Robust and precise chromatographic analytical methods are key for efficient development of mRNA production process. Three different analytical methods, which utilize three different column chemistries, are embedded in a ready-to-use PATfix™ mRNA analytical platform to support mRNA process development and product quantification and characterization.
First chromatographic step in industrial platform using monoliths is a capture step, where rAAV is selectively bound to strong cation exchanger (SO3) in acidic pH conditions (1,2). In this step majority of host cell contaminants are removed and rAAV is strongly bound to the matrix. Productivity of the step is therefore strongly influenced by the dynamic binding capacity (DBC). DBC is dependent on the sample preparation prior to chromatography and the availability of the binding sites at the chromatographic matrix.
Due to increasing demand for rAAV quantity and quality for clinical manufacturing, large volumes of upstream produced material are being purified on industrial scale. At this point, rAAV capture is a first chromatographic step to be optimized for optimal selection of the chromatographic parameters. Doing this, SO3 monolithic columns can be used with their full potential. To reduce the development time using multi-factor screening and increase comparability during downstream process development, CIM® SO3 monolithic 96-well plates were introduced to Sartorius BIA Separations portfolio. Obtained results on small scale can be applied to the CIMmultus™ line, which is scalable to large industrial volume.
Affinity-based chromatographic isolation of mRNA is robust and simple, lending itself as a useful industrial platform. mRNA constructs contain a 3’ polyA tail to increase stability in vivo, thereby affording the possibility of affinity purification using oligo-deoxythymidinic acid (Oligo dT) probes covalently coupled to a solid support. Poly-adenylated mRNA forms a stable hybrid with Oligo dT under high-salt conditions which is destabilized when the salt is removed, allowing mRNA to be released.
Due to an increasing productivity of IVT reaction, finding conditions that increase binding capacity of Oligo dT has been an intense focus of development. Multi-parallel approaches, such as screening in 96-well plate format, can significantly cut the development time by screening multiple conditions at once. 96-well plates can then be scaled-up to preparative scale, such as CIMmultus™ product line operated by chromatographic skids.
CIMac™ pDNA Analytical Column is powerful tool for pDNA quantification for in-process control or in a QC laboratory. The column can separate pDNA isoforms from each-other and from RNA impurities. Monitoring of pDNA production leads to a controlled and robust process, and can result in consistent high quality of the final product.
Optimised methods are a key component of a well-functioning analytical system, sometimes requiring time-consuming method development and steep learning curves. The following two methods described in this quick start guide can provide a starting point for pDNA purity and isoform analysis.
Optimized analytical methods are key components of a well-functioning analytical system, while method development usually comes with a time-consuming learning curve and optimization.
PATfix pDNA analytics platform, designed for in-process control of linear pDNA production, enables monitoring of pDNA linearization progression, as shown in Figure 1. Fully optimized and validated analytical methods, as well as guidelines for buffer and sample preparation come as part of the PATfix system, allowing users to focus on their specific application. In addition, the PATfix pDNA analytical package includes a pDNA calibration standard, which enables accurate quantification of the pDNA species of interest.
Miniaturised immobilised enzymatic reactors can be used for small scale digestion of proteins. There is need for such devices; small scale devices are used either for processing of analytical sample quantities, or as proof of concept before protein digestion at larger scale. This application note compares the performance of a flow through miniaturised immobilised enzymatic reactor (μIMER) with in-solution batch digestion of simple proteins and complex matrices. Automation of peptide analysis by coupled LC-MS is explored as an option to increase throughput. In the cases evaluated, the miniaturised immobilised enzymatic reactor offered comparative results to overnight in-solution digestion, within less than 10 minutes.
Pre-activated CIMmic™ monolithic columns with 100 μL bed volume were immobilised with trypsin from bovine pancreas. This small format allows coupling to HPLC for on-line protein digestion, as well as syringe (manual) operation of the IMER. Pre-treated samples (denatured, alkylated and ultra-filtered) are injected into the column, and the eluate (tryptic digests) are subjected to LC-ESI-MS-MS analysis for protein identification and post-translational modification (PTM) determination.
