Production Of Monoclonal Antibodies Research Articles

Production and characterization of a human antisperm monoclonal antibody against CD52g for topical contraception in women.

BackgroundApproximately 40% of human pregnancies are unintended, indicating a need for more acceptable effective contraception methods. New antibody production systems make it possible to manufacture reagent-grade human monoclonal antibodies (mAbs) for clinical use. We used the Nicotiana platform to produce a human antisperm mAb and tested its efficacy for on-demand topical contraception.MethodsHeavy and light chain variable region DNA sequences of a human IgM antisperm antibody derived from an infertile woman were inserted with human IgG1 constant region sequences into an agrobacterium and transfected into Nicotiana benthamiana. The product, an IgG1 mAb [“Human Contraception Antibody” (HCA)], was purified on Protein A columns, and QC was performed using the LabChip GXII Touch protein characterization system and SEC-HPLC. HCA was tested for antigen specificity by immunofluorescence and western blot assays, antisperm activity by sperm agglutination and complement dependent sperm immobilization assays, and safety in a human vaginal tissue (EpiVaginal™) model.FindingsHCA was obtained at concentrations ranging from 0.4 to 4 mg/ml and consisted of > 90% IgG monomers. The mAb specifically reacted with a glycan epitope on CD52g, a glycoprotein produced in the male reproductive tract and found in abundance on sperm. HCA potently agglutinated sperm under a variety of relevant physiological conditions at concentrations ≥ 6.25 µg/ml, and mediated complement-dependent sperm immobilization at concentrations ≥ 1 µg/ml. HCA and its immune complexes did not induce inflammation in EpiVaginal™ tissue.InterpretationHCA, an IgG1 mAb with potent sperm agglutination and immobilization activity and a good safety profile, is a promising candidate for female contraception.FundingThis research was supported by grants R01 HD095630 and P50HD096957 from the National Institutes of Health.

A safe and effective mucosal RSV vaccine in mice consisting of RSV phosphoprotein and flagellin variant.

Respiratory syncytial virus (RSV) is a major cause of serious acute lower respiratory tract infection in infants and the elderly. The lack of a licensed RSV vaccine calls for the development of vaccines with other targets and vaccination strategies. Here, we construct a recombinant protein, designated P-KFD1, comprising RSV phosphoprotein (P) and the E.-coli-K12-strain-derived flagellin variant KFD1. Intranasal immunization with P-KFD1 inhibits RSV replication in the upper and lower respiratory tract and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ Tcells provoked by P-KFD1 intranasal (i.n.) immunization either reside in or migrate to the respiratory tract and mediate protection against RSV infection. Single-cell RNA sequencing (scRNA-seq) and carboxyfluorescein succinimidyl ester (CFSE)-labeled cell transfer further characterize the Th1 and Th17 responses induced by P-KFD1. Finally, we find that anti-viral protection depends on either interferon-γ (IFN-γ) or interleukin-17A (IL-17A). Collectively, P-KFD1 is a promising safe and effective mucosal vaccine candidate for the prevention of RSV infection.

Conformation-specific monoclonal antibodies recognizing the native structure of G protein-coupled receptor (GPCR)

It is quite difficult to generate monoclonal antibodies that recognize the three-dimensional structures of the antigens of interest. To address this limitation, we developed a new hybridoma technology termed “optimized stereospecific targeting (SST)”. Here we aimed at generating stereospecific monoclonal antibodies against a G protein-coupled receptor (GPCR). The optimized SST technique enabled the efficient production of conformation-specific monoclonal antibodies against human corticotropin-releasing hormone receptor 1 (huCRHR1). Hybridoma cells secreting stereospecific monoclonal antibodies were selectively cloned by a limiting dilution method and the target monoclonal antibodies were purified by protein A column chromatography. They specifically cross-reacted with native huCRHR1 expressed on the surface of CHO cells, whereas they showed no affinity for MDA-MB-231 cancer cells, which abundantly express EphA2 on the cell surface. Furthermore, immunofluorescence analysis revealed that treatment of huCRHR1-expressing CHO cells with 4% paraformaldehyde led to a decrease in the affinity of purified monoclonal antibodies for intact huCRHR1 on the cell surface. In addition, purified monoclonal antibodies showed no cross-reactivity with huCRHR1 expressed on Sf9 insect cells. These results strongly suggest that monoclonal antibodies generated by the optimized SST technique feature specific binding to the intact form of the target GPCR on mammalian cells.

