Accelerated national lot release on COVID-19 vaccines in Republic of Korea.

Independent quality testing of samples from vaccine lots is part of quality assurance, especially to ensure the consistency of production lot by lot. Effective national lot release system that ensures the quality of each lot of vaccine before it is on the market is important because vaccines are intended to healthy people. In order to respond more quickly to public health crises such as the COVID-19 pandemic, the MFDS implements accelerated national lot release for rapid vaccination in Republic of Korea. For the accelerated system, improvement has been made in terms of timing of application for lot release and required documents. In addition, the processing period has been shortened and sampling method and test items have been streamlined. A thorough preparation for accelerated lot release has been developed by establishing test methods for a new platform in advance. As a result, a total of 43.88 million doses have been released within eight days on average. The accelerated lot release system has contributed significantly to rapid COVID-19 vaccination in Korea.

Points to consider for COVID-19 vaccine quality control and national lot release in Republic of Korea: focus on a viral vector platform.

Due to the global public health crisis caused by the coronavirus disease 2019 (COVID-19) pandemic, the importance of vaccine development has increased. In particular, a rapid supply of vaccines and prompt deployment of vaccination programs are essential to prevent and overcome the spread of COVID-19. As a part of the vaccine regulations, national lot release is regulated by the responsible authorities, and this process involves the assessment of the lot before a vaccine is marketed. A lot can be released for use when both summary protocol (SP) review and quality control testing are complete. Accelerated lot release is required to distribute COVID-19 vaccines in a timely manner. In order to expedite the process by simultaneously undertaking the verification of quality assessment and application for approval, it is necessary to prepare the test methods before marketing authorization. With the prolonged pandemic and controversies regarding the effectiveness of the COVID-19 vaccine against new variants, public interest for the development of a new vaccine are increasing. Domestic developers have raised the need to establish standard guidance on the requirements for developing COVID-19 vaccine. This paper presents considerations for quality control in the manufacturing process, test items, and SP content of viral vector vaccines.

Establishment of the 2nd Korean national biological reference standard for blood coagulation factor VIII:C concentrate.

Since the 1st Korean national biological reference standard for factor (F)VIII concentrate, established in 2001, has shown declining potency, we conducted this study to replace this standard with a 2nd Korean national biological reference standard for blood coagulation FVIII concentrate. The candidate materials for the 2nd standard were prepared in 8000 vials with 10 IU/ml of target potency, according to the approved manufacturing process of blood coagulation Factor VIII:C Monoclonal Antibody-purified, Freeze-dried Human Blood Coagulation Factor VIII:C. Potency was evaluated by one-stage clotting and chromogenic methods and the stability was confirmed to meet the specifications during a period of 73 months. Since the potencies obtained by the two methods differed significantly (P < 0.015), the values were determined separately according to the geometric means (8.9 and 7.4 IU/vial, respectively). The geometric coefficients of interlaboratory variability were 3.4% and 7.6% by the one-stage clotting and chromogenic assays, respectively.

Improved quantification of protein in vaccines containing aluminum hydroxide by simple modification of the Lowry method.

Aluminum (Al) components in vaccines are known to act as adsorbents that interfere with accurate protein quantification by the Lowry method. Therefore, certain modifications based on the characteristics and compositions of the vaccine are required for determination of protein contents. We investigated the effects of an additional centrifugal separation and found that protein contents were overestimated by up to 238% without centrifugation through a collaborative study performed with hepatitis B vaccines containing Al. However, addition of a centrifugation step yielded protein concentrations that were similar to the actual values, with small coefficients of variation (CVs). Proficiency testing performed in 11 laboratories showed that four laboratories did not have satisfactory results for vaccines containing aluminum hydroxide, although all laboratories were proficient in protein analysis when samples did not contain aluminum hydroxide. Incomplete resuspension of aluminum hydroxide solution with alkaline copper solution was the major cause of insufficient proficiency in these laboratories.

Standardization of the methods and reference materials used to assess virus content in varicella vaccines.