A purification of synthetic oligonucleotides by using CIM™ monolith was evaluated. In this case study, the CIM™ anion exchange column had the capability to resolve oligonucleotides with small difference in comparative chain length.
A crude reaction mixture of synthetic oligonucleotide was loaded onto the CIM™ anion exchange column. Sample elution was achieved by salt concentration gradient. In comparison with conventional media, CIM™ monolith indicated higher resolution for major impurities.
Advantages of the characteristic properties of the CIM™ monolith were evaluated based on the high throughput purification of oligonucleotides under the identified gradient separation conditions. Over 99 % HPLC purity for the target oligoDNA was achieved by one-step purification from the crude reaction mixture.
Pre-activated CIMmic™ monolithic columns are cost efficient tools for screening of immobilisation conditions and small scale proof-of-concept testing of custom affinity columns and enzymatic reactors. Each column is assembled from a dedicated housing and discs containing a chromatography medium. With a bed volume of 100 μL, sample requirements are minimal, while inserting multiple discs in the housing adapts the column volume to application requirements. Different surface modifications of the discs enable immobilisation of a wide variety of ligands.
The increasing demand for messenger RNA (mRNA) as a therapeutic product requires larger production scales, and in turn more efficient extraction techniques. One of the most convenient techniques for its extraction is the use of oligo deoxythymidine (dT) coupled to a solid support . Oligo dT hybridises to the poly-adenylated tail which is present on most eukaryotic mRNAs, or synthetised onto the molecule during IVT, while other contaminant impurities (proteins, unreacted nucleotides, plasmid DNA, CAP analogues, partial transcripts, dsRNA side products and enzymes) lack the poly-A moiety and do not adhere to the solid support.
Polyclonal anti-HSA antibodies were immobilised on CIMmic™ HDZ (hydrazide chemistry, 100 μL bed volume), following an optimised protocol. This CIMmic™ α-HSA column was prepared to quantitatively remove human serum albumin (HSA), the most abundant plasma protein in humans, and obtain albumin-free samples which can be further processed.
The CIMmic™ α-HSA column is optimized to selectively bind and remove HSA from plasma; albumins from other sources are bound with low efficiency. HSA depletion is carried out in two steps: (i) a loading step in which the diluted and filtered plasma sample is loaded into the column, HSA is bound and HSA-depleted plasma is collected downstream of the column and (ii) a regeneration step in which the bound HSA is removed and the column is regenerated.
The increasing demand for messenger RNA (mRNA) as therapeutic product requires larger production scales, and in turn more efficient extraction techniques. Messenger RNA can be produced by in vitro transcription reactions (IVT) or isolated from eukaryotic cells. One of the most convenient techniques for its extraction is the use of oligo deoxythymine (dT) coupled to a solid support. Oligo dT hybridises to the poly-adenylated tail which is present on most eukaryotic mRNAs, or synthetised onto the molecule during IVT. Contaminant impurities, such as proteins, unreacted nucleotides, plasmid DNA, CAP analogues, partial transcripts, dsRNA side products and enzymes lack the poly-A moiety and are not retained on the solid support.
Chromatography using a solid phase consisting of large channels, such as monoliths, allows high flow rates and low shear forces. This can have a positive impact on recovery and productivity in purification of biologics. In addition, chromatography offers a closed system to minimise the risk of cross-contamination or exposure to RNase degradation, and an easily scalable platform.
CIMmultus™ Oligo dT is a chromatography column with Oligo dT ligands covalently bound on its surface. The sample containing poly-adenylated mRNA is loaded onto the column in a high salt concentration buffer. Salt ions screen the electrostatic repulsion between the negatively charged backbones and allow interaction between the Oligo dT and poly-adenylated tail of mRNA. Before product elution, a wash step at reduced salt concentration removes unspecifically bound contaminants. Elution of messenger RNA occurs under mild conditions in low conductivity buffer at neutral pH. In the absence of salt, electrostatic repulsion between the negatively charged backbones of Oligo dT and poly-adenine destabilises the T–A pairs and releases mRNA from the column.
