On May 12th, the biaseparations.com website will be retired and migrated tosartorius.com.Learn moreabout our combined offering today!
2021

Optimizing processing steps in sc pDNA isolation is critical for obtaining good process yields as well as high product purity. PATfix platform with convective chromatography media (e.g. monolith) offers a rapid analytical method to characterize complex biomolecular mixtures and gives immediate feedback during process development. E coli lysis represents such a challenging step, where multiple critical quality attributes need to be identified and critical processing parameters optimized. This approach leads to better yields and product purity, allowing for simplified downstream steps. A new PATfix analytical platform presented here uses CIMac pDNA column, to separate and characterize plasmid from impurities, allowing for easy optimization of key parameters such as RNA removal.

Attachments

Full view

AAV9 clarified harvest from Sf9 cells was concentrated and purified using combination of tangential flow filtration, nuclease treatment and cation exchange capture. First part was TFF coupled with Kryptonase treatment. Capture step was performed on CIMmultus SO3 cation exchange column. AAV elution fraction was cleared of DNA and protein contaminants and prepared for final polishing – empty capsid removal. PrimaT separation mechanism is based on ligand multimodality, one of them being metal-chelating ability. This was sucessfully exploited for AAV capsid separation.

Attachments

Full view

2020

Separation of empty and full AAV capsids is important analytically as a means of monitoring the effects of different transfection strategies, cell culture conditions, lysis methods, sample preparation and purification methods. It is at least as important on a prepartive level because it offers the possibility of removing empty capsids without ultracentrifugation. This poster introduces a new column for performing separation of empty and full capsids.

CIMac PrimaS™ (AAV) beta employs a new ion exchange-hydrogen bonding multimodal ligand that provides a new orthogonal option for separation of empty and full capsids. Gross selectivity is similar to strong (QA) anion exchangers eluted with salt gradients, but it generally provides better resolution. Its distinct separation mechanism is documented by the fact that it provides its best results when eluted with increasing pH gradients. This is directly opposite to QA exchangers where increasing pH causes capsids to bind more strongly.

CIMac PrimaS™ (AAV) beta can also be eluted with salt gradients and often provides better resolution than QA, but its best resolution is obtained with pH gradients. Separation of a free capsid protein (CP) occurs only with the pH elution format. CIMac PrimaS™ (AAV) beta can be used instead of classical anion exchange, for example following capture by cation exchange chromatography or affinity. Its distinct separation mechanism also makes it possible to perform orthogonal separations in which it is combined with QA. 

Attachments

Full view

Bioreactor cell supernatants or post lysis materials entering downstream purification are heterogeneous mixtures of empty, full, and misassembled AAV capsids mixed with host cell proteins, genomic DNA, chromatin complexes, and other contaminants. Monolith- based HPLC columns provide high resolution among these species but overlap of elution position among capsids and contaminants makes it impossible to estimate the relative content of full and empty capsids by UV absorbance. Simultaneous monitoring with multiple detectors however enables quantitative insights that extend far beyond the limitations of UV absorbance.


This poster demonstrates how the combination of fast high resolution separations with monoliths can be combined with multiple monitors to obtain much deeper characterization than traditional assays. Anion exchange fractionation of filtered lysate and cation exchange- purified AAV 8 was monitored by UV absorbance at 260 nm and 280 nm, simultaneously with tryptophan fluorescence to differentially detect proteins without interference by nucleic acids, and with Multi Angle Light Scattering (MALS) to detect capsids.

Attachments

Full view

2019

Fast, accurate, and meaningful characterization of cell culture harvests and in process samples is critical for both process development and for documentation of in process control. UV analysis of chromatography profiles has been a valuable tool for decades, but it has major limitations with respect to sensitivity and its ability to discriminate the product of interest from particular contaminant classes.
In this study we use filtered lysate containing AAV 8 to demonstrate the ability of a strong cation exchange monolith (CIMac ™ SO3-0.1) coupled with multiple monitors to enable high sensitivity detection of AAV capsids while characterizing the relative distributions of DNA and protein contaminants. This approach can be used to evaluate cell culture methods, influence of harvest time, lysis methods, and effectivity of purification methods across a process.

Attachments

Full view

AAV vector lots are generally a heterogeneous mixture of empty particles (i e do not contain DNA) and full particles (i.e. contain DNA). Different spectrometric based methods can be used to establish the ratio between full and empty AAV particles, but accurate evaluation of empty/full ratio is often obstructed due to complex spectroscopic behavior of empty and full AAV particles, such as poor separation and impurity overlapping. An approach that takes difference in physical chemical properties between empty and full capsids into account overcomes limitations of spectrometric based evaluation of empty and full AAV particle ratio.

Chromatographic separation of empty and full AAV 2 8 capsids was achieved on the CIMac AAV full/empty analytical column (strong anion exchanger, QA quaternary amine chemistry) with the PATfix™ system using a linear NaCl gradient at pH 9.0 Signal response from three different detectors connected in series was analyzed fluorescence (excitation 280 nm emission 348 nm), light scattering 90 angle, LS) and UV absorbance 260 nm and 280 nm).

