The role of formulation in insulin comparability assessments.

The concept of comparability can be applied when changes are made to manufacturing processes for biotechnology products subsequent to pivotal clinical trial studies. For many process changes, comparability can be demonstrated based entirely on relevant in vitro data provided that a detailed knowledge of the process/product exists, suitable analytical methodology is employed, and historical data are available for the assessment. Insulin provides an excellent model system to illustrate many important considerations when dealing with comparability exercises for biotechnology products. The physicochemical properties of insulin demonstrate the numerous chemical reactions and physical transformations that are exclusive to proteins. These properties are heavily influenced by formulation conditions and must be carefully evaluated when process changes are made. In addition, physical and chemical testing performed on representative formulations can provide valuable insight when assessing the comparability between pre- and post-change materials. This paper reviews our experience with manufacturing changes involving insulin emphasizing the important role of formulation in the comparability exercise for protein biopharmaceuticals.     

Potency assay development for cellular therapy products: an ISCT review of the requirements and experiences in the industry

The evaluation of potency plays a key role in defining the quality of cellular therapy products (CTPs). Potency can be defined as a quantitative measure of relevant biologic function based on the attributes that are linked to relevant biologic properties. To achieve an adequate assessment of CTP potency, appropriate in vitro or in vivo laboratory assays and properly controlled clinical data need to be created. The primary objective of a potency assay is to provide a mechanism by which the manufacturing process and the final product for batch release are scrutinized for quality, consistency and stability. A potency assay also provides the basis for comparability assessment after process changes, such as scale-up, site transfer and new starting materials (e.g., a new donor). Potency assays should be in place for early clinical development, and validated assays are required for pivotal clinical trials. Potency is based on the individual characteristics of each individual CTP, and the adequacy of potency assays will be evaluated on a case-by-case basis by regulatory agencies. We provide an overview of the expectations and challenges in development of potency assays specific for CTPs; several real-life experiences from the cellular therapy industry are presented as illustrations. The key observation and message is that aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development.     

Comparability testing of a humanized monoclonal antibody (Synagis) to support cell line stability, process validation, and scale-up for manufacturing.

Biochemical and functional testing of a humanized monoclonal antibody directed against Respiratory Syncytial Virus (Synagis) has been performed to evaluate cell line stability, support process validation, and to demonstrate "comparability" during the course of process development. Using a variety of analytical methods, product manufactured at different sites and in bioreactors from 20 litres to 10,000 litres was shown to be biochemically and functionally equivalent. The biochemical testing for microheterogeneity found on Synagis included evaluation of changes in post-translational modifications such as deamidation, truncation, and carbohydrate structure. Studies were also performed to support cell line stability assessment and cell culture process validation. Cell culture conditions were deliberately varied in an attempt to determine if this would have an impact on the microheterogeneity of the product. In these studies Synagis was produced from cells cultured beyond the population doublings achieved at the maximum manufacturing scale, under conditions of low glucose, and using harvest times outside of the historical manufacturing operating range. Results showed that there was a different pattern of glycosylation during the early stages of bioreactor culture. No other changes in microheterogeneity were apparent for the other culture conditions studied. In summary, comparability assessment demonstrated that the Synagis manufacturing process is robust and consistent resulting in a predictable and reproducible monoclonal antibody product.     

Review of regulation of biological and biotechnological products in Latin American and Caribbean countries

The regulation of the biological and biotechnological products constitutes a significant challenge, since they are part of a sector of the pharmaceutical industry that is currently experiencing rapid growth. Unlike conventional medicines, the manufacture of these products involves the use of living organisms and processes that impede manufacturing consistency. Even though there are numerous international reference documents related to biotechnological product regulation, there is no consensus by official entities that are considered reference institutions, with regard to the most important definitions used and the mechanisms for product regulation.The Pan-American Health Organization (PAHO), through the Technology, Health Care and Research Area, has developed a series of activities that are described in this document. The objective of this publication is to present the current picture of biotechnological and biological product regulation in the Latin American and Caribbean Region, in order to offer guidance that will facilitate the regulation of these products in a harmonized manner among the countries of the Member States, as well as responding to the request from some regulatory agencies to address the growing demand for licensing applications of these products.     

