Recombinant Drug Production Research Articles

Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients.

Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.

СПЕЦИФИКА МОЛЕКУЛЫ МОРОКТОКОГА АЛЬФА: РЕЗУЛЬТАТЫ ПРОСПЕКТИВНОГО МНОГОЦЕНТРОВОГО НАБЛЮДАТЕЛЬНОГО ИССЛЕДОВАНИЯ ЭФФЕКТИВНОСТИ И БЕЗОПАСНОСТИ У ПОДРОСТКОВ С ГЕМОФИЛИЕЙ A

The main method of treatment for hemophilia A is replacement therapy with drugs of blood coagulation factors VIII (FVIII). The active use of biotechnological methods in the production of recombinant drugs contributes to the development and registration of new FVIII drugs. The results of clinical trials of drugs in children usually include a limited number of patients for a specific period of time. Post-marketing observational studies provide additional information on the results of using a new drug in patients in clinical practice. The objectives of the study were to collect and analyze data on the efficacy and safety of domestic recombinant FVIII with a deleted B-domain, moroctocog alfa (Octofactor, GENERIUM JSC) in the treatment of adolescents aged 12–18 years with hemophilia A in routine clinical practice. Materials and methods of research: a prospective multicenter open-label observational study of the results of using the Octofactor in adolescents with hemophilia A (№ OCF-HPA-N01) included 24 male patients with severe hemophilia A aged 12 to 18 years (mean age 14,8±1,7 years), who received moroctocog alfa in routine clinical practice. Enrollment in the study was carried out after the signing of the informed consent form by the parent of the minor patient and the minor patient aged 14 to 18 years, taking into account the results of the screening examination. The follow-up period was 52±4 weeks, which, presumably, was sufficient to achieve at least 100 days of moroctocog alfa administration. To assess the effectiveness, we analyzed the incidence of spontaneous bleeding that occurred within 48–72 h after drug administration; the severity of spontaneous bleeding against the background of prophylactic treatment with moroctocog alfa, the number of injections and the dose of the study drug for prophylactic treatment, as well as for treatment at the request of one episode of bleeding, taking into account its severity; the researcher's determination of the response to treatment of acute hemarthrosis according to the scale of the World Federation of Hemophilia (2020). To assess safety, the frequency and characteristics of adverse events (AEs) associated with drug administration were analyzed, including the frequency of formation of an inhibitor to FVIII. Results: during the study, 59 bleedings were recorded, of which 21 (36%) were spontaneous and 38 (64%) were post-traumatic. Among spontaneous bleeding episodes, 5 (24%) episodes occurred within 48–72 hours after administration of the study drug. Spontaneous bleeding within 48–72 hours after administration of Octofactor was absent against the background of prophylactic treatment in most patients (81%) and was observed rarely during the observation period (1–3 times) in the remaining 19% of patients. The median number of bleeding within 48–72 h after study drug administration was 1 episode over the follow-up period. The proportion of mild to moderate bleeding was 97 [88; 100]% among all types of bleeding and 100 [84; 100]% among spontaneous bleeding within 48–72 hours after administration of the Octofactor drug. The median of a single dose of Octofactor for preventive treatment was 2000 [1500; 2000] IU or 31,7 [25,6; 38,5] IU/kg, and with treatment on demand ‒ 2000 [2000; 3000] IU or 34,1 [28,8; 38,5] IU/kg per single injection. To stop the resulting bleeding required 1 [1; 2] introduction in a single dose of 3000 [2000; 4000] IU; the average dose was 4490 ÷ 4993 IU. When doctors subjectively assess the response to treatment of acute hemarthrosis on the scale of the World Federation of Hemophilia, an excellent response was noted in 9 (27%) episodes, good and moderate ‒ in 2 (6%) and 22 (67%) episodes, respectively. Lack of response to treatment of acute hemarthrosis with moroctocog alfa was not revealed in the study. During the study, 23 AEs were recorded in 13 (54%) patients not related to the study drug. Conclusion: thus, the obtained results of the study indicates the efficacy and safety of the Octofactor both the prophylactic treatment and treatment of on-demand bleedings in 12 to 18 year old patients with severe hemophilia A.

Postmarketing Safety of Biosimilars: Current Status, Challenges, and Opportunities in the Spontaneous Reporting System.