Attachments

Full view

One of the handicaps of working with bacteriophages is the long duration required to perform plaque assays. Plaque assays also impose questions about accuracy and precision relative to the scale and experience of the persons performing and interpreting them. This poster presents a pair of high precision, high accuracy chromatography-based assays that permit determination of phage concentration in less than 1 hour. Sensitivity of UV absorbance is poor because of the low concentration of phages. However, phage sensitivity is strongly amplified by monitoring the chromatogram with either fluorescence or MALS. Fluorescence works by measuring the fluorescence emission from tryptophan residues of the phage proteins. MALS works by passing a laser beam through the sample and reading the scatter produced when it encounters a particle. Larger species generate more scatter.

Attachments

Full view

Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants. The most common technique for purification is ultracentrifugation using cesium chloride gradients. This technique is elaborate, cumbersome, expensive and difficult to scale-up.
Alternative techniques for purification are usually time consuming and affect phage recovery and/or viability. In this study we present efficient two-step chromatographic purification method with binding phages to a stationary phase - Convective Interaction Media (CIM®) monoliths. The aim of the study was to develop robust, fast and effective virus purification platform that can be used for several types of bacteriophages for any application. In this work bacterial lysate with bacteriophage T4 (host E.Coli) was used.

Attachments

Full view

Serotype 10 adeno-associated virus (AAVrh_10mCherry) was analysed on the PATfix™ system with the CIMac™ AAV full/empty analytical column to estimate the ratio of empty and full AAV particles based on the peak area of the chromatogram given with three different detectors. AAV included a protein capsid containing single stranded DNA. CIMac™ AAV column consisted of a strong anion exchanger with QA chemistry (quaternary amine).

Poster was prepared by Blaz Goricar and presented at ISBioTech 9th Spring Meeting where it was awarded the first prize. Congratulations!

Attachments

Full view

2018

This poster presents fully scalable non-affinity purification strategy that has been proven to be effective for all AAV serotype tested to date. Cell lysate is directly subjected to column purification after removal of cell debris without requiring a concentration step using tangential flow filtration. The process consists of three chromatographic steps. Hydrophobic interaction chromatography on a CIMmultus OH monolith is used for initial virus capture and purification. Precipitating salts are used at 1.0–2.0 M to achieve virus binding. Most of the small molecule contaminants and proteins are eliminated in the flow-through. AAV co-elutes with a highly reduced population of contaminating proteins. DNA-protein complexes are very strongly retained and require NaOH for removal. Intermediate polishing is performed with a CIMmultus SO3 cation exchange monolith. The AAV fraction from the capture step is titrated to a pH value of 3.5—5.0 and diluted to binding conditions. Sugars and surfactants are added to suppress non-specific interactions with tubing and containers, and the product is eluted in a salt gradient. Final polishing is conducted on a CIMmultus QA anion exchange monolith which separates empty capsids from full capsids. This is achieved in a salt gradient at alkaline pH. For more information please refer to BIA Application note A048 (www.biaseparations.com/applications).

Attachments

Full view

Immunoaffinity columns using antibodies as ligands against mammalian proteins could be used for different applications in protein expression control and, if a standard available, for direct protein quantification in complex sample solutions. Additionally, these columns are ideal for polishing step of recombinant proteins, such as mammalian receptor Fc fusion proteins. Most importantly, such columns could extract a significant amount of a single membrane protein from native source, suitable for downstream analyses, such as mass spec analysis of their glycans. Immunoaffinity chromatographic monoliths against RAE-1 GPI anchored glycoprotein were developed (CIMmic HDZ - @RAE-1 column) as a part of Glycomet project with the main goal to analyze the antigen glycoprofile.

Attachments

Full view

Hydrazide-activated (HDZ) columns were proven to be a product of choice for making the most effective immunoaffinity columns. They take advantage of a special hydrazide linkage that binds antibodies through the carbohydrate residues on their Fc regions. This leaves the antigen-binding domains fully accessible to enable the most effective capture of desired target (Figure bellow).
CIMac™ HDZ monoliths make HDZ-immobilized antibody columns even more effective. Because of their large channel size and the efficiency of convective mass transport, they eliminate the long loading residence times that are required for affinity chromatography on porous particle columns. Flow rates of 5–10 column volumes per minute allow complete purifications in a few minutes, even when the source material contains a low concentration of antigen. The same performance is achieved whether a small peptide or a large bio-assemblage like a virus particle or extracellular vesicle is isolated. The combination of HDZ monoliths and the immobilization protocol offers a strong tool for fast antigen isolation from complex biological sample (plasma, lysate, etc.) and consequently sensitive antigen quantification. An example of CIMac™ HDZ application is a purification of fibrinogen from human plasma.

Attachments

Full view

Chromatography is a useful purification method for large biomolecules and virus manufacturing and it is easily scalable to large production volumes. Convective Interaction Media (CIM) monolithic columns constitute of large flow-through channels and consequently have high surface accessibility of binding sites. Preferences of CIM monolithic columns are flow independent performance, resulting in fast separation, concentration, purification, impurities removal, and analytics of biopharmaceuticals.
The aim of the study was to develop Influenza virus purification platform, which can be used for several virus strains. The main objective was to develop a process with as little as possible of intermediate steps, especially omitting Tangential Flow Filtration (TFF) or other sample pre-treatments with high host-cell DNA and protein removal, as well as to achieve high binding capacity of the Influenza virus per mL of monolithic support.

