Live attenuated and inactivated viral vaccine formulation and nasal delivery: potential and challenges.

ADR-12667; No of Pages 23 Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr

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José L. Tlaxca a, Scott Ellis b, Richard L. Remmele Jr. a,⁎ a

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Keywords: Vaccines Drug delivery Formulation Stability Nasal Live-attenuated Killed-inactivated

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Vaccines are cost-effective for the prevention of infectious diseases and have significantly reduced mortality and morbidity. Novel approaches are needed to develop safe and effective vaccines against disease. Major challenges in vaccine development include stability in a suitable dosage form and effective modes of delivery. Many live attenuated vaccines are capable of eliciting both humoral and cell mediated immune responses if physicochemically stable in an appropriate delivery vehicle. Knowing primary stresses that impart instability provides a general rationale for formulation development and mode of delivery. Since most pathogens enter the body through the mucosal route, live-attenuated vaccines have the advantage of mimicking natural immunization via noninvasive delivery. This presentation will examine aspects of formulation design, types of robust dosage forms to consider, effective routes of delivery (invasive and noninvasive), and distinctions between live attenuated or inactivated vaccines. © 2014 Published by Elsevier B.V.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Live-attenuated vaccines . . . . . . . . . . . . . . . . . 1.2. Killed-inactivated vaccines . . . . . . . . . . . . . . . . 1.3. Immune response . . . . . . . . . . . . . . . . . . . . Instability and formulation considerations . . . . . . . . . . . . . 2.1. Stresses . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Formulation type and stability . . . . . . . . . . . . . . . 2.3. Activity assessment . . . . . . . . . . . . . . . . . . . . Dosage forms . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Liquid . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Lyophilized . . . . . . . . . . . . . . . . . . . . . . . 3.3. Spray-dried . . . . . . . . . . . . . . . . . . . . . . . 3.4. Foam dried . . . . . . . . . . . . . . . . . . . . . . . . RSV live attenuated vaccine dosage form stabilization . . . . . . . . 4.1. Lyophilization issues-RSV . . . . . . . . . . . . . . . . . 4.2. Spray-drying RSV . . . . . . . . . . . . . . . . . . . . . 4.3. Frozen-liquid RSV . . . . . . . . . . . . . . . . . . . . Administration route . . . . . . . . . . . . . . . . . . . . . . 5.1. Nasal delivery . . . . . . . . . . . . . . . . . . . . . . 5.2. Validating API . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Applying a proper model . . . . . . . . . . . . . 5.2.2. Pharmaceutical factors affecting nasal vaccine delivery 5.2.3. Deposition . . . . . . . . . . . . . . . . . . . .

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MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA Gilead Sciences Inc., 333 Lakeside Drive, Foster City, CA 94404, USA

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Live attenuated and inactivated viral vaccine formulation and nasal delivery: Potential and challenges☆

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☆ This review is part of the Advanced Drug Delivery Reviews theme issue on "Protein stability in drug delivery applications". ⁎ Corresponding author. Tel.: +1 301 398 1889. E-mail address: [email protected] (R.L. Remmele).

http://dx.doi.org/10.1016/j.addr.2014.10.002 0169-409X/© 2014 Published by Elsevier B.V.

Please cite this article as: J.L. Tlaxca, et al., Live attenuated and inactivated viral vaccine formulation and nasal delivery: Potential and challenges, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/j.addr.2014.10.002

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J.L. Tlaxca et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

6. Non-clinical toxicology considerations 7. Device closure . . . . . . . . . . . 8. Conclusions . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . . . . .

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Live-attenuated vaccines generate strong humoral as well as cellmediated immune responses (CMI). Disease-producing (“wild”) viruses or bacteria are manipulated in vitro to reduce pathogenicity. Some attenuation in pathogenicity can occur through genetic alterations leading to the absence of toxins that make up the vaccine [2]. A small dose of the attenuated pathogen is required to replicate in an inoculated individual, capable of triggering an immune response with minimum virulence. They are usually administered by the natural route of infection. Moreover, the attenuated pathogens induce lengthy immune responses without adjuvants (an immunological agent that boosts immunogenicity) and are able to survive the low pH, enzymatic environment, of the stomach. In theory, these types of vaccines are safe and devoid of side effects, although there have been vaccine-associated

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1.2. Killed-inactivated vaccines

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Killed-inactivated vaccines are made from disease-causing microbes that have lost or attenuated their ability to infect through physical, chemical or radiation processes, without compromising the antigenicity of the microbial agent. The term killed commonly refers to bacterial vaccines, whereas inactivated relates to viral vaccines. These types of vaccines do not generally elicit a full cellular immune response [10]. Syringes are the most commonly used method of administration worldwide. To overcome weak immune responses, some inactivated vaccines (e.g., protein subunit varieties) are often co-administered with an adjuvant with large and more frequent doses than would be anticipated for live-attenuated vaccines. Moreover, adjuvants could cause local reactions at the vaccination site or contribute to other aspects of reactogenicity [11]. The major challenges of the inactivated vaccines are formulation stability, ability to generate long-term immunity and safety.

