Journal Pre-proof

Manufacturing challenges and rational formulation development for

AAV viral vectors

Arvind Srivastava , Krishna M.G. Mallela , Nandkumar Deorkar ,

Ger Brophy

PII: S0022-3549(21)00193-3
DOI: https://doi.org/10.1016/j.xphs.2021.03.024
Reference: XPHS 2371

To appear in: Journal of Pharmaceutical Sciences

Received date: 23 December 2020

Revised date: 19 March 2021
Accepted date: 30 March 2021

Please cite this article as: Arvind Srivastava , Krishna M.G. Mallela , Nandkumar Deorkar ,
Ger Brophy , Manufacturing challenges and rational formulation development for AAV viral vectors,
Journal of Pharmaceutical Sciences (2021), doi: https://doi.org/10.1016/j.xphs.2021.03.024

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Manufacturing challenges and rational formulation development for AAV viral vectors

Arvind Srivastava1,* , Krishna M.G. Mallela2,*, Nandkumar Deorkar1 and Ger Brophy1

1
Biopharma Production, Avantor, Inc., 1013 US Highway, 202/206, Bridgewater, New Jersey,

United States
2
Center for Pharmaceutical Biotechnology, Department of Pharmaceutical Sciences, Skaggs

School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical

Campus, 12850 East Montview Boulevard, MS C238-V20, Aurora, Colorado 80045, United

States.

*Corresponding Authors

Arvind Srivastava, Avantor, Inc., E-mail: arvind.srivastava@avantorsciences.com

Krishna Mallela, University of Colorado Anschutz Medical Campus, E-mail:

krishna.mallela@cuasnchutz.edu

1
ABSTRACT

Adeno-associated virus (AAV) has emerged as a leading platform for gene delivery for treating

various diseases due to its excellent safety profile and efficient transduction to various target

tissues. However, the large-scale production and long-term storage of viral vectors is not

efficient resulting in lower yields, moderate purity, and shorter shelf-life compared to

recombinant protein therapeutics. This review provides a comprehensive analysis of upstream,

downstream and formulation unit operation challenges encountered during AAV vector

manufacturing, and discusses how desired product quality attributes can be maintained

throughout product shelf-life by understanding the degradation mechanisms and formulation

strategies. The mechanisms of various physical and chemical instabilities that the viral vector

may encounter during its production and shelf-life because of various stressed conditions such as

thermal, shear, freeze-thaw, and light exposure are highlighted. The role of buffer, pH,

excipients, and impurities on the stability of viral vectors are also discussed. As such, the aim of

this review is to outline the tools and a potential roadmap for improving the quality of AAV-

based drug products by stressing the need for a mechanistic understanding of the involved

processes.

Keywords: AAV, antioxidants, chemical stability, deamidation, excipients, formulation, gene

therapy, oxidation, physical stability, viral vector.

Abbreviations: aap, assembly; AAV, adeno-associated viruses; AEX, anion exchange

chromatography; ATR, attenuated total reflectance; cap, capsid; DSF, differential scanning

fluorometry; DSC, differential scanning calorimetry; GOI, gene of interest; IEC, ion exchange

2
chromatography; ITR, inverted terminal repeat; rep, replication; SVHC, substance of very high

concern; TFF, transient flow filtration; Tm, thermal melting temperature; TRS, terminal

resolution site; VR, variable region.

3
1. Introduction

Significant technological advancements have been made over several decades providing

option to treat and control life threatening diseases. One of the most revolutionary advances in

this new era is gene therapy. At its most basic definition, gene therapy (also called human gene

transfer) is the therapeutic delivery of the gene of interest into a patient’s cells as a therapy to

treat a life-threatening disease. The clinical success of gene therapy depends in large part on the

efficient delivery of the genetic material to the target cells. Several different viral-based vectors

and non-viral systems have been evaluated for gene delivery, including nanoparticles, liposomes,

lentivirus, adenovirus, and adeno-associated virus (AAV).1,2 In comparison to other delivery

systems for gene therapy,1,2 AAV has emerged as the predominant vector due to many desirable

attributes, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells

and sustained maintenance of the viral genome. 3,4 Two FDA-approved gene therapy products

currently exist on the market based on AAV delivery technologies. In 2017, FDA approved

Luxturna® as an AAV2-based gene therapy for a rare, genetic form of blindness. 5 Similarly, in

2019, an AAV9-based therapy, Zolgensma®, was approved to treat spinal muscular atrophy

(SMA).6 In addition, hundreds of AAV based gene therapy products are being tested at different

phases of clinical trials. Table 1 provides some examples. 7 These breakthrough milestones in

AAV therapies have encouraged pharmaceutical companies to increase investments in gene

therapy product development and expansion of manufacturing capacities to meet

clinical/commercial demand and supply. However, despite significant clinical and commercial

successes, the cost-effective manufacturing of viral vectors yet remains a challenge, mainly

because of the lack of a clear mechanistic understanding of how various processes impact AAV

product quality and shelf-life. This is because clinical development of AAV gene therapy has

4
outpaced CMC, manufacturing, and formulation development. This review discusses the various

manufacturing steps and challenges encountered during AAV production and storage and

provides a roadmap to improve the efficiency in manufacturing workflow and improve product

shelf-life.

2. Adeno-associated virus

2.1 The virus and its structure

AAV viral vector consists of a protein shell protecting a small, single-stranded DNA

genome of approximately 4.7 kilobases (kb). 2,8,17,18,9–16 AAV viral vectors belong to the

parvovirus family. Efficient replication and viral gene expression of AAV genome requires co-

infection with a second helper virus, such as adenovirus or herpesvirus. 19 Figure 1 illustrates the

capsid surface and domain structure of AAV viral vector. AAVs have an eight-stranded

antiparallel -barrel structure and an -helix. The -barrel forms the core of the capsid, with the

-sheet forming the interior surface providing the enclosure for the packaged ssDNA.20 The

single-stranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap

(Assembly). These coding sequences are flanked by inverted terminal repeats (ITRs) that are

required for genome replication and packaging. The Rep gene encodes four proteins (Rep78,

Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging. The

Cap gene encodes three overlapping structural viral capsid proteins (VP1, VP2 and VP3), which

form the outer capsid shell that protects the viral genome, as well as being actively involved in

cell binding and internalization.12,15,29,30,21–28 The capsid protein viral coat is comprised of 60

proteins arranged into an icosahedral structure with the capsid proteins in an approximate molar

ratio of 1:1:10 (VP1:VP 2:VP3).4,9,10,12,16,21,31 Among capsid proteins, VP1 is the largest (87

kDa), followed by VP2 (72 kDa) and VP3 (62 kDa). 22 The three capsid proteins contain a

5
common β-barrel domain but different N-terminal extensions.14 Variable loops create specific

surface topologies for each AAV variant that mediate the molecular interactions responsible for

cell association, entry, and immunological properties. The aap gene encodes the assembly

activating protein (AAP) in an alternate reading frame overlapping the cap gene. This nuclear

protein provides a scaffolding function for capsid assembly. 27

There are over 100 naturally occurring AAVs that have been isolated in humans and

animals,32 and each differ in their capsid components and display different cellular tropism,

transduction efficiency and immunogenicity. 33 Rep proteins from one AAV serotype can

complement the ITRs from another AAV serotype. The exception is AAV5, which has the least

homology with any of the known AAV serotypes, with its ITR containing a unique terminal

resolution site (TRS).9,34 SDS-PAGE and negative stain electron microscopy (EM) of various

AAV serotypes are shown in Figure 2A.26 AAVs also differ from each other in their stability

(Figure 2B), determined by differential scanning fluorometry (DSF) thermal melting studies. 26

This Tm difference might be associated with the structural difference amongst serotypes. The

study has also demonstrated that the full and empty capsids have similar Tm values for all the