Selection of CHO host and recombinant cell pools by inhibition of the proteasome results in enhanced product yields and cell specific productivity

Chinese hamster ovary (CHO) cells are the leading mammalian cell expression platform for biotherapeutic recombinant molecules yet some proteins remain difficult to express (DTE) in this, and other, systems. In recombinant cell lines expressing DTE proteins, cellular processes to restore proteostasis can be triggered when the folding and modification capabilities are exceeded, including the unfolded protein response and ER-associated degradation (ERAD) and proteasomal degradation. We therefore investigated whether the proteasome activity of CHO cells was linked to their ability to produce recombinant proteins. We found cell lines with diverse monoclonal antibody (mAb) productivity show different susceptibilities to inhibitors of proteasome activity. Subsequently, we applied selective pressure using proteasome inhibitors on mAb producing cells to determine the impact on cell growth and recombinant protein production, and to apply proteasome selective pressure above that of a metabolic selection marker during recombinant cell pool construction. The presence of proteasome inhibitors during cell pool construction expressing two different model molecules, including a DTE Fc-fusion protein, resulted in the generation of cell pools with enhanced productivity. The increased productivities, and ability to select for higher producing cells, has potential to improve clonal selection during upstream processes of DTE proteins.

Synthesis of hapten, production of monoclonal antibody, and development of immunoassay for ribavirin detection in chicken.

Ribavirin (RBV) is an effective antiviral drug, whose use is prohibited in animal husbandry worldwide. In this work, a novel immunizing hapten of RBV, named Hapten 4, was designed by comparing the conformational and electronic properties of RBV and haptens based on computational chemistry. Hapten 4 was synthesized and conjugated with carrier proteins to produce monoclonal antibody (mAb). The obtained mAb 4C3 for RBV exhibited an IC50 value of 6.24ng/ml in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and displayed no cross-reaction with five other antiviral drugs, including amantadine. The applicability of the developed icELISA was verified in chicken, with a calculated limit of detection of 4.23µg/kg. The recoveries in spiked chicken were 79.2%-107.3% with a coefficient of variation less than 15.9%. The results indicated that the produced antibody from the new hapten was reliable and would be useful for RBV screening in chicken. PRACTICAL APPLICATION: RBV is a broad-spectrum antiviral drug, which is commonly used illegally in poultry farms. A high-affinity mAb 4C3 against RBV was produced and used to develop icELISA with acceptable sensitivity and accuracy. The constructed icELISA has excellent performance for detecting RBV residues in chicken.

Effect of iron addition on mAb productivity and oxidative stress in Chinese hamster ovary culture.

Trace metals play a critical role in the development of culture media used for the production of therapeutic proteins. Iron has been shown to enhance the productivity of monoclonal antibodies during Chinese hamster ovary (CHO) cell culture. However, the redox activity and pro-oxidant behavior of iron may also contribute toward the production of reactive oxygen species (ROS). In this work, we aim to clarify the influence of trace iron by examining the relationship between iron supplementation to culture media, mAb productivity and glycosylation, and oxidative stress interplay within the cell. Specifically, we assessed the impacts of iron supplementation on (a) mAb production and glycosylation; (b) mitochondria-generated free hydroxyl radicals (ROS); (c) the cells ability to store energy during oxidative phosphorylation; and (d) mitochondrial iron concentration. Upon the increase of iron at inoculation, CHO cells maintained a capacity to rebound from iron-induced viability lapses during exponential growth phase and improved mAb productivity and increased mAb galactosylation. Fluorescent labeling of the mitochondrial hydroxyl radical showed enhanced environments of oxidative stress upon iron supplementation. Additional labeling of active mitochondria indicated that, despite the enhanced production of ROS in the mitochondria, mitochondrial membrane potential was minimally impacted. By replicating iron treatments during seed train passaging, the CHO cells were observed to adapt to the shock of iron supplementation prior to inoculation. Results from these experiments demonstrate that CHO cells have the capacity to adapt to enhanced environments of oxidative stress and improve mAb productivity and mAb galactosylation with minimal perturbations to cell culture.