In speciation analysis of anticancer metallodrugs, size exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometer (ICP-MS) is frequently used. The size exclusion coupled ICP-MS is troubled by low resolution, long time of analysis (up to 60 min) and tendency of some metals to irreversibly adsorb on the surface of the stationary phase. Higher resolution can be achieved by ion-exchange particle columns, but this doesn’t address time frame of the analysis and brings in additional bottlenecks in the form of the potential clogging issues connected to the particle pore diameter.
Alternatively, monolithic ion-exchangers can replace the particle-based columns. Monoliths enable improved protein separation facilitated by increased mass permeability, allowing for higher flow rates while preserving separation efficiency at low backpressures. Subsequently, time of the chromatographic separation can be shortened2 – making kinetic studies less troublesome. In addition, monolithic columns are more robust, can undergo rigorous cleaning procedures and enable analyses of large series of clinical samples.
Pre-activated CIMmic™ Monolithic Columns are used as a basis for preparation of small volume affinity chromatographic columns as well as enzyme reactors. Small bed volume and flexible design makes them a powerful tool for screening purposes and immobilization protocol optimizations. Range of covalently bound ligands is wide and includes diverse set of proteins, peptides, nucleotides and other affinity ligands. The covalent nature of the bond between the ligand and matrix reduces leaching and improves stability and reusability. Reaction conditions must cater to their specific physiochemical nature.
Successful preparation of an affinity column includes a decision on the appropriate matrix chemistry and determination of an optimal immobilization protocol. Presented case study explores the basics of a coupling protocol optimization using covalent immobilization of Recombinant Prokaryotic Lectins (RPL-Gal1) on CIMmic CDI-0.1 and CIMmic ALD-0.1 columns, as an example. Carboxy imidazole (CDI) and aldehyde (ALD) activated CIMmic™ columns are used for covalent immobilization of amine or thiol containing molecules.
CIMmic™ Monolithic Columns combine the advantages of the CIM® stationary phase with a flexible design and the possibility to operate with syringe. Discs containing the stationary phase can be easily interchanged inside the custom designed housing. Pre-activated chemistries enable immobilisation of numerous ligands and can be used for preparation of affinity chromatographic columns or enzyme reactors. Their small bed volume is particularly suitable for screening purposes and to optimise immobilisation protocols due to economic usage of often expensive ligands.
Carboxy imidazole (CDI) monolithic chromatographic columns are used for covalent immobilisation of proteins, peptides and other amine or thiol containing molecules. The covalent nature of the carbamate bond between the ligand and matrix reduces leaching and improves stability and reusability.
An example described below shows the feasibility of CIMmic™ CDI-0.1 utilisation for covalent immobilisation of recombinant protein A (r-pA). Additionally the example was used for the evaluation of the reproducibility of CIMmic™ CDI columns. The dynamic binding capacity for human polyclonal immunoglobulin (IgG) was used as metric for comparison of the affinity columns.
Coupling trypsin enzyme onto chromatographic supports provides a platform to reuse the enzyme and automate the hydrolysis process. A monolithic chromatographic support, such as Convective Interaction Media (CIM®), enables mass transfer of molecules within its channels exclusively by convective flow. This results in enzymatic conversion which is not limited by diffusion, making CIM® monoliths ideal for the preparation of immobilised monolith enzymatic reactors (IMERs). BIA Separations offers CIMac™ trypsin IMER with a bed volume of 0.1 mL as analytical platform for mass spectrometry (MS)-based proteomics. Larger volume IMERs (up to 80 mL) are available for industrial scale production of protein hydrolysates. The following example describes the enzymatic production of β-Lactoglobulin (β-Lg) hydrolysates using monoliths with 2 μm (N1) or 6 μm (N2) average channel diameter.
Bottom-up proteomic approach based on tandem mass spectrometry (MS/MS) is a method of choice for identification and quantitation of proteins in the complex biological samples. Followed by quantitative analysis of a protein sample, this approach enables the identification of putative biomarkers for early pathology diagnostics and differentiation. The main challenges confronting this analysis are suppression of low-abundance ions and the limited dynamic range of MS/MS.
