGF have a good efficacy in the treatment of osteoarthritis (OA), low back pain and chronic pain, which may be a promising therapy. In this study, DNA sequences of NGF-his and NGF-hFc were synthesized using eukaryotic expression system and subcloned into pTT5 expression vector. After that, NGF proteins were expressed by transient expression in HEK293E cells. We immunized mice with NGF-hFc protein and fused mouse spleen cells to prepare hybridomas. NGF-His protein was used to screen out the hybridoma supernatant that could directly bind to NGF. Antibodies were purified from hybridioma supernatant. Futhermore, via surface plasmon resonance (SPR) screening, six anti-NGF mAbs were screened to block the binding of NGF and TrkA receptor in the treatment of chronic pain. Among them, 58F10G10H showed high affinity (KD = 1.03 × 10-9 M) and even better than that of positive control antibody Tanezumab (KD = 1.53 × 10-9 M). Moreover, the specific reactivity of 58F10G10H was demonstrated by TF-1 cell proliferation activity experiments, competitive binding Enzyme-linked immunosorbent assay (ELISA) and the arthritis animal models in mice, respectively. In conclusion, in this study, a method for the preparation of high-yield NGF-HFC and NGF-His proteins was designed, and a high-affinity monoclonal antibody against NGF with potential for basic research and clinical application was prepared.

Potential Applications of Fluorescence-Activated Cell Sorting (FACS) and Droplet-Based Microfluidics in Promoting the Discovery of Specific Antibodies for Characterizations of Fish Immune Cells.

Akin to their mammalian counterparts, teleost fish possess a complex assortment of highly specialized immune cells that are capable of unleashing potent innate immune responses to eradicate or mitigate incoming pathogens, and also differentiate into memory lymphocytes to provide long-term protection. Investigations into specific roles and functions of fish immune cells depend on the precise separation of each cell type. Commonly used techniques, for example, density gradient centrifugation, rely on immune cells to have differing sizes or densities and thus fail to separate between similar cell types (e.g. T and B lymphocytes). Furthermore, a continuously growing database of teleost genomic information has revealed an inventory of cellular markers, indicating the possible presence of immune cell subsets in teleost fish. This further complicates the interpretation of results if subsets of immune cells are not properly separated. Consequently, monoclonal antibodies (mAbs) against specific cellular markers are required to precisely identify and separate novel subsets of immune cells in fish. In the field of fish immunology, mAbs are largely generated using the hybridoma technology, resulting in the development of mAbs against specific cellular markers in different fish species. Nevertheless, this technology suffers from being labour-intensive, time-consuming and most importantly, the inevitable loss of diversities of antibodies during the fusion of antibody-expressing B lymphocytes and myeloma cells. In light of this, the focus of this review is to discuss the potential applications of fluorescence-activated cell sorting and droplet-based microfluidics, two emerging technologies capable of screening and identifying antigen-specific B lymphocytes in a high-throughput manner, in promoting the development of valuable reagents for fish immunology studies. Our main goal is to encourage the incorporation of alternative technologies into the field of fish immunology to promote the production of specific antibodies in a high-throughput and cost-effective way, which could better allow for the precise separation of fish immune cells and also facilitate the identification of novel immune cell subsets in teleost fish.

Highly active engineered IgG3 antibodies against SARS-CoV-2.

Monoclonal antibodies (mAbs) that efficiently neutralize SARS-CoV-2 have been developed at an unprecedented speed. Notwithstanding, there is a vague understanding of the various Ab functions induced beyond antigen binding by the heavy-chain constant domain. To explore the diverse roles of Abs in SARS-CoV-2 immunity, we expressed a SARS-CoV-2 spike protein (SP) binding mAb (H4) in the four IgG subclasses present in human serum (IgG1-4) using glyco-engineered Nicotiana benthamiana plants. All four subclasses, carrying the identical antigen-binding site, were fully assembled in planta and exhibited a largely homogeneous xylose- and fucose-free glycosylation profile. The Ab variants ligated to the SP with an up to fivefold increased binding activity of IgG3. Furthermore, all H4 subtypes were able to neutralize SARS-CoV-2. However, H4-IgG3 exhibited an up to 50-fold superior neutralization potency compared with the other subclasses. Our data point to a strong protective effect of IgG3 Abs in SARS-CoV-2 infection and suggest that superior neutralization might be a consequence of cross-linking the SP on the viral surface. This should be considered in therapy and vaccine development. In addition, we underscore the versatile use of plants for the rapid expression of complex proteins in emergency cases.

Development of Monoclonal Antibody PMab-269 Against California Sea Lion Podoplanin.