AAVs tested. 26 In another similar study,15 physical properties of the various serotypes of AAV,

in particular AAV1, AAV2, AAV5, and AAV8, were compared to determine the factors that

govern their thermodynamic stability. Thermal stability measurements using differential

scanning fluorimetry (DSF) showed that capsid melting temperatures differed by more than 20°C

between the least and most stable serotypes, AAV2 and AAV5, respectively. The relative sizes

of different virial proteins and their quaternary packing is the major factor determining the

stabilities of individual AAVs.1526

2.2 AAV for in-vivo gene transfer

6
The choice of a particular AAV to use as a gene transfer vector depends on the following

criteria: (1) target cell/tissue types; (2) safety profile associated with the delivered gene; (3)

choice of systemic versus local delivery; and (4) the use of tissue-specific or constitutively active

promoters. The differences in sugar-binding inclinations encoded in capsid sequence in AAV

variants mediate the molecular interactions responsible for cell association, entry, and

immunological properties.2,10,12 After initial binding to cell surface glycans (e.g., heparin sulfate,

N-linked sialic acids, galactose, etc.), the entry of AAV into target cells is mediated by

interactions with co-receptors such as fibroblast growth factor receptor, epidermal growth factor

receptor, and platelet-derived growth factor receptor.2,10,12 For example, AAV9 has a preference

for primary cell binding through galactose as a result of unique amino acid differences in its

capsid sequence.35 It has been postulated that this preferential galactose binding could confer

AAV9 with the unique ability to cross the blood–brain barrier (BBB) and infect cells of the

central nervous system (CNS), including primary neurons. 36,37 In addition to the primary

carbohydrate interactions, secondary receptors have been identified that also play a role in viral

transduction and contribute to cell and tissue selectivity of viral variants. AAV2 uses the

fibroblast/hepatocyte growth factor receptor and the integrins aVb5 and a5b1; AAV6 utilizes the

epidermal growth factor receptor, whereas AAV5 utilizes the platelet-derived growth factor

receptor. AAV8 has been shown to effectively transduce and deliver genes to the liver of rodents

and non-human primates, and is currently being explored in clinical trials to deliver genes for

hemoglobinopathies and other diseases. 38 Similarly, AAV1 and AAV9 have been shown to be

effective at delivering genes to cardiac and skeletal muscle in various animal models. 1,18,39–44

Engineered AAV1 is currently being explored as the gene transfer factor in clinical trials for

heart failure, and has been approved for the treatment of lipoprotein lipase deficiency.45

7
Although different AAV vectors have been identified that preferentially transduce many

different cell types, there still exist certain cell types for which AAVs have proven difficult to

transduce.46 Zolgensma® is an AAV9 and arguably the most successful gene therapy to-date.

However, its formulation may not be the most stabilizing based on its limited shelf-life of 12

months under frozen conditions and only 14 days of stability at 2-8C.6 The AAV2 serotype has

been used for gene transfer to the liver and muscle in clinical trials for hemophilia B 47 and has

been approved as Luxturna® to be used in the retina for the treatment of Leber congenital

amaurosis.48 The rational modification of the capsid of these AAV serotypes resulted in

promising clinical translation in the near future. 49–52

3. Viral vector manufacturing

Production of AAV viral vectors is a complex process and requires innovative

approaches to meet safety and efficacy requirements, clinical and market demands, and cost of

goods targets. Preparing stable viral vectors, preventing their degradation during manufacturing,

handling, and storage, and maintaining their long-term stability and efficacy are major challenges

for the AAV manufacturer. Combinations of traditional approaches and novel technologies are

needed to develop scalable and robust manufacturing processes for gene therapy products. Viral

vector manufacturing workflow is shown in Figure 3, which includes upstream, downstream

formulation and fill/finish processing unit operations.30,53–58 The manufacturing unit operations

workflow and associated challenges are discussed below.

3.1 Upstream Processing

8
3.1.1 Upstream process Workflow

Unit operations in upstream viral vector production include: (1) plasmid development and

production where a cis-plasmid (that encodes a gene of interest (GOI) flanked by the AAV

inverted terminal repeats (ITRs)), a trans-plasmid (that encodes the AAV rep and cap genes), and

a helper plasmid (that encodes adenovirus (Ad) helper genes, E2A, E4, and VA RNA) are

designed and produced, (2) cell expansion where E1 transduced cells (e.g. adherent HEK293T or

suspension HEK293) are expanded to a desired cell density, (3) plasmid transfection where

plasmids are introduced into cells after it has reached a desired cell density, and (4) viral vector

production where transiently transfected cells are then allowed to produce the virus for several

days.

3.1.2 Challenges in upstream processing

Plasmid development

The plasmid DNA is essentially a starting material in the AAV production. Plasmid DNA

is commonly produced in recombinant E. coli fermentation, during which the appropriate genetic

sequences are amplified and then harvested, purified, and tested for safety. 57–59 The lot-to-lot

consistency in the fermentation, along with the large lot-to-lot variability in yield and purity of

the resulting plasmid, are a major concern in AAV production.60 Producing GMP grade plasmid

DNA with greater than 95 percent purity and free from process-related impurities and variants

remains a challenge in plasmid DNA production. 60 Low yield of plasmid DNA production is also

a concern.60 Therefore, in order to meet the rising demand of AAV therapies, plasmid

9
manufacturing process needs to be improved for better yield and product consistency by using

well defined media and process controls.

Cell expansion

The widely used HEK293-based transient transfection process for viral vector production

is easy to perform at lab scale. However, the scale-up process produces a lot of variability.
13,17,60–62
Scale-up in vector production by adherent cells is performed by increasing the surface

area for cell culture, which in practice is accomplished by the addition of parallel culture plates

(a scale-out approach).62 The use of adherent cells comes with many challenges including,

scaling up, risk of contamination during handling of the culture systems, and difficulty with

monitoring and regulation of culture conditions such as oxygen concentration, pH, etc.

Suspension-based cell cultures, on the other hand, provide scalability; however, cell densities on

a per volume basis are generally lower as compared with adherent cells. 63 Three-plasmid

transfection system remains somewhat inefficient in suspension cell culture as not all cells

receive optimal ratios of the plasmids required for efficient packaging. Plasmid imbalance may

also contribute to the variation in empty-to-full capsid ratios between vector batches. 17 Full

capsids refer to capsids that contain genomic materials and empty capsids refer to the capsids

that lack or contain fragmented genomic materials. The use of animal-based products such as

serum can be a source for adventitious agents that serve as contaminants to the product;

therefore, there is an absolute need to minimize adventitious agent (AA) contaminations (viruses,

microbial) in the cell culture step. Since the viral vectors are similar in size and characteristics to

the adventitious viruses, it is impossible to separate the two without affecting product yield and

efficacy. Better-defined chemical media, as well as downstream platform separation technology,

10
would significantly reduce the risk of contamination. 60 The development of stable cell lines

carrying the sequences of the serotype-specific capsid and the gene of interest into the

genome of the cell would be an ideal solution.

Plasmid transfection

Most used method is transient triple transfection of mammalian cells or infection of

mammalian (such as HEK293) and insect cells (such as baculovirus). 64 The transfection

efficiency and the high cost of the DNA and transfection reagents are major challenges in vector

production.60 Typically, AAV vectors are produced in HEK293 cells following transfection of

three DNA plasmids, a cis (encoding the AAV ITRs and GOI), a trans (encoding the AAV rep

and cap genes), and a helper (encoding Ad helper genes). Successful transfection of all three

plasmids is necessary to produce AAV vectors, which otherwise results in low titer and

incomplete viral packaging. There are several methods for plasmid transfection; however, each

has its own limitations. Calcium phosphate methods are subject to significant batch to batch

variability due to both reagent purity and pH sensitivity. 54 Liposome transfections are highly

efficient and minimally cytotoxic, but reagents are much more costly, particularly when used at a

scale required for commercial AAV production. 54 The most widely used polyethyleneimine

(PEI) is toxic to producing cells and its performance is sensitive to changes in pH. 65

3.2 Downstream processing

3.2.1 Downstream process workflow

The downstream process includes purification of viral particles from process-and

product-related impurities, including host cell material, plasmid DNA, and empty capsids.

