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.     

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.     

Regulatory perspectives from Japan - comparability of biopharmaceuticals.

In Japan there is no official guideline about comparability assessment of biotechnological products at present. However, there is some notifications which should be referred to, when the manufacturer changes the manufacturing process. Here, regulatory perspectives from Japan on the comparability assessment are presented. When establishing the comparability of biotechnological products derived from different manufacturing processes and the validity of modified manufacturing process, rational step-by-step approaches based on both product and process aspects would be useful. At first, relevant physicochemical and biological properties of products including purity, impurity profiles and stability should be compared before and after the manufacturing change, depending on the type and nature of the desired products. It is also necessary to examine the capacities of the new manufacturing process for ensuring the consistent production of the active protein product as well as the anticipated elimination of potential impurities and contaminants. Further relevant assessment of preclinical and clinical comparability of product may be necessary in some cases.     

Bioanalytical strategy used in development of pharmacokinetic (PK) methods that support biosimilar programs

The development of biosimilar products is expected to grow rapidly over the next five years as a large number of approved biologics reach patent expiry. The pathway to regulatory approval requires that similarity of the biosimilar to the reference product be demonstrated through physiochemical and structural characterization, as well as within in vivo studies that compare the safety and efficacy profiles of the products. To support nonclinical and clinical studies pharmacokinetic (PK) assays are required to measure the biosimilar and reference products with comparable precision and accuracy. The most optimal approach is to develop a single PK assay, using a single analytical standard, for quantitative measurement of the biosimilar and reference products in serum matrix. Use of a single PK assay for quantification of multiple products requires a scientifically sound testing strategy to evaluate bioanalytical comparability of the test products within the method, and provide a solid data package to support the conclusions. To meet these objectives, a comprehensive approach with scientific rigor was applied to the development and characterization of PK assays that are used in support of biosimilar programs. Herein we describe the bioanalytical strategy and testing paradigm that has been used across several programs to determine bioanalytical comparability of the biosimilar and reference products. Data from one program is presented, with statistical results demonstrating the biosimilar and reference products were bioanalytically equivalent within the method. The cumulative work has established a framework for future biosimilar PK assay development.     

Bioanalytical strategy used in development of pharmacokinetic (PK) methods that support biosimilar programs

The development of biosimilar products is expected to grow rapidly over the next five years as a large number of approved biologics reach patent expiry. The pathway to regulatory approval requires that similarity of the biosimilar to the reference product be demonstrated through physiochemical and structural characterization, as well as within in vivo studies that compare the safety and efficacy profiles of the products. To support nonclinical and clinical studies pharmacokinetic (PK) assays are required to measure the biosimilar and reference products with comparable precision and accuracy. The most optimal approach is to develop a single PK assay, using a single analytical standard, for quantitative measurement of the biosimilar and reference products in serum matrix. Use of a single PK assay for quantification of multiple products requires a scientifically sound testing strategy to evaluate bioanalytical comparability of the test products within the method, and provide a solid data package to support the conclusions. To meet these objectives, a comprehensive approach with scientific rigor was applied to the development and characterization of PK assays that are used in support of biosimilar programs. Herein we describe the bioanalytical strategy and testing paradigm that has been used across several programs to determine bioanalytical comparability of the biosimilar and reference products. Data from one program is presented, with statistical results demonstrating the biosimilar and reference products were bioanalytically equivalent within the method. The cumulative work has established a framework for future biosimilar PK assay 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.     

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.     

Quantitation and pharmacokinetic modeling of therapeutic antibody quality attributes in human studies

A thorough understanding of drug metabolism and disposition can aid in the assessment of efficacy and safety. However, analytical methods used in pharmacokinetics (PK) studies of protein therapeutics are usually based on ELISA, and therefore can provide a limited perspective on the quality of the drug in concentration measurements. Individual post-translational modifications (PTMs) of protein therapeutics are rarely considered for PK analysis, partly because it is technically difficult to recover and quantify individual protein variants from biological fluids. Meanwhile, PTMs may be directly linked to variations in drug efficacy and safety, and therefore understanding of clearance and metabolism of biopharmaceutical protein variants during clinical studies is an important consideration. To address such challenges, we developed an affinity-purification procedure followed by peptide mapping with mass spectrometric detection, which can profile multiple quality attributes of therapeutic antibodies recovered from patient sera. The obtained data enable quantitative modeling, which allows for simulation of the PK of different individual PTMs or attribute levels in vivo and thus facilitate the assessment of quality attributes impact in vivo. Such information can contribute to the product quality attribute risk assessment during manufacturing process development and inform appropriate process control strategy.     