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1.3. Immune response

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Besides creating stable vaccine formulations, the delivered antigenic agents need to trigger the immune system to produce neutralizing antibodies particular to that disease. The immune system can be divided into innate and adaptive roles. The innate system (non-specific involving

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Vaccines are antigenic substances that elicit an immune response, providing protective immunity against a specific or closely related pathogen. A hallmark of an effective vaccine is a long-term protection to the immunized individual from the pathogen upon re-exposure to that agent at a later time. The immune system responds in the generation of neutralizing antibodies (humoral response) against a specific pathogen. Vaccines may be of bacterial or viral in origin. Few live attenuated bacterial vaccines have become commercially available primarily because of safety and poor stability issues in such cases [1]. A vaccine contains an agent resembling a disease-causing microorganism with attenuated virulence and potent antigenic properties. Some vaccines are made from live-attenuated, killed, fractions or substances produced by the same pathogenic organism. This work will primarily focus on viral vaccines and emphasize the live attenuated and inactivated viral vaccine types. Nevertheless, much of the information discussed pertaining to viral vaccines may be applied to live and inactivated bacterial vaccines recognizing that compositional differences need to be taken into account. Table 1 shows an assortment of the commercially available live attenuated vaccines currently on the market. Some general observations from the table are that most listed are formulated as dry solids with influenza and rotavirus being the exceptions as liquid dosage forms. There are formulated cases (Rotarix, Vivotif, and RotaTeQ) that can be delivered orally. Polio vaccine is a historic example of another live attenuated dosage form that can be administered orally but the disease has since been eradicated within the United States. Among the orally administered dosage forms, two are solid (Rotarix and Vivotif) and two are liquid (RotaTeQ, Rotarix). Only FluMist™ is administered intranasally using an Accuspray device, the rest by scarification, intravesicular or subcutaneous routes. Dose volume ranges are also listed in the table and range from 0.0025 mL (bifurcated vaccination needle; ACAM2000) to 53 mL (intravesicular; TheraCys) with corresponding dosing frequency. Table 2 shows a listing of commercially available killed (inactivated) vaccines. Approximately 16% are solid and 84% are liquid dosage forms. Most of the liquid dosage forms are inactivated influenza viral vaccines, while the solid dosage forms involve a variety of immunizations against other diseases (e.g., rabies, meningitis, diphtheria, tetanus, poliovirus, and pertussis). Most are administered intramuscularly with few administered via subcutaneous or intradermal routes. Dose volumes range from 0.1 mL (Fluzone-ID) to 1 mL.

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effects reported. For example, paralytic polio has been linked with an oral polio vaccine (now discontinued) and intussusception associated with a recalled rotovirus vaccine (RotaShield) [3,4]. There are practical challenges associated with some live-attenuated vaccines. For example, refrigerated storage and distribution (i.e. “cold chain”) requirements for liquid formulations are a significant hurdle in third world countries. Fig. 1 illustrates the differences between the component parts of different virus types and their respective compositions. From the figure it can be seen that there are enveloped and non-enveloped viruses. The enveloped types have lipid bilayer membranes with an assortment of protein receptors transmembraneously attached to the surface. The lipid bilayer houses the capsid protein (and other proteins) and genome materials (Fig. 1A). In contrast, the non-enveloped variety lack the lipid membrane but have capsid and other proteins that house the genome material and may have a variety of different protein compositions with receptors for docking and fusing with host cells. Thus for enveloped viral vaccines, the general composition includes lipid structure, proteins, and genome (either DNA or RNA) in contrast to proteins and genome for non-enveloped vaccines (Fig. 1B). The type of viral vaccine and molecular compositions should be taken into account when considering vulnerability to a variety of stresses and instability. Lipid bilayers can be composed of glyco-lipids, glyco-proteins, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. They may also contain cholesterol and oleic acid. Enveloped viruses derive their assorted phospholipid and other compositions from the host cells they bud from [5,6]. In enveloped viruses the integrity of the lipid bilayer can be important for infection. Some evidence has been reported in the case of myxoviruses (influenza and paramyxoviruses) regarding the role of the membrane in fusion processes [7,8]. Furthermore, a high cholesterol to phospholipid molar ratio within viral envelopes has been noted as a requirement for infectivity [9]. Therefore, stresses that impact this membrane integrity can also impair the efficacy of the viral vaccine.

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153 154 155 156 157 158 159 160 161 162 163 164

167 168 169

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Table 1 Commercially available live attenuated vaccines.