Chemometrics applications in biotech processes: assessing process comparability

A typical biotech process starts with the vial of the cell bank, ends with the final product and has anywhere from 15 to 30 unit operations in series. The total number of process variables (input and output parameters) and other variables (raw materials) can add up to several hundred variables. As the manufacturing process is widely accepted to have significant impact on the quality of the product, the regulatory agencies require an assessment of process comparability across different phases of manufacturing (Phase I vs. Phase II vs. Phase III vs. Commercial) as well as other key activities during product commercialization (process scale-up, technology transfer, and process improvement). However, assessing comparability for a process with such a large number of variables is nontrivial and often companies resort to qualitative comparisons. In this article, we present a quantitative approach for assessing process comparability via use of chemometrics. To our knowledge this is the first time that such an approach has been published for biotech processing. The approach has been applied to an industrial case study involving evaluation of two processes that are being used for commercial manufacturing of a major biosimilar product. It has been demonstrated that the proposed approach is able to successfully identify the unit operations in the two processes that are operating differently. We expect this approach, which can also be applied toward assessing product comparability, to be of great use to both the regulators and the industry which otherwise struggle to assess comparability.     

Biosimilars advancements: Moving on to the future

Many patents for the first biologicals derived from recombinant technology and, more recently, monoclonal antibodies (mAbs) are expiring. Naturally, biosimilars are becoming an increasingly important area of interest for the pharmaceutical industry worldwide, not only for emergent countries that need to import biologic products. This review shows the evolution of biosimilar development regarding regulatory, manufacturing bioprocess, comparability, and marketing. The regulatory landscape is evolving globally, whereas analytical structure and functional analyses provide the foundation of a biosimilar development program. The challenges to develop and demonstrate biosimilarity should overcome the inherent differences in the bioprocess manufacturing and physicochemical and biological characterization of a biosimilar compared to several lots of the reference product. The implementation of approaches, such as Quality by Design (QbD), will provide products with defined specifications in relation to quality, purity, safety, and efficacy that were not possible when the reference product was developed. Actually, the need to prove comparability to the reference product by the biosimilar industry has increased the knowledge about the product and the production‐process associated by the use of powerful analytical tools. The technological challenges to make copies of biologic products while attending regulatory and market demands are expected to help innovation in the direction of attaining more productive manufacturing processes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1139–1149, 2015     

Good Manufacturing Practice–compliant change of raw material in the manufacturing process of a clinically used advanced therapy medicinal product–a comparability study

The development of medicinal products often continues throughout the different phases of a clinical study and may require challenging changes in raw and starting materials at later stages. Comparability between the product properties pre- and post-change thus needs to be ensured. Here, we describe and validate the regulatory compliant change of a raw material using the example of a nasal chondrocyte tissue-engineered cartilage (N-TEC) product, initially developed for treatment of confined knee cartilage lesions. Scaling up the size of N-TEC as required for the treatment of larger osteoarthritis defects required the substitution of autologous serum with a clinical-grade human platelet lysate (hPL) to achieve greater cell numbers necessary for the manufacturing of larger size grafts. A risk-based approach was performed to fulfill regulatory requirements and demonstrate comparability of the products manufactured with the standard process (autologous serum) already applied in clinical indications and the modified process (hPL). Critical attributes with regard to quality, purity, efficacy, safety and stability of the product as well as associated test methods and acceptance criteria were defined. Results showed that hPL added during the expansion phase of nasal chondrocytes enhances proliferation rate, population doublings and cell numbers at passage 2 without promoting the overgrowth of potentially contaminant perichondrial cells. N-TEC generated with the modified versus standard process contained similar content of DNA and cartilaginous matrix proteins with even greater expression levels of chondrogenic genes. The increased risk for tumorigenicity potentially associated with the use of hPL was assessed through karyotyping of chondrocytes at passage 4, revealing no chromosomal changes. Moreover, the shelf-life of N-TEC established for the standard process could be confirmed with the modified process. In conclusion, we demonstrated the introduction of hPL in the manufacturing process of a tissue engineered product, already used in a late-stage clinical trial. Based on this study, the national competent authorities in Switzerland and Germany accepted the modified process which is now applied for ongoing clinical tests of N-TEC. The described activities can thus be taken as a paradigm for successful and regulatory compliant demonstration of comparability in advanced therapy medicinal products manufacturing.     

