International standards for monoclonal antibodies to support pre- and post-marketing product consistency: Evaluation of a candidate international standard for the bioactivities of rituximab

The intrinsic complexity and heterogeneity of therapeutic monoclonal antibodies is built into the biosimilarity paradigm where critical quality attributes are controlled in exhaustive comparability studies with the reference medicinal product. The long-term success of biosimilars will depend on reassuring healthcare professionals and patients of consistent product quality, safety and efficacy. With this aim, the World Health Organization has endorsed the need for public bioactivity standards for therapeutic monoclonal antibodies in support of current controls. We have developed a candidate international potency standard for rituximab that was evaluated in a multi-center collaborative study using participants' own qualified Fc-effector function and cell-based binding bioassays. Dose-response curve model parameters were shown to reflect similar behavior amongst rituximab preparations, albeit with some differences in potency. In the absence of a common reference standard, potency estimates were in poor agreement amongst laboratories, but the use of the candidate preparation significantly reduced this variability. Our results suggest that the candidate rituximab standard can support bioassay performance and improve data harmonization, which when implemented will promote consistency of rituximab products over their life-cycles. This data provides the first scientific evidence that a classical standardization exercise allowing traceability of bioassay data to an international standard is also applicable to rituximab. However, we submit that this new type of international standard needs to be used appropriately and its role not to be mistaken with that of the reference medicinal product.     

Present status and future prospects for the hybridoma technology

Summary A somatic cell genetic technique has recently been developed that makes it possible to obtain very large amounts of homogeneous antibodies and to replenish the supply of the exact same antibodies whenever they are needed. This hybridoma technology has already contributed to major scientific advances and will surely improve the diagnosis and treatment of many diseases. Because the technology itself is relatively simple and inexpensive, it has captured the attention of basic scientists, clinicians, and industrial managers and investors. This work was supported by National Institutes of Health Grants AI 10702 and AI 5231, National Science Foundation Grant PCM77-25635, and American Cancer Society Grant NP-317.     

Biosimilars entering the clinic without animal studies: A paradigm shift in the European Union

The concept of biosimilars has spread from Europe to other regions throughout the world, and many regions have drafted regulatory guidelines for their development. Recently, a paradigm shift in regulatory thinking on the non-clinical development of biosimilars has emerged in Europe: In vivo testing should follow a step-wise approach rather than being performed by default. To not require animal testing at all in some instances can well be seen as a revolutionary, but science-based, step. Here, we describe the internal discussions that led to this paradigm shift. The mainstay for the establishment of biosimilarity is the pharmaceutical comparability based on extensive physicochemical and biological characterization. Pharmacodynamic comparability can be evaluated in in vitro assays, whereas pharmacokinetic comparability is best evaluated in clinical studies. It is considered highly unlikely that new safety issues would arise when comparability has been demonstrated based on physicochemical and in vitro comparative studies.     

Biosimilars in oncology and inflammatory diseases: current and future considerations for clinicians in Latin America

Biological therapies have revolutionized the treatment of several cancers and systemic immune-mediated inflammatory conditions. Expiry of patents protecting a number of biologics has provided the opportunity to commercialize highly similar versions, known as biosimilars. Biosimilars are approved by regulatory agencies via an independent pathway that requires extensive head-to-head comparison with the originator product. Biosimilars have the potential to provide savings to healthcare systems and expand patient access to biologics. In Latin American countries, regulatory frameworks for biosimilar approval have been introduced in recent years, and biosimilars of monoclonal antibody and fusion protein therapies are now emerging. However, the situation in this region is complicated by the presence of “non-comparable biotherapeutics” (also known as “intended copies”), which have not been rigorously compared with the originator product. We review the considerations for clinicians in Latin American countries, focusing on monoclonal antibody biosimilars relevant to oncology, rheumatology, gastroenterology, and dermatology.     