O

R

t1:3 t1:4

Therapeutic Molecule category name

Virus product

t1:5

Infection

FluMist

Influenza

Liquid; 2–8 °C; 18 weeks

Intranasal

t1:6

Infection

RotaTeQ

Rotavirus

Liquid; 2–8 °C; 24 months

Oral

t1:7

Infection

DryVax

Smallpox

Scarification (Percutaneous)

t1:8

Infection

ACAM2000

Smallpox

Solid; 2–8 °C; replaced by ACAM2000 Solid; −25 °C to −15 °C; 72 months

t1:9

Infection

Zostavax

Herpes zoster (Shingles)

Solid; −50 to −15 °C; 18 months

Subcutaneous

t1:10

Infection

YF-VAX

Yellow fever

Solid; 2–8 °C; 12 months

Subcutaneous

t1:11

Infection

ATTENUVAX

Measles (Rubeola)

Subcutaneous

t1:12

Infection

MERUVAX II

Rubella

t1:13

Infection

ProQuad

Measles, Mumps, Rubella and Varicella

Solid; 2–8 °C or colder; 24 months when stored at 2–8 °C Solid; 2–8 °C or colder; 24 months when stored at 2–8 °C Solid; −15 °C or colder; 18 months

t1:14

Infection

VARIVAX

Varicella

Solid; 2–8 °C or colder; 24 months

Subcutaneous

t1:15

Infection

M-M-R-II

Measles, Mumps

Solid; −50 °C to +8 °C;

Subcutaneous

Formulation type, storage condition, shelf- life

Mode of delivery

Dose of API

Administration

Target population

Device

0.2 mL dose contains 106.5–7.5 FFUa each of H1N1, H3N2, B/Massachusetts/2/2012 and B/Brisbane/60/2008 virus strains 2 mL dose contains 2–2.8 × 106 IUb each of G1P7, G2P7, G3P7, G4P7 and G6P1A 1.0 mL dose of reconstituted vaccine contains 100 million living vaccinia viruses 2.5 μL dose contains 2.5–12.5 × 105 PFUc of live vaccinia virus 0.65 mL of reconstituted vaccine contains no less than 19,400 PFUc 0.5 mL of reconstituted vaccine contains no less than 5.04 log10 PFUc

1 dose, 2 doses

2–49 years

BD Accuspray Nasal Spray System

3 doses

6–32 weeks

Persons determined to be at risk

12 months and older

Persons determined to be at risk

12 months and older

1 dose

50 years and older

1 dose, revaccination if at risk condition

9 months and older

Pre-filled squeezable dose tube Bifurcated vaccinating needle Bifurcated vaccinating needle Administration with needle and syringe Administration with needle and syringe Administration with needle and syringe Administration with needle and syringe Administration with needle and syringe

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E

C

Scarification (Percutaneous)

Subcutaneous

Subcutaneous

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D

P

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0.5 mL of reconstituted vaccine contains not less than 1000 TCIDd50 of measles virus 0.5 mL of reconstituted vaccine contains not less than 1000 TCIDd50 of rubella virus 0.5 mL of reconstituted vaccine contains not less than 3 log10 TCID50 of measles virus, 4.3 log10 TCID50 of mumps virus, 3 log10 TCIDd50 of rubella virus and 3.99 log10 PFUc of varicella virus 0.5 mL of reconstituted vaccine contains a minimum of 1,350 PFUc 0.5 mL of reconstituted vaccine

O

1 dose

1 dose

1 dose, 2 doses

F

12 months and older followed by with M-M-R IIe 12 months and older followed by with M-M-R IIe 12 months to 12 years

1 dose, 2 doses

12 months and older

1dose, 2 dose

12 months



t1:1 t1:2

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Administration with needle and syringe Administration 3

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Therapeutic Molecule category name

Mode of delivery

Virus product

Formulation type, storage condition, shelf- life

and Rubella

24 months when stored at 2–8 °C

t1:16

Infection

(Preparation of ATTENUVAX, MUMPSVAX and MERUVAX II) ROTARIX

t1:17

Infection

Vivotif

Typhoid (Salmonella typhi)

t1:18

Cancer

⁎OncoTICE

Primary or recurrent flat urothelial cell carcinoma in situ of the urinary bladder


Treatment and prophylaxis of carcinoma in situ of the urinary bladder and for the prophylaxis of primary or recurrent stage Ta and/or T1 papillary tumors following transurethral resection


t1:19

t1:20 t1:21 t1:22 t1:23 t1:24Q1 t1:25 t1:26 t1:27

Bladder Disease

a

⁎TheraCys

U

Rotavirus

N

Fluorescent focus units (FFU). Infectious units (IU). c Plaque forming units (PFU). d Tissue culture infectious dose (TCID). e Median cell culture infective dose (CCID50). e Measles, Mumps, and Rubella Virus Vaccine Live (M-M-R II). f Colony forming units (CFU). ⁎Bacterial vaccine. b