Characterizing biological products and assessing comparability following manufacturing changes

Changes in production methods of a biological product may necessitate an assessment of comparability to ensure that these manufacturing changes have not affected the safety, identity, purity, or efficacy of the product. Depending on the nature of the protein or the change, this assessment consists of a hierarchy of sequential tests in analytical testing, preclinical animal studies and clinical studies. Differences in analytical test results between pre- and post-change products may require functional testing to establish the biological or clinical significance of the observed difference. An underlying principle of comparability is that under certain conditions, protein products may be considered comparable on the basis of analytical testing results alone. However, the ability to compare biological materials is solely dependent on the tests used, since no single analytical method is able to compare every aspect of protein structure or function. The advantages and disadvantages of any given method depends on the protein property being characterized.     

Application of multivariate analysis toward biotech processes: case study of a cell-culture unit operation

This paper examines the feasibility of using multivariate data analysis (MVDA) for supporting some of the key activities that are required for successful manufacturing of biopharmaceutical products. These activities include scale-up, process comparability, process characterization, and fault diagnosis. Multivariate data analysis and modeling were performed using representative data from small-scale (2 L) and large-scale (2000 L) batches of a cell-culture process. Several input parameters (pCO2, pO2, glucose, pH, lactate, ammonium ions) and output parameters (purity, viable cell density, viability, osmolality) were evaluated in this analysis. Score plots, loadings plots, and VIP plots were utilized for assessing scale-up and comparability of the cell-culture process. Batch control charts were found to be useful for fault diagnosis during routine manufacturing. Finally, observations made from reviewing VIP plots were found to be in agreement with conclusions from process characterization studies demonstrating the effectiveness of MVDA as a tool for extracting process knowledge.     

Guidelines for analytical method development and validation of biotechnological synthesis of drugs. Production of a hydroxyprogesterone as model

In connection with biotechnological synthesis of pharmaceutical drugs, validated methods for quantification of both product and substrate at different time intervals are essential for proper calculation of rate coefficients. In this field, there still exist no guidelines for analytical validation, unlike the situation in the bioanalytical field. Therefore, in this study the detailed guidelines by FDA for bioanalytical method validation were applied to a typical biotechnological process; the enzymatic synthesis of 9alpha-hydroxyprogesterone in E. coli using progesterone as substrate. The process liquid was extracted and analyzed using an HPLC-DAD system. The quality control (QC) samples of the product demonstrated excellent precision (C.V.<1.5%) and accuracy between 99.3 and 107%. The study showed that the recommendations and the validation terms for bioanalytical methods can be used also for biotechnological production, but with some important exceptions. The tolerances (C.V. values) of the validation terms should be much narrower; the internal standard (I.S.) must be present in the process liquid before the start of the process and must be much different in structure from the substrate (so as not to participate in the biotechnological process). In addition, the selectivity must be checked very frequently during the process due to the changes in the blank process liquid with time.     