Production of monoclonal shark-derived immunoglobulin new antigen receptor antibodies using Chinese hamster ovary cell expression system

Cartilaginous fishes such as sharks have adaptive immune systems based on immunoglobulins similar to those in mammals. During their evolution, cartilaginous fishes individually have acquired their adaptive immune system called immunoglobulin new antigen receptor (IgNARs). IgNARs maintain their functions in the harsh environment of shark serum, which contains a high concentration of urea to prevent water loss in seawater. Therefore, IgNARs have high structural stability, and are expected to be used as next-generation antibodies in applications different from those of conventional IgG antibodies. However, no recombinant expression system for IgNAR, which has a molecular weight of approximately 147kDa as a dimer and multiple N-glycosylation sites, has yet been constructed. This has stalled research into IgNAR development. Here, we constructed a recombinant expression system for IgNAR using Chinese hamster ovary (CHO) cells, widely used as hosts for IgG antibody production. Using this system, IgNAR was successfully expressed and purified as a human IgG Fc fusion protein and showed antigen-binding ability. After Protein A affinity purification, followed by specific cleavage and removal of the human Fc-region, the final yield of IgNAR was 1.07 mg/L-medium. Moreover, this CHO cell expression system modified IgNAR with various N-glycans, including high-mannose and complex types. This expression system will allow us to analyze the structure, physicochemical properties, and biological functions of IgNAR. This fundamental information will advance the development of IgNARs for industrial and biotechnological applications.

Research contributions of Leo A. Behie to chemical and biomedical engineering

AbstractLeo Augustus Behie was known as a dedicated teacher and mentor, a team builder, and an outstanding researcher and innovator. He had a most productive and illustrious career, spanning five decades, that covered both industrial and academic sectors. His academic research career started with tackling classical chemical engineering challenges and, with time, shifted to biotechnology and biomedical engineering. He was the founding director of a unique research laboratory for undertaking innovative biotechnology and biomedical engineering research at the University of Calgary, called the Pharmaceutical Production Research Facility (PPRF). This paper presents an overview of his academic and research accomplishments, which also included the training of 17 doctoral students, 21 master's degree students, and many postdoctoral scholars. His main research areas under the classical chemical engineering theme included the investigation of the grid region in gas–solid fluidized bed reactors; gas–liquid and liquid–solid multiphase systems; fluidized bed reactors for metals recovery from coal ash; spouted fluid‐bed reactors; kinetics of pyrolysis chemical reactions; transport phenomena in rotary drums; and reactions, thermodynamics, and reactor modelling of sulphur systems. His main research areas under the biotechnology and biomedical engineering theme included advances in biotechnology and bioreactor design; production of antibiotic and monoclonal antibody; protein production in insect cells; process development for the production of neural stem cells, cancer stem cells, and islet cells; mesenchymal stem cells; and cellular secretome studies aimed at developing a cure for Parkinson's disease.

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy.

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.

In silico molecular dynamics of human glycophorin A (GPA) extracellular structure

Background: Glycophorin A (GPA) is one of two glycoproteins found on the surface of human red blood cells (RBCs) that constitute the MNS blood group system. The structure of GPA’s extracellular domain is unknown despite previous attempts using X-ray crystallography and NMR spectroscopy. As a result, there is a knowledge gap regarding antigen presentation. This hinders the production of monoclonal antibodies (mAbs) against MNS antigens.

Requirement of Fc-Fc Gamma Receptor Interaction for Antibody-Based Protection against Emerging Virus Infections.

Identification of therapeutics against emerging and re-emerging viruses remains a continued priority that is only reinforced by the recent SARS-CoV-2 pandemic. Advances in monoclonal antibody (mAb) isolation, characterization, and production make it a viable option for rapid treatment development. While mAbs are traditionally screened and selected based on potency of neutralization in vitro, it is clear that additional factors contribute to the in vivo efficacy of a mAb beyond viral neutralization. These factors include interactions with Fc receptors (FcRs) and complement that can enhance neutralization, clearance of infected cells, opsonization of virions, and modulation of the innate and adaptive immune response. In this review, we discuss recent studies, primarily using mouse models, that identified a role for Fc-FcγR interactions for optimal antibody-based protection against emerging and re-emerging virus infections.

Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

Identification of and Surveillance for the SARS-CoV-2 Variants B.1.427 and B.1.429 - Colorado, January-March 2021.

