Journal: Pharmaceutics

Article Title: Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

doi: 10.3390/pharmaceutics13050748

Figure Lengend Snippet: Overview of the five tested purification strategies to purify and to separate the empty and full recombinant adeno-associated virus (rAAV). For each experiment, only one harvesting method mentioned in the first arrow was performed. The abbreviations (in white) on the right side are also used throughout the text and refer here to the shown purification processes. AC, affinity chromatography; AEX, anion exchange chromatography; CEX, cation exchange chromatography; HIC, hydrophobic interaction chromatography; PEG, polyethylene glycol; TFF, tangential flow filtration.

Article Snippet: We therefore explored another preparative AEX column (CIMmultus PrimaS (AAV), BIA separations) for full/empty separation.

Techniques: Purification, Recombinant, Virus, Affinity Chromatography, Chromatography, Hydrophobic Interaction Chromatography, Filtration

Journal: Pharmaceutics

Article Title: Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

doi: 10.3390/pharmaceutics13050748

Figure Lengend Snippet: Comparison of HIC-CEX-AEX and AC-AEX for purification of the rAAV2/8 vectors. Each chromatogram shows the respective ultra-violet (UV) signal in blue (scale on the left Y -axis) and the concentration of buffer B depicted as a dashed green line (scale on the right). ( A – C ) Chromatograms obtained with HIC-CEX-AEX in three subsequent steps on a HIC ( A ), a CEX ( B ), and an AEX ( C ) column. ( D ) SV-AUC analysis of rAAV2/8 of the indicated samples from different purification states. ( E , F ) Chromatograms obtained with AC-AEX in two subsequent steps on an AC ( E ) and an AEX ( F ) column. ( G ) Magnification view of the AEX chromatogram shown in F (focusing on the 120–150-mL range) to better visualize the peaks of the empty and full rAAVs. ( H ) SV-AUC analysis after AC and AEX. ( I ) Silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the rAAV material after TFF, and the intermediate steps from the purification downstream processes HIC-CEX-AEX and AC-AEX. AC, affinity chromatography; AEX, anion exchange chromatography; CEX, cation exchange chromatography; HIC, hydrophobic interaction chromatography; PEG, polyethylene glycol; rAAV, recombinant adeno-associated virus; TFF, tangential flow filtration; VP, viral protein.

Article Snippet: We therefore explored another preparative AEX column (CIMmultus PrimaS (AAV), BIA separations) for full/empty separation.

Techniques: Comparison, Purification, Concentration Assay, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Affinity Chromatography, Chromatography, Hydrophobic Interaction Chromatography, Recombinant, Virus, Filtration

Journal: Pharmaceutics

Article Title: Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

doi: 10.3390/pharmaceutics13050748

Figure Lengend Snippet: Effect of different harvesting methods on the rAAV2/8 vector production yield and pureness of the human embryonic kidney (HEK) 293T cell DNA . ( A – F ) Graphs showing the yield (total vector genomes, vg) for the rAAV2/8 vectors obtained with the two strategies, with materials harvested from ( A , B ) the cell culture supernatant and cell pellet lysate filtered, concentrated, and buffer-exchanged by tangential flow filtration (TFF) ( n = 3). ( C , D ) Culture supernatant by polyethylene glycol (PEG) precipitation ( C ), followed by HIC-CEX ( n = 2) and ( D ) followed by AC-AEX ( n = 5) or ( E , F ) cell pellet lysate ( E ) followed by HIC-CEX ( n = 2) and ( F ) followed by AC-AEX ( n = 5). ( G , H ) Graphs showing the removal efficiency of the strategies for HEK cell DNA at all the purification steps. AC, affinity chromatography; AEX, anion exchange chromatography; CEX, cation exchange chromatography; HIC, hydrophobic interaction chromatography; PEG, polyethylene glycol; rAAV, recombinant adeno-associated virus.

Article Snippet: We therefore explored another preparative AEX column (CIMmultus PrimaS (AAV), BIA separations) for full/empty separation.

Techniques: Plasmid Preparation, Cell Culture, Filtration, Purification, Affinity Chromatography, Chromatography, Hydrophobic Interaction Chromatography, Recombinant, Virus

Journal: Pharmaceutics

Article Title: Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

doi: 10.3390/pharmaceutics13050748

Figure Lengend Snippet: Purification of the rAAV2/8 vectors using the CEX-AEX process. ( A – C ) Chromatograms obtained with cell pellet materials in only two steps on the CEX ( A ) and AEX ( B ) columns. ( A ) CEX with a linear buffer B gradient. ( B ) AEX with a linear buffer B gradient. Magnification view of the AEX chromatogram shown in B. Each chromatogram shows the respective UV signal in blue (scale on the left Y -axis) and the concentration of buffer B depicted as a dashed green line (scale on the right). ( C ) Graph showing the yield (total vector genomes, vg) for the rAAV2/8 vectors obtained in CEX-AEX with the material harvested by lysis of the cell pellet. The residual HEK cell DNA amount is shown in ( D ). Due to matrix effects in the qPCR, the starting amount cannot be shown here. AAV, recombinant adeno-associated virus; AC, affinity chromatography; AEX, anion exchange chromatography; CEX, cation exchange chromatography.