Manufacturing models permitting roll out/scale out of clinically led autologous cell therapies: regulatory and scientific challenges for comparability

Manufacturing of more-than-minimally manipulated autologous cell therapies presents a number of unique challenges driven by complex supply logistics and the need to scale out production to multiple manufacturing sites or near the patient within hospital settings. The existing regulatory structure in Europe and the United States imposes a requirement to establish and maintain comparability between sites. Under a single market authorization, this is likely to become an unsurmountable burden beyond two or three sites. Unless alternative manufacturing approaches can be found to bridge the regulatory challenge of comparability, realizing a sustainable and investable business model for affordable autologous cell therapy supply is likely to be extremely demanding. Without a proactive approach by the regulators to close this “translational gap,” these products may not progress down the development pipeline, threatening patient accessibility to an increasing number of clinician-led autologous cellular therapies that are already demonstrating patient benefits. We propose three prospective manufacturing models for the scale out/roll out of more-than-minimally manipulated clinically led autologous cell therapy products and test their prospects for addressing the challenge of product comparability with a selected expert reference panel of US and UK thought leaders. This paper presents the perspectives and insights of the panel and identifies where operational, technological and scientific improvements should be prioritized. The main purpose of this report is to solicit feedback and seek input from key stakeholders active in the field of autologous cell therapy in establishing a consensus-based manufacturing approach that may permit the roll out of clinically led autologous cell therapies.     

Quality, safety and efficacy of follow-on biologics in Japan

Recently, WHO, EU, Japan and Canada have published guidelines on biosimilar/follow-on biologics. While there seems to be no significant difference in the general concept in these guidelines, the data to be submitted for product approval are partially different. Differences have been noted in the requirements for comparability studies on stability, prerequisites for reference product, or for the need of comparability exercise for determination of process-related impurities. In Japan, there have been many discussions about the amount and extent of data for approval of follow-on biologics. We try to clarify the scientific background and rational for regulatory pathway of biosimilar/follow-on biologics in Japan in comparison with the guidelines available from WHO, EU and Canada. In this article, we address and discuss the scientific background underlying these differences to facilitate the harmonization of follow-on biologic principles in the guidelines in future.     

Practical considerations in clinical strategy to support the development of injectable drug-device combination products for biologics

The development of an injectable drug-device combination (DDC) product for biologics is an intricate and evolving process that requires substantial investments of time and money. Consequently, the commercial dosage form(s) or presentation(s) are often not ready when pivotal trials commence, and it is common to have drug product changes (manufacturing process or presentation) during clinical development. A scientifically sound and robust bridging strategy is required in order to introduce these changes into the clinic safely. There is currently no single developmental paradigm, but a risk-based hierarchical approach has been well accepted. The rigor required of a bridging package depends on the level of risk associated with the changes. Clinical pharmacokinetic/pharmacodynamic comparability or outcome studies are only required when important changes occur at a late stage. Moreover, an injectable DDC needs to be user-centric, and usability assessment in real-world clinical settings may be required to support the approval of a DDC. In this review, we discuss the common issues during the manufacturing process and presentation development of an injectable DDC and practical considerations in establishing a clinical strategy to address these issues, including key elements of clinical studies. We also analyze the current practice in the industry and review relevant and status of regulatory guidance in the DDC field.     

Analytical lessons learned from selected therapeutic protein drug comparability studies

The successful implementation of process and product changes for a therapeutic protein drug, both during clinical development and after commercialization, requires a detailed evaluation of their impact on the protein's structure and biological functionality. This analysis is called a comparability exercise and includes a data driven assessment of biochemical equivalence and biological characterization using a cadre of analytical methodologies. This review focuses on describing analytical results and lessons learned from selected published therapeutic protein comparability case studies both for bulk drug substance and final drug product. An overview of the currently available analytical methodologies typically used is presented as well as a discussion of new emerging analytical techniques. The potential utility of several novel analytical approaches to comparability studies is discussed including distribution and stability of protein drugs in vivo, and enhanced evaluation of higher-order protein structure in actual formulations using hydrogen/deuterium exchange mass spectrometry, two-dimensional nuclear magnetic resonance fingerprinting or empirical phase diagrams. In addition, new methods for detecting and characterizing protein aggregates and particles are presented as these degradants are of current industry-wide concern. The critical role that analytical methodologies play in elucidating the structure–function relationships for therapeutic protein products during the overall assessment of comparability is discussed.     