Cardiac myofibrillar proteins: Biochemical markers to estimate myocardial injury

Ischaemic heart disease represents the most common of the serious health problems in the contemporary society and acute myocardial infarction (AMI) is the major cause of cardiovascular morbidity and death. The accurate localization and determination of the infarct size and the volume of myocardium at risk at the time of insult is crucial and vital for the choice of treatment. Initially the ischaemic cells are reversibly injured. However, if these changes are not reverted at the earliest, it results in the death of the myocyte. This irreversible myocyte necrosis travels transmurally towards epicardium in the form of a wavefront [1]. A timely intervention during evolving infarct could reduce and delimit the infarct and preserve the left ventricular function [2].Enzyme analysis and electrocardiography (ECG) along with the clinical history of the patient is still considered to constitute a reliable triad in the diagnosis of myocardial infarction (MI) [3]. Efforts have been made to relate infarct size with the serum enzyme level changes without much success. In addition, a number of specialist techniques such as planar radioisotope imaging, single photon emission computed tomography (SPECT), positron emission tomography (PET), Echocardiography, Ventriculography and nuclear magnetic resonance (NMR) imaging have been devised to support diagnosis in the patients who show ambiguous symptoms and ECG findings. However most of these procedures are unavailable to the patients due to economic reasons while others have suffered due to non-availability of ideal radiopharmaceuticals. Major advances have been made in the methods based on immunological techniques to improve the detection and estimation of infarct. These methods are exclusively based upon the production and availability of specific antibodies against intracellular, cardiac specific components [4].     

Monoclonal antibodies in solid tumours: approaches to therapy with emphasis on gynaecological cancer

Monoclonal antibodies have progressed from the laboratory to the clinic. Although recognised in diagnosis there are still problems as far as their therapeutic use is concerned. This review looks at the history, principles of active specific immunotherapy, clinical experience with monoclonal antibodies in therapy of solid tumours, in particular the development of new bispecific monoclonal antibodies, and trials in ovarian, breast and colorectal cancer. Immunoconjugates, linked with radionuclides and cytotoxic drugs, indicate future developments. Conditions for successful therapy, especially with adjuvants in patients with small tumour residues, are also described.     

Glycosylation of monoclonal antibody products: Current status and future prospects

Protein based therapeutics have started to dominate the pharmaceutical landscape primarily due to the high efficacy that they have demonstrated against complex diseases. Despite the significant success, issues with regards to safety, efficacy and quality of biotherapeutics have been a point of considerable debate and concern among the various stakeholders. The correlation between the glycoform profile and the safety and efficacy of a drug, in particular, has garnered significant attention of researchers worldwide. An escalated emphasis is presently given to develop an understanding of how the various process parameters as well molecular biology considerations contribute to glycan heterogeneity. This paper aims to review the major developments that have occurred in this area over the last decade. The impact of the various process parameters on glycan expression, methods for obtaining a pre‐determined glycan levels/profiles of a protein therapeutic and developments in the area of real‐time glycan monitoring and control are discussed. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1091–1102, 2016     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional ¹H,¹⁵N and ¹H,¹³C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-¹⁵N, 20%-¹³C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.     

Moving through three‐dimensional phase diagrams of monoclonal antibodies

Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three‐dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1103–1113, 2014     

Limitations to the structure-based design of HIV-1 vaccine immunogens

In spite of 25 years of intensive research, no effective human immunodeficiency virus type 1 (HIV-1) vaccine has yet been developed. One reason for this is that investigators have concentrated mainly on the structural analysis of HIV-1 antigens because they assumed that it should be possible to deduce vaccine-relevant immunogens from the structure of viral antigens bound to neutralizing monoclonal antibodies. This unwarranted assumption arises from misconceptions regarding the nature of protein epitopes and from the belief that it is justified to extrapolate from the antigenicity to the immunogenicity of proteins. Although the structure of the major HIV-1 antigenic sites has been elucidated, this knowledge has been of little use for designing an HIV-1 vaccine. Little attention has been given to the fact that protective immune responses tend to be polyclonal and involve antibodies directed to several different epitopes. It is concluded that only trial and error, empirical investigations using numerous immunization protocols may eventually allow us to identify which mixtures of immunogens are likely to be the best candidates for an HIV-1 vaccine.     