Recombinant drug products successfully treat many life-threatening and chronic diseases. The high cost of these drugs makes them inaccessible to the patients particularly in developing countries. Patent expiration of innovator recombinant drug products has led to the development of biosimilars or similar biologics by several manufacturers. Unlike generics, these are not identical to their innovator products because of the differences in the manufacturing process; however, they are similar in quality characteristics, biological activity, safety, and efficacy. The regulatory procedures used for generic drugs cannot be applied for biosimilars as they are large complex structures produced from living cells and can produce potential risk of immune-based adverse reactions. Out of several safety issues related to biosimilars, two main safety concerns are variable potency and immunogenicity, for which a robust long-term pharmacovigilance system is needed. Various guidelines have been issued for the regulatory approval and pharmacovigilance of biosimilars by USFDA, EU, and pharma-emerging countries like China and India. The article includes the pharmacovigilance plan of biosimilars in these countries, discusses the challenges and opportunities in pharmacovigilance through spontaneous reporting systems, and suggests amendments in the existing suspected adverse event reporting form of the Pharmacovigilance Programme of India.

Postmarketing Safety of Biosimilars: Current Status, Challenges, and Opportunities in the Spontaneous Reporting System.

Recombinant drug products successfully treat many life-threatening and chronic diseases. The high cost of these drugs makes them inaccessible to the patients particularly in developing countries. Patent expiration of innovator recombinant drug products has led to the development of biosimilars or similar biologics by several manufacturers. Unlike generics, these are not identical to their innovator products because of the differences in the manufacturing process; however, they are similar in quality characteristics, biological activity, safety, and efficacy. The regulatory procedures used for generic drugs cannot be applied for biosimilars as they are large complex structures produced from living cells and can produce potential risk of immune-based adverse reactions. Out of several safety issues related to biosimilars, two main safety concerns are variable potency and immunogenicity, for which a robust long-term pharmacovigilance system is needed. Various guidelines have been issued for the regulatory approval and pharmacovigilance of biosimilars by USFDA, EU, and pharma-emerging countries like China and India. The article includes the pharmacovigilance plan of biosimilars in these countries, discusses the challenges and opportunities in pharmacovigilance through spontaneous reporting systems, and suggests amendments in the existing suspected adverse event reporting form of the Pharmacovigilance Programme of India.

A new and updated resource for codon usage tables

BackgroundDue to the degeneracy of the genetic code, most amino acids can be encoded by multiple synonymous codons. Synonymous codons naturally occur with different frequencies in different organisms. The choice of codons may affect protein expression, structure, and function. Recombinant gene technologies commonly take advantage of the former effect by implementing a technique termed codon optimization, in which codons are replaced with synonymous ones in order to increase protein expression. This technique relies on the accurate knowledge of codon usage frequencies. Accurately quantifying codon usage bias for different organisms is useful not only for codon optimization, but also for evolutionary and translation studies: phylogenetic relations of organisms, and host-pathogen co-evolution relationships, may be explored through their codon usage similarities. Furthermore, codon usage has been shown to affect protein structure and function through interfering with translation kinetics, and cotranslational protein folding.ResultsDespite the obvious need for accurate codon usage tables, currently available resources are either limited in scope, encompassing only organisms from specific domains of life, or greatly outdated. Taking advantage of the exponential growth of GenBank and the creation of NCBI’s RefSeq database, we have developed a new database, the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs), to present and analyse codon usage tables for every organism with publicly available sequencing data. Compared to existing databases, this new database is more comprehensive, addresses concerns that limited the accuracy of earlier databases, and provides several new functionalities, such as the ability to view and compare codon usage between individual organisms and across taxonomical clades, through graphical representation or through commonly used indices. In addition, it is being routinely updated to keep up with the continuous flow of new data in GenBank and RefSeq.ConclusionGiven the impact of codon usage bias on recombinant gene technologies, this database will facilitate effective development and review of recombinant drug products and will be instrumental in a wide area of biological research. The database is available at hive.biochemistry.gwu.edu/review/codon.

Seroprevalence for 2117-like vesiviruses in Italian household dogs

In 2003, a novel calicivirus, the vesivirus (VeV) strain 2117, was identified incidentally as a contaminant in Chinese Hamster Ovary (CHO) cell cultures by a German pharmaceutical company. Similar contaminations have been documented in three additional episodes, in bio-reactors used for production of recombinant drugs. More, recently 2117-like VeVs have also been identified at high prevalence in the stools from asymptomatic kennel dogs and only sporadically in diarrhoeic and healthy household dogs. In this study, antibodies for 2117-like viruses were detected in 21.5% of sera from household dogs, indicating that they are common viruses in the canine host.

Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli.

BackgroundProduction of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free proteins, no data has been so far recorded regarding how the system performs in the fabrication of smart nanostructured materials.ResultsWe have here explored the nanoarchitecture and in vitro and in vivo functionalities of CXCR4-targeted, self-assembling protein nanoparticles intended for intracellular delivery of drugs and imaging agents in colorectal cancer. Interestingly, endotoxin-free materials exhibit a distinguishable architecture and altered size and target cell penetrability than counterparts produced in conventional E. coli strains. These variant nanoparticles show an eventual proper biodistribution and highly specific and exclusive accumulation in tumor upon administration in colorectal cancer mice models, indicating a convenient display and function of the tumor homing peptides and high particle stability under physiological conditions.DiscussionThe observations made here support the emerging endotoxin-free E. coli system as a robust protein material producer but are also indicative of a particular conformational status and organization of either building blocks or oligomers. This appears to be promoted by multifactorial stress-inducing conditions upon engineering of the E. coli cell envelope, which impacts on the protein quality control of the cell factory.

Trace element associated reduction of norleucine and norvaline accumulation during oxygen limitation in a recombinant Escherichia coli fermentation

BackgroundNorleucine and norvaline belong to a group of non-canonical amino acids which are synthesized as byproducts in the branched chain amino acid metabolism of Escherichia coli. The earlier observed misincorporation of these rare amino acids into recombinant proteins has attracted increasing attention due to the rising use of protein based biopharmaceuticals in clinical application. Experimental data revealed pyruvate overflow inducing conditions, which typically occur in oxygen limited zones of large-scale fermentations as a major reason leading to norvaline and norleucine synthesis during E. coli cultivation. Previous approaches to suppress misincorporation of norleucine and norvaline considered growth media supplementation with the relevant canonical isostructural compounds, but no research was performed on the impact of the overflow metabolism related trace elements molybdenum, nickel and selenium. These elements form essential parts of the formate hydrogen lyase (FHL) metalloprotein complex, which is a key enzyme of anaerobic pyruvate metabolism in E. coli and could therefore represent a crucial connection to the pyruvate accumulation associated biosynthesis of rare amino acids.ResultsIn this study, the trace element associated response of recombinant antibody producing E. coli to oxygen limitation at high glucose concentration with a special focus on non-canonical amino acids was analysed. During fed-batch cultivation with provoked oxygen limitation and glucose excess norleucine and norvaline were only accumulated in the absence of molybdenum, nickel and selenium. In contrast, the trace element supplemented stress fermentation showed significantly reduced concentrations of these rare amino acids and the major signature fermentation product formate, supporting the correlation between a functional formate hydrogen lyase complex and low unspecific amino acid synthesis under oxygen limitation at high glucose concentration.ConclusionsThe formation of norleucine and norvaline by recombinant E. coli during cultivation with provoked oxygen limitation and glucose excess can be reduced to levels at the detection limit by adding the trace elements molybdenum, selenium and nickel to the fermentation medium. Even under the metabolic burden during induction phase the physiologically available concentrations of non-canonical amino acids remained low. Since our results allow facile process changes that can be easily implemented to avoid the undesirable accumulation of norleucine and norvaline, we consider this study highly interesting for improved process development in E. coli based recombinant drug production and the future development of possible mechanisms to reduce misincorporation events into protein based biopharmaceuticals.

Risks of transmission of spongiform encephalopathies by reproductive technologies in domesticated ruminants

This paper considers whether transmissible spongiform encephalopathies (TSEs or prion diseases) could be spread by artificial insemination, embryo transfer and other more advanced reproductive technologies which are used for genetic improvement and also for purposes such as production of recombinant drugs for medical use. Although the technologies are most used in cattle, they are increasingly used in sheep, goats and deer as well, all of which can be naturally affected by TSEs. In general, provided appropriate precautions are taken, the risks of TSE carriage specifically by the gametes (spermatozoa and oocytes) or by in-vivo-derived embryos per se appear to be negligible, but further research, some of which is already in progress, will be helpful to give assurance on this point. Greater concerns relate to the many biological products that are used in the technologies, e.g. pituitary hormones used for the superovulation of donors, and various tissues and blood products used in semen and embryo culture/transport media, some of which have the potential to carry TSE infectivity if derived from infected animals. The myriad instruments and items of technical equipment that are used also give cause for concern because if they become contaminated with TSEs they may, due to their construction, be impossible to sterilise properly.

