ChromaChats™ Recap

May 2023 ChromaChats™: Process Improvement & Next-Generation Purification Platforms of AAVs

We had an incredible group for our second ChromaChats™ meeting on Wednesday, May 31 st . We had 35 industry leaders in attendance and our discussion focused on “Process Improvement and Next-Generation Purification Platforms of AAVs”. This roundtable was led by ChromaTan Founder Oleg Shinkazh, and featured presentations from six fabulous leaders in the AAV
purification space. We’ve included a few bullet points from each presentation below:

Dr. Richard Braatz – Professor at Massachusetts Institute of Technology

We mapped out regions of pH and precipitant concentrations in which crystallization of full and empty capsids occurs
Preferential crystallization has potential as an alternative method for the purification of full capsids

Dr. Xiaotong Fu – Head of Downstream at Resilience

A reproducible, fully automated, and scalable AEX manufacturing process was developed to remove empty capsid from half-loaded AAV.
With proper process optimization, AEX chromatography processes can deliver ultra-pure material (greater than 90%full by AUC) with good yield (65%+ step yield).
Process scalability from bench scale to GMP scale were confirmed and consistent process performance and product quality were observed.
AUC is still the gold standard for empty/full measurement, but CDMS and mass photometry also show good alignment.

Oleg Shinkazh – Founder at ChromaTan

Chromatan’s CCTC platform is a continuous and single-use technology very well suited for AAV capture purification
The column-less system can capture AAVs using POROS AAVX off the shelf resin with very high recovery (95% vs 60-70% in columns)
The platform can cycle the resin very aggressively and thus reduce resin consumption 10-20-fold for AAV capture
The platform has shown higher purity / less HCP in product effluent (below detection)
Chromatan is looking for partners to continue the work with various serotypes of AAV
Future plans include empty vs full separation and fully integrated AAV purification process

John Taylor – Process Development Scientist at MilliporeSigma
Separation of AAV Capsids Using Fractogel ® Resin

Fractogel® TMAE (S) resin offered higher resolution of AAV8 capsids compared to Fractogel® TMAE (M) resin.
A “hybrid gradient” elution strategy selectively removes empty capsids in a gradient wash, followed by full capsid recovery in a simple step elution.
A254/A280 ratio improved from 0.85 in feed to 1.05 in elution.
Full capsid purity improved from 15-20% to 42-53% * . Greater than 2.5x enrichment achieved.
Full capsid yield 36-42% * .
Compared to linear gradient elution, the “hybrid gradient” approach enables collection of a single elution pool, as opposed to many fractions across a gradient. Manufacturing friendly.
Adding 2mM MgCl 2 to the empty capsid wash buffer may facilitate a step wash, rather than a gradient. Step wash offered higher purity (58%) but lower yields (24%) compared to a gradient wash strategy.
Wash with 28% Buffer B ** provided optimal performance in this case. Wash conditions may differ for other AAV feeds and serotypes. Linear gradient PD studies must be performed for each feed type.

Dr. Tony Tavares – Principal Scientist at Teknova
‘Optimized AEX Buffer Formulations for AAV Full Capsid Enrichment’ and the AAV·Tek™ AEX Buffer Screening Kit: AAV2

Teknova can accelerate the introduction of diagnostics, novel vaccines, and next-generation therapies to Clients by providing Clients with high-quality, custom products with short turnaround times
Utilizing DoEs with significant experimentation and analytics, the R&D team at Teknova has determined optimized buffer formulations that can effectively separate empty and full capsids of AAV during the AEX chromatography polishing step
The factor contribution levels (base buffer type and concentration, elution salt, detergent type and concentration, stabilizer type and concentration, acidity/alkalinity, and excipient presence and concentration) associated with maximum and robust quality attributes (infectivity, peak separation, full capsid recovery, and purity) change for each serotype/elution salt combination
Teknova has determined and demonstrated a panel of AEX buffers that are versatile in the capacity to resolve empty and full capsids from different serotypes, USP/DSP platforms, and different transgene constructs
The AAV·Tek™ AEX Buffer Screening Kit: AAV2 is commercially available and contains 7 paired sets of 1 L equilibration and elution buffers (14 bottles total) that can save months in development time and provide the confidence to scale from research through process development into commercialization
AEX Buffer Screening Kits for AAV6, AAV8, and AAV9 will shortly be commercially available

Kevin Roberts – Sr. Manager at Thermo Fisher Scientific

ThermoFisher Scientific is offering free of charge service AAV affinity capture process optimization
Using customer criteria designated at project on-set, TFS scientists will optimize a process using customer provided feed stock material
The output of this process will be material, methods, report, and process for the affinity capture and elution of target molecule as per Statement of Work established by collaboration with TFS and the customer.

April 2023 ChromaChats™: Challenges in the Purification of AAVs

We had a wonderful turnout for our first ChromaChats™ meeting on Wednesday, April 26 th . We had 30 industry leaders in attendance for a discussion focused on “Challenges in the Purification of AAVs”. This roundtable was co-led by Chromatan CEO Oleg Shinkazh and Dr. Peter Abbink, Managing Director at Batavia. The extensive discussion addressed many topics ranging from purification operations, pain points such as high cost and low recovery, scalability challenges, as well as what the future of AAV manufacturing looks like. We cherrypicked some points below, but only the attendees get the full scoop.

Topic: Pain Points for AAV Purification – Loss in Recovery for AAV Capture

Meat of the problem = scalability & chromatography.
One thing Oleg from Chromatan learned in their process is that a small addition of poloxamer made a significant difference. They were running a process where for the first 30 minutes, product was getting absorbed somewhere on the tubing or in the system, but when they added 0.01%, they were able to recover much faster.
AAV protein concentration is quite low. One attendee discussed how when protein concentration is low, you notice how much more it sticks to surfaces. Using products to combat surface adherence can boost yields.

Topic: Do certain resins work better to optimize elution profiles?

An attendee on the call discussed her company’s experience and the importance of finding good resin:

With all the new resins coming in, she explained how they don’t want to miss an opportunity to find one, but that it’s a hard balance between using material, using time, and also screening the resins thoroughly. One of the main methods they employ is very small-scale screening using a liquid handler.
Small-scale screening has worked well and has scaled up well. With the liquid handle, it takes a lot of preparation to make sure it can act like the platform you use. Important to match it to your system as much as possible.
Volume for experiment depends on load and concentration you are starting with. The attendee’s company uses very little because liquid handler they use has 200μL and 600μL columns.
Another pain point: just because they have a nice separation process for one certain type of capsid doesn’t mean it will always work for the others.

Topic: Any guidance from regulatory and what they are looking for full percentage?

One attendee shares his experience and thoughts:

Attendee discussed how what a regulatory agency is looking for but won’t tell you is “is your product safe”? That’s ultimately what you are trying to achieve and it’s not something any regulatory agency can tell you as they themselves don’t know.
There are so many factors that come into play with product safety – it’s very product specific, it depends on what tissue you are trying to deliver it to, and what your delivery method is. Up to you to show why you think the distribution profile of your product is safe and to achieve empty/full ratios that you can safely demonstrate in animal and human models.

Topic: Where is the value on the triangle of product quality, yield, and cost?

One attendee shares how as a process development person, he tends to stray towards product quality, but that there is a direct trade-off in gene therapy with product quality and yield surrounding empty/full separation.
How do you increase yield while maintaining product quality? Attendee shares that the key to achieving both is to develop a technology that can be both high yielding and support high purity.