The development of protein-specific antibodies is essential for understanding a wide variety of biological phenomena. Parasitic and viral infections and cancers are known to occur within California sea lion (Zalophus californianus) populations. However, sensitive and specific monoclonal antibodies (mAbs) for the pathophysiological analysis of California sea lion tissues have not yet been developed. A type I transmembrane glycoprotein, podoplanin (PDPN), is a known diagnostic marker of lymphatic endothelial cells. We have previously developed several anti-PDPN mAbs in various mammalian species, with applications in flow cytometry, Western blotting, and immunohistochemistry. In this study, we established a novel mAb against California sea lion PDPN (seaPDPN), clone PMab-269 (mouse IgG1, kappa), using a Cell-Based Immunization and Screening method. PMab-269 is specifically detected in seaPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells using flow cytometry and Western blotting. Moreover, PMab-269 clearly identified pulmonary type I alveolar cells, renal podocytes, and colon lymphatic endothelial cells in California sea lion tissues using immunohistochemistry. These findings demonstrate the usefulness of PMab-269 for the pathophysiological analysis of lung, kidney, and lymphatic tissues of the California sea lion.

CellectSeq: In silico discovery of antibodies targeting integral membrane proteins combining in situ selections and next-generation sequencing.

Synthetic antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal Abs against human antigens. Yet, they typically depend on purified proteins, which exclude integral membrane proteins that require the lipid bilayers to support their native structure and function. Here, we present an Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins on native cells in complex environment. As proof of concept, we targeted three transmembrane proteins linked to cancer, tetraspanin CD151, carbonic anhydrase 9, and integrin-α11. First, we performed in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders. Then, we designed next-generation sequencing procedures to explore Ab diversities and abundances. Finally, we developed motif-based scoring and sequencing error-filtering algorithms for the comprehensive interrogation of next-generation sequencing pools to identify Abs with high diversities and specificities, even at extremely low abundances, which are very difficult to identify using manual sampling or sequence abundances.

Functional Immunostimulating DNA Materials: The Rising Stars for Cancer Immunotherapy.

Cancer immunotherapy has risen as a promising method in clinical practice for cancer treatment and DNA-based immune intervention materials, along with DNA nanotechnology, have obtained increasing importance in this field. In this review, various immunostimulating DNA materials are introduced and the mechanisms via which they exerted an immune effect are explained. Then, representative examples in which DNA is used as the leading component for anticancer applications through immune stimulation are provided and their efficacy is evaluated. Finally, the challenges for those materials in clinical applications are discussed and suggestions for possible further research directions are also put forward.

4-(2,5-Dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide improves monoclonal antibody production in a Chinese hamster ovary cell culture.

There is a continuous demand to improve monoclonal antibody production for medication supply and medical cost reduction. For over 20 years, recombinant Chinese hamster ovary cells have been used as a host in monoclonal antibody production due to robustness, high productivity and ability to produce proteins with ideal glycans. Chemical compounds, such as dimethyl sulfoxide, lithium chloride, and butyric acid, have been shown to improve monoclonal antibody production in mammalian cell cultures. In this study, we aimed to discover new chemical compounds that can improve cell-specific antibody production in recombinant Chinese hamster ovary cells. Out of the 23,227 chemicals screened in this study, 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide was found to increase monoclonal antibody production. The compound suppressed cell growth and increased both cell-specific glucose uptake rate and the amount of intracellular adenosine triphosphate during monoclonal antibody production. In addition, the compound also suppressed the galactosylation on a monoclonal antibody, which is a critical quality attribute of therapeutic monoclonal antibodies. Therefore, the compound might also be used to control the level of the galactosylation for the N-linked glycans. Further, the structure-activity relationship study revealed that 2,5-dimethylpyrrole was the most effective partial structure of 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N-(2,5-dioxopyrrolidin-1-yl) benzamide on monoclonal antibody production. Further structural optimization of 2,5-dimethylpyrrole derivatives could lead to improved production and quality control of monoclonal antibodies.

Anti-glycan antibodies: roles in human disease.

Carbohydrate-binding antibodies play diverse and critical roles in human health. Endogenous carbohydrate-binding antibodies that recognize bacterial, fungal, and other microbial carbohydrates prevent systemic infections and help maintain microbiome homeostasis. Anti-glycan antibodies can have both beneficial and detrimental effects. For example, alloantibodies to ABO blood group carbohydrates can help reduce the spread of some infectious diseases, but they also impose limitations for blood transfusions. Antibodies that recognize self-glycans can contribute to autoimmune diseases, such as Guillain-Barre syndrome. In addition to endogenous antibodies that arise through natural processes, a variety of vaccines induce anti-glycan antibodies as a primary mechanism of protection. Some examples of approved carbohydrate-based vaccines that have had a major impact on human health are against pneumococcus, Haemophilus influeanza type b, and Neisseria meningitidis. Monoclonal antibodies specifically targeting pathogen associated or tumor associated carbohydrate antigens (TACAs) are used clinically for both diagnostic and therapeutic purposes. This review aims to highlight some of the well-studied and critically important applications of anti-carbohydrate antibodies.

Generation and characterization of a high-affinity chimeric anti-OX40 antibody with potent antitumor activity.

OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist-based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti-OX40 antibody (mAb035-hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4+ T cells and similar potent antitumor activity compared with BMS 986178, an anti-OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen-specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies.

A novel anti-human IL-1R7 antibody reduces IL-18-mediated inflammatory signaling.

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans-induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.

Rapid and reliable hybridoma screening method that is suitable for production of functional structure-recognizing monoclonal antibody.

Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer.

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media.

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.