Assessing the Risk of Herbicides to Terrestrial Non-Target Plants Using Higher-Tier Studies

Risk assessment for non-target plants is based on single species phytotoxicity tests. This approach may not reflect relevant ecological processes in terrestrial ecosystems. The current risk assessment scheme is based on endpoints measured at the species level and the assessment of ecological effects relies on the extrapolation from one species to another or from a single species to a community. This extrapolation contains many uncertainties that may be reduced by adopting more realistic testing approaches. However, currently higher-tier plant studies are not obligatory in herbicide risk assessment. We reviewed the published literature and found that potential higher-tier approaches for terrestrial non-target plants are extremely limited. Sixteen studies were found that assessed the effects of herbicides on non-target plants by performing microcosms, mesocosms, or field studies. These studies showed that microcosms might provide useful data and help to reduce uncertainties associated with single-species tests. However, due to the limited number of available studies, much work is required to develop appropriate testing methods for regulatory processes. In addition, field experiments are necessary to establish baseline knowledge concerning the effects of herbicides on natural plant communities and to compare data generated in tiered testing approaches with data obtained from natural systems.     

Guidelines for safety tests and registration of bacterial pesticides

Guidelines have been formulated for safety tests and registration of new microbial pesticides containing pathogenic or inhibitory bacteria. These guidelines are intended for the advice of interested firms and governmental agencies. They are based on over a decade's experience and intense recent activity in this field. For the registration of a new microbial product, information is required on the identity of the new bacterium, its biological properties, production, formulation, quality control, application and efficacy. For safety assessment a series of tests on the infectivity, toxicity and allergenicity etc. in laboratory mammals is arranged in 3 tiers. Entirely negative results in tier 1 indicate acceptance of the product as safe. Positive results may lead to its rejection or to further lengthier tests in tiers 2 and 3, culminating in rejection or acceptance after a risk-benefit analysis, possibly with restrictions concerning its use required on the label. Data on residues, inactivation and degradability may be necessary. Information on exposure of humans during production and use are particularly valuable. The intended use of the product greatly influences both safety test requirements and tests on non-target organisms and wildlife, e.g. bees, important predators and parasites of the target species, earthworms, birds and fish. Also phytotoxicity and phytopathogenicity must be considered. Aspects of current interest are critically discussed. Any fundamental studies not justified as mandatory requirements for testing a new product should be funded by non-industrial sources.     

Evaluation of the structural,physicochemical, and biological characteristics of SB4, a biosimilar of etanercept

A biosimilar is a biological medicinal product that is comparable to a reference medicinal product in terms of quality, safety, and efficacy. SB4 was developed as a biosimilar to Enbrel® (etanercept) and was approved as Benepali®, the first biosimilar of etanercept licensed in the European Union (EU). The quality assessment of SB4 was performed in accordance with the ICH comparability guideline and the biosimilar guidelines of the European Medicines Agency and Food and Drug Administration. Extensive structural, physicochemical, and biological testing was performed with state-of-the-art technologies during a side-by-side comparison of the products. Similarity of critical quality attributes (CQAs) was evaluated on the basis of tolerance intervals established from quality data obtained from more than 60 lots of EU-sourced and US-sourced etanercept. Additional quality assessment was focused on a detailed investigation of immunogenicity-related quality attributes, including hydrophobic variants, high-molecular-weight (HMW) species, N-glycolylneuraminic acid (NGNA), and α-1,3-galactose. This comprehensive characterization study demonstrated that SB4 is highly similar to the reference product, Enbrel®, in structural, physicochemical, and biological quality attributes. In addition, the levels of potential immunogenicity-related quality attributes of SB4 such as hydrophobic variants, HMW aggregates, and α-1,3-galactose were less than those of the reference product.     

