Quality Of Biopharmaceuticals Research Articles

Optimization of continuous spin-freeze-drying: The role of spin-freezing on quality attributes and drying efficiency of a model peptide formulation

Continuous spin-freeze-drying is an innovative pharmaceutical manufacturing approach offering real-time monitoring and control at the individual vial level, unlike conventional batch lyophilization. A central feature of this technology is spin-freezing, which involves rapidly spinning liquid-filled vials under a precisely controlled cold gas flow, resulting in a thin, uniform frozen product layer. Using a model peptide formulation, we investigated the impact of different cooling and crystallization rates on quality attributes (QA) and primary drying duration. Key QAs included monomer content, peptide assay, moisture content, and pore structure. The monomer content, peptide content, and primary drying duration remained consistent across all spin-freezing conditions. However, scanning electron microscopy (SEM) and Karl Fischer titration revealed that freezing parameters significantly influenced pore structure and residual moisture content. Samples with smaller pores displayed lower residual moisture, as larger surface areas facilitate moisture desorption. Variations in freezing parameters also significantly impacted desorption kinetics during secondary drying. Slower crystallization rates led to more cracks and less shrinkage in the cake structure, while faster rates resulted in more uniform, stable cakes. Although specific to the product under study, these findings highlight the crucial role of spin-freezing in enhancing freeze-drying efficiency and product quality of biopharmaceuticals.

An Overview of Biotechnological Drug’s Various Techniques of Downstream Process, Guideline’s And Different Chromatographic Analysis

Abstract: Biotechnology, a field discovered in 1919, unites biology and engineering to harness living organisms for medical purposes. Fueled by using DNA's discovery in the 1950s, biotechnology has converted through genetic engineering, yielding impactful merchandise regulated by means of entities like the FDA. The manufacturing involves upstream and downstream processing including the various techniques involved in the downstream processing of biotechnological drugs, along with relevant guidelines and chromatographic analysis methods. The biotechnological industry, which integrates biological science with engineering, has significantly advanced since the discovery of DNA's structure, leading to the development of biopharmaceuticals. These drugs, including monoclonal antibodies, recombinant proteins, and gene therapies, are produced using living organisms and hold the potential for treating complex diseases. The downstream process, a crucial phase in biopharmaceutical production, involves the purification and formulation of drug products to meet stringent regulatory standards. Traditional techniques such as centrifugation, filtration, and chromatography are employed to extract and purify biopharmaceuticals. Chromatographic techniques, including ion exchange, affinity, and size exclusion chromatography, play a pivotal role in achieving the desired purity levels. However, these methods are often time-- consuming and expensive, necessitating continuous advancements in the field. The paper highlights the importance of regulatory guidelines, including cGMP, in ensuring the quality and safety of biopharmaceuticals. It also discusses the significant role of organizations such as the FDA and EMA in regulating biotechnological drug production. The evolution of downstream processing techniques and the development of novel methods promise greater efficiency, scalability, and cost-effectiveness in biopharmaceutical production. Understanding these advancements is essential for continued growth and innovation in the industry, ultimately contributing to improved patient care and pharmaceutical innovation.

Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs.

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.

Platform development for high-throughput optimization of perfusion processes:Part I: Implementation of cell bleeds in microwell plates.

The promise of continuous processing to increase yields and improve product quality of biopharmaceuticals while decreasing the manufacturing footprint is transformative. Developingand optimizing perfusion operations requires screening various parameters, which is expensive and time-consuming when using benchtop bioreactors. Scale-down models (SDMs) are the most feasible option for high-throughput data generation and condition screening. However, new SDMs mimicking perfusion are required, enabling experiments to be run in parallel. In this study, a method using microwell plates (MWP) operating in semi-perfusion mode with an implemented cell bleed step is presented. A CHO cell line was cultivated in a 24-well MWP (Vw = 1.2 mL) and grown at four high cell density (HCD) setpoints. Quasi steady-state condition was obtained by manually performing cell bleeds followed by a total medium exchange after centrifugation. Further, two HCD setpoints were scaled up (VW = 30 mL), comparing a squared six-well deepwell plate (DWP) to shake flasks (SF). This evaluation showed comparable results between systems (DWP vs.SF) and scales (MWP vs.DWP + SF). The results show that the well-plate-based methods are suitable to perform HCD and quasi steady-state cultivations providing a robust solution to industrially relevant challenges such as cell clone and media selection.