Solid, Liquid; 2–8 °C; 36 months

Dose of API

Oral

C

O


Oral

R

Catheter via the urethra into the bladder

R

E

C

Intravesicular

Administration

contains not less than 1000 TCIDd50 of measles virus, 12,500 TCIDd50 of mumps virus and 1000 TCIDd50 of rubella virus 1 mL of reconstituted vaccine (or 1.5 mL of the liquid formulation) contains at least 106 CCIDe50 of human rotovirus RIX4414 strain One capsule per dose, contains viable S. typhi Ty21a 2–6.8 × 109 CFUf and non-viable S. typhi Ty21a 5–50 × 109 bacterial cells 50 mL of reconstituted vaccine contains 1–8 × 108 CFUf of Bacillus Calmette-Guérin (BCG), strain TICE

T

53 mL of reconstituted vaccine contains 10.5 ± 8.7 × 108 CFUf of BCG

E

D

Target population

Device

and older

with needle and syringe

2 doses

6 weeks and older

Oral applicator

4 doses

6 years and older

Oral Capsules

Induction treatment: weekly one instillation for 6 weeks Maintenance treatment: additional instillations at week 8 and 12 and monthly from months 4–12 Induction treatment: weekly one instillation for 6 weeks Maintenance treatment: one dose given 3, 6, 12, 18 and 24 months following the initial dose

18 years and older

Urethral Catheter

TheraCys has not be given to a pregnant women, nursing mothers or pediatric use

Urethral Catheter

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O

F



Table 1 (continued)

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Table 2 Commercially available killed (inactivated) vaccines.

O

t2:3 t2:4

Therapeutic category

Molecule name

Virus product

t2:5

Infection

Pentacel

t2:6

Infection

Menomune-ACYW-135

⁎Diphtheria, ⁎tetanus, ⁎pertussis, poliomyelitis and ⁎Haemophilus influenzae type b ⁎Meningitidis (Serogroups A,C, Y and W-135)

R

Mode of delivery

Formulation type, storage, shelf-life

R



E

Intramuscular

C

Subcutaneous

Infection

Imovax

Rabies


Intramuscular

t2:8

Infection

Rabipur

Rabies

Intramuscular

t2:9

Infection

Infanrix IPV

⁎Diphtheria, ⁎tetanus, ⁎pertussis

Solid; 2–8 °C; 48 months Liquid; 2–8 °C; 36 months

Intramuscular

and poliomyelitis

Infection

INFANRIX

t2:11

Infection

PEDIARIX

⁎Diphtheria, ⁎tetanus and ⁎pertussis ⁎Diphtheria, ⁎tetanus, ⁎pertussis, Hepatitis

Liquid; 2–8 °C; 12 months Liquid; 2–8 °C; 12 months

Intramuscular Intramuscular

B and poliovirus

t2:12

Infection

t2:13

Administration

Target population

Device

0.5 mL of reconstituted vaccine per dose

4 doses

6 weeks and older

Administration with needle and syringe

0.5 mL dose of reconstituted vaccine contains 50 μg from each serogroup. 1 mL dose of reconstituted vaccine is equal or greater than 2.5 IUa of rabies virus 1.0 mL per dose contains no less than 2.5 IUa of rabius virus 0.5 mL of vaccine contains no less than 30 IUa of diptheria, 40 IUa of tetanus, 25ug of pertussis and 40 DUb, 8 DUb and 32 DUb poliovirus type 1, 2 and 3 respectively 0.5 mL per dose

1 dose

2 years and older


Pre-exposure: 3 doses Post-exposure: 6 doses

All ages


Pre-exposure: 3 doses Post-exposure: 5 doses 3 doses

All ages

Administration with needle and syringe Administration with needle and syringe

T

t2:7

t2:10

Dose of API

Influenza (monovalent) Hepatitis A and Hepatitis B


Intramuscular

Infection

Influenza (H5N1) Sanofi Pasteur TWINRIX

t2:14

Infection

⁎TYPHIM Vi

Typhoid fever

°C;

Intramuscular

t2:15

Infection

VAQTA

Hepatitis A

°C;

Intramuscular

t2:16

Infection

IXIARO

Japanese Encephalitis

°C;

Intramuscular

t2:17

Infection

IPOL

Poliovirus

Liquid; 2–8 36 months Liquid; 2–8 36 months Liquid; 2–8 24 months Liquid; 2–8 12 months

°C;

Intramuscular

Intramuscular

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0.5 mL of vaccine contains 25 lf of diphteria, 10 lf of tenanus, 25 μg of pertussis, 10 μg of HBsAg and 40 DUb, 8 DUb and 32 DUb poliovirus type 1, 2 and 3 respectively 1 mL of vaccine contains 90 μg haemagglutinin of strain H5N1 1 mL of vaccine contains 720 ELISA units of hepatitis A virus and 20 μg of recombinant HBsAg protein 0.5 mL of vaccine contains 25 μg of purified Vi polysaccharide 1 mL of vaccine contains 50U of hepatitis A virus 0.5 mL of vaccine contains 6 μg of Japanese Encephalitis vaccine proteins 0.5 mL of vaccine contains 40 DUb, 8 DUb and 32 DUb poliovirus type 1, 2 and 3 respectively