Validation of a Method for Automated Nucleotide Sequence Analysis and Estimation of the Limits of Detection of Variant Sequences in a Heterogenic DNA Sample

The assessment of the genetic stability of an expression-system used for the production of recombinant drugs and the verification of the nucleotide sequence of expression plasmids makes it necessary to estimate a limit of detection for variant sequences. Therefore, a series of experiments was performed in which defined mixtures of two different plasmid-DNAs were subjected to nucleotide sequence analysis using an automatic sequencer (ABI 377) and PCR-based sequencing reactions. A plasmid, pGR201 (Fig. 1a), encoding recombinant non-glycosylated human prourokinase (Saruplase), was spiked in varying ratios (90:10; 80:20; 70:30; 60:40) with a related plasmid, pSJ41 (Fig. 1a), differing in a single base-substitution and a cluster of multiple mutations, respectively. Each of these mixtures was sequenced six times in a 50 bp-region covering the variant sequences in order to estimate a limit of detection for point mutations and clusters of multiple mutations. The results are of general interest since we analysed enough point-mutations as well as deletion-mutations to allow a statistical assessment. It was shown that a single point mutation resulting in a base-substitution will be detected by sequencing in one direction if at least 20 % of the plasmids within the DNA sample are mutated, while the sequencing method is sensitive enough to detect a cluster of multiple mutations when at least 10 % of the sequenced plasmid DNA molecules carry the altered sequence. In contrast to other publications the sequencing-procedure presented in this paper results in electropherograms with small background signals although only a very simple purification of the sequencing reaction products was used in order to present a method that is not time-consuming.

A two-component expression system that responds to inflammatory stimuli in vivo.

A therapeutic dilemma often complicates the management of inflammatory diseases; the benefits gained from reducing inflammation must be balanced against the potentially harmful consequences of chronic immunosuppression. Gene therapy might address this dilemma by producing anti-inflammatory proteins in response to a patient's endogenous signals, so that recombinant drug production is linked to the intensity and duration of the inflammatory condition. To test this, we have developed inflammation-inducible systems for regulating recombinant protein production in vivo. We describe a two-component expression construct in which (1) the murine complement factor 3 (C3) promoter regulates production of the human immunodeficiency virus (HIV) transactivator of transcription (Tat), and (2) the Tat protein then stimulates protein expression from genes inserted downstream of the the HIV promoter. When incorporated into a nonreplicating adenovirus (Ad.C3-tat/HIV-luc) and studied in a murine model, the construct produces large amounts of recombinant protein in vivo in response to two different inflammatory stimuli.

Drug products of recombinant DNA technology

Presented are the steps in creating a recombinant DNA molecule, examples of recombinant drug products, a description of DNA fingerprinting methods for diagnosing diseases, a discussion of the patenting of recombinant drugs, and a look to the future of this revolutionary biotechnology. Constructing a recombinant DNA molecule involves cutting the DNA into fragments with restriction endonucleases and rejoining the fragments in novel arrangements with ligase. Propagating the molecule in a microorganism, or cloning, is necessary to increase the number of gene copies to facilitate detection of the gene of interest and to produce the protein it encodes. Recombinant DNA drug products have been developed that represent the communicator, structural, and modifier classes of proteins. Recombinant communicator proteins include interferons alfa-2a and alfa-2b and granulocyte-macrophage colony-stimulating factor (immune system modulators); epidermal growth factor and erythropoietin (tissue repair promoters); and human insulin, growth hormone, and atrial peptide (metabolism modulators). Recombinant structural proteins include hepatitis B virus vaccine and CD4 protein, and recombinant modifier proteins include tissue plasminogen activator and superoxide dismutase (agents that split or splice organic molecules). In the future, gene defects associated with genetic diseases will be unraveled, leading to the production of new therapeutic agents designed to counteract or actually reverse those defects. Recombinant protein drugs will be further tailored to enhance their activity and specificity. These advances are so novel and momentous that patent protection has been extended not only to recombinant drugs but to the recombinant microorganisms in which they are manufactured. In cloning genes, investigators directly use the protein designs that occur naturally. Basic research will soon lead to the engineering of novel proteins with specified functions.