Development of a stable low-dose aglycosylated antibody formulation to minimize protein loss during intravenous administration

The physical and chemical integrity of a biopharmaceutical must be maintained not only during long-term storage but also during administration. Specifically for the intravenous (i.v.) delivery of a protein drug, loss of stability can occur when the protein formulation is compounded with i.v. bag diluents, thus modifying the original composition of the drug product. Here we present the challenges associated with the delivery of a low-dose, highly potent monoclonal antibody (mAb) via the i.v. route. Through parallel in-use stability studies and conventional formulation development, a drug product was developed in which adsorptive losses and critical oxidative degradation pathways were effectively controlled. This development approach enabled the i.v. administration of clinical doses in the range of 0.1 to 0.5 mg total protein, while ensuring liquid drug product storage stability under refrigerated conditions.     

Challenges and new frontiers in analytical characterization of antibody-drug conjugates

Antibody-drug conjugates (ADCs) are a growing class of biotherapeutics in which a potent small molecule is linked to an antibody. ADCs are highly complex and structurally heterogeneous, typically containing numerous product-related species. One of the most impactful steps in ADC development is the identification of critical quality attributes to determine product characteristics that may affect safety and efficacy. However, due to the additional complexity of ADCs relative to the parent antibodies, establishing a solid understanding of the major quality attributes and determining their criticality are a major undertaking in ADC development. Here, we review the development challenges, especially for reliable detection of quality attributes, citing literature and new data from our laboratories, highlight recent improvements in major analytical techniques for ADC characterization and control, and discuss newer techniques, such as two-dimensional liquid chromatography, that have potential to be included in analytical control strategies.     

Boosting antibody developability through rational sequence optimization

The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.     

Boosting antibody developability through rational sequence optimization

The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.     

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.     

Stability engineering of the human antibody repertoire

Human monoclonal antibodies often display limited thermodynamic and colloidal stabilities. This behavior hinders their production, and places limitations on the development of novel formulation conditions and therapeutic applications. Antibodies are highly diverse molecules, with much of the sequence variation observed within variable domain families and, in particular, their complementarity determining regions. This has complicated the development of comprehensive strategies for the stability engineering of the human antibody repertoire. Here we provide an overview of the field, and discuss recent advances in the development of robust and aggregation resistant antibody therapeutics.     

The new ParaDIgm: IgM from bench to clinic

The inaugural IgM event entitled “The new ParaDIgm: IgM from bench to clinic” brought together the increasingly active and growing IgM antibody community to discuss recent advances and challenges facing the discovery and development of IgM antibody therapies and technologies. Researchers, clinicians and biomanufacturing experts delivered 21 talks on the basic science and isolation of IgM, upstream and downstream development, and formulation and clinical development of the molecules. Participants networked around topics aimed at exploring the full potential of IgM antibodies. The meeting was held at DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e. V. (Society for Chemical Engineering and Biotechnology), a non-profit scientific and technical society based in Frankfurt am Main, Germany. The meeting was sponsored by Patrys, Laureate Biopharma, Bio-Rad Laboratories, BIA Separations, Percivia and the Bio Affinity Company (BAC). The second New ParaDIgm: IgM from bench to clinic meeting, will be held on April 23–24, 2013 in Frankfurt, Germany.     

The new ParaDIgm: IgM from bench to clinic: November 15–16, 2011, Frankfurt,Germany

The inaugural IgM event entitled “The new ParaDIgm: IgM from bench to clinic” brought together the increasingly active and growing IgM antibody community to discuss recent advances and challenges facing the discovery and development of IgM antibody therapies and technologies. Researchers, clinicians and biomanufacturing experts delivered 21 talks on the basic science and isolation of IgM, upstream and downstream development, and formulation and clinical development of the molecules. Participants networked around topics aimed at exploring the full potential of IgM antibodies. The meeting was held at DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e. V. (Society for Chemical Engineering and Biotechnology), a non-profit scientific and technical society based in Frankfurt am Main, Germany. The meeting was sponsored by Patrys, Laureate Biopharma, Bio-Rad Laboratories, BIA Separations, Percivia and the Bio Affinity Company (BAC). The second New ParaDIgm: IgM from bench to clinic meeting, will be held on April 23–24, 2013 in Frankfurt, Germany.     