Biosimilars entering the clinic without animal studies

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 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 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     

Tools to evaluate the conformation of protein products

Production of recombinant proteins is a process intensively used in the research laboratory. In addition, the main biotechnology market products are recombinant proteins and monoclonal antibodies. The biological (and clinical) properties of the protein product strongly depend on the conformation of the polypeptide. Therefore, assessment of the correct conformation of the produced protein is crucial. There is no single method to assess every aspect of protein structure or function. Depending on the protein, the methods of choice vary. There are general methods to evaluate not only mass and primary sequence of the protein, but also higher-order structure. This review outlines the principal techniques for determining the conformation of a protein from structural (biophysical methods) to functional (in vitro binding assays) analyses.     

Regionalization of agri-food life cycle assessment: a review of studies in Portugal and recommendations for the future

Purpose

One of the main trends in life cycle assessment (LCA) today is towards increased regionalization in inventories and impact assessment methods. LCA studies require the collection of activity data but also of increasingly region-specific background data to accurately depict supply chain processes and enable the application of an increasing number of geographically explicit impact assessment models. This is particularly important for agri-food products. In this review, we assess progress in Portugal towards this goal and provide recommendations for future developments.

Methods

We perform a comprehensive review of available LCA studies conducted for Portuguese agri-food products, in order to evaluate the current state of Portuguese agri-food LCA. Among other issues, we assess availability of data, methods used, level of regionalization, impact assessment model relevance and coherence for inter-product comparability. We also provide conclusions and recommendations based on recent developments in the field.

Results and discussion

We found 22 LCA studies, covering 22 different products. The analysis of these studies reveals limitations in inter-study comparability. The main challenges have to do with a lack of country-specific foreground data sources applied consistently in the studies found, with discrepancies in impact assessment categories, and with the use of simple functional units that may misrepresent the product analyzed.

Conclusions

We conclude that Portuguese agri-food LCA studies do not have a systematic and country-scale approach in order to guarantee regional accuracy and comparability. We propose a research strategy to engage the Portuguese agri-food LCA community in devising a consistent framework before practical application studies are conducted.

     

Evaluation of analytical similarity between trastuzumab biosimilar CT-P6 and reference product using statistical analyses

The evaluation of analytical similarity has been a challenging issue for the biosimilar industry because the number of lots for reference and biosimilar products available at the time of development are limited, whilst measurable quality attributes of target molecule are numerous, which can lead to potential bias or false negative/positive conclusions regarding biosimilarity. Therefore, appropriate statistical analyses are highly desirable to achieve a high level of confidence in the similarity evaluation. A recent guideline for the risk-based statistical approaches recommended by the US Food and Drug Administration provides useful tools to systematically evaluate analytical similarity of biosimilar products compared with reference products. Here, we evaluated analytical similarity of CT-P6, a biosimilar product of trastuzumab, with the reference products (EU-Herceptin® or US-Herceptin®) following these statistical approaches. Various quality attributes of trastuzumab were first ranked based on the clinical impact of each attribute and subsequently adjusted to one of three tiers (Tier 1, Tier 2 and Tier 3) considering the characteristics of the assay, the level of attribute present and the feasibility of statistical analysis. Two biological activities with highest potential clinical impact were evaluated by an equivalent test (Tier 1), and other bioactivities and structural/physicochemical properties relevant to the clinical impact were evaluated by a quality range approach (Tier 2). The attributes with low risk ranking or qualitative assay were evaluated by visual comparison (Tier 3). Analytical similarity assessment analyzed by the three tiers clearly demonstrated that CT-P6 exhibits highly similar structural and physicochemical properties, as well as functional activities, compared with the reference products. There were small differences observed in a few quality attributes between CT-P6 and the reference products, but the differences were very minor, and unlikely to impact on clinical outcome. The recently reported equivalent clinical efficacy of CT-P6 with the reference product further supports that CT-P6 is highly similar compared with the reference product in the view of totality-of-evidence.     