11
Several purification processes have been developed in recent years to prepare AAV vectors,

which differ considerably from one another. 66–68 A typical downstream process includes: (1) cell

lysis to release viruses by detergent, mechanical stress, hypertonic shock, or freeze-thaw

procedures; (2) nucleic acid removal where lysates are digested with endonucleases to reduce

nucleic acid contaminants; (3) solid removal by centrifugation or microfiltration to remove cell

fragments and debris prior to chromatographic purification; (4) affinity chromatography to

remove host cell proteins (HCPs) and any serum protein impurities; (5) separation of full gene-

containing infectious viruses from empty, non-infectious viruses, either by cesium chloride

gradient ultracentrifugation procedures or ion-exchange chromatography, and (6) final

purification to further reduce HCPs or other low molecular weight contaminants using core-bead

adsorbents.

3.2.2 Challenges in downstream processing

Downstream processing can make up a large part of the total cost of virus production;

therefore, an effective and reproducible method of generating highly pure and homogeneous

population of viral vectors is necessary for cost effective manufacturing of AAVs. 69 Major

challenges in downstream workflow include cell lysis, filtration, and purification, where there is

a lack of an effective and reproducible platform for the separation of full capsids from the empty

capsids.

Cell lysis

There are several mechanical and chemical methods for cell lysis. 70 The rudimentary

mechanical technique to release AAV vectors from cells is repeated freeze/thaw followed by a

12
low speed centrifugation step.30,71,72 However, this technique is not appropriate for large scale

purification because it is difficult to scale-up. Mechanical homogenization such as French press

is another lysis method where cells in media are forced through an orifice using high pressure. 70

Disruption of the membrane occurs due to high shear force as the cells are subjected to

compression while entering the orifice and expansion upon discharge. Although this method is

scalable, it often leads to product loss due to shear stress-induced aggregation and precipitation.

Chemical lysis using Triton X-100 has been used as the primary detergent for cell lysis and has

provided sufficient overall yield in viral vector purification processes.73 It is easy to scale-up;

however, recent research has shown that Triton X-100 has created some acute oral toxicity, eye

damage, skin irritation, and chronic aquatic toxicity. 74 As a result, the detergent was listed as the

substance of very high concern (SVHC) in December 2016 by the European Chemicals Agency

under REACH regulations after several years of debate regarding its safety. 74 Therefore, an

alternative cell lysis method is needed that is not only able to lyse cells, but can also protect

AAV vectors from various stresses during the purification process.

Filtration

Filtration is the most expensive unit operation in the AAV downstream processing. It can

result in AAV particles to aggregate or lose functionality as a result of shear stress during the

filtration process.75 Cell lysis generates significant amount of cell debris, which is difficult to

pass through filters. Optimizing a filter for high throughput recovery remains a challenge as it

also depends on the AAV serotype being processed. Continuous filtration as a separation

technique might reduce filter clog, but the industry is yet to implement the technique in their

13
workflow. Large holdup volume and product loss during filtration due to the lack of appropriate

size filters for gene therapy products is also a concern. 76

Platform purification method

The current state of the downstream operations results in very low yields in terms of virus

recovery due to the lack of robust purification processes. 77 Typical chromatographic methods

include affinity and ion exchange chromatography (IEC). While affinity chromatography is

capable of generating high yields of purified AAV, it cannot discriminate between empty and

full viral capsids.69 One of the biggest challenges is that each AAV serotype requires a subtype

specific purification approaches to achieve optimal yield while maintaining product potency and

integrity. For downstream processing, isolation of viral particles and reduction of process-and

product-related impurities need to be achieved while preserving potency and yield targets. The

industry needs a platform purification process that can be utilized for varieties of viral vectors

and can potentially reduce the number of unit operations in the purification process. In addition,

it is important to minimize both physical and chemical degradation (such as aggregation,

proteolysis, oxidation and deamidation) of capsid proteins at all stages of the downstream

processes, so that the virus infection efficiency should not be affected.2 One of the reasons for

huge losses and significant damages is the use of sub-optimal buffer conditions which cannot

preserve the integrity of the AAV during the complex AAV manufacturing process. Flow-

through processes that do not involve viral product capture and elution could increase both

productivity and product yield. Novel resins that can selectively capture host cell proteins and

other impurities would also contribute greatly to achieving highly pure product.

14
Separation of empty capsids from full capsids

Empty capsids are considered impurities because they either completely lack genomic

material or contain only fragments of the genome. The presence of empty capsids could affect

the efficacy and safety of AAV vector products because of their risk of increased

immunogenicity of the end product. Separation of empty from full capsids is challenging because

they are similar in charge and size. 78 The two widely used methods to separate full and empty

capsids are cesium chloride and anion exchange chromatography (AEX). 31 The cesium chloride

gradient based method is the primary method for early product development and generates

prurient material.30,69,79 However, scalability is difficult, the equipment is expensive, and the

technique is intolerant of even minor operator errors. These issues continue to push the industry

toward the more familiar approach of chromatography-based fractionation, but this represents a

very significant challenge. The degree of surface differentiation between empty and full capsids

is modest at the best and varies by serotypes. 80 IEC, which is currently used to separate empty

and full viral capsids, often uses elution conditions such as extreme pH and high conductivity,

which can sometimes damage the viral vectors. 81 Significant overlap between empty and full

capsid peaks in IEC chromatogram is commonly observed, which means that some full capsids

need to be sacrificed to achieve complete elimination of empty capsids using this method.

Exposure to extreme pH values during separation can lead to compromised capsid stability,

which is a potential concern.82 Scale-up to industrial chromatography skids with linear-gradient

capability is simple and robust, but performance may be compromised on chromatography skids

with only step-gradient capability.78 Very few resins are commercially available that are capable

of separating full-from empty-capsids. Clearly, there is a need for a platform separation

15
technology that is robust, suitable for a variety of AAVs and that can improve overall product

yields.83

3.3 Formulation, Fill &Finish

3.3.1 Formulation, Fill/Finish workflow

Formulation goals for AAV vectors include maintaining vector stability and activity

during manufacturing and storage shelf-life, and achieving optimal target tissue transduction in

vivo. Formulation and fill/finish processes include formulation design, concentration and

diafiltration using transient flow filtration (TFF) into final formulation and fill/finish. 30,84

Formulation development workflow includes identification of the buffer and pH associated with

maximal stability followed by excipient screening for optimal stability and efficacy. 85 These

studies include forced degradation under various stressed conditions to understand the

degradation pathways and develop stability indicating methods.2,26,86 Typical stressed conditions

used are accelerated temperature, freeze/thaw, exposure to light, low and high pH, shear stress,

forced oxidation and deamidation. The choice of the excipients in the formulation depends on the

identification of the degradation mechanisms.

3.3.2 Challenges in formulation and fill/ finish

The main challenges in formulation and fill/finish unit operations are to identify the

solution conditions that are suitable to the different routes of administration of gene therapy

products (e.g., intravenous (IV), subcutaneous, intrathecal, subretinal) and to minimize product

degradation during manufacturing, aseptic fill/finish operations, and storage.

16
Product degradation

Aggregation and loss of efficacy during manufacturing, storage and use are major

concerns for AAVs. Because of poor structural stability and sub-optimal formulation buffer

conditions, AAV products in many cases are currently stored under frozen conditions.5,6 The

concentrated AAV stocks are prone to aggregation, which can lead to purification losses and

inconsistencies in the testing of AAVs. 87–89 Aggregation and surface adsorption of AAV particles

may occur during manufacturing depending on the process and solution conditions. 90,91

Additionally, aggregation can alter bio-distribution or immunogenicity of the vector upon in vivo

administration.92 Compared to multi-dose recombinant protein therapeutics, immunogenicity risk

for AAVs may be less in those cases where only a single dose of AAV therapy is required, but

cannot be ignored for those patients whose immunity is already compromised. Increase in

aggregation and viscosity with increase in AAV product concentration makes these products

difficult to administer in small volumes of concentrated vector to certain sites, such as the central

nervous system.2 Other degradation pathways for AAVs include proteolysis and/or chemical

modifications such as oxidation and deamidation. Viral vectors may unfold and denature after

nonspecific binding to various surfaces during production, purification, and administration. Non-

specific adsorption and unfolding may also occur during product delivery through an

administrative device.79 Using excipients to stabilize AAV drug products is critical, especially

for clinical applications in which small amounts of highly concentrated vectors are introduced

into confined spaces such as the eye or the brain. 93–96 Well characterized excipients with low

impurities and endotoxins to control degradation rate are expected to serve as better alternates in