BACKGROUND: In Korea, every vaccine lot is tested by the National Center for Lot Release (NCLR) in accordance with the national lot release procedures to ensure the safety and efficacy of vaccines. These quality tests examine the virus content in varicella vaccines via plaque assays (either the agar overlay method [AOM] or plaque staining method [PSM]), according to the procedures suggested by the Korean Reference Material for the Varicella Vaccine (KRMVV) or the manufacturer's standard in-house protocol. AIM: To standardize the virus content tests, viral titers in the KRMVV were measured using the PSM at four participating laboratories in a collaborative study. With the aim of developing a standardized method using the KRMVV as a positive control, we compared the ability of the two test methods, AOM and PSM, to accurately and reproducibly determine the virus content of two commercial varicella vaccines. RESULTS: The results showed that the standardized method (PSM) was more suitable for quality control analysis of the varicella vaccine. CONCLUSION: Use of a standardized method (PSM) according to the Korean reference material will improve the reliability and objectivity of lot release testing.

Review of the current use and evaluation of cell substrates for producing biologicals in selected countries.

In 2010, the WHO guidance document for the evaluation of cell substrates for producing biologicals was replaced with updated recommendations and in May 2013 an implementation workshop on the new recommendations was held in Beijing, China. As part of this workshop, a survey of the use and evaluation of cell substrates for producing biologicals was undertaken and the information obtained was updated in June 2014. The purpose of survey was to capture the status of national requirements related to cell substrates in various countries with particular emphasis on whether or not the updated WHO recommendations had been, or were to be, incorporated into national requirements. This paper reports the outcome of the survey and is based on information provided by regulators in eleven countries. Since the publication of the updated WHO recommendations, several activities such as the implementation workshop and publications have been undertaken by the WHO. The aim of these activities, including the publication of this article, is to contribute to the implementation of WHO recommendations so as to reduce regulatory gaps between national requirements and globally agreed expectations.

Src activates HIF-1α not through direct phosphorylation of HIF-1α specific prolyl-4 hydroxylase 2 but through activation of the NADPH oxidase/Rac pathway.

Hypoxia-Inducible Factor (HIF)-1α/ß heterodimer is a master transcription factor for several genes involved in angiogenesis, glycolysis, pH balance and metastasis. These HIF-1 target genes help tumors to overcome forthcoming metabolic obstacles as they grow. Under normoxic condition, the HIF-1α subunit is hydroxylated by its specific prolyl-4 hydroxylase 2, given the acronym PHD2. Hydroxylated HIF-1α becomes a target for von Hippel-Lindau (VHL), which functions as an E3 ubiquitin ligase. Src prevents hydroxylation-dependent ubiquitinylation of HIF-1α, thus stabilizing it under normoxic conditions. We found that active Src does not directly phosphorylate any tyrosine residue of PHD2. In vitro hydroxylation reaction showed that the presence of the purified active Src protein does not inhibit the hydroxylation activity of the purified PHD2 enzymes. Instead of directly inhibiting PHD2, Src recruits several downstream-signaling pathways to intercept hydroxylation-dependent ubiquitinylation of HIF-1α. Using biochemical and genetic inhibition, we demonstrated that Src requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/Rac complex for stabilization of HIF-1α. We found that excess vitamin C treatment attenuates Src-induced HIF-1α activation. HIF-1α-hydroxylation-dependent VHL pull-down assay showed that Src inhibits cellular PHD2 activity by inducing ROS production in a mechanism involving Rac1-dependent NADPH oxidase. Src-induced ROS reduces cellular vitamin C, which is required for the activity of PHD2, thus Src can block VHL recruitment of HIF-1α, leading to stabilization of HIF-1α.

The zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, increases the level of nonfunctional HIF-1alpha protein in normoxic cells.