6 weeks and older

5 doses

6 weeks and older

3 doses

6 weeks to 7 years

O

2 doses

F


18–64 years



t2:1 t2:2

U


3 doses, 4 doses

18 years and older

1 dose

2 years and older

2 doses

12 months and older

2 doses

17 years and older

4 doses

6 weeks and older

Administration with needle and syringe Administration with needle and syringe Administration with needle and syringe Administration with needle and syringe

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Therapeutic category

Molecule name

Virus product

Formulation type, storage, shelf-life

Mode of delivery

Dose of API

Administration

Target population

Device

t2:18

Infection

Fluzone Intradermal

Influenza


Intradermal

1 dose

18–64 years


t2:19

Infection

Fluzone High


Intramuscular

1 dose

65 years and older


t2:20

Infection

Fluzone

Influenza


Intramuscular

1 dose, 2 doses

6 months and older


t2:21

Infection

Influenza A (H1N1) Novartis

Influenza (monovalent)

Intramuscular

1 dose, 2 doses

4 years and older


t2:22

Infection

Influenza (monovalent)

Intramuscular

0.5 mL of vaccine contains 15 μg haemagglutinin of H1N1

1 dose, 2 doses

6 months and older


t2:23

Infection

Influenza A (H1N1) Sanofi Pasteur FLUARIX

Liquid; 2–8 °C; Novartis recalled H1N1 vaccine Liquid; 2–8 °C; 18 months

0.1 mL of vaccine contains 9 μg haemagglutinin of each H1N1, H3N2 and B/Massachusetts/2/2012 0.5 mL of vaccine contains 60 μg haemagglutinin of each H1N1, H3N2 and B/Massachusetts/2/2012 0.5 mL of vaccine contains 15 μg haemagglutinin of each H1N1, H3N2 and B/Massachusetts/2/2012 0.5 mL of vaccine contains 15 μg haemagglutinin of H1N1


3 years and older


Infection

AGRIFLU

Influenza


Intramuscular

1 dose

18 years and older


t2:25

Infection

AFLURIA

Influenza


Intramuscular

1 dose, 2 doses

5 years and older


t2:26

Infection

FLULAVAL

Influenza


Intramuscular

1 dose

18 years and older


t2:27

Infection

FLUVIRIN

Influenza


Intramuscular

1 dose, 2 doses

4 years and older


t2:28

Infection


1 dose, 2 doses

6 months and older

Infection

Influenza (Pandemic) Influenza (Pandemic)

Intramuscular

t2:29

Pandemrix (H1N1) Celvapan (H1N1)

0.5 Ml of vaccine contains 15 μg haemagglutinin of each H1N1, H3N2 and B/Massachusetts/2/2012 0.5 mL of vaccine contains 15 μg haemagglutinin of each H1N1, H3N2 and NYMC BX-39 0.5 mL of vaccine contains 15 μg haemagglutinin of each H1N1, H3N2, NYMC X-233 and B/Massachusetts/2/2012 0.5 mL of vaccine contains 15 μg haemagglutinin of each H1N1, H3N2 and B/Massachusetts/2/2012 0.5 mL of vaccine contains 15 μg haemagglutinin of each H1N1, H3N2 and B/Massachusetts/2/2012 0.5 mL of vaccine contains 3.5 μg of H1N1

1 dose, 2 doses

t2:24

R

Intramuscular

Intramuscular

0.5 mL of vaccine contains 7.5 μg of H1N1

2 doses

6 months and older


t2:30 t2:31 t2:32

a

International units (IU). D-antigen units (DU). ⁎ Bacterial vaccine. b

U

Influenza

N

Influenza

C

O

R

E

C

T

E

D

P

R O

O

F



Table 2 (continued)


A

7

B

F

VP7

Matrix (M) protein

dsRNA Segment

VP6

RNP Complex

P

VP2

SH G

Lipid bilayer

VP4

F

-SS genomic RNA

L

VP1/VP3

O

N

184 185 186 187

+

CD8 T-cell

D

P

down antigenic protein into peptide fragments that can be presented as class I or II depending on the type of T-cell receptor (TCR) that interacts with either major histocompatibility complex I (MHC I that interacts specifically with CD8+ T-cells) or by MHC II that interacts specifically with CD4+ T-cells. The capture, breakdown, and presentation of antigenic peptides to T-cells in the lymph nodes serve the purpose of immunization and discrimination between pathogenic and innocuous substances [13,14]. The CD8+ T-cell pathway leads to cell death and is referred to as the CTL (cytotoxic T-lymphocyte) pathway. This pathway is important for the elimination and control of viral infection. The CD4+ T-cell pathway can elicit both a cell mediated immunity (CMI and phagocyte-dependent), protective response and/or a humoral (antigenic) response to the antigen. Cell mediated responses can be classified by the kinds of cytokines secreted by two different forms of activated helper T-cells. The helper T-cell 1 response (Th1) can produce the cytokines, interferon-γ (IFN-γ), IL-2 and TNF-α which activate macrophages and are responsible for CMI in contrast to helper T-cell 2 (Th2) that can be identified by secretion of cytokines, IL-4, IL-5, IL-10, and IL-13. The Th2