17
resolving these issues as they have been demonstrated to be very effective for other

biotherapeutics.97

The top three degradation pathways for AAVs are: (1) Freeze/thaw induced unfolding

and activity loss, (2) AAV aggregation at low ionic strength, and (3) Shear induced unfolding,

aggregation, and precipitation. AAVs stability is sensitive to freeze/thaw. Viral vectors denature,

form aggregates and loose activity as a result of freeze/thaw. Freeze-thaw stability could also be

related to change in solution pH during freezing due to the crystallization of the buffer

components, such as sodium phosphate buffers, or temperature dependence of buffer pKa. The

structural change related to pH was reported for AAV1 and AAV6 where the α-helical structure

of capsid proteins was gradually lost when pH was decreased from 7.5 to 4.0. 21 AAV vectors

tend to aggregate in low ionic strength solutions. In one report,91 hydrodynamic radius of AAV2

decreased with the increase in ionic strength of the solution before it plateaued suggesting

electrostatic attractions as the main driving force for the AAV aggregation. AAV viral vectors

unfold and aggregate when exposed to shear stress at air-water interfaces. Shear induced

damages can be reduced by using non-ionic surfactants in AAV formulations. At least there are

two reports demonstrating the inhibition of AAV2 viral product loss when formulated with

nonionic surfactants.79,98

Stability of the genomic materials encapsulated in viral capsids is also a concern while

manufacturing AAV drug products. Nucleic acids are not stable at in an aqueous solution under

non-physiological temperature; pH and ionic strength disrupt the DNA helix and cause

denaturation.99 Other challenges includes serum nuclease susceptibility, rapid renal clearance,

phagocyte uptake.100 One study reported that the genomic material in AAV8 capsids was

sensitive to DNase I impurity in the manufacturing process.101

18
Fill/finish

Viral vectors can aggregate and form particulates as a result of shear stress during

fill/finish operations. Filling facilities commonly used for manufacturing small volume gene

therapy products are semi-automated, raising concerns regarding errors associated with crimping

and sealing. Low-shear filling operations such as peristaltic pump technology in closed systems

would be advantageous. These closed filling devices need to ensure sterility, given that repetitive

sterile filtrations are impossible to execute for viral vectors due to product loss. Real-time

sterility testing and closed end-to-end processes would offer a potential solution to these issues

for the long-term.

4. Degradation mechanisms of AAVs

Viral vectors can undergo various challenges during manufacturing, storage, shipping,

and handling, which can impact the safety and efficacy of AAV products. AAV degradation

pathways can be divided into two major categories-physical and chemical instabilities. The

degradation of the viral vectors depends on the solution conditions, in particular pH, ionic

strength and raw material/excipient contaminants, and external factors such as temperature, shear

stress, freeze/thaw, and light.2,26,79,82,90–92

4.1 Physical Instability

Physical instability refers to the change in the higher order structure of AAVs. Physical

degradation does not involve covalent modification of capsid proteins. Three major pathways

exist for physical instabilities-denaturation/unfolding, aggregation, and surface adsorption.

19
Physical instability of vectors can adversely impact the stability and transduction efficiency of

the AAV product. If the degradation products are toxic or immunogenic because of their non-

native structures, the safety of the AAV preparation is also compromised. 87

4.1.1 Denaturation/unfolding

Denaturation refers to the loss of native structure of the AAVs either in terms of the loss

of secondary, tertiary, or quaternary structures. 15,21,22 Unfolding/refolding can occur during

capsid protein expression and production. The viral vector may denature and unfold because of

shear stress. It may denature upon adsorption to the surfaces during manufacturing, and storage.

The product could also denature due to the dilution during dose preparation or absorption to the

infusion/injection devices during administration to the patient. Unfolded capsid proteins are

more prone to aggregate during the refolding process and may lead to loss of titer during

manufacturing.2,102,103 Many factors such as temperature, protein concentration, type and

concentration of denaturant, pH, ionic strength, refolding catalysts, thiol/disulfide agents, and

miscellaneous additives have been found to affect protein aggregation during refolding.104–107

Shear stress induced loss of viral particle integrity and reduced virus recovery were reported.87–
89,108
Thermal and pH induced change in the denaturation of AAV1 was reported where a local

unfolding of VP proteins occurred rather than the whole capsid degradation.21 The pH induced

denaturation was found to be reversible in this case. In another report, thermal induced

denaturation of capsid proteins was observed for AAVrh32.33, AAV9 and AAV5. 82 In a relevant

study on the denaturation of four different types of AAVs, 15 AAV2 was found to be the least

thermally stable, followed by AAV8, AAV1, and AAV5.

20
4.1.2 Aggregation

Aggregation is a complex phenomenon that can occur through many different

mechanisms. Aggregation may occur from both the association of unfolded or largely

unstructured proteins or from the self-association or oligomerization of the native

proteins.103,109,110 The concentrated AAV stocks are prone to aggregation, which can lead to

purification losses and inconsistencies in the testing of AAVs. 87–89 Aggregation may also occur

due to change in solution or environmental conditions. Protein aggregates are a major concern

for biologics because of their potential to cause immunogenic response. 2,91,92 The intrinsic

tendency of viral vectors for agglomeration within a composition leads to inhomogeneous size

distribution associated with increased polydispersity and subsequent loss of infectivity. This in

turn results in a significant loss of therapeutic efficacy and can even lead to adverse effects. 2

Solution conditions significantly controlled the aggregation of AAV2. 111 The AAV aggregation

is also associated with lower ionic strength, suggesting a need of minimum ionic strength to

reduce AAV clustering.91 Immune responses that limit transgene expression following AAV

vector administration were also reported. 112–114 Aggregation was also reported for PEGylated

AAV through cross-linking of the polymer with multiple virus capsids. 115 High polydispersity

was associated with high viscosity of AAV formulations. 91,116 Such compositions are also

expected to elicit problems with syringability and injectability. 91,116 The efficient removal of

residual vector surface host cell DNA by treatment with nucleases was reported to be an

effective strategy in reducing aggregation. 90

4.1.3 Surface Adsorption

21
 Viral vectors can adsorb on tubing, glass, plastic, and stainless-steel surfaces during

manufacturing and to the drug product container/closure, resulting in surface-induced

aggregation. These physical degradation processes lead to accumulation and adhesion of protein

molecules to the surfaces. During this process, protein molecules change their physical state and

conformation.117–120 Different mechanisms of surface adsorption exist and some of the major

factors driving adsorption include intra-molecular forces, hydrophobicity, ionic, and electrostatic

interactions.85,121 Viral vector capsid proteins, like other protein therapeutics, can adsorb to a

variety of surfaces that include interfacial stresses leading to protein unfolding, aggregation and

precipitation, and reduce the AAV concentration in solution. 85,122,123 Misfolded capsid proteins

may reveal hydrophobic residues that facilitate protein aggregation through hydrophobic

interactions. These interactions may lead to the formation of small aggregates, which in turn may

nucleate further AAV aggregation, ultimately generating visible particles in solution. In one

study, up to 75% of vector loss occurred and a significant reduction in dose was reported due to

the surface adsorption of AAV vector when product was administered using 1 mL syringe.79 In

another preclinical study, AAV5 exhibited significant adsorption to glass and plastic surfaces. 29

Non-specific absorption of rAAV2-REP1 (Rab Escort Protein-1) on administration devices was

observed during dose preparation and administration. 98

4.2 Chemical Instability

Chemical degradation refers to a process that involves protein modification via chemical

bond formation or dissociation, resulting in a new chemical entity. 124 Chemical modification of

AAV capsid proteins could impact vector safety and efficacy. The major chemical degradation

pathways in proteins include disulfide exchange, deamidation, oxidation and isomerization.