The hypoxia-inducible factor-1alpha (HIF-1alpha) subunit is activated in response to lack of oxygen. HIF-1alpha-specific prolyl hydroxylase and factor inhibiting HIF-1alpha (FIH-1) catalyze hydroxylation of the proline and asparagine residues of HIF-1alpha, respectively. The hydroxyproline then interacts with ubiquitin E3 ligase, the von Hippel-Lindau protein, leading to degradation of HIF-1alpha by ubiquitin-dependent proteasomes, while the hydroxylation of the asparagine residue prevents recruitment of the coactivator, cAMP-response element-binding protein (CBP), thereby decreasing the transactivation ability of HIF-1alpha. We found that the Zn-specific chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), enhances the activity of HIF-1alpha-proline hydroxylase 2 but the level of HIF-1alpha protein does not fall because TPEN also inhibits ubiquitination. Since the Zn chelator does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1alpha, its presence leads to the accumulation of HIF-1alpha that is both prolyl and asparaginyl hydroxylated and is therefore nonfunctional. In hypoxic cells, TPEN also prevents HIF-1alpha from interacting with CBP, so reducing expression of HIF-1alpha target genes. As a result, Zn chelation causes the accumulation of nonfunctional HIF-1alpha protein in both normoxia and hypoxia.

Inhibition of the catalytic activity of hypoxia-inducible factor-1alpha-prolyl-hydroxylase 2 by a MYND-type zinc finger.

Hypoxia-induced gene expression is initiated when the hypoxia-inducible factor-1 (HIF-1) alpha subunit is stabilized in response to a lack of oxygen. An HIF-1alpha-specific prolyl-hydroxylase (PHD) catalyzes hydroxylation of the proline-564 and/or -402 residues of HIF-1alpha by an oxygen molecule. The hydroxyproline then interacts with the ubiquitin E3 ligase von Hippel Lindau protein and is degraded by an ubiquitin-dependent proteasome. PHD2 is the most active of three PHD isoforms in hydroxylating HIF-1alpha. Structural analysis showed that the N-terminal region of PHD2 contains a Myeloid translocation protein 8, Nervy, and DEAF1 (MYND)-type zinc finger domain, whereas the catalytic domain is located in its C-terminal region. We found that deletion of the MYND domain increased the activity of both recombinant PHD2 protein and in vitro-translated PHD2. The zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine augmented the activity of wild-type PHD2-F but not that of PHD2 lacking the MYND domain, confirming that the zinc finger domain is inhibitory. Overexpression of PHD2 lacking the MYND domain caused a greater reduction in the stability and function of HIF-1alpha than did overexpression of wild-type PHD2, indicating that the MYND domain also inhibits the catalytic activity of PHD2 in vivo.

Hepatitis B virus X protein enhances transcriptional activity of hypoxia-inducible factor-1alpha through activation of mitogen-activated protein kinase pathway.

Hepatitis B virus X protein (HBx) of the hepatitis B virus was strongly implicated in angiogenesis and metastasis during hepatocarcinogenesis. Here, we explored the possibility of cross-talk between HBx and hypoxia-inducible factor-1alpha (HIF-1alpha), a potent transcriptional inducer of angiogenic factors. First, we showed that stability of HIF-1alpha protein was increased by HBx in HBx-inducible Chang liver cells as well as in transient HBx expression system of non-hepatic cells. Immunofluorescence studies revealed that the HBx-induced HIF-1alpha was partially translocated into the nucleus in majority of cells while additional CoCl2-induced hypoxic condition caused complete nuclear translocation. Second, HBx induced both phosphorylation of HIF-1alpha and activation of p42/p44 mitogen-activated protein kinases (MAPKs), which were synergistically enhanced in the presence of CoCl2. Furthermore, HBx enhanced transcriptional activity of HIF-1alpha in the reporter genes encoding hypoxia response element or VEGF promoter. Either treatment of MEK inhibitor PD98059 or coexpression of dominant-negative MAPK mutants abolished the HBx-induced transcriptional activity and protein stability as well as nuclear translocation of HIF-1alpha, suggesting that HBx activates HIF-1alpha through MAPK pathway. Third, the association of HIF-1alpha with von Hippel-Lindau was decreased but the association with CREB-binding protein was enhanced in the presence of HBx, suggesting the molecular mechanism by which HBx enhances the protein stability and transactivation function of HIF-1alpha. Finally, we demonstrated that expression of HIF-1alpha and vascular endothelial growth factor was increased in the liver of HBx-transgenic mice, suggesting that the cross-talk between HIF-1alpha and HBx may lead to transcriptional activation of HIF-1alpha target genes, which play a critical role in hepatocarcinogenesis.

Insulin and hypoxia share common target genes but not the hypoxia-inducible factor-1alpha.

Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.