Deep learning-based image analysis for in situ microscopic imaging of cell culture process

Mammalian cell culture is an important bioprocess that directly affects the quality and yield of biopharmaceuticals. Traditionally, condition monitoring of the operation is based on sampling periodically and off-line analysis, which is labor intensive, time consuming, and causing time delays. In this work, in situ microscope is investigated for on-line real-time monitoring of the culture process of Chinese hamster ovary cells with focus on investigation of deep learning-based Mask R-CNN algorithm for image analysis. The model is trained by 184 images with 183,040 cells using data augmentation methods and transfer learning technique. Mask R-CNN segmented the clustered cells more effectively than the conventional one combining edge detection, intensity thresholding, and advanced watershed method as well as the multi-scale edge detection method. Its Dice score, accuracy, precision, sensitivity, F1 score, specificity, and relative volume difference reach 0.862, 0.945, 0.901, 0.827, 0.862, 0.977, and 0.082, respectively. The evolution of geometrical features of cells were further analyzed, including equivalent diameter, circularity, aspect ratio, and eccentricity. The result demonstrated the great potential of deep learning technology in analysis of on-line images for optimization and control of the cell culture process.

Post-approval quality-related regulatory actions for biopharmaceuticals approved in the European Union and the United States between 1995 and 2019

The quality of biopharmaceuticals is carefully monitored by manufacturers and regulators to ensure safety and efficacy throughout the entire product life cycle. Quality defects can lead to post-approval regulatory actions (RAs) to inform healthcare professionals (HCPs). The present study identified quality-related RAs for biopharmaceuticals approved in the European Union and United States between 1995 and 2019. Quality-related RAs were issued due to various quality defects and required different actions by HCPs. The quality defects were not identified due to a negative impact on efficacy and/or safety, which is reassuring. The findings reflect the capability of the stringent regulatory system and quality control to capture and counter various quality defects before the affected product and batches can harm patients.

Identification of an Oxidizing Leachable from a Clinical Syringe Rubber Stopper.

Leaching of toxic or reactive chemicals from polymeric materials can adversely affect the quality and safety of biopharmaceuticals. It was therefore the aim of the present study to analyze leachables from a disposable clinical administration syringe using a polysorbate-containing surrogate solution and to assess their chemical reactivity. Analytical methods did include (headspace) GC-MS, Fourier-transform-infrared spectroscopy, a ferrous oxidation-xylenol orange assay, and nuclear magnetic resonance analysis. In the syringe leachables solution, the carcinogenic 1,1,2,2-tetrachloroethane (TCE) was detected in concentrations above the ICH M7-derived analytical evaluation threshold. TCE was shown to be an oxidation product of dichloromethane used during sample preparation. Since TCE was only isolated from incubations with the contained rubber stopper, we hypothesized that a stopper-derived leachable acted as a reactive oxidant promoting this chemical reaction. Subsequently, the leachable was identified to be the polymerization initiator Luperox® 101. Combining different analytical approaches led to the structural elucidation of a chemical reactive oxidant, which has the potential to interact and alter drug products. We conclude that chemically reactive compounds, such as the newly identified rubber stopper leachable Luperox® 101, may be of concern and therefore should be routinely considered if a prolonged exposure of polymers with drug products can be anticipated.