E

T

182 183

C

180 181

E

178 179

R

176 177

R

174 175

CD4+ T-cell

TCR

Activated Helper T-cell: Th1: IFN- , IL-2

N C O

172 173

antigen presenting cells) is a first-line of defense against pathogenic agents, while the adaptive system (specific to T and B lymphocytes) acts as a second line of defense, providing protection against reexposure to the same pathogen [12]. Immunity against an infectious disease can be acquired artificially through immunization or it can be mediated naturally from attack of a disease causing pathogen or from the transmission of antibodies from the mother to the fetus and newborn infant via the placenta or breast milk, respectively. An immune response may take from days to weeks to develop and can provide a long lasting or even a lifelong protection that is active immunity. On the other hand, a short-lived protection, ranging from several weeks up to 4 months, is an example of passive immunity. Fig. 2 shows a schematic of components and pathways of the innate and adaptive immune system that can be activated with an antigenic insult. The antigen (Ag) is first taken up (via phagocytosis) by an antigen presenting cell (APC) that could be a dendritic cell (DC), neutrophil, or macrophage. These cells have the ability to stimulate naïve (immature) T-cells to proliferate and secrete cytokines. At this point the APC breaks

Th2: IL-4, IL-5, IL-10, IL-13

MHC I

Resting B-cell

MHC II APC

U

170 171

R O

Fig. 1. (A) Respiratory syncytial virus (RSV) is an example of an enveloped virus. The “G” and fusion “F” glycoproteins are responsible for viral attachment and fusion to host cells. The “SH” membrane protein is involved in a Th1 response that inhibits antiviral responses. The genome is housed inside the lipid bilayer with associated nucleocapsid “N,” phosphoprotein “P” and Large “L” protein required for RSV replication. (B) Non-enveloped, icosahedral viruses (e.g., rotavirus; 11-segment double stranded RNA genome) with its compositional viral proteins (VPs).

Activated B-cell

Target Cell BCR Antigen

Dead Target

CTL Pathway

Antibodies

Fig. 2. The immune system is divided into innate and adaptive portions. Antigen is presented to an antigen presenting cell (APC) and subsequently phagocytosed and broken down within the APC (innate, first line of defense). The produced antigenic peptides are processed and displayed on either a major histocompatibility complex type I (MHC I) or MHC II surface molecules. The CD8+ T-cell activation is governed by the T-cell receptor (TCR)–peptide/MHC I complex promoting a cytotoxic function to the target cell. The CD4+ T-cells have TCRs with affinity to class II MHC molecules. Following antigenic peptide stimulation, CD4+ T-cells can differentiate into either Th1 or Th2 type helper T-cells, where both cytokine secretion and B-cell stimulation can occur in the lymph nodes. The activated B-cell proliferates to produce a population of plasma cells and memory cells. B-cells can also be stimulated when specific antigens bind to their B-cell receptors (BCR), followed by a co-stimulation signal from an activated T cell. The MHCI and II pathways make up the adaptive part of the immune system (second line of defense).


188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205

238 239

Most attenuated or virally inactive vaccine types are impacted by stresses that would usually affect the stability or efficacy of the virus itself that causes the disease. The types of stresses that need to be considered are temperature [20,21] (heating and chilling), pH, dehydration (osmotic), chemical agents, agitation, and radiation. All of these have been known to have some measurable impact on the stability of a given viral entity. For example, heating hepatitis A virus shows a nearly linear logarithmic inactivation with temperature going from 40 to 70 °C [22]. Acidic pH has been noted in the literature to inactivate viruses with some resistance to disinfection in aggregated forms [23]. Furthermore, there is cited evidence of a relationship observed between acidic pH instability and reduction in immunogenicity in the case of influenza vaccine [24]. There have been reports of measles virus instability as a result of inactivation by dehydration stress [25]. Some chemical agents, like solvents/detergents [26,27], excipients like arginine [28,29], and metallic ions/complexes [30–33], have been shown to inactivate viruses. Stresses associated with interfaces as might be achieved with agitation can potentially lead to viral inactivation [34]. Radiation induced genome damage has also been cited to inactivate viruses [35–37]. Instability can involve physical and chemical pathways of degradation. Physical pathways may include aggregation and colloidal protein–protein events induced by the stresses already cited above. Chemical pathways generally refer to changes in the molecular properties via oxidation (e.g., methionine, tryptophan, and cysteines), deamidation (e.g., asparagines), hydrolysis, glycation (e.g., L-lysines), β-elimination (e.g., cystines) and racemization. It should be noted here that in the case of live enveloped vaccines, chemical degradation of lipids can also occur and impart some change on efficacy. For example, in lipid peroxidation, the formation and propagation of lipid radicals, the uptake of oxygen, and rearrangement of double bonds in unsaturated lipids can lead to a variety of breakdown products that include alcohols, ketones, aldehydes

