Posters

Quaternary amine (QA) modified monolithic columns are frequently used for purification of therapeutically relevant 
adeno-associated virus (AAV) vectors, used in gene therapy. Beside process- and sample-derived variability, 
chromatographic material variability can influence the efficiency and scalability of AAV downstream processing. This study 
presents the evaluation of highly reproducible (HR) QA-modified CIM monolithic columns, where the goal is to elute AAV 
capsids in a very narrow conductivity range, regardless of the batch and size of the the column used. 
We have developed a chromatographic test method for proving the intra- and inter-batch homogeneity of the material 
through different column sizes. The method is based on separation of AAV2/8 capsids in ascending KCl gradient on 
CIMmultus QA 1 mL columns and specimen 0.2 mL units, taken from large-scale QA monoliths up to 8000 mL in size.

Lentiviral vectors are efficient tools for transfer and stable integration of large gene inserts into the genomes of both dividing and non-dividing cells. Third generation of lentiviral vectors, which uses a fraction set of HIV genes, is replication incompetent and self-inactivating, offering a relatively safe tool for academic and industrial use, while offering a larger gene transfer capabilities compared to more commonly used AAV. As a result, several therapies using lentiviral vectors are already approved or in clinical trials, with primarily ex-vivo use. Limitations for more widespread use, as well as development of in-vivo therapies, however remain, with one of the causes being downstream purification of lentiviruses. Here we showcase a process development for CIM monolithic columns for use in downstream purification of lentiviruses. Several CIM monolithic chemistries have been tested to determine their performance for lentiviral purification, with three chemistries showing potential for further use. Of the three chemistries, two have been selected for further development. Several modifications of the two chemistries have been prepared and tested on CIMmic columns with promising initial results. The chemistries have been successfully upscaled to CIMmultus monoliths and the results from initial findings confirmed. Additional experiments are required to confirm the findings and perform optimization. The full downstream process will be developed once the most suitable chemistry is selected. During preparative, multi-angle light scatter (MALS) was used as indicator of viral presence, while analytics, used in the process development, were ddPCR and infectivity test.

Several recombinant AAV (rAAV)-mediated therapies have been approved so far, and many more are in clinical trials, yet developing an efficient rAAV process platform remains a challenge. First obstacle arises from frequent practice of upstream (USP) and downstream (DSP) process being developed separately. USP optimization activities are usually focused on achieving high viral titer, which does not always translate into maximal purity and recovery in DSP. Second significant challenge is to bridge USP and DSP into a single bioprocess, due to lack of appropriate analytical methodology capable of producing reliable and unbiased results on both sides of the bridge. Furthermore, rAAV-based gene therapy vectors require the removal of process-specific combined with product-specific impurities, as they represent serious safety threats. Here we showcase the example of AAV8 USP optimization directed towards good performance in DSP. AAV8 is produced in HEK suspension cells via plasmid transfection. Based on the screening results, the best performing set of conditions was successfully scaled up to a 5L stirred-tank bioreactor. Harvested material was then evaluated in DSP, comprised of pre-capture, and two ion-exchange chromatographic steps on CIM monolithic columns. As bridging analytics of choice, we relied on PATfix platform, (d)dPCR and Comassie Bradford protein assay measurements for thorough assessment of relevant parameters such as titer, percentage of full AAV8 capsids and total protein concentration.