The B.1.427 and B.1.429 variants of SARS-CoV-2, the virus that causes COVID-19, were first described in Southern California on January 20, 2021 (1); on March 16 they were designated variants of concern* (2). Data on these variants are limited, but initial reports suggest that, compared with other lineages, they might be more infectious (1,2), cause more severe illness (2), and be less susceptible to neutralizing monoclonal antibody products such as bamlanivimab, an investigational treatment for mild-to-moderate COVID-19 (1-3). On January 24, the Colorado Department of Public Health and Environment (CDPHE) identified the first Colorado case of COVID-19 attributed to these variants. B.1.427 and B.1.429 were considered a single variant described as CAL.20C or B.1.427/B.1.429 in the 20C clade (1,3); in this report "B.1.427/B.1.429" refers to B.1.427 or B.1.429 lineage, including those reported as B.1.427/B.1.429 without further differentiation.

A novel approach to residence time distribution characterization in a mAb continuous process.

Residence time distribution modeling of integrated perfusion to capture process can elucidate the impact of product quality excursions and filter fouling on monoclonal antibody production. In this case study, a glycosylation inhibitor and fluorescently labeled antibody are applied to the continuous process to study protein quality modulation, perfusion filter fouling, and unit operation hold times. The unit operations were modeled as continuous-stirred tank reactors and the residence time distribution of a small molecule glycan inhibitor and impact on glycosylation were characterized. A fluorescently labeled antibody was applied as a tracer molecule and confirmed the impact of packed cell volume and filter fouling. This study demonstrates how a biologics continuous process can be modeled and characterized through residence time distribution to ensure a robust, well-understood process.

Freeze thaw and lyophilization induced alteration in mAb therapeutics: Trastuzumab as a case study

Long-term stability of therapeutic monoclonal antibody (mAb) products is necessary for their successful commercialization. Freeze-thaw (F/T) operations are often performed for a mAb product during processing, storage and distribution. Lyophilization (Lyo) is another unit operation that is commonly used for drug product manufacturing of mAbs. This paper aims to explore the impact of these operations on structure and function of a mAb therapeutic, as well as of biosimilars. Trastuzumab innovator and its five biosimilars were analysed for aggregation, charge heterogeneity, secondary structure, binding kinetics, and potency after each freeze-thaw and lyophilization cycle. It is observed that both F/T and Lyo induce protein aggregation, which in turn causes perturbations in the biological potency of the mAb therapeutic. The average value of the percentage of aggregation increased from 0.6 % (week 1) to 5.3 % (week 10) in F/T study and from 0.8 % (week 1) to 10.1 % (week 10) in Lyo study. The acidic pool increased from 26.5 % (week 1) to 44.4 % (week 10) and the basic variants from 13.9 % (week 1) to 24.0 % (week 10) in F/T study. Similarly, acidic pool increased from 27.1 % (week 1) to 42.0 % (week 10) and basic variants from 14.8 % (week 1) to 24.4 % (week 10) in Lyo study. The average percentage of beta-sheet increased from 58.4 % (week 1) to 60.9 % (week 10) in F/T study and from 59.7 % (week 1) to 72.6 % (week 10) in Lyo study. Lower binding affinity was found in week 7 as compared to week 1 in Lyo study whereas no change in binding affinity was observed in the F/T study. The average potency value gradually decreased from 0.97IU/ ml (week 1) to 0.75IU/ ml (week 10) in F/T study and from 1.0IU/ ml (week 1) to 0.66IU/ ml (week 10) in Lyo study. Results indicate that lyophilization has a bigger impact on binding affinity than freeze thaw and as expected, the impact was comparable across the innovator and biosimilar products.

Clinical efficiency of Nigella sativa Linn in novel covid-19: A review study

Objective: To evaluate the efficiency of N. sativa in prevention and management of COVID-19. Background: A novel SARS-CoV-2 which is a severe acute respiratory syndrome, was acknowledged as SARS endemic, started in Asia, with the common of cases occurring in China and the Asia–Pacific countries. According to Ministry of Health and Family Welfare, GOI, since the outbreak, of COVID-19 global pandemic, on March 11, 2020, presently at the end of September 2020, total 940441 cases were active and 5187825 cases were cured, and 97497 were dead. Hence counting the number of cases, including mild cases, is necessary to standardize the epidemic response. Most drug designs against 2019-nCoV have been focused on immunomodulators (such as corticosteroids and interferons), monoclonal antibody production, and inhibitory agents against viral proteinase, helicase, and polymerases. However, to date, few specific antiviral treatment or vaccine has been approved by the FDA for COVID-19, but not hundred percent effectiveness has been achieved. Considering the success of AYUSH systems in managing several epidemics and restoring health, AYUSH system recommends some herbs for the treatment of COVID-19. Among them Nigella sativa is one which is Known by the common name

