Posters

Extracellular vesicles (EV) are lipid bound products secreted by cells. Among them, exosomes have great potential for clinical applications. Animal and human-derived components used in cell culture, such as fetal bovine serum (FBS), naturally contain exosomes that can cross-contaminate the desired product. In order to study exosomes derived from cells of interest, multiple producers have come up with exosome-depleted FBS (EV (-) FBS) generated using different approaches. In this work we evaluated commercially available EV (-) FBS supplements for residual exosome content and tested their performance in upstream exosome production process. The analysis was performed with PATfix high pressure liquid chromatography system using PATfix size exclusion (SEC) analytical method.

Removal of impurities generated in the production of adeno associated virus (AAV) is an important step as they may pose a serious health risk, as well as deteriorate the economics of the production process. The most critical subsets of these impurities include: host cell nucleic acids, host cell proteins, chromatin, capsid aggregates, capsid DNA complexes and empty capsids.
This poster introduces two new column s for performing separation of empty (E) and full (F) capsids using multimodal approach. A PATfix® system with three different detectors, i. e. absorbance, fluorescence, and light scattering in combination with three analytical columns traded as CIMac ™ AAV Empty/Full, CIMac PrimaS ™ (AAV) - Beta, and CIMac ™ PrimaT - Beta by BIA Separations Sartorius. The separation columns were used to determine and evaluate empty AAV capsids as one of the critical impurities in AAV samples. The analytical results using CIMac PrimaS™ (AAV)-Beta and CIMac™ Prima T- Beta show that other fast and reliable orthogonal HPLC methods to the CIMac™ AAV full/empty column can also be used for the separation of empty and full capsids with monolithic columns.

Adenovirus has after two decades gained new consideration and is now used as a COVID-19 vaccine delivery vehicle. To reduce side effects of the vaccine it’s purity is of utmost importance. Constant enhancement of the vaccine purity and improvement of the impurity detection methods is therefore necessitated. In this work we present second generation adenoviral vectors purification procedure based on monolith chromatography using CIMmultus QA to secure safer product, as well the accompanying analytical tools. The novel industrial process secures better purity at higher yields. The robustness of the process was verified using different upstream materials. Manufacturing of the vaccines in large quantities due to pandemic represent great challenges, mainly in terms of production time and costs. Higher capacity of the CIMmultus QA columns used in this process overcomes the raw material supply bottlenecks.

Density gradient ultracentrifugation (DGUC) is a well established tool for Empty/ Full AAV capsid separation based on density differences between AVV sub-populations. However DGUC practice is laborious and lacks any detection options, therefore fractions must be collected manually and analyzed later. Both of these shortcomings can be addressed by coupling post DGUC workflow to PATfix analytical system. BIA Separations PATfix platform provides sufficient tools for liquid extraction and fractionation as well as a comprehensive detector suite for precise fraction characterization. Baseline separation of capsid species was achieved in a density gradient of CsCl, producing a centrifugram that reveals information traditional DGUC and anion exchange chromatography cannot.

Removal of empty capsids is a particular goal of AAV purification. Multimodal PrimaT ligand offers new options for removal of empty and also
damaged capsids. Besides it also contributes to better clearance of contaminating DNA. PrimaT works for different AAV serotypes - for example AAV 2/8 and AAV 2/9. AAV 2/8 or AAV 2/9 clarified harvest from Sf 9 cells was first processed by tangential flow filtration (TFF) coupled with Kryptonase treatment to reduce host cell DNA. Initial AAV capture step was performed on CIMmultus SO3 cation exchange column. After elution with sodium chloride gradient AAV fraction was cleared of DNA and protein contaminants. Separation of empty and full AAV capsids was performed by multimodal metal affinity chromatography with CIMac and CIMmultus PrimaT.

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.

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.

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. 

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.

Removal of host cell DNA is essential for all human-injectable biologics. This poster shows a method for achieving low host cell levels in preparations of exosomes. Purified exosome samples were prepared with anion exchange chromatography (AEC) and pre-treated with tangetial flow filtration (TFF) and nuclease treatment. Results are compared with an experimental control using TFF and size exclusion chromatohraphy (SEC).

The steps in purification process are illustrated by analytical size exclusion chromatography (SEC) on PATfix system with in-line UV, MALS and fluorescence detectors and by staining with Picogreen reagent. This technique visualizes sample composition by size, UV, light scattering and fluorescent properties.

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.

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).

Exosomes fulfill a critical role as communicators among cells, with targeting and message content depending on their surface receptors and payload. This makes them obvious candidates for an extensive range of diagnostic, therapeutic applications and a need for a fast, robust and scalable purification procedure.

CIMmultus™ monolithic columns are designed to meet the special fractionation needs of very large biologics like exosomes.

We show examples of exosome purification from cell culture with CORNERSTONE Exosome Process Development Pack and analysis of exosomal vesicle populations by Image stream flow cytometry.

This poster shows how Multi-Angle Light Scattering detector and Fluorescence detector couppled to PATfix analytical system can be used to track extracellular vesicles through purification process. Samples were analyzed by analytical size exclusion chromatography (SEC). On SEC cell culture components diffuze into pores of chromatographic media and are separated (mostly) based on size. Particles larger than the media pore size are excluded in the void peak. This peak represents extracellular vesicles including apoptosomes, microvesicles and exosomes as well as cell debris and aggregates.

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.

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.

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!

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).

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.

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.