Assessing product environmental performance with PEF methodology: reliability,comparability, and cost concerns

Purpose

The Product Environmental Footprint (PEF) method was launched by the European Commission to harmonize the measurement of the product’s environmental performance. The PEF aims to increase the supply of green products in EU markets by lowering the cost of measuring the product’s environmental performance, enabling reliable environmental claims, and conducting proper product comparison. This study assesses whether the methodological choices of PEF enable reliable and comparable PEF studies to be conducted in a cost-efficient way.

Methods

The PEF was compared with ISO 14044, ISO/TS 14067, and the Greenhouse Gas Protocol Product Standard to identify the differences in the methodological choices. From a practical perspective, the possible challenges for PEF that could not be identified by a method comparison were obtained via interviews. The interviewees were company representatives participating in a 3-year PEF pilot phase to create Product Environmental Footprint Category Rules (PEFCR). To discover the possible advantages and disadvantages of the PEF, the methodological choices for PEF itself were assessed from the standpoints of reliability, comparability, and cost. In conclusion, some proposals for improvements have been suggested.

Results and discussion

The impact assessment phase, i.e., some of the predefined impact assessment methods, together with a selected normalization method include disadvantages as regards reliability and comparability. They are not sufficiently mature to properly reflect both the product studied and its potential environmental impact. From a cost perspective, the inventory analysis phase increases the workload due to the cutoff ban, the numerous predefined impact assessment categories, and the data quality assessment of all data. However, the predefined issues, e.g., requirements for data quality to be determined in PEFCRs, could also increase the comparability and lower the costs of the PEF study, since these need not be determined separately for each study. Additionally, the reliability maybe increased due to the data quality assessment of all data.

Conclusions

Currently, in the PEFs 3-year pilot phase, both advantages and disadvantages exist as regards reliability, costs, and comparability. Since PEF aims are important, the Commission and PEFCR developers should devote time to finding the most appropriate methodological choices and continue developing the method further. It is important to find a balance between comparability, reliability, and costs. Additionally, the current issues and characteristics of EU green products market should be taken into account when implementing PEF.

     

Detection of Innate Immune Response Modulating Impurities in Therapeutic Proteins

Therapeutic proteins can contain multiple impurities, some of which are variants of the product, while others are derived from the cell substrate and the manufacturing process. Such impurities, even when present at trace levels, have the potential to activate innate immune cells in peripheral blood or embedded in tissues causing expression of cytokines and chemokines, increasing antigen uptake, facilitating processing and presentation by antigen presenting cells, and fostering product immunogenicity. Currently, while products are tested for host cell protein content, assays to control innate immune response modulating impurities (IIRMIs) in products are focused mainly on endotoxin and nucleic acids, however, depending on the cell substrate and the manufacturing process, numerous other IIRMI could be present. In these studies we assess two approaches that allow for the detection of a broader subset of IIRMIs. In the first, we use commercial cell lines transfected with Toll like receptors (TLR) to detect receptor-specific agonists. This method is sensitive to trace levels of IIRMI and provides information of the type of IIRMIs present but is limited by the availability of stably transfected cell lines and requires pre-existing knowledge of the IIRMIs likely to be present in the product. Alternatively, the use of a combination of macrophage cell lines of human and mouse origin allows for the detection of a broader spectrum of impurities, but does not identify the source of the activation. Importantly, for either system the lower limit of detection (LLOD) of impurities was similar to that of PBMC and it was not modified by the therapeutic protein tested, even in settings where the product had inherent immune modulatory properties. Together these data indicate that a cell-based assay approach could be used to screen products for the presence of IIRMIs and inform immunogenicity risk assessments, particularly in the context of comparability exercises.     