A practical strategy for using miniature chromatography columns in a standardized high‐throughput workflow for purification development of monoclonal antibodies

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high‐throughput process development (HTPD) strategy implementing several high‐throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high‐throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high‐throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high‐throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:626–635, 2014     

Proteins of the acrosomal region in mouse sperm: Immunological probes reveal post-testicular modifications

Effects of reduced glutathione (GSH), oxidized glutathione (GSSG), or glutathione reductasc (GR) supply were studied on the ability of hamster oocytes to be fertilized by human sperm. Zona-free oocytes were pretreated with these compounds prior to sperm insemination. Oocyte pretreatment with high concentrations of GSH or GSSG (50 or 100 mM. 30 min) significantly increased the penetrated oocyte rate (PR). Polyspermy was not increased except when high concentrations of GSH (100 mM) were used. Incubation of oocytes with GR (1 or 10IU/ml) prior to sperm insemination induced increasing dose-dependent PR. Polyspermy increased significantly with 10 mM GR in oocyte incubation medium. Oocyte incubation for 30 min with the sulfhydryl blocking agent iodoacetamide (1 mM) led to a drastic decrease in oocyte penetration and in polyspermy. Our results demonstrate an original way to increase the efficacy of human-hamster heterospecific fertilization. Various hypotheses are discussed explaining these observations which open new investigations for heterospecific and homospecific in vitro fertilization.     

A monoclonal antibody to swine erythrocytes recognizes the B blood group on the major glycophorin

Recently monoclonal antibodies (mAbs) to swine red blood cells have been described. One of them (1AC11) was specific for the major swine glycoprotein with a molecular weight of 45 kDa and another mAb, 2G2, recognized the B ^a allele in the B system of swine blood groups. Immunoblotting experiments to characterize the mAb 2G2 indicated that it reacts with an antigen of 45 kDa, present on the aqueous phase, glycophorin fraction, of swine red blood cells with the B ^a allele and does not react with B ^bB^b homozygous cells. The antigen recognized by 2G2 has the same characteristics as the major glycophorin recognized by 1AC11, so we can conclude that the B system of the swine blood group is on the major glycophorin of swine erythrocyte membranes.     

2nd PEGS Annual Symposium on Antibodies for Cancer Therapy

The 2nd Annual Antibodies for Cancer Therapy symposium, organized again by Cambridge Healthtech Institute as part of the Protein Engineering Summit, was held in Boston, USA from April 30^th to May 1^st, 2012. Since the approval of the first cancer antibody therapeutic, rituximab, fifteen years ago, eleven have been approved for cancer therapy, although one, gemtuzumab ozogamicin, was withdrawn from the market. The first day of the symposium started with a historical review of early work for lymphomas and leukemias and the evolution from murine to human antibodies. The symposium discussed the current status and future perspectives of therapeutic antibodies in the biology of immunoglobulin, emerging research on biosimilars and biobetters, and engineering bispecific antibodies and antibody-drug conjugates. The tumor penetration session was focused on the understanding of antibody therapy using ex vivo tumor spheroids and the development of novel agents targeting epithelial junctions in solid tumors. The second day of the symposium discussed the development of new generation recombinant immunotoxins with low immunogenicity, construction of chimeric antigen receptors, and the proof-of-concept of ‘photoimmunotherapy’. The preclinical and clinical session presented antibodies targeting Notch signaling and chemokine receptors. Finally, the symposium discussed emerging technologies and platforms for therapeutic antibody discovery.     

The effect of low intensity ultraviolet‐C light on monoclonal antibodies

As part of an investigation to identify potential new viral reduction strategies, ultraviolet‐C (UV‐C) light was examined. Although this technology has been known for decades to possess excellent virus inactivation capabilities, UV‐C light can also introduce significant unwanted damage to proteins. To study the effect on monoclonal antibodies, three different antibodies were subjected to varying levels of UV‐C light using a novel dosing device from Bayer Technology Services GmbH. The range of fluencies (or doses) covered was between 0 and 300 J/m² at a wavelength of 254 nm. Product quality data generated from the processed pools showed only minimal damage done to the antibodies. Aggregate formation was low for two of the three antibodies tested. Acidic and basic variants increased for all three antibodies, with the basic species increasing more than the acidic species. Peptide maps made for the three sets of pools showed no damage to two of the three antibody backbones, whereas the third antibody had very low levels of methionine oxidation evident. Samples held at 2–8°C for 33 days showed no increase in aggregates or charge variants, indicating that the proteins did not degrade and were not damaged further by reactive or catalytic species that may have been created on exposure to UV‐C light. Overall, UV‐C light was shown to induce very little damage to monoclonal antibodies at lower fluencies and appears to be a viable option for viral inactivation in biotechnology applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Monoclonal antibody to fibulin-1 generated by genetic immunization