22
Chemical degradation often alters the physical properties of proteins such as electrostatics,

hydrophobicity, secondary and/or tertiary structure, and the thermodynamic and kinetic

unfolding/folding barriers.85,121,125

4.2.1 Disulfide Formation/Exchange

Incorrect disulfide bond formation/exchange is one of the common chemical degradation

pathways for protein therapeutics. This degradation pathway can occur readily in various

processing steps. Free cysteine residues in proteins can be oxidized to form disulfide bond

linkages or cause disulfide exchanges, causing protein aggregation or polymerization. 126,127

Disulfide formation is pH dependent, usually resulting from an increase in formulation pH. 85 The

AAV capsid protein contains five highly conserved cysteines at sequence positions 230, 289,

361, 394 and 482 that remain mostly buried within the capsid. Molecular modeling studies

suggested that Cys 230 and 394 are fully conserved, but Cys 289, 361 and 482 might be

dispensable for disulfide formation. 128 It is conceivable that disulfides can potentially be formed

due to the observed proximity between certain cysteine residues in the VP subunit.129 Disulfide

bonds may or may not have any impact on the function of the protein depending on their location

in protein and capsid structures.129

4.2.2 Deamidation

Capsid proteins can undergo deamidation under extreme solution conditions, thereby

resulting in a loss of activity. Deamidation of capsid proteins has the potential to introduce vector

heterogeneity. Deamidation occurs when the amide group of an asparagine or, less frequently, a

glutamine side chain is lost after nucleophilic attack from an adjacent main-chain amide. This

23
process leads to a succinimidyl intermediate130 that, via hydrolysis, results in a mixture of

aspartic acid and isoaspartic acid (or glutamic acid and isoglutamic acid). 131 Deamidation

kinetics depend on the local flexibility of the peptide chain, overall protein structure, solvent

accessibility, buffer identity, pH, and temperature. 2,132,133 Deamidation of selected amino acids

modulates the stability and the immune response to the recombinant protein therapeutics.85 The

deamidation of asparagine residues was recently reported in AAV1, AAV3B, AAV4, AAV5,

AAV7, AAV8, AAV9, and Rh32.33 serotypes, 124 and it was noted that the extent of deamidation

was dependent on the vector’s age and multiple factors including amino acid sequence and three-

dimensional capsid protein structure.124 However, the extent of deamidation was largely

independent of the vector recovery and purification conditions. The loss of vector transduction

activity was correlated with the rate of deamidation of AAV8. 124 An LC-MS analysis of AAV8

also demonstrated deamidation of capsid proteins. 134

4.2.3 Oxidation

Oxidation is another commonly occurring chemical modification that can impact viral

vector safety and efficacy. Capsid proteins have the ability to oxidize during downstream

processing or storage and can thus impact the virus infection efficiency. 30 Amino acids that are

susceptible to oxidation include mainly Met, Tyr, Trp, His, and Cys. Nonenveloped enteric

viruses such as AAV could be injured by exogenous stresses in the natural environment, and thus

the potential damage to viral capsid protein can lead to an inability of the viruses to recognize

cellular receptors to initiate a viral life cycle. 135 Studies have shown that the main cause of

damage of viral particles during long-term storage for 24 months at 5C is free-radical oxidation,

which was limited by the addition of combinations of metal chelators and hydroxyl radical

24
scavengers such as EDTA and histidine.91,136,137 The methionine oxidation of AAV8 was

reported in an LS-MS study.134

4.2.4 Isomerization

The most common isomerization in proteins is iso-aspartic acid formation, which results

from the isomerization of Asp through the hydrolysis of a succinimide intermediate. Succinimide

is the intermediate step in Asp going to iso-Asp. The pH-dependent formation of the succinimide

intermediate can occur either from Asn deamidation or Asp dehydration. 138 Isomerization is

prevalent in AAV viral vectors and has been reported in AAV5 under various stress

conditions.139

5 Rational formulation design using excipients.

Currently, most of the clinical and commercial AAV products are stored frozen because

they do not have long-term storage stability under refrigerated conditions. For example,

Luxturna® and Zolgensma® are stored frozen and they have only one year of shelf-life under

frozen conditions.5,6 Because of time-consuming and expensive freeze-thaw procedures and the

potential aggregation and efficacy loss during freeze-thaw,137,140,141 refrigerated storage of viral

vectors is highly desirable.

For example, stability of an AAV2 was measured in terms of virus particle size

distribution after 5 days storage at 4C and after 1, 5 and 10 freeze-thaw cycles at -20C and -

80C (Figure 4). The particle size increased by 2-, 6- and 10- fold after 5 days of storage 4C,

25
after 10 freeze-thaw cycles at -20C, and after 10 freeze-thaw cycles at -80C, respectively.91 In

another study, stability of AAV1 vectors showed 20% efficacy reduction after storage at 4C for

7 weeks.140 However, systematic efforts to optimize liquid viral vector formulations and stability

are rarely reported.142–147 Lyophilized formulation may be another option to achieve desired

product quality attributes and storage conditions for AAV products,2 but it has not been

significantly explored yet. This might be because the area of gene therapy is new, and developers

are rushing to bring it to the clinic rather than spending time and efforts to understand the

mechanistic details and optimize the AAV manufacturing processes. A rational formulation

design chosen by optimizing solution conditions and the addition of excipients can significantly

increase AAV stability and thus shelf-life. The usefulness of excipients on recombinant proteins

has been extensively reported,97,104,105,107,119,121,123,148 and excipients can in a similar way play a

major role in the safety and efficacy of viral vectors. However, there is a lack of a rational,

methodologically structured and systematic method of excipient selection. 87,91,141,149 Excipients

increase solubility and reduce physical and chemical instability of biologics. 150 The stabilizers

encompass a wide variety of molecules including sugars, salts, polymers, surfactants, and amino

acids. The effects of these excipients on protein stability in solution are mainly caused by their

interaction with the protein and the container surface, and with water. Some excipients stabilize

proteins in solution by direct binding, while others involve indirect interactions with protein

molecules. In the dry state lyophilized formulation, stabilization occurs through direct interaction

of excipients with protein molecules. Similar mechanisms have been observed for excipients

stabilizing the viral capsid protein assembly. 2,88,111

5.1 Osmolytes

26
 Osmolytes are small organic compounds, widely used to stabilize proteins151 against

denaturation and aggregation.8 Proteins in an aqueous solution exists in equilibrium between the

folded (F) and unfolded (U) states.152,153 Stabilization occurs by a preferential exclusion

mechanism121,154 where osmolytes shift the equilibrium towards the F-state. 153,155 The most

widely used osmolytes in the biopharmaceutical formulations are sucrose, glycine, mannitol,

histidine, dextrose, arginine, trehalose and lactose.156 Similar mechanisms exist for osmolytes

stabilizing AAVs. Sugars can also significantly improve the vector production yield. For

example, the presence of 0.2 M sucrose in a cell culture media has increased the yield by 1.9-

and 1.5-fold for AAV2 and rAAV2-retro serotypes, respectively (Figure 5).157 This could be the

result of a sucrose stabilizing AAVs during harsh downstream purification process. Intriguingly,

no effect of sucrose treatment was observed on AAV5 yields. 157 Given the wide

availability, low cost, and ease of use, sucrose can be adopted by AAV manufacturers to improve

the manufacturing yield and stability. 97

5.2 Buffering agents and pH

Buffer and pH strongly influence conformational and colloidal stabilities of viral capsid

proteins. Proteins are stable only in a very narrow pH range. The pH determines the net charge

on the protein molecule and the nature of electrostatic interactions. Generally, the higher the net

charge, the lower will be the aggregation propensity due to electrostatic repulsions, and higher

will be the colloidal stability.158–163

The buffer type and pH can impact vector stability and infectivity. Structural changes in

the AAV capsids with pH were reported.21 The α-helical structure of VP1 domain of capsid

protein in AAV1 and AAV6 was gradually lost when pH was decreased from 7.5 to 4.0; this

structural change was reversed when pH was increased back to 7.5.21 The dilution of AAV in

27
low concentration buffer may also cause vector aggregation, as it was reported in the case of

AAV2 when it was diluted in a low concentration phosphate buffer. 91 The poor stability at low

pH makes phosphate buffers unsuitable for viral vector when stored at frozen conditions because

the pH of the phosphate buffer could decrease by as much as 3 pH units upon freezing due to

selective precipitation of the disodium phosphate below -20C.164

Buffers can significantly affect the thermal stability and transfection efficiency of AAVs