Molecular basis of high viscosity in concentrated antibody solutions: Strategies for high concentration drug product development

Effective translation of breakthrough discoveries into innovative products in the clinic requires proactive mitigation or elimination of several drug development challenges. These challenges can vary depending upon the type of drug molecule. In the case of therapeutic antibody candidates, a commonly encountered challenge is high viscosity of the concentrated antibody solutions. Concentration-dependent viscosity behaviors of mAbs and other biologic entities may depend on pairwise and higher-order intermolecular interactions, non-native aggregation, and concentration-dependent fluctuations of various antibody regions. This article reviews our current understanding of molecular origins of viscosity behaviors of antibody solutions. We discuss general strategies and guidelines to select low viscosity candidates or optimize lead candidates for lower viscosity at early drug discovery stages. Moreover, strategies for formulation optimization and excipient design are also presented for candidates already in advanced product development stages. Potential future directions for research in this field are also explored.     

Formation of acylated growth hormone-releasing peptide-6 by poly(lactide-co-glycolide) and its biological activity

The purpose of this study was to investigate the formation of acylated impurity resulting from a chemical reaction between the growth hormone-releasing peptide-6 (GHRP-6) and poly(lactide-co-glycolide) (PLGA) and the effect of peptide acylation on the in vivo biological activity of GHRP-6. The peptide acylation pattern of GHRP-6 by hydrophilic PLGA polymers with different molecular weights was characterized by reversed-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Higher levels of acylated GHRP-6 were produced with the higher molecular weight PLGA, which might be due to the slower degradation rate of the polymer. The evaluation of the biological activity in rats showed that the acylated GHRP-6 had a much lower activity than the intact GHRP-6. This finding suggests that the acylation reaction would decrease the effectiveness of the GHRP-6 formulation such as PLGA microspheres. There-fore, a strategy for stabilizing the GHRP-6 will be necessary for the development of a successful formulation of PLGA microspheres. Published: June 8, 2007     

Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies

The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.     

Floating drug delivery systems: A review

The purpose of writing this review on floating drug delivery systems (FDDS) was to compile the recent literature with special focus on the principal mechanism of floatation to achieve gastric retention. The recent developments of FDDS including the physiological and formulation variables affecting gastric retention, approaches to design single-unit and multiple-unit floating systems, and their classification and formulation aspects are covered in detail. This review also summarizes the in vitro techniques, in vivo studies to evaluate the performance and application of floating systems, and applications of these systems. These systems are useful to several problems encountered during the development of a pharmaceutical dosage form. Published: October 19, 2005     

细胞培养工艺条件对抗体异质性影响的研究进展

抗体药物具有靶向明确、副作用小等优势,近年来受到国内外药企的广泛关注。然而,动物细胞大规模培养和抗体质量分析已然成为我国的抗体药物产业化的主要限制因素,尤其是细胞培养工艺条件对抗体异质性的影响非常显著,如何有效通过优化工艺条件来满足抗体药物开发要求成为迫在眉睫的问题。文中简要综述了近年来细胞培养工艺条件对抗体异质性影响的技术进展,并对未来国内抗体药物开发作了展望。     

Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies

The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.     

Functional monolithic platforms: Chromatographic tools for antibody purification

Polymer monoliths are an efficient platform for antibody purification. The use of monoclonal antibodies (mAbs) and engineered antibody structures as therapeutics has increased exponentially over the past few decades. Several approaches use polymer monoliths to purify large quantities of antibody with defined clinical and performance requirements. Functional monolithic supports have attracted a great deal of attention as they offer practical advantages for antibody purification, such as more rapid analysis, smaller sample volume requirements and the opportunity for a greater target molecule enrichment. This review focuses on the development of synthetic and natural polymer-based monoliths for antibody purification. The materials and methods employed in monolith production are discussed, highlighting the properties of each system. We also review the structural characterization techniques available using monolithic systems and their performance under different chromatographic approaches to antibody capture and release. Finally, a summary of monolithic platforms developed for antibody separation is presented, as well as expected trends in research to solve current and future challenges in this field. This review comprises a comprehensive analysis of proposed solutions highlighting the remarkable potential of monolithic platforms.     

Hole Transport Materials in Conventional Structural (n–i–p) Perovskite Solar Cells: From Past to the Future

With the application of organic–inorganic hybrid perovskites to liquid‐type solar cells, the unprecedented development of perovskite solar cells (Per‐SCs) has been boosted by the introduction of solid‐state hole transport materials (HTMs). The removal of liquid electrolyte has lead to improved efficiency and stability. Supported by high‐quality perovskite films, the certified efficiency of Per‐SCs has reached 25.2%. For Per‐SCs assembled in a conventional structure (n–i–p), the hole transport layer (HTL) plays an extra role in preventing the perovskite layer from external stimuli. In summary, the successful design and fabrication of the HTL must meet various requirements in terms of solubility, hole transport, recombination prevention, stability, and reproducibility, to name but a few. Many research strategies are focused on the development of high‐performance HTMs to meet such requirements. Such strategies for the development of HTMs employed in conventional n–i–p solar cells are reviewed herein. A vision of the future HTMs is proposed in this review based on the already proposed solutions and current trends.     