Practical Use of Analytical Tools for Glycan Structures Related to Functionality and Safety of Biopharmaceuticals

It is known that glycosylation of biopharmaceuticals relates to drug potency and half-life in blood. Since glycan modification is essential for the effector effect in antibody drugs, biopharmaceuticals are produced by using biosynthesis of mammalian cells but not microorganisms like as Escherichia coli. In particular, a biantennary N-glycan attached on Fc region of antibody is an important factor for interaction with its receptor FcRIIIa, and lack of a core-fucose residue attached to innermost GlcNAc at reducing end of N-glycan dramatically enhances interaction between FcRIIIa and IgG. Recently, it is reported that a particular galactose residue at non-reducing end of biantennary N-glycan also promotes the interaction with FcRIIIa receptor. On the other hand, glycan structure of post-translational modification by the use of mammalian-derived cells for production of biopharmaceuticals is different from that of human in some cases, and non-human type glycans is observed sometimes. For instance, high-mannose N-glycan is observed in unpreferable process step of manufacturing production and non-human type glycans are found depend on host cell type. Antigenicity of α1,3galactose (α1,3Gal) antigen and N-glycolylneuraminic acid (NeuGc) are observed at non-human type glycans well-known as xenoantigen. Because human lacks the enzyme activities of α1,3Gal and NeuGc synthesis for during the process of evolution with acquired gene mutations, immune response may act as a exogeneous antigen when they are injected to human patients. Therefore, contamination of antigenic glycosylation is considered to affect the safety of biopharmaceuticals. Currently, general standards have not been established regarding methods for glycan analysis from the viewpoint of the functionality and safety of biopharmaceuticals. Furthermore, it seems that the comprehensive supply system of glycan standards and recognition probes is still partial. We will report our developed tools regarding glycan standards and recognition probes. The glycan standards are available for quantitative analysis of reducing terminal core fucose and non-reducing terminal galactose (G2 and 6-G1 glycans) to evaluate the effector function of antibody drugs. The recognition probes derived from chicken are expected to be applied for qualitative analysis of an allergy risk factor caused by xenoantigen (α1,3Gal and NeuGc epitopes). The tools developed in this study would contribute to the analysis of glycan structures that affect the quality of biopharmaceuticals.

Utilization of Glucocorticoids as Additives for Enhanced Sialylation of Fc-fusion Protein in CHO Cell Cultures

Sialylation, which is considered as a critical quality attribute, plays important roles in pharmacokinetics of recombinant proteins. Terminal sialic acids of glycoproteins are modulated by intracellular sialyltransferase (ST) and extracellular sialidase. Since CHO cells producing recombinant proteins can only transfer α2,3-sialic acid to a nascent oligosaccharide, inactivation of α2,6-ST results in a different composition of glycan with human proteins, which have predominantly α2,6-linkages. To overcome this problem and maximize total sialic acid contents, we used glucocorticoids (GCs) that are classified as a steroid hormone in CHO cells expressing both α2,3-ST and α2,6-ST. This study was performed to determine the effect of GCs on CHO cell cultures and sialylation of Fc-fusion protein. We observed that all cultures supplemented with GCs reduced the titer of the Fc-fusion protein, but enhanced the sialylation level, when compared to those of control. Especially, addition of corticosterone increases the levels of α2,6-sialylation as well as the total contents of sialic acid. Enhanced sialic acid contents are thought to be due to the intracellular improvement by sialylatransferase and the inhibition of sialidase. In conclusion, GCs can be utilized as an effective medium additive to improve the quality of biopharmaceuticals using CHO cell cultures.

Multiplex secretome engineering enhances recombinant protein production and purity

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

Sensitive detection of glucagon aggregation using amyloid fibril-specific antibodies.

Sensitive detection of protein aggregates is importantfor evaluatingthe quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay-which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates-can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5-1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.