Attachments

Full view

During recombinant adeno associated virus (rAAV) downstream processing, a large amount of host-cell and product related impurities needs to be removed from the product. Succesful process on laboratory scale, such as Cesium chloride purification, lacks scalability when the process is due to be transfered to larger industrial scale. The aim of the study was to develop robust, fast and effective rAAV virus purification platform, which can be used for several AAV serotypes with various inserts. Lysed harvest and supernatant of rAAV9 were first captured and concentrated on CIMmultus™ OH column, followed by intermediate step on CIMmultus™ SO3 column and further polishing on CIMmultus™ QA column. Derived purity of industrial scale monolith purification product was compared to laboratory scale purification.

Attachments

Full view

CIM® chromatographic monoliths enable high 1) productivity of pDNA downstream process (DSP) due to high dynamic binding capacity for pDNA in small elution volumes and short chromatographic runs; 2) high resolution power due to convective-based mass transfer.

Sample displacement mode utilizes different relative binding affinities of components in a sample mixture and separates pDNA isoforms under overloading conditions - where sc pDNA isoform acts as a displacer of oc or linear pDNA isoform.

Attachments

Full view

2017

Preparative scale chromatographic separation of open-circular (oc) from supercoiled (sc) plasmid DNA (pDNA) isoforms has been already established on CIM® C4 with high ligand density (C4 HLD) monolithic columns with sample loading in 3.0 M ammonium sulphate (AS). The process requires high molarity of AS, increasing the overall cost of the process. Sample displacement chromatography (SDC) can be used as an alternative to decrease the AS concentration required during loading onto hydrophobic chromatographic supports. This study compares three chromatographic monoliths with different hydrophobic ligands on the surface (C4 HLD, pyridine and histamine) for the purification of different pDNA vectors in SD mode.

Attachments

Full view

New vaccines against Influenza A are required each year to keep up with the most virulent evolving strains. This highlights a need for predictive analytical tools that can aid purification process development and validation. Rapid and reliable quantification of Influenza A virus is therefore of the utmost importance for enabling good yields and controlling the costs of the downstream processing. Here we demonstrate the ability of monolithic chromatography media to produce process predictive profiles that can document ability to remove impurities and obtain high product recoveries.

CIMac™ Analytical Columns are short bed high performance monolithic columns offering all the advantages of CIM® monolithic technology. Their small volume and short column length allow the operation at high volumetric flow rates enabling to receive the information about the product quantity and purity in just a few minutes. Hence, the CIMac™ Analytical Columns can be effectively used for the in-process and final control of various samples from different purification process steps.

Attachments

Full view

2016

Adeno-associated virus (AAV) vectors of various serotypes are considered to have high potential for gene therapy applications. Currently, manufacturing of AAV vectors faces the challenge of co-production of incompletely formed particles lacking a recombinant viral genome. Empty capsids increase the dose of total AAV administered for efficient transduction and are thought to cause unwanted immunological reactions against the virus.Removal of empty capsids during manufacturing, as well as analysis of empty/full AAV particle content is therefore a critical requirement for any AAV production process. This poster demonstrates how CIMmultus™ QA monolithic columns can be used to remove empty AAV capsids from the product chromatographically in a single step.

Attachments

Full view

The upstream and downstream monoclonal antibody (mAb) bioprocessing makes them susceptible to physical and chemical modifications. In the biotechnological production process of mAbs, structural variations may arise due to some enzymatic activity. Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity and cation-exchange chromatography (CEX) is one of the typical approaches for mAb charge variant analyses. We tested several CEX columns under different conditions and the best column for isotype separation was weak cation-exchanging CIMac COOH chromatographic monolith in pH gradient. We have proven a flow independent separation of mAb charge variants and in this way, a resolution comparable to classical CEX particulate-based analytical columns was achieved in only 6 min analysis time.

Attachments

Full view

Since plasmid DNA (pDNA) as a pharmaceutical product has stringent requirements of purity and efficacy, one or more chromatographic steps are often used in the downstream processing train. High ligand density butyl-modified (C4 HLD) monolithic support is currently used in a polishing step of a pDNA purification process (1) and is mainly focused to supercoiled (sc) pDNA isoform separation from the open circular (oc) and linear pDNA isoform as well as for removal of remaining gDNA and RNA. The goal of the study was to compare the productivities of two variations of the polishing chromatographic process employing monoliths – classical bind-elute (BE) versus recently described (2) sample displacement purification (SDP). Classical purification requires high concentration of ammonium sulphate (AS) during loading step and elution is then achieved by descending AS gradient. SDP utilises different relative binding affinities of components in a sample mixture and separates pDNA isoforms under overloading conditions, where sc pDNA isoform acts as a displacer of oc or linear pDNA isoform.

Attachments

Full view