231 232 233 234

240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268

C

229 230

E

227 228

R

225 226

R

223 224

O

221 222

C

219 220

N

217 218

U

215 216

2.2. Formulation type and stability

275

From the collection of stresses noted above, a stabilization strategy of live attenuated virus and inactivated virus particles would need to take into account the properties of the virus particle (whether enveloped or non-enveloped) regarding stresses that could impact membrane, genome and protein integrity. Thus, such a strategy would need to guard against such stresses and remain viable for several years in a commercializable dosage form. Thus excipients would need to be considered that stabilize the membrane (if enveloped) or proteins if non-enveloped. Non-reducing disaccharide sugars have been noted to afford some membrane and protein protection to accompany freezing and dehydration induced stresses [41]. These sugars are noted cryoprotectants as well because of the glass forming properties during freezing and drying [42,43]. Since degradation rates in aqueous environments are generally more rapid than in solid dosage form environments, the latter become more advantageous in consideration of longevity and durability for supply chain management. Formulation design must take into account these potential areas in context with instability that may impact the activity and ultimately the efficacy of the vaccine. So buffers must be chosen to provide capacity within optimal pH regions where physical and chemical instability are minimized. Optimal pH conditions for most viral and bacterial [1] vaccines should be in the range from approximately pH 5.5 to 8. Thus, suitable buffers for this range would include histidine, succinate, citrate, phosphate, and Tris. Other stabilizing or viscosity modifying agents could also be added to the mixture (e.g., polymers, salts, and gelatin) as needed or required to achieve suitability with the device used to administer the drug product. Excipients that have been shown to improve stabilization against thermal stress include albumin and pluronic polymer (F-127) for a live-attenuated flavivirus vaccine [44]. Table 3 shows an assortment of live and inactivated vaccines with corresponding excipients found in their respective compositions. From the ingredients listed, the body of the compositions includes buffering agents, stabilizers (amino acids, gelatin, disaccharides, polyols, polysaccharides, salt, and proteins), preservatives (phenol), surfactants (polysorbate 80), vitamins (e.g., ascorbic acid), antibiotics (neomycin, gentamicin, streptomycin, chlorotetracycline), chelating agents (EDTA) and residuals (fetal bovine serum, neomycin, amphotericin B, egg protein, DNA, xantham). Surfactants can act as wetting agents, reduce or eliminate drug precipitation, decrease degradation and facilitate drug uptake. Lipids may be used like surfactants or emulsifying agents that include egg or soy phosphatidylcholine, soy phosphatidylethanolamine, oleic acid, and monoglycerides. It should be noted that although arginine can inactivate some viruses, it is not always the case since it is an agent found in FluMist (LAIV, marketed by MedImmune). It therefore is desirable to carry out stability evaluations on a case by case basis to reveal the true impact that a potential stabilizer might have on a given viral vaccine. Stresses related to viral inactivation as a consequence of contact with different surfaces or interfacial environments should be assessed for formulation and environmental mitigation strategies. If, for example, metal ion leaching into the drug product occurs, then a suitable anti-oxidant could be used in the formulation (e.g., methionine) or chelating agent to mop up potential leached metal ions. Surfactants can be used to minimize interfacial stresses like those involving air/water. The strategy should also minimize exposure to radiation (e.g., fluorescent lights) and require some relevant photo-stability testing to understand how light impacts potency. Thus, a successful formulation strategy would need to assess the role of an assortment of contact stresses on the

276

F

2.1. Stresses

213 214

O

237

212

R O

2. Instability and formulation considerations

210 211

269 270

P

236

208 209

and ethers [38]. Thermal stress at oxidative conditions can lead to the formation of aldehydes and cis/trans isomerization events [39]. Lipids can also degrade by thermal stress at non-oxidative conditions from dehydration, decarboxylation, ester hydrolysis, double bond conjugation, polymerization, dehydro-cyclization, aromatization, dehydrogenation, and degradation by carbon–carbon cleavage mechanisms [40].