Optimising the new ucoe models as higher direct transgene expression profile for effective gene therapy applications

Background & Aim: Our new chromatin opening models which free from potential mutation sites that reduce the size of the current UCOE models used in gene therapy and recombinant protein biotechnology studies, tested on various cell groups, including mouse stem cell groups. As a result, it has been demonstrated in terms of replacing existing UCOE models that these new UCOE candidates we have developed are more efficient than the previous ones and that they are also a safer profile model for clinical gene therapy studies since they are free from additional cassette areas. To see the potentiality of our new generation UCOE designs in gene therapy studies, it was tested whether the universal chromatin opening abilities will be retained stable of activation on human induced pluripotent stem cells by differentiating them into different tissue cell types as done before on mouse embryonic stem cells. Methods, Results & Conclusion: After transfection of induced human pluripotent stem cells with Lentiviral vectors carrying the UCOE [Figure presented] [Figure presented] candidates, the activation of UCOE elements which carry a reporter gene, were measured by flowytometry, immunofluorescence staining and DNA methylation analyses for two months without differentiation and also after tissue type differentiation into nerve, hepatocyte and cardiomycet type cells. In the light of the obtained results, it has been observed that the new UCOE designs (1.2kb and 1.7kb UCOE chromatin elements) that we developed, have maintained their expression levels stably on human iPS cells before and after differentiation into three different tissue type cells. And additionally, they also showed their potential on monoclonal antibody production with CHO cells as producing mg and gr level of recombinant antibodies into two months of period in another parallel study. In addition, our new 0.5kb design (0.5kb UCOE), produced from another CpG density region of HNRPA2B1 gene, has been observed to display a stable expression level compared to our other designs and even a more stable profile. The new 0.5kb, 1.2kb 1.7kb (without expression enhancing cassette regions, thus eliminating the potential mutation problem) with a length of 10, 6 and 3 times shorter than the current standard A2UCOE models showed to be more advantageous for gene therapy and recombinant protein production studies as well. Recently, we have been using these new models now on producing the two of the target antigens of COVID-19 to provide a potential recombinant vaccine candidate.

Monoclonal Antibodies for Prevention of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of hospitalizations in infants worldwide. Palivizumab, a humanized monoclonal antibody against the RSV F protein, is the only licensed agent for prevention of severe RSV infection in high-risk infants. Palivizumab is administered intramuscularly, every month during the RSV season, usually 5 doses are required. In recent years, the resolution of the structure of the RSV F protein, with identification of potent neutralizing epitopes, and new technologies for production of monoclonal antibodies (mAbs) have facilitated the development of new alternative strategies for the prevention of RSV infections. One promising approach is a new generation of mAbs directed to new neutralizing epitopes and with prolonged half life. These enhanced mAbs are expected to provide adequate protection during the complete RSV season with a single intramuscular (IM) dose. The long-term goal of this approach is to provide passive immunization for the prevention of RSV lower respiratory tract infection to all infants (preterm and full term) in the first months of life before their initial exposure to RSV.

Opportunities in an Evolving Pharmaceutical Development Landscape: Product Differentiation of Biopharmaceutical Drug Products.

The current perspective reviews the biopharmaceutical market until end of 2020 and highlights the transforming biopharmaceutical landscape during the recent decade. In particular, the rise of biosimilars and the development of new therapeutic modalities through recent advancement in molecular biology research sustainably change the product scenery. The present manuscript describes opportunities for pharmaceutical technical development, highlighting concepts such as product differentiation to succeed in a competitive product landscape. Product differentiation offers the opportunity for numerous life-cycle options and market exclusivity through incremental improvements in standard of care treatment. In particular, different formulation options and formulation-device combinations are described, focusing on systemic delivery of monoclonal antibody products and patient-centered development. The concept of product differentiation is exemplified in a case study about HER2+ breast cancer therapy, underlining pharmaceutical technical solutions and major improvements for the patient.