Article Snippet: We therefore explored another preparative AEX column (CIMmultus PrimaS (AAV), BIA separations) for full/empty separation.

Techniques: Purification, Concentration Assay, Plasmid Preparation, Lysis, Recombinant, Virus, Affinity Chromatography, Chromatography

Journal: Pharmaceutics

Article Title: Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

doi: 10.3390/pharmaceutics13050748

Figure Lengend Snippet: Purification of the rAAV2/8 vectors using CEX-AEX(E/F). ( A – C ) Chromatograms obtained with a PEG-precipitated material and a CEX-AEX(E/F) process in only two steps on a CEX ( A ) and a novel CIMmultus PrimaS (AAV) ( B , C ) column. ( A ) CEX with a linear buffer B gradient. ( B ) CIMmultus PrimaS (AAV) with a linear buffer B gradient. ( C ) CIMmultus PrimaS (AAV) with a step gradient of 25%, 40%, and 55% buffer B. Each chromatogram shows the respective UV signal in blue (scale on the left Y -axis) and the concentration of buffer B depicted as a dashed green line (scale on the right). ( D ) Graph showing the yield (total vector genomes, vg) for the rAAV2/8 vectors obtained in the CEX-AEX(E/F) downstream process with material harvested by PEG precipitation ( n = 2) from the cell culture supernatant and cell pellet lysate ( n = 3). ( E ) Residual human embryonic kidney (HEK) cell DNA amount determined by real-time quantitative polymerase chain reaction (qPCR). ( F ) Silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the material from each step of the purification CEX-AEX(E/F) downstream process. AEX, anion exchange chromatography; CEX, cation exchange chromatography; PEG, polyethylene glycol; rAAV, recombinant adeno-associated virus; TFF, tangential flow filtration; VP, viral protein.

Article Snippet: We therefore explored another preparative AEX column (CIMmultus PrimaS (AAV), BIA separations) for full/empty separation.

Techniques: Purification, Concentration Assay, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Chromatography, Recombinant, Virus, Filtration

Journal: bioRxiv

Article Title: Extensive length and homology dependent chimerism in pool-packaged AAV libraries

doi: 10.1101/2025.01.14.632594

Figure Lengend Snippet: (a) A complex doubly barcoded cloning dock with an a priori determined valid barcode pairs dictionary serves as the starting point to clone libraries of barcoded inserts varying length and class (homologous & non-homologous) within an AAV cargo. (b) Barcoded libraries were separately packaged in AAVs, and submitted for direct long-read sequencing, in parallel with corresponding insert size-selected from the plasmid digest. (c) Quantification of the fraction of discordant barcode pairs as a function of the full-length BC-to-BC average size. Each point corresponds to swap quantification from 6 separate libraries ([short, mid, long] × [homologous, non-homologous]) for both plasmid-derived (square) and AAV-derived (circle) material (n=1 replicate per library). For each datapoint, we analyzed full length BC-to-BC reads passing quality control filters and having separately valid BC1 and BC2 ( , ). Right panel corresponds to a zoomed inset showing the y axis range from 0 to 0.1. Quantifications derived from library p153 × corresponding to the pool of multiply-sized inserts in a single AAV-packaged sample are marked by an ×. Vertical error-bars correspond to 20 th to 80 th percentiles from bootstrap resampling. Horizontal error-bars to the 10th to 90th percentile of the BC-to-BC length distribution from plasmid digest inserts. Swaps are significantly higher (one-sided bootstrap FDR: ***<10 −5 ) for the homologous vs. their respective size-matched non-homologous libraries. (d) Same as (c), but with additional packaging conditions with library p153 × corresponding to AAV2 serotype and sparse co-packaging with carrier DNA. With both PHP.eB and AAV2, and for the three sizes of inserts, sparse packaging reduces swapping significantly (one-sided bootstrap FDR: *<0.005, **<0.0005, ***<10 −5 ) albeit incompletely. (e) Comparison of the fraction of discordant barcode pairs from PCR-derived libraries (x-axis) vs. direct [same data as panel (c)] (y-axis), see also . AAV samples only include the PHP.eB serotype with standard packaging conditions. Errorbars correspond to 20 th to 80 th percentiles from bootstrap resampling of reads as in panel (c).

Article Snippet: The AAV sequencing library preparation from Plasmidsaurus, based on a recent protocol , relies on annealing of the ssAAV DNA followed by end-repair and adapter ligation.