In every adeno associated virus (AAV) downstream process the key steps are the enrichment of full capsids and the in-process empty/full analytics. A common approach for enrichment of full capsids is liquid chromatography using ion exchange or multimodal chemistries, which based on particles’ surface charge differences, enables separation of full (F) capsids and product related impurities. Removal of impurities is an important step as they may pose a serious health risk, as well as deteriorate the economics of the production process. A PATfix® triple detector set in combination with an CIMac™ AAVEmpty/Full analytical column by Sartorius BIA Separations was used to determine E/F ratio. The column is a strong anion exchanger which exploits capsid charge differences for separation when employing an ascending salt gradient. Usually, NaCl with the addition of 2 mM magnesium is used, as the presence of magnesium has been shown to improve the peak shape and the removal of empty capsids. According to the Hofmeister series salts have different impact on non-covalent interactions among capsid themselves and between columns and capsids. For example, antichaotrope salts were shown to improve the empty/full capsid ratio, possibly by inducing the hydrophobic interactions between them. Quaternary alkyl ammonium salts (QAAS) are known to have antichaotropic properties and they differ in the substituents. Wang et al. reported that QAASs produced a better empty/full separation than NaCl. This poster presents a screening of different eluents for empty/full analytics. Beside NaCl as a reference salt, two QAAS were chosen: tetramethylammonium chloride- TMAC and a possible nonhazardous alternative choline chloride, which is bifunctional and contains both a quaternary ammonium and hydroxyl groups. The influence of magnesium on E/F separation was also tested.

Adenovirus is well-know gene therapy tool that gained attention as a promising vaccine delivery vehicle, specially during Covid-19 pandemics, where it was used to deliver sequence for protein S (S). Multiple serotypes have been tested in clinical trials for various applications, the most common one being human adenovirus serotype 5 (Ad5). With this in mind, we chose Ad5-S construct with GFP tag as a model vector to develop upstream process (USP) and supporting analytical tools.

Endotoxins are robust and persistent impurity, which are native to majority of phage substrates. Two anion exchangers, CIMmultus PrimaS and H-Bond, were tested for their capacity for endotoxin removal in comparison to well known strong anion exchanger, CIMmultus QA. 

Monoliths are commonly used for separation of empty and full AAV capsids on analytical and preparative scale. PrimaS approach for AAV separation employs an ascending pH gradient, which can be sensitive towards small changes in chromatographic parameters. In present work 200 µL testing units extracted from large CIMmultus PrimaS monolithic columns, were employed for the adjustment of critical chromatographic parameters.

Recombinant adeno-associated viral (rAAV) vectors are the leading gene delivery tool for treatment of a variety of diseases. While several rAAV mediated therapies have been approved so far, and many more are in clinical trials, rAAV production still faces many challenges. Key goal of rAAV upstream process development is achievieng high viral titer together with a sufficient percentage of full capsids. Moreover, analysis of complex upstream harvest samples can be challenging. Classical analytical methods such as ddPCR/ELISA offer limited information due to differences in sample preparation and basic principles for detecting empty and full capsids. Method is also time consuming and therefore less useful for following rAAV production process in real time. To overcome these limitations, we developed a PATfix Valve Switch analytical method that is based on ion exchange biochromatography and can be successfully applied for analysis of empty/full ratios in crude upstream samples.

Standard 96-well design offers a great advantage for screening many samples or conditions and supports process automatization. Our approach was multi-parallel screening of different mobile phases for rAAV capture step using CIM® SO3 0.05 mL Monolithic 96-well Plates. Buffers of different pH, sodium chloride concentrations and use of Poloxamer 188 were screened to purify AAV2/9 clarified lysate obtained from Sf9 cells. Sample was pretreated by tangential flow filtration (TFF) coupled with nuclease treatment – Kryptonase. It was shown that the optimal conditions were buffers of pH 3.5, 500 mM NaCl, with addition of Poloxamer 188. Verification of results with selected buffer resulted in high capacity (1.44E14 capsids/mL SO3), great recovery (87.7 %) and excellent protein and DNA reduction (99.98 and 99.25 %).

In each adeno associated virus (AAV) downstream process one of the key steps is enrichment of full capsids. This could be achieved by density gradient ultracentrifugation however a main drawback is its scalability. A more common approach is liquid chromatography using ion exchange chemistries, which based on particles’ charge differences, enables separation of full (F) capsids and product related impurities including non functioning AAV capsids (empty, partially filled, misfolded and wrongly packaged genome or other DNA containing subspecies).

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.

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.

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).

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.