Looking inside the ‘black box’: Freezing engineering to ensure the quality of freeze-dried biopharmaceuticals

The freezing step plays a central role in reaching the most stringent requirements of quality, homogeneity and standardization of freeze-dried products. In this paper, a systematic procedure has been proposed to obtain a quantitative estimation of the pore-size variability of lyophilized products resulting from uncontrollable variations of the nucleation temperature. This procedure consisted in collecting the nucleation temperature from a statistically significant number of samples and correlating each nucleation temperature to the corresponding product morphology, using a mathematical model, to obtain a statistical description of the lyophilized product structure. This approach can also be used to obtain an estimation of the variability of the mass transfer resistance to vapor flow and, finally, of the drying time. Two different freezing configurations, i.e., conventional and suspended-vial freezing, have been used as case studies since they can produce significantly different freezing rates.

Coupling of on-column trypsin digestion-peptide mapping and principal component analysis for stability and biosimilarity assessment of recombinant human growth hormone.

Peptide mapping (PM) is a vital technique in biopharmaceutical industry. The fingerprint obtained helps to qualitatively confirm host stability as well as verify primary structure, purity and integrity of the target protein. Yet, in-solution digestion followed by tandem mass spectrometry is not suitable as a routine quality control test. It is time consuming and requires sophisticated, expensive instruments and highly skilled operators. In an attempt to enhance the fuctionality of PM and extract multi-dimentional data about various critical quality attributes and comparability of biosimilars, coupling of PM generated using immobilized trypsin followed by HPLC-UV to principal component analysis (PCA) is proposed. Recombinant human growth hormone (rhGH); was selected as a model biopharmaceutical since it is available in the market from different manufacturers and its PM is a well-established pharmacopoeial test. Samples of different rhGH biosimilars as well as degraded samples: deamidated and oxidized were subjected to trypsin digestion followed by RP-HPLC-UV analysis. PCA of the entire chromatograms of test and reference samples was then conducted. Comparison of the scores of samples and investigation of the loadings plots clearly indicated the applicability of PM-PCA for: i) identity testing, ii) biosimilarity assessment and iii) stability evaluation. Hotelling's T2 and Q statistics were employed at 95% confidence level to measure the variation and to test the conformance of each sample to the PCA model, respectively. Coupling of PM to PCA provided a novel tool to identify peptide fragments responsible for variation between the test and reference samples as well as evaluation of the extent and relative significance of this variability. Transformation of conventional PM that is largely based on subjective visual comparison into an objective statiscally-guided analysis framework should provide a simple and economic tool to help both manufacturers and regulatory authorities in quality and biosimilarity assessment of biopharmaceuticals.

Boosted structured additive regression for Escherichia coli fed-batch fermentation modeling.

The quality of biopharmaceuticals and patients' safety are of highest priority and there are tremendous efforts to replace empirical production process designs by knowledge-based approaches. Main challenge in this context is that real-time access to process variables related to product quality and quantity is severely limited. To date comprehensive on- and offline monitoring platforms are used to generate process data sets that allow for development of mechanistic and/or data driven models for real-time prediction of these important quantities. Ultimate goal is to implement model based feed-back control loops that facilitate online control of product quality. In this contribution, we explore structured additive regression (STAR) models in combination with boosting as a variable selection tool for modeling the cell dry mass, product concentration, and optical density on the basis of online available process variables and two-dimensional fluorescence spectroscopic data. STAR models are powerful extensions of linear models allowing for inclusion of smooth effects or interactions between predictors. Boosting constructs the final model in a stepwise manner and provides a variable importance measure via predictor selection frequencies. Our results show that the cell dry mass can be modeled with a relative error of about ±3%, the optical density with ±6%, the soluble protein with ±16%, and the insoluble product with an accuracy of ±12%. Biotechnol. Bioeng. 2017;114: 321-334. © 2016 Wiley Periodicals, Inc.

Shaping the quality of biopharmaceuticals – a multiscale perspective on the utilization of big data for efficient bioprocess development

Shaping the quality of biopharmaceuticals – a multiscale perspective on the utilization of big data for efficient bioprocess development

Molecular simulations of the pairwise interaction of monoclonal antibodies.