(Figure 6). Different buffers have different effects on each serotype, with no single buffer having

a consistent stabilization or destabilization on all viruses. 26 For AAV8, transfection efficiency

was found to be independent of the buffer type. 26

The effect of pH on the thermal stability was evaluated for AAV1, AAV2, AAV5,

AAV6.2, AAV8, and AAV9 viral vectors formulated in citrate buffer at various pH values,

ranging from 7 to 3 (Figure 6).82 The melting temperature of AAV vectors was strongly affected

by changes in pH in a serotype-dependent manner. The melting temperatures of AAV1, AAV6.2,

and AAV9 were comparable between pH 5 and pH 7 and significantly decreased between pH 3

and pH 5.82 The melting temperatures of AAV2 and AAV8 reached a maximum at pH 5,

whereas the melting temperature of AAV5 displayed a progressive decrease between pH 7 and

pH 3.82 In another study, pH dependent thermal melting temperature of AAV1, AAV2, AAV5

and AAV8 was determined using differential scanning fluorimetry (DSF). 86 The thermal

temperature varies with pH for all AAV serotypes. The same study examined the transfection

efficiency of AAV1, AAV2, AAV5 and AAV8 as a function of pH and storage time by

Luciferase Assay System (Promega). Results are shown in Figure 7. Cellular transduction by

rAAV-Luciferase vectors following a 24 h pre-incubation at different pHs and temperatures

showed a similar trend in transduction efficiency for all AAV serotypes. 86 The highest

28
transduction level was observed at pH 7.4 and 6.0 for the samples stored at -80C and 4C. The

higher temperature along with low pH storage conditions has reduced transduction efficiency by

over 80%. Among all serotypes, rAAV5 was the most susceptible to low pH storage conditions.
86
At pH 2.5, all the viruses lost transduction efficiency by at least 20% when stored at -80C.

Higher storage temperatures further reduced the loss of transduction efficiency, with no

detectable transfection for samples stored at 37C for 24 hours. These data indicate that the

pH of the formulation can have a major impact on the AAV stability.

5.3 Salts

The effect of salts on capsid protein stability is complex. Salts may have stabilizing,

destabilizing, or no effect on protein stability depending on the type and concentration of salt,

nature of ionic interactions, and the electrostatic interactions between charged residues in

proteins.165 Salt effects on protein stability could be due to non-specific (Debye-Hückel)

electrostatic shielding, and/or by specific ion binding to the protein. At low concentrations, salts

affect electrostatic interactions in proteins. Therefore, this effect could be stabilizing when there

are repulsive interactions leading to protein unfolding, or destabilizing when there are stabilizing

salt bridges or ion pairs in the protein. At high salt concentrations, electrostatic interactions are

saturated; the dominant effect of salt is on solvent properties of the solution. The stabilizing salts

increase surface tension at water-protein interface and strengthen hydrophobic interactions by

keeping hydrophobic groups away from water molecules, inducing preferential hydration of

proteins.121 The salt effect strongly depends on the salt concentration and solution pH, as pH

determines the charged state of ionizable amino acids in protein groups. 121,123

29
 Detailed analysis of AAV2 vector aggregation as a function of the salt concentration and

type was performed (Figure 8).91 Salts of multivalent ions were required at lower concentrations

to prevent aggregation than high concentrations of NaCl.91 For example, magnesium sulfate at

~200 mOsm prevented aggregation, while NaCl was required at ~350 mOsm to achieve a similar

effect.91 Sodium salts of citrate, sulfate, and phosphate were intermediate in their potency. These

data suggested that the ionic strength of the solution, a parameter that depends on charge valency

as well as salt concentration, was the excipient characteristic affecting vector aggregation. 91

Improvement in solubility by Mg2+ (20 mM) was also reported for AAV2 (pH 4.5 to 7.5);

however, no improvement in solubility with a multi-charge anion (citrate3+) was observed. 89

5.4 Surfactants

Proteins can unfold, aggregate or even precipitate upon shear stress or when exposed to

air-water interface,79 resulting in the formation of soluble and insoluble aggregates.104–106

Shaking increases the area of air/water interface in solution. Since air/water interface is

hydrophobic, proteins re-orient themselves at the interface and expose the hydrophobic regions

in order to maximize their interaction with the interface. 166 Exposure of the hydrophobic regions

in proteins increases the chance of inter-molecular protein–protein interaction and hence, the

chances of increased protein aggregation.167 Aggregation of capsid proteins can cause

immunogenic response in AAV products.2,92 Nonionic surfactants such as polysorbate 80,

polysorbate 20, and poloxamer 188 can protect proteins against surface-induced damage by

competing with proteins for adsorption sites on surfaces.168 Both marketed AAV products,

Luxturna® and Zolgensma® use poloxamer 188 in their formulation. 5,6 However, caution is

needed regarding their use as impurities in the surfactant can adversely affect the quality of bio

30
therapeutics.104–106,169,170 Additionally, quality of surfactants has been suspected to cause

injection site pain and hypersensitivity reactions in patients. 171,172

In another case study, AAV2 vector recovery was assessed in the presence of Pluronic

F68 (PF-68) following vector dilution from a concentrated stock solution in PBS buffer with and

without PF-68 and then passed through three different injection devices (Figure 9). 79 There was

106%, 104% and 96% recovery with formulation containing 0.001% PF-68 compared to 51%,

24% and 27% without PF-68, respectively.79 In another example, an AAV9 containing 0.001%

Pluronic F-68, 5% D-sorbitol, and 0.25% bovine serum albumin (BSA) has been shown to

inhibit adsorption of AAV capsids to container and instrument surfaces in hemophilia B and

Leber congenital amaurosis (LCA) gene therapy clinical trials.173 58,174 In another example, the

biocompatibility and stability of rAAV2-REP1 (Rab Escort Protein-1) was assessed before and

after passage through the injection device over a period of time to mimic the clinical scenario. 98

The dose was prepared using either formulation buffer that contained 0.001% of a non-ionic

surfactant (PF-68) or balanced salt solution (BSS). The PF-68 formulation dose was made at two

different dose levels; however, BSS buffer was tested only at one dose. The percent loss in titer

from the base line was measured (Figure 9). 98 Significant losses in the genomic titer of samples

diluted with BSS occurred for all time points. The addition of 0.001% PF-68 prevented loss at

both dose levels.98

5.5 Antioxidants/ Free radical scavengers

Peptide oxidation is a major cause of chemical instability and also sometimes linked to

physical instability.121 Amino acids such as methionine, cysteine, histidine, tyrosine and

tryptophan in peptides are susceptible to oxidation. Capsid proteins can oxidize upon exposure to

31
light and due to metal ion impurities in the raw materials and excipients, 139 leading to a loss in

functionality.136 Oxidation can be prevented with the use of free amino acids such as methionine

and histidine and metal ion scavengers such as ethanol, EDTA and DTPA.175–180 Nature of metal

ion can also significantly affect the quality of AAV formulations. In another example, stability of

AAV5 was tested on the thermal stability. The AAV5 was sensitive to methionine oxidation,

data also showed significant reduction in the infectively loss when stored at 37C.139

6 Viral vector stabilization by chemical modifications

The stability and clinical performance of viral vectors can be desirably changed by

chemical modifications.4,50,180–184 Mutation of serine, threonine and lysine residues in the AAV2

capsid with alanine or arginine has shown to enhance transduction efficiency. 185 The introduction

of an azide moiety into the AAV capsid by mutating the VP3 sequence and introducing unnatural

amino acids was reported to modify AAV specificity.4 Recently, grafting of a functionalized

RGD peptide onto the capsid of a genetically modified AAV to specifically target tumor cells

was reported.186

PEGylation is often used for improving the pharmacokinetics and pharmacodynamics of

protein pharmaceuticals,187–190 and has been investigated in viral vector protection, including

AAV production.115,182 Incorporation of the azide moiety into the AAV capsid protein followed

by orthogonal and stoichiometric conjugation of a variety of polyethylene glycols (PEGs)

through click chemistry has shown to improve the stability by 1.7 to 2.4-fold in pooled human

serum and a nearly two-fold reduction in antibody recognition. 183 PEGylated AAV (AAV2)

particles via amine functionalities have also been developed to protect the virus from

neutralization and enable significant levels of gene expression upon re-administration without

compromising the patient's immune system. 181,191 The rAAV2 conjugated to PEG 2000 showed a

32
2.3-fold increase in transduction efficiency at a ratio of 1000:1 in the presence of neutralizing

antibodies.191

7 Summary

This review summarized the current understanding of AAV production workflow and the

challenges that are encountered to make cost effective manufacturing of the AAV drug products.