Fibulin-1 (Fbln-1) is an extracellular matrix (ECM) and plasma glycoprotein. Considering the growing evidence indicating that Fbln-1 plays a role in cancer we sought to develop monospecific antibodies to better facilitate further studies of the function of Fbln-1 in breast cancer. Using a plasmid expression vector encoding full-length human Fbln-1D as an immunogen and CpG oligodeoxyribonucleotides as adjuvant a monoclonal antibody (MAb) against Fbln-1 was produced. This MAb, designated MEM-2 was of IgM isotype and reacted with bacterially expressed Fbln-1. Furthermore, MEM-2 reacted with Fbln-1 expressed in the ECM released by cultured human breast carcinoma SKBR-3 cells in ELISA, and also with Fbln-1 present in SKBR-3 cell extract in immunoprecipitation and Western blotting. MEM-2 also reacted with Fbln-1 in human breast carcinoma specimens. These findings illustrate the utility of genetic immunization as a means of generating monoclonal antibodies to tumor-related ECM proteins. MEM-2 represents a useful new tool for the study of Fbln-1 in breast cancer.     

Challenges and opportunities for the future of monoclonal antibody development: Improving safety assessment and reducing animal use

The market for biotherapeutic monoclonal antibodies (mAbs) is large and is growing rapidly. However, attrition poses a significant challenge for the development of mAbs, and for biopharmaceuticals in general, with large associated costs in resource and animal use. Termination of candidate mAbs may occur due to poor translation from preclinical models to human safety. It is critical that the industry addresses this problem to maintain productivity. Though attrition poses a significant challenge for pharmaceuticals in general, there are specific challenges related to the development of antibody-based products. Due to species specificity, non-human primates (NHP) are frequently the only pharmacologically relevant species for nonclinical safety and toxicology testing for the majority of antibody-based products, and therefore, as more mAbs are developed, increased NHP use is anticipated. The integration of new and emerging in vitro and in silico technologies, e.g., cell- and tissue-based approaches, systems pharmacology and modeling, have the potential to improve the human safety prediction and the therapeutic mAb development process, while reducing and refining animal use simultaneously. In 2014, to engage in open discussion about the challenges and opportunities for the future of mAb development, a workshop was held with over 60 regulators and experts in drug development, mechanistic toxicology and emerging technologies to discuss this issue. The workshop used industry case-studies to discuss the value of the in vivo studies and identify opportunities for in vitro technologies in human safety assessment. From these and continuing discussions it is clear that there are opportunities to improve safety assessment in mAb development using non-animal technologies, potentially reducing future attrition, and there is a shared desire to reduce animal use through minimised study design and reduced numbers of studies.     

Accelerated CMC workflows to enable speed to clinic in the COVID-19 era: A multi-company view from the biopharmaceutical industry

The COVID-19 pandemic has placed unprecedented pressure on biopharmaceutical companies to develop efficacious preventative and therapeutic treatments, which is unlikely to abate in the coming years. The importance of fast progress to clinical evaluation for treatments, which tackle unmet medical needs puts strain on traditional product development timelines, which can take years from start to finish. Although previous work has been successful in reducing phase 1 timelines for recombinant antibodies, through utilization of the latest technological advances and acceptance of greater business risk or costs, substantially faster development is likely achievable without increased risk to patients during initial clinical evaluation. To optimize lessons learned from the pandemic and maximize multi-stakeholder (i.e., patients, clinicians, companies, regulatory agencies) benefit, we conducted an industry wide benchmarking survey in September/October 2021. The aims of this survey were to: (i) benchmark current technical practices of key process and product development activities related to manufacturing of therapeutic proteins, (ii) understand the impact of changes implemented in COVID-19 accelerated Ab programs, and whether any such changes can be retained as part of sustainable long-term business practices and (iii) understand whether any accelerative action(s) taken have (negatively) impacted the wider development process. This article provides an in-depth analysis of this data, ultimately highlighting an industry perspective of how biopharmaceutical companies can sustainably adopt new approaches to therapeutic protein development and production.