Molecular simulations are employed to compute the free energy of pairwise monoclonal antibodies (mAbs) association using a conformational sampling algorithm with a scoring function. The work reported here is aimed at investigating the mAb-mAb association driven by weak interactions with a computational method capable of predicting experimental observations of low binding affinity. The simulations are able to explore the free energy landscape. A steric interaction component, electrostatic interactions, and a nonpolar component of the free energy form the energy scoring function. Electrostatic interactions are calculated by solving the Poisson-Boltzmann equation. The nonpolar component is derived from the van der Waals interactions upon close contact of the protein surfaces. Two mAbs with similar IgG1 framework but with small sequence differences, mAb1 and mAb2, are considered for their different viscosity and propensity to form a weak interacting dimer. mAb1 presents favorable free energy of association at pH 6 with 15 mM of ion concentration reproducing experimental trends of high viscosity and dimer formation at high concentration. Free energy landscape and minimum free energy configurations of the dimer, as well as the second virial coefficient (B22) values are calculated. The energy distributions for mAb1 are obtained, and the most probable configurations are seen to be consistent with experimental measurements. In contrast, mAb2 shows an unfavorable average free energy at the same buffer conditions due to poor electrostatic complementarity, and reversible dimer configurations with favorable free energy are found to be unlikely. Finally, the simulations of the mAb association dynamics provide insights on the self-association responsible for bulk solution behavior and aggregation, which are important to the processing and the quality of biopharmaceuticals.

Process analytical technology (PAT) tools for the cultivation step in biopharmaceutical production

The process analytical technology (PAT) initiative is now 10 years old. This has resulted in the development of many tools and software packages dedicated to PAT application on pharmaceutical processes. However, most applications are restricted to small molecule drugs, mainly for the relatively simple process steps like drying or tableting where only a limited number of parameters need to be controlled. A big challenge for PAT still lies in applications for biopharmaceuticals and then especially in the cultivation process step, where the quality of a biopharmaceutical product is largely determined. This review gives an overview of the currently available tools for monitoring and controlling the biopharmaceutical cultivation step and of the main challenges for the most common cell platforms (i.e. Escherichia coli, yeast, and mammalian cells) used in biopharmaceutical manufacturing. The real challenge is to understand how intracellular mechanisms (from synthesis to excretion) influence the quality of biopharmaceuticals and how these mechanisms can be monitored and controlled to yield the desired end product quality. Modern “omics” tools and advanced process analyzers have opened up the way for PAT applications for the biopharmaceutical cultivation process step.

Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells

Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times.

Overview of the nonclinical quality and toxicology testing for recombinant biopharmaceuticals produced in mammalian cells

There was a mistake in the running title of the paper that was published in Journal of Applied Toxicology in issue 2010: 30: 387–396 (DOI 10.1002/jat.1551). “DNA damage and behavioral deficits induced by amantadine” should be “Quality and Toxicology Testing of Biopharmaceuticals”. The publisher apologises for this error.

A new CZE method for profiling human serum albumin and its related forms to assess the quality of biopharmaceuticals

We present a new CZE method, which uses a polyethylene oxide-coated capillary to separate native HSA from more than five of its structural variants. These variants include oxidized, truncated, and cysteinylated forms of HSA which can all be found in biopharmaceutical products. Both CE and MS confirmed the high degree of heterogeneity of HSA preparations. Recovery studies demonstrated that adsorption of HSA on the capillary was significantly reduced under the conditions we developed, which led to a satisfactory repeatability (RSD for migration times and relative peak areas were less than 0.2 and 7.0%, respectively). Assignment of the main peaks was attempted using in vitro degraded/stressed HSA. We used our method to test batch-to-batch comparability and detected slight quantitative differences in the proportion of native HSA in batches produced from different fractionation methods.