The AAV vectors undergo various stress conditions during production, storage, shipping, and

handling, resulting in sub-optimal product quality and shorter shelf-life. The review also

discussed the challenges that the AAV viral vector manufacturers experience during production

and long-term storage, and the possible mitigation strategies. The challenges that gene therapy

manufacturers encounter today are similar to the ones monoclonal antibody manufacturers

experienced when antibody therapeutics were new. For example, monoclonal antibodies were

also challenged with low titer, product and process related impurities and degradation during

manufacturing storage and handling. Although the risk associated with process related impurities

may be lower for single dose AAV products compared to recombinant mAbs, it cannot be

ignored and will depend on the type of impurity, dose, and route of administration. Because of

these similarities, there is an opportunity for drug manufacturers, chemical, and excipient

suppliers to collaborate and develop innovative solutions that enable robust and cost effective

AAV product manufacturing. To utilize the full potential of the gene therapy products,

developing new and better technologies in the manufacturing process workflow are warranted.

Some of the major challenges that AAV manufacturers currently experience, and possible

mitigation strategies are discussed below:

Major Challenges Mitigation Strategy

33
 Major Challenges
Upstream process involving adherent cell lines
and triple transfection results in low titer and
high variability in product quality. It also limits cell lines that do not require triple transfection
scale up supporting only limited increase in
capacity with parallel culture plates which
cannot be a long-term solution.
Serotype specific purification strategy is a
challenge for the manufacturer. At present
there does not exist a uniform purification
approach that works for all varieties of AAV
serotypes. This has resulted in lower yield,
higher cost, and inferior quality of the product.
This in effect has led to delays in clinical
research.
There is limited understanding on the
degradation mechanisms of AAV and an
absence of any systematic formulation
development approach. Despite cases of
aggregation and efficacy loss during
manufacturing and storage has been reported,
discussions about minimizing or controlling it
have been limited.
Shelf-life of AAV based products are limited,
34
Mitigation Strategy
Simplification of upstream process either by
developing stable AAV producer suspension
or by utilizing other innovative technologies
that is plasmid-free. Innovative transfection
technology that significantly improves titer and
full/empty capsid ratio could also be an
attractive alternative as well.
Develop serotype-independent robust platform
for purification process that is suitable for
varieties of AAV serotypes. Current state of
the affinity chromatography technology does
not differentiate between full and empty
capsids. Resins with selectivity for full capsids
will be a breakthrough in the viral vector
downstream workflow. It will eliminate the
need for empty capsid separation unit operation
in the downstream workflow.
Conduct forced degradation studies under
various stressed condition to understand the
degradation mechanism of AAV viral vector.
Once sufficient knowledge has been gathered,
it is possible to build effective formulation
development strategies to control degradation
rates under selected storage conditions.
Develop innovative formulation strategies by
 Major Challenges
typically 12-18 months and needs frozen
conditions for storage and shipping. This
makes inventory management and supply-
chain very complicated.
Mitigation Strategy
exploring existing excipients or developing
novel excipients to minimize the rate of
degradation. This will provide longer shelf-life
to finished product and possibly enable storage
under refrigerated conditions, shipping, and
handling.

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Figure Legends

Figure 1: Schematic representation of AAV genomic structure. (a) Exterior capsid

surfaces: Exterior capsid surfaces are radially color cued (from capsid center to surface: blue to

green to red to yellow; ~80 to 140 Å). The white triangles depict the viral asymmetric units

bounded by a 5-fold (5f) axis and two 3-fold (3f) axes divided by a line through a 2-fold (2f)

axis. Example surface features that differ among serotypes are VR-IV, VR-VII, DE loop, and HI

loop. (b) Cross-section: Cross-sections showing their interior surfaces. The dark blue regions

show the -strand and -sheet secondary-structure elements. (C) AAV has a linear single-

stranded DNA (ssDNA) genome of approximately 4.7-kilobases (kb), with two 145 nucleotide-

58
long inverted terminal repeats (ITR) at the termini and the three arrows indicate each of the three

promoters at positions 5, 19 and 40. Regulatory proteins (Rep78, Rep68, Rep52 and Rep40) are

encoded by rep gene. Rep52 and Rep40 are expressed from p19 promoter while Rep78 and

Rep68 are transcribed from p5 promoter. Rep68 is a splice variant from Rep78, and the splice

site is common for Rep52 and Rep40 transcripts. Structural proteins are encoded by cap gene.

There are three capsid proteins VP1 (virion protein 1), VP2, VP3 transcriptionally regulated by

p40 promoter, with molecular weights of 87, 72 and 62 kDa, respectively. These capsid proteins

assemble into a near-spherical protein shell of 60 subunits. Two mRNAs result from p40

expression, the first one encodes for VP1 and the second one is a splice variant that encodes for

both VP2 and VP3. Ribosome read-through produces both VP2 and VP3 in stoichiometric

amounts. All AAV transcripts share the same polyadenylation signal (polyA). AAP facilitates

nuclear import of the major VP3 capsid protein and promotes assembly and maturation of the

capsid, but AAP is not present in the mature capsid. The aap gene encodes the assembly

activating protein (AAP) in an alternate reading frame overlapping the cap gene. This nuclear

protein provides a scaffolding function for capsid assembly.

59
Figure 2: Sample evaluation and stability of full rAAV1-rAAV9-gfp and rAAVrh.10-gfp

(packaging the green florescent protein transgene) vectors in PBS. A) Coomassie stained SDS

PAGE (left) and negative-stained EM (right) of rAAV1-rAAV9 and rAAVrh.10 as indicated

above each panel. A 100 nm scale bar is shown in the AAVrh.10 EM image. B) Thermal profile

(shown as normalized relative fluorescence units, RFU) versus temperature (T (ºC)) of rAAV1-

rAAV9 and rAAVrh.10 obtained by DSF analysis. Each profile is colored according to the

serotype as shown on the right-hand side. Data summarized from Bennett et. al. 26

60
Figure 3: Viral vector manufacturing process workflow, which involves various upstream,

downstream, formulation, and fill/finish steps. Results were summarized and challenges that

need to be overcome were discussed in the review.

Figure 4: Freeze-thaw stability of AAV2 vectors: The stability of AAV2 was monitored in

terms of particle size in 140 mM NaCl, 10 mM sodium phosphate, 5% sorbitol, pH 7.3 buffer at

4C and after 1, 5 and 10 freeze-thaw cycles at -20C and -80C. Data summarized from Wright

et. al. 91

61
Figure 5: Effect of sucrose on the yield of AAV production after sucrose treatment in a

large-scale production. AAVpro 293T cells were triple transfected with a transfer plasmid, a

plasmid encoding rep and serotype specific cap genes and a plasmid encoding the adenoviral

helper sequences. Media was replaced the following day with fresh DMEM with or without 0.1

M sucrose. For each serotype, the range and average number of AAV genome copies (GC) from

different preparations were calculated and plotted. Data summarized from Rego et. al.157

62
Figure 6: (a) Thermal melting temperature of different AAV serotypes in commonly used buffer

as determined by DSF. Desecrate dots represent the distribution of melting temperature for each

AAV serotype in the different buffers. The compositions of the buffers were PBS: 10 mM

Na2HPO4, 1.8 mM KH2PO4 pH 7.4, 27 mM KCl and 137 mM NaCl; CiPO4: 0.2 M Na2HPO4,

and 0.1 M Citric acid pH 7.4; HEPES: 50 mM HEPES pH7.4, 150 mM NaCl, and 2 mM MgCl2 ;

Tris: 20 mM Tris-HCl pH 7.4, 150 mM NaCl, and 2 mM MgCl; LR: 27.7mM Sodium Lactate

pH 6.5, 102.7mM NaCl, 4.0mM KCl, and 1mM CaCl2; BSS: 109.5 mM NaCl, 10.1 mM KCl,

3.3 mM CaCl2, 1.4 mM MgCl2, 28 mM Sodium Acetate and 5.8 mM Sodium Citrate pH 7.4,

0.02% Tween and UB: 20 mM HEPES, 20 mM MES, 20 mM Sodium Acetate, pH 7.4, 150 mM

NaCl, and 5 mM CaCl2. (b) Thermal melting as a function of pH for viral vectors AAV1, AAV2,

AAV5, AAV6.2, AAV8 and AAV9 in citrate buffer as measured by fluorescence method.