Techniques: Cloning, Sequencing, Plasmid Preparation, Derivative Assay, Control, Comparison

Journal: bioRxiv

Article Title: Extensive length and homology dependent chimerism in pool-packaged AAV libraries

doi: 10.1101/2025.01.14.632594

Figure Lengend Snippet: Similar to with related modifications. (a) Table showing long-read counts retention at different filtering steps in our pipeline (schematic at right indicate filter nature), e.g., presence of 4/4 signpost sequences and (separate) exact matches to BC1 and BC2 from our dictionary. (b) Cumulative distribution of alignment scores to the signposts. Thresholds for detection (75% match) are shown as dashed lines at top. (c) Two-dimensional histogram showing the Levenshtein distance to the best and second best matches to insert indices. Decision boundary to deem insert demultiplexing as unambiguous is shown by the red dashed line. (d) Cumulative distribution of the BC-to-BC length (from middle of both barcodes) as determined by the detected signpost positions from the reads passing signpost quality control steps and with separate exact matches to BC1 and BC2. Each panel comes from a separate AAV packaging sample, and was indexed separately (Plasmidsaurus) for ONT sequencing. For p153 × samples, different inserts were demultiplexed using the internal insert index. Line type is related to insert size category (parental, short, mid-sized, long), color to insert class (black: parental, light blue: homologous, orange: non-homologous). Vertical lines indicate 1% and 99% percentiles from the plasmid libraries (same as ) used to call a read ‘full BC-to-BC length’. Note the substantial fraction of reads with shorter than expected lengths for the long insert libraries. (e) Similar to panel (d), but for total read length. Vertical lines used to consider whether the read is full ITR-to-ITR or not (p153 × -short: 500 to 750 nt, p153 × -mid: 1100 to 1400 nt, all others: 2250 to 2750 nt) for analysis of Table S3. (f) Table of counts of valid reads stratified by demultiplexed libraries (based on insert index), tallying proportion of reads with valid barcode pairs. Quantiles (1%, 10%, 90%, 99%) of the BC-to-BC length across the libraries is also shown. (g) Plasmidsaurus (order ID L9RV6B) virtual gel for confirmation of p153, detecting only a single product. (h) Agarose gel (low and high contrast) of undigested p153, showing evidence of possible lower molecular weight products (arrows). (i) PCR from both AAV template from p153 [serotype PHP.eB, standard condition, sample 5] (left) and plasmid (right) with respectively primers o949+o950 (ITR-to-ITR) and o949+o936 (BC-to-BC). We note that band intensities are probably not representative of species abundance due to possible length-bias of amplification. The three products are of the expected sizes for libraries with short, mid-sized, and long inserts respectively, consistent with their detection in the long-read data.

Article Snippet: The AAV sequencing library preparation from Plasmidsaurus, based on a recent protocol , relies on annealing of the ssAAV DNA followed by end-repair and adapter ligation.

Techniques: Control, Sequencing, Plasmid Preparation, Agarose Gel Electrophoresis, Molecular Weight, Amplification

Journal: bioRxiv

Article Title: Extensive length and homology dependent chimerism in pool-packaged AAV libraries

doi: 10.1101/2025.01.14.632594

Figure Lengend Snippet: (a) Flowchart illustrating the different types of libraries considered (starting material: plasmids & AAV packaged DNA), method (PCR-based vs. direct, i.e., PCR-free, corresponding to digestion & size selection for plasmids, and annealing fpr AAVs, prior to end-repair & adapter ligation). PCR libraries were pooled prior to submission and library identity assigned by demultiplexing based on the internal index (see ). (b) Similar to , now also showing the PCR-derived libraries (open symbols). This data is represented as a comparison between direct/PCR libraries in . (c) and (d) Table showing long-read counts retention at different filtering steps in our pipeline for PCR libraries prepared from AAV packaged DNA and plasmids respectively. (e) Cumulative distribution of the BC-to-BC length (from middle of both barcodes) as determined by the detected signpost positions from the reads passing signpost quality control steps and with separate exact matches to BC1 and BC2. Line type corresponds to template type (full: plasmid, dashed: AAV), color to insert class (black: parental, light blue: homologous, orange: non-homologous). Vertical lines indicate 1% and 99% percentiles of the length distributions respectively from the plasmid digest (and used to consider a long-read in the AAV-packaged data as ‘full BC-to-BC length’). Panels correspond to different insert libraries. Parental inserts from the AAV sample is not shown as it cannot be reliably attributed to a sample (came from residual empty plasmids packaged from all libraries). (f) Similar to panel (e), but for the full read length. Grey arrows indicate ‘dimer reads’ (see Methods ) that were roughly twice the length of the expected full length reads. (g) Tapestation (D5000) of pooled PCR-prepared libraries submitted for ONT sequencing, showing predominant expected product (≈2.2 kb), and minor empty product from p146 (≈900 bp), and lack of product >4 kb corresponding to ‘dimer reads’ seen in (f). (h) Table of read counts with valid barcode pairs and valid fraction across libraries originating from plasmids or AAV packaged DNA (different rows), and direct (PCR-free) [central columns] vs. PCR-derived libraries [right columns]. The data from the direct libraries is reproduced from and for plasmid digest and AAVs respectively.

Article Snippet: The AAV sequencing library preparation from Plasmidsaurus, based on a recent protocol , relies on annealing of the ssAAV DNA followed by end-repair and adapter ligation.

Techniques: Size Selection, Adapter Ligation, Derivative Assay, Comparison, Control, Plasmid Preparation, Sequencing