Affinity depletion of abundant proteins is an important stage in routine sample preparation prior to tandem mass spectrometry (MS/MS) analysis of biological samples. One such protein is Human Serum Albumin (HSA). In this study, polyclonal αHSA antibodies were immobilised onto a chromatography support to use as immunoaffinity-based column (CIMac™ HDZ -αHSA) for HSA depletion from embryo culture media for in vitro fertilization (IVF).
As an alternative to conventional particle-packed columns for liquid chromatography, CIM® monolithic supports can be used.1-3 They are extremely permeable and allow very efficient mass transport at low back pressures and good separation efficiency at high flow rates. Consequently, the time of chromatographic separation can be shortened. Such characteristics are very valuable in speciation analysis where preservation of the integrity of individual chemical species of a given element is of crucial importance.4,5 CIM® monolithic disks can be placed together in one housing forming so-called conjoint liquid chromatography (CLC), which combines two different chromatographic modes in one step.
For better understanding of anticancer therapy with Pt-based chemotherapeutics, the quality of pharmaceutical formulations must be assured and the behaviour of the drugs studied by separation and detection of the intact drug and its individual biotransformation species in clinical samples at therapeutically relevant levels. To study the interactions of Pt-based chemotherapeutics with serum proteins, two-dimensional chromatographic separation of serum proteins was suggested, combining size-exclusion and CIM DEAE monolithic.6 Since this procedure is time consuming the potential of the use of CLC was investigated.
The application describes separation of Ni species by assembling four weak CIM DEAE anion-exchange disks into a monolithic column. The concentrations of the Ni species eluted from the column were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID)-ICP-MS. The Ni binding ligands eluted under the chromatographic peaks were identified off-line by tandem electro spray mass spectrometry (ESI-MS-MS), scanning for negative ions.
The mild chromatographic conditions of the CIM DEAE disks preserved chemical species and enabled separation of negatively charged Ni complexes.4 NH4NO3 was chosen as eluent since it enabled separation of Ni species and is compatible with ICP-MS and mass spectrometry detectors.
Human coronavirus OC43 (HCoV-OC43) is a frequent cause of respiratory tract illness, ranging from common cold to severe disease. The research on coronaviruses and medical application of coronaviral vectors/vaccines requires a quality material of high purity. Unfortunately, virus preparations are highly contaminated with cell debris and purification requires laborious, cost-ineffective procedures.
Here, we report a simple and efficient method for coronavirus concentration and purification by the example of HCoV-OC43. To achieve this, virus chromatography was performed on CIM QA monolithic columns (BIA Separations), with immobilized positively charged quaternary amines. The quality of the obtained virus stock was assessed with SDS Page electrophoresis, followed by Western blot analysis. Finally, infectivity of recovered virus was evaluated by titration.
Sample displacement chromatography exploits the different relative binding affinities of components in a sample mixture to achieve accummulation of a desired substance on the column before elution. In pharmaceutical applications, requirements for purity and efficacy of plasmid DNA (pDNA) as a therapeutic product are stringent. The separation of linear, supercoiled (sc) and open-circular (oc) pDNA isoforms has already been established on CIM® butyl (C4 HLD) monolithic columns at preprative scale. This process requires high concentration of ammonium sulphate for loading which increases the overall production requirements. Competing adsorption in sample displacement chromatography utilises the binding capacity of the chromatographic resin more efficiently and increases productivity of the chromatographic step.
This application note investigates three monolithic chromatographic supports with different hydrophobicities regarding their applicability for sample displacement of pDNA. CIMac™ C4 HLD (butyl, high ligand density) as a commercial product and pyridine and histamine as custom immobilised columns are compared.
Influenza vaccines are still predominantly produced in embryonated chicken eggs and the purification processes barely have changed during the years. There is a growing need for fast, efficient and economical vaccine production.
So far, monolithic supports have been used successfully in virus purification and concentration, as well as in the purification of virus-like particles (VLP) propagated in cell cultures.
Therefore, our aim was to prove the applicability of monoliths in purification of influenza virus A propagated in embryonated chicken eggs.