Results are given as mean ± SD of the mean, obtained from three independent experiments. Data

summarized from Bennett el. al.26 and Pacouret et. al.82

63
Figure 7: Effect of pH and temperature on AAV transduction.: (a) rAAV1, (b) rAAV2, (c)

rAAV5, and (d) rAAV8 (all packaging the luciferase gene) in HEK293 cells infected with virus

incubated for 24 h in citrate-phosphate buffer at the indicated pH and storage temperature. The

transduction efficiency for each AAV serotype is shown relative to virus stored at pH 7.4 and -

80C. The experiments were performed in triplicate and are displayed mean+SD (n = 3).

Data summarized from Lins-Austin et. al.86

64
Figure 8: Effect of salt type and concentration on AAV aggregation: on ionic strength of

selected excipients. The average particle radius of AAV2 vectors was measured by dynamic

light scattering (DLS) following vector dilution in 10 mM sodium phosphate pH 7.5 containing

different salts. Filled circles: sodium chloride; open circles: sodium citrate; filled squares:

sodium phosphate; open squares: sodium sulfate; inverted filled squares: magnesium sulfate;

open diamonds: glycerol. Data summarized from Wright et. al. 91

65
 Figure 9: (a) Recovery of adeno-associated virus 2 (AAV2) vector following dilution and

passage through administration devices. Concentrated stock AAV2 vector was diluted into

phosphate-buffered saline (PBS) that contained 0.001% Pluronic F-68 (+PF68) or without (-

PF68) and drawn into 1-mL syringes. The vector solution was passed through three different

devices (A, B and C). After passes, the concentration of recovered vector was measured by

quantitative-polymerase chain reaction (Q-PCR). (b) Titer of rAAV2-REP1 vector samples

following dilution and passage through surgical devices at several time points was measured.

Difference of the mean titer to baseline at each time point for all samples collected. Symbols

represent mean of three replicates ± SD, except for 1E+12 particles/mL dose, where only two

replicates were considered. Data summarized from Bennicelli et. al. 79 and Patrício et. al.98

Table 1: Examples of ongoing clinical trials using AAV viral vectors.

AAV Serotype Disease Sponsor Clinical Phase

Applied Genetic Technologies

AAV1 Alpha-1 Antitrypsin Deficiency Phase 2
Corp

AAV1 Ischemic Cardiomyopathy Celladon Corporation Phase 2

Limb Girdle Muscular Dystrophy

AAV1 Genethon Phase 1
Type

AAV1 Chronic Heart Failure Patients Imperial College London Phase 2

AAV1 Duchenne Muscular Dystrophy Jerry R. Mendell Phase 2

AAV1 Becker Muscular Dystrophy Nationwide Children's Hospital Phase 1

66
AAV Serotype Disease Sponsor Clinical Phase

Charcot-Marie-Tooth Neuropathy
AAV1 Nationwide Children's Hospital Phase 2
Type 1A

University of Massachusetts,
AAV1 Alpha 1-Antitrypsin Deficiency Phase 1
Worcester

Applied Genetic Technologies

AAV2 Achromatopsia Phase 2
Corp

Applied Genetic Technologies

AAV2 Leber Congenital Amaurosis Phase 2
Corp

Applied Genetic Technologies

AAV2 X-Linked Retinitis Pigmentosa Phase 2
Corp

Applied Genetic Technologies

AAV2 X-linked Retinoschisis Phase 2
Corp

AAV2 Pompe Disease Asklepios Biopharmaceutical, Inc. Phase 2

AAV2 Parkinson's Disease Brain Neurotherapy Bio, Inc. Phase 1

AAV2 Choroideremia Byron Lam Phase 2

AAV2 Parkinson's Disease Ceregene Phase 1

AAV2 Acute Intermittent Porphyria Digna Biotech S.L. Phase 2

AAV2 Mucopolysaccharidosis Type VI Fondazione Telethon Phase 2

AAV2 AADC Deficiency Krystof Bankiewicz Phase 1

67
AAV Serotype Disease Sponsor Clinical Phase

AAV2 Osteoarthritis, Knee Mayo Clinic Phase 1

Radiation-Induced Parotid Gland

AAV2 MeiraGTx UK II Ltd Phase 1
Hypofunction

National Institute of Neurological

AAV2 Parkinson's Disease Phase 1
Disorders and Stroke (NINDS)

NightstaRx Ltd, a Biogen

AAV2 Choroideremia Phase 3
Company

AAV2 Hemophilia A Pfizer Phase 3

AAV2 Choroideremia Spark Therapeutics Phase 1

CHM (Choroideremia) Gene

AAV2 Spark Therapeutics Phase 2
Mutations

Inherited Retinal Dystrophy Due to

AAV2 Spark Therapeutics Phase 3
RPE65 Mutations

AAV2 Sanfilippo Syndrome B UniQure Biopharma B.V. Phase 2

AAV2 Pompe Disease University of Florida Phase 1

University of Massachusetts,
AAV2 Alpha 1-Antitrypsin Deficiency Phase 1
Worcester

AAV2 Choroideremia University of Oxford Phase 2

AAV3 Xerostomia Due to Radiotherapy MeiraGTx UK II Ltd Phase 2

68
AAV Serotype Disease Sponsor Clinical Phase

AAV3 Muscular Dystrophies Nationwide Children's Hospital Phase 1

NightstaRx Ltd, a Biogen

AAV3 Choroideremia
Company

St. Jude Children's Research

AAV3 Hemophilia B Phase 1
Hospital

Batten Disease/Late Infantile Weill Medical College of Cornell

AAV3 Phase 1
Neuronal Ceroid Lipofuscinosis University

Radiation-Induced Parotid Gland

AAV4 MeiraGTx UK II Ltd Phase 1
Hypofunction

AAV4 Duchenne Muscular Dystrophy Nationwide Children's Hospital Phase 1

NightstaRx Ltd, a Biogen

AAV4 Choroideremia Phase 2
Company

AAV4 Choroideremia STZ eyetrial Phase 2

AAV5 Choroideremia Ian M. MacDonald Phase 1

AAV5 Xerostomia Due to Radiotherapy MeiraGTx UK II Ltd Phase 2

AAV5 Hemophilia B UniQure Biopharma B.V. Phase 2

AAV5 Huntington Disease UniQure Biopharma B.V. Phase 1

AAV6 X-Linked Retinitis Pigmentosa MeiraGTx UK II Ltd Phase 1

69
AAV Serotype Disease Sponsor Clinical Phase

AAV6 Hemophilia B UniQure Biopharma B.V. Phase 2

AAV7 Hemophilia B UniQure Biopharma B.V. Phase 2

HIV-1 Infected Adults With National Institute of Allergy and

AAV8 Phase 1
Controlled Viremia Infectious Diseases (NIAID)

NightstaRx Ltd, a Biogen

AAV8 X-Linked Retinitis Pigmentosa Phase 3
Company

AAV8 Hemophilia B Spark Therapeutics Phase 1

Ornithine Transcarbamylase
AAV8 Ultragenyx Pharmaceutical Inc Phase 1
(OTC) Deficiency

AAV8 Hemophilia B UniQure Biopharma B.V. Phase 3

National Human Genome

AAV9 Lysosomal Diseases Phase 2
Research Institute (NHGRI)

70