Physicochemical and biological characterization of SB2, a biosimilar of Remicade® (infliximab)

A biosimilar is a biological medicinal product that contains a version of the active substance of an already authorized original biological medicinal product. Biosimilarity to the reference product (RP) in terms of quality characteristics, such as physicochemical and biological properties, safety, and efficacy, based on a comprehensive comparability exercise needs to be established. SB2 (Flixabi® and Renflexis®) is a biosimilar to Remicade® (infliximab). The development of SB2 was performed in accordance with relevant guidelines of the International Conference on Harmonisation, the European Medicines Agency, and the United States Food and Drug Administration. To determine whether critical quality attributes meet quality standards, an extensive characterization test was performed with more than 80 lots of EU- and US-sourced RP. The physicochemical characterization study results revealed that SB2 was similar to the RP. Although a few differences in physicochemical attributes were observed, the evidence from the related literature, structure-activity relationship studies, and comparative biological assays showed that these differences were unlikely to be clinically meaningful. The biological characterization results showed that SB2 was similar to the RP in terms of tumor necrosis factor–α (TNF-α) binding and TNF-α neutralization activities as a main mode of action. SB2 was also similar in Fc-related biological activities including antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, neonatal Fc receptor binding, C1q binding, and Fc gamma receptor binding activities. These analytical findings support that SB2 is similar to the RP and also provide confidence of biosimilarity in terms of clinical safety and efficacy.     

Quality attributes of recombinant therapeutic proteins: an assessment of impact on safety and efficacy as part of a quality by design development approach

Quality by Design (QbD) is a new approach to the development of recombinant therapeutic protein products that promotes a better understanding of the product and its manufacturing process. The first step in the QbD approach consists in identifying the critical quality attributes (CQA), i.e., those quality attributes of the product that have an impact on its clinical efficacy or safety. CQAs are identified through a science-based risk assessment taking into consideration a combination of clinical and nonclinical data obtained with the molecule or other similar molecules or platform products, as well as the published literature. The purpose of this article is to perform a comprehensive review of the published literature, supporting an assessment of the impact on safety and efficacy of the quality attributes commonly encountered in recombinant therapeutic proteins, more specifically those produced in mammalian cell expression systems. Quality attributes generally observed in biopharmaceutical proteins including product-related impurities and substances, process-related impurities, product attributes, and contaminants are evaluated one by one for their impact on biological activity, pharmacokinetics and pharmacodynamics, immunogenicity, and overall safety/toxicity.     

转基因植物对传粉蜂类影响的研究进展

随着转基因作物的大规模商业种植,其对非靶生物的安全性问题已经成为转基因生物风险评估的重要内容。有些转基因植物是需要蜂类传粉的显花植物．有些则可作为蜂类的食物来源。抗性转基因植物可能对蜂类产生直接和间接的影响。转基因植物的花粉或者花蜜可能对蜂类产生直接影响,测试化学杀虫剂安全性的相同方法被用来评估这种影响。很多研究测试了纯化的转基因蛋白质(如Bt,蛋白酶抑制剂)对蜜蜂和熊蜂的影响。间接影响来自于遗传转化可能使转基因植物的表型产生的异常变化,特别是花的改变。这种影响是用转基因植物进行评估的。转基因植物对蜂类的影响研究所测定的参数包括蜂类的肠道生理、取食和嗅觉学习行为、经口毒性和寿命等。对在实验室中以幼蜂或者成年蜂为对象、在封闭条件下针对蜂群的研究以及蜂类在转基因植物上的取食行为方面的研究进行了总结。结果表明,转基因植物是否对传粉蜂类产生影响以及影响的大小主要取决于转基因植物的生物学特征、转基因蛋白的性质和表达量。一般来说,Bt毒素蛋白和葡聚糖酶等蛋白质对蜂类没有影响;而某些蛋白酶抑制剂和几丁质酶则可能对蜂类产生不利影响,蜂类所摄取到这类蛋白质的剂量将决定影响的程度。然而,由于采用的研究方法各不相同,难以对不同的研究结果进行分析和比较,并得出明确的结论。