Development and qualification of a representative scale-down model of automated Ficoll-based processing of a cell-based therapeutic according to quality by design principles

Background aimsThis article describes the development of a small-scale model for Ficoll-based cell separation as part of process development of an advanced therapy medicinal product and its qualification. Because of the complexity of biological products, their manufacturing process as well as characterization and control needs to be accurately understood. Likewise, scale-down models serve as an indispensable tool for process development, characterization, optimization and validation. This scale-down model represents a cell processor device widely used in advance therapies. This approach is inteded to optimise resources and to focus its use on process characterisation studies under the paradigm of the Quality by design. A scale-down model should reflect the large manufacturing scale. Consequently, this simplified system should offer a high degree of control over the process parameters to depict a robust model, even considering the process limitations. For this reason, a model should be developed and qualified for the intended purpose.MethodsProcess operating parameters were studied, and their resulting performance at full scale was used as a baseline to guide scale-down model development. Once the model was established, comparability runs were performed by establishing standard operating conditions with bone marrow samples. These analyses showed consistency between the bench and the large scale. Additionally, statistical analyses were employed to demonstrate equivalence.ResultsThe process performance indicators and assessed quality attributes were equivalent and fell into the acceptance ranges defined for the large-scale process.ConclusionsThis scale-down model is suitable for use in process characterization studies.     

Regulatory affairs and biotechnology in Europe: III. Introduction into good regulatory practice — Validation of virus removal and inactivation

This paper provides for an overview on the practical consequences of the EC guideline (III/8115/89): Validation of Virus Removal and Inactivation. This guideline can only be used as a blueprint in combination with other specific guidelines, especially those concerned with EC recommendations during production and quality control for various biotech products.A potential risk associated with the production and use of biological products is viral contamination. This contamination may be present in the source material, eg. human blood, human or animal tissues, cell banks, or introduced in the manufacturing process through the use of animal sera (eg. foetal calf serum or trypsin) in cell culture supernatant.The objectives of validation are to establish — ideally both qualitatively as well as quantitatively — the overall level of virus clearance. Evidence of viral clearance must be obtained in all stages of purification and adequate viral removal and/or inactivation must be proven. The method used when validating viral removal and /or inactivation is by challenging the system through the deliberate addition (spiking) of significant amounts of virus into the crude material to be purified and to different fractions obtained during the various purification stages. Removal or inactivation of the virus during the subsequent stages of purification and /or inactivation is thereafter determined.Such a quality system is by no means a simple one: it is estimated that in some production lines around 600 Standard Operating Procedures are necessary to guarantee the quality and the safety of the desired biotechnological product. Small companies will probably not be able to perform all procedures needed for obtaining the desired quality of the product. Then, external laboratories may take over a part of the Part II development requirements, which may not be representative for the total of internal Quality Assurance. New developments in the production and quality control of biotechnological products may require that companies should introduce novel, sophisticated methods such as: polymerase chain reaction (PCR), as yet not recommended by the CPMP in detail.Abbreviations III/8115/89 EC     

Comparability studies of new 3rd generation recombinant human factor VIII GreenGene F after improvement of formulation and viral inactivation/removal process

A new 3rd generation recombinant factor VIII (rFVIII), GreenGene F (WHO INN: beroctocog alfa), which is a highly homogenous B-domain deleted FVIII protein comprising of two peptides as heavy chain (A1 and A2 domain) and light chain (A3, C1, and C2 domain) at 80 and 90 kDa, was developed from its predecessor product GreenGene (2nd generation product previously approved by Korea FDA after clinical studies in South Korea) by process improvements of i) addition of Solvent/Detergent treatment for virus inactivation, ii) nanofiltration (20 nm pore size) for viral removal and iii) alterations to an albumin-free formulation to minimize the risk of viral contamination. An assessment of comparability between the two products was made to see if process improvements for safer product manufacturing affected the rFVIII structural and functional characteristics. Physicochemical and physiological characteristics were observed, in vivo efficacy following a single intravenous administration to FVIII knock-out mice and toxicity by various GLP in vivo tests were evaluated. All results showed equivalence, proving that no changes in protein characteristics of rFVIII occurred from process changes in formulation, viral inactivation, and viral removal which minimize the risk of pathogen transmission to enhance safety.