Polymer-Based Sustained-Release Dosage Forms for Protein Drugs, Challenges, and Recent Advances

While the concept of using polymer-based sustained-release delivery systems to maintain therapeutic concentration of protein drugs for extended periods of time has been well accepted for decades, there has not been a single product in this category successfully commercialized to date despite clinical and market demands. To achieve successful systems, technical difficulties ranging from protein denaturing during formulation process and the course of prolonged in vivo release, burst release, and incomplete release, to low encapsulation efficiency and formulation complexity have to be simultaneously resolved. Based on this updated understanding, formulation strategies attempting to address these aspects comprehensively were reported in recent years. This review article (with 134 citations) aims to summarize recent studies addressing the issues above, especially those targeting practical industrial solutions. Formulation strategies representative of three areas, microsphere technology using degradable hydrophobic polymers, microspheres made of water soluble polymers, and hydrophilic in vivo gelling systems will be selected and introduced. To better understand the observations and conclusions from different studies for different systems and proteins, physicochemical basis of the technical challenges and the pros and cons of the corresponding formulation methods will be discussed.     

Advances in liquid formulations of parenteral therapeutic proteins

Liquid formulation of therapeutic proteins is a maturing technology. Demand for products that are easy to use in the clinic or that are amenable to self-administration make a ready to use liquid formulation desirable. Most modern liquid formulations have a simple composition; comprising a buffer, a tonicity modifier, a surfactant, sometimes a stabiliser, the therapeutic protein and water. Recent formulations of monoclonal antibodies often use histidine or acetate as the buffer, sucrose or trehalose as the tonicity modifier and polysorbate 20 or 80 as the surfactant with a pH of 5.7 +/? 0.4. The mechanisms for the behaviour of excipients is still debated by academics so formulation design is still a black art. Fortunately, a statistical approach like design of experiment is suitable for formulation development and has been successful when combined with accelerated stability experimentation. The development of prefilled syringes and pens has added low viscosity and shear resistance to the quality attributes for a successful formulation. To achieve patient compliance for self-administration, formulations that cause minimal pain and tissue damage is also desirable.     

l-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (IVIG) solutions

Liquid intravenous immunoglobulin (IVIG) products offer improved convenience in preparation but often lack sufficient stability to allow room temperature storage. Furthermore, clinical tolerability may be affected due to formation of idiotype/anti-idiotype IgG dimers and/or aggregates. Here we report on the development of a 10% IVIG formulation with optimized stability achieved by the use of l-proline. The stability of concentrated liquid IVIG was strongly pH dependent. Aggregate formation, yellowish discoloration of the solution and loss of anti-hepatitis B surface antigen (HBs) antibody activity was minimal at intermediate pH (pH 4.8–5.3). Fragmentation of IgG was highest at low pH (pH 4.1). Idiotype/anti-idiotype IgG dimer formation was highest at neutral pH and was reduced with decreasing pH. The presence of l-proline further improved stability by inhibiting protein aggregation, reducing loss of anti-HBs antibody activity and decreasing coloring, particularly compared with glycine formulations. The IgG dimer content was up to 30% lower in solutions containing l-proline compared with those containing glycine or other stabilizers. In conclusion, a weakly acidic pH of approximately 5 and l-proline as stabilizer are optimal conditions for long-term stability of a liquid IVIG. l-proline, an amphiphilic, naturally occurring amino acid, is superior to glycine in restricting IgG dimer formation.     

GM-CSF-based cellular vaccines: a review of the clinical experience

Immunotherapy is playing an increasing role in the treatment of many cancers. The recent advances in antibody therapy gives much optimism that both passive (antibody therapy) as well as active (vaccine therapy) immunotherapeutic interventions will acquire an increasing presence in oncology. Granulocyte macrophage-colony stimulation factor (GM-CSF)-based vaccines have now been tested in several diseases in a variety of formulations. The success and broad applicability of such an approach rests on the development of an ideal vaccine formulation administered in the appropriate clinical context. This review summarizes the results from the clinical trials performed to date and discusses the future directions of GM-CSF-based cellular vaccine strategies aimed at maximizing the therapeutic benefit.