RESUMO
The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic.
Assuntos
DNA , RNA , Humanos , RNA Mensageiro/genética , Cromatografia Líquida de Alta Pressão/métodos , Plasmídeos/genética , DNA/genéticaRESUMO
The COVID-19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed-batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti-reverse cap analog (ARCA)-capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed-batch approach achieved productivity of 9 mg/ml with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed-batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA.
Assuntos
COVID-19 , Pandemias , Humanos , Cromatografia Líquida de Alta Pressão , RNA Mensageiro/genéticaRESUMO
Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl) in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers. It also improves resolution among sc, linear, and multimeric/aggregated forms. Sharper elution peaks with less tailing increase sensitivity about 30%. However, elution with an exclusively Gdn gradient to 900 mM causes more than 10% loss of plasmid. Elution with a sodium chloride gradient while maintaining Gdn at a level concentration of 300 mM achieves close to 100% recovery of sc plasmid while maintaining the separation improvements achieved by exclusively Gdn elution. Corresponding improvements in separation performance are not observed on a strong (quaternary amine) anion exchanger. Other chaotropic salts do not produce a favorable result on either exchanger, nor does the inclusion of surfactants or EDTA. Selectivity of the diethylaminoethyl-Gdn method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than CE, and resolution approaching CE.
Assuntos
Cromatografia por Troca Iônica , DNA , Etanolaminas , Guanidinas , Plasmídeos , Ânions , Guanidina , Cloreto de SódioRESUMO
The plasma-derived IgG used either for diagnostic purpose or intravenous application (in form of IVIG) in various medical therapies is certainly gaining more and more attention on annual basis. Different manufacturing processes are used to isolate immunoglobulins from human plasma. However, a quest for alternative paths in IgG isolation not only requires development of the most efficient isolation process, but also a rapid and reliable analytics to track the purification. Fast and reliable fingerprint-based method for characterization of IgG prepared from Cohn I+II+III paste is presented in this paper. The fingerprint method bases on partial separation of proteins in linear gradient on CIMac™ quaternary amine, strong anion exchange group (QA) 0.1 mL column. Partial separation of proteins does not allow simple quantitative analysis of the samples during the IgG isolation from Cohn I+II+III fraction paste, but very accurate qualitative information about the composition of the sample can be obtained in less than 5 min. From the differences in the chromatograms of various samples, the ratio between IgG and impurities in each sample can be easily assessed. The method is suitable for input material control, in-line monitoring of the downstream processing, final control of the products, as well as in stability studies and enables taking fast and accurate decisions during fractionation process.
Assuntos
Proteínas Sanguíneas/química , Cromatografia Líquida de Alta Pressão/métodos , Imunoglobulinas Intravenosas/sangue , Imunoglobulinas Intravenosas/isolamento & purificação , Humanos , Imunoglobulinas Intravenosas/química , Reprodutibilidade dos TestesRESUMO
The human papillomavirus (HPV) minor capsid protein L2 plays important roles in the generation of infectious viral particles and in the initial steps of infection. Here we show that HPV-16 L2 protein is sumoylated at lysine 35 and that sumoylation affects its stability. Interestingly, the sumoylated form of L2 cannot bind to the major capsid protein L1, suggesting a mechanism by which capsid assembly may be modulated in an infected cell. Additionally, L2 appears to modulate the overall sumoylation status of the host cell. These observations indicate a complex interplay between the HPV L2 protein and the host sumoylation machinery.
Assuntos
Proteínas do Capsídeo/metabolismo , Papillomavirus Humano 16/química , Proteínas Oncogênicas Virais/metabolismo , Proteína SUMO-1/metabolismo , Humanos , Lisina/metabolismo , Ligação Proteica , Estabilidade ProteicaRESUMO
HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation.
RESUMO
Oligonucleotide aptamers are selected from libraries typically comprising up to 10(15) different sequences by an iterative process of binding, separation, amplification and purification, called SELEX. During this process, the diversity of the oligonucleotide pool decreases until, presumably, only sequences with highest binding affinities towards chosen targets remain. This selection technique is time-consuming, labor-intensive and expensive. Though well posed in principles, the SELEX procedure is noise sensitive, due to amplification of unspecific-binding sequences, and it is not surprising that aptamer selection is often not successful in practice. In view of that, a follow-up of the progress of selection during its course with simple yet reliable methods is necessary. In this paper, we describe five independent assays to estimate the sequence complexity of SELEX pools including qualitative restriction fragment length polymorphism analysis, melting curve analysis, quantitative fluorescence intensity measurements of bound ssDNA, real time PCR quantification and pool dissociation constant analysis during the progress of aptamer selection against streptavidin. Properties and features of each method are discussed and compared. Pool dissociation constant analysis and sequencing serve as reference methods.