T

235

response also favors a humoral response (phagocyte-independent) that promotes the generation of neutralizing polyclonal antibodies through interaction with resting and activated B-cells. Moreover, while TCRs can only recognize an antigen peptide when it has interacted with a MHC presented fragment by an APC, B-cell receptors (BCRs) can also respond to antigenic stimulation and generate a burst of plasma cells that contribute to a marked but transient elevation of serum antibodies [15]. The adaptive immune response can react to a live or inactivated vaccine through the pathways described above where the antigen is the vaccine entity. Fig. 1 shows that virus vaccines can be composed of an assortment of protein compositions. The recognition of protein epitopes is important toward generating a protective response that is linked to the magnitude of the humoral and/or cellular immune pathways [16]. The B-cells make up the antibody secreting plasma cells. Upon activation by cytokines (T-cell response), B-cells differentiate into memory B-cells (long-lived antigen-specific B-cells). B-cells by themselves cannot prevent infection by a pathogen, but can regulate the spread by rapidly differentiating into antibody secreting plasma cells [17]. Memory B-cells may play a role in long-term immunity [18]. Antigenic fragments that activate a Th2 response can induce B-cell activation that leads to high levels of different immunoglobulin (IgG) molecules associated with the infection [10]. Some major disadvantages of killed/inactivated vaccines are as follows: multiple doses (boosters) may be required to produce adaptive immunity, local reactions mediated by co-administered adjuvants to increase antigenicity, usually little to low cell-mediated immunity and little mucosal/local immunity (IgA) [10]. Live attenuated vaccines, on the other hand, can produce antigenic fragments that lead to production of plasma cells, humoral IgG and local IgA antibodies and memory B-cells while not usually needing an adjuvant. For example, LAIV has been shown to induce both systemic and mucosal immune responses [19].

D

206 207


E

8


271 272 273 274

277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332

J.L. Tlaxca et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx Table 3 Vaccine products and formulation compositions.

t3:3

Product

t3:4

FluMist (influenza)

t3:15

t3:16 t3:17 t3:18 t3:19

t3:20

333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358

F

P

R O

O

exhaustive, of primary degradation challenges and their respective potential resolution. In the process of achieving suitable longevity, dosage forms should be considered that mitigate or reduce degradation kinetics so that they achieve some minimal stable period conducive to the manufacture and market profiles for a given drug product. For example, if in the case of seasonal flu epidemics, a period of 1 year or less would be satisfactory given that the flu strain will likely change nullifying an extension into the next season. In this case a short-term stability profile would be acceptable and a liquid dosage form easier to obtain. If on the other hand a vaccine that is to be stable across several years is desired, a more stable dosage form, like a dry solid, should be considered.

D

t3:14

pH optimization pH optimization, sugars, salts, amino acids, surfactants, protect from light Clips pH optimization, remove protease (s)& impurities, add chelator Oxidation Excipient purity, free radical scavenger (mannitol, sorbitol), competitive inhibitor (L-methionine), EDTA, citric acid, antioxidant, protect from light Surface denaturation, adsorption Surfactants, excipient proteins, polymers, lipids Dehydration Sugars, amino acids, polymers, lipids

a

Bacterial vaccine.

vaccine candidate being developed and import appropriate stabilizing excipient remedies where possible. The mode of delivery also needs to be taken into account when considering pH. If nasally administered, the dosage form could be as a liquid or solid preparation and the pH for the delivered preparation should reside within the range from 4.5 to 6.5 to minimize nasal irritation [45]. Based on animal studies, oral, intravenous, and intramuscular routes can have formulated compositions within the pH 2–9 ranges, taking into consideration that dosing such solutions with a pH that is either too acidic or alkaline can cause discomfort and pain on injection [46]. Tonicity modifiers may be required depending upon mode of administration (e.g., ~ 300 mOsmol/kg for subcutaneous injection, ~ 600 or 700 mOsmol/kg if nasal administered). Frozen-liquid stabilization has been a suitable dosage form strategy for FluMist, where the product is thawed prior to nasal administration. In this example storage and stabilization with regard to freezing is important for longevity while allowing a thawed intra-nasal liquid administration that is user friendly. Conversely, dried powder nasal delivery applications can also be a suitable alternative approach. This could be achieved with either a solid dosage form that lends itself to reconstitution prior to administration as a liquid, or as a powder-spray using a suitable device (to be discussed further below). If the dosage form is a dry solid, then bulking agents may be required. Finally, an assortment of stabilizers can be added to mitigate physical and chemical degradation. Table 4 provides a snapshot of stability problems and potential resolutions. This is a brief summary, although not

t4:3 t4:4 t4:5 t4:6 t4:7

t4:8 t4:9

359 360 361 362 363 364 365 366 367 368 369

2.3. Activity assessment

370

The most critical assay for assessment of potency is the activity assay that can measure properties of the virus vaccine. This is important as it should be correlated to an appropriate standard and thus stability of the active pharmaceutical ingredient (API). Here stability is defined as the ability of the vaccine to retain its chemical, physical, microbial, and biological properties within specific limits throughout its shelf-life [47]. Thus, the activity assay must take into account the envelope (morphology), protein and genome integrity assessments. However, the ability to assess live attenuated or inactivated vaccine stability can be in and of itself quite challenging since they are measured against the appropriate immune response they are intended to stimulate. Moreover, stability cannot be fully characterized by physicochemical methods alone. It then becomes necessary to establish vaccine stability based on a change of activity property (direct or indirect) as indication of vaccine immunogenicity or efficacy. Assays that are commonly used for live and attenuated viral vaccines include the fluorescent-focus assay (FFA), tissue culture 50% infectious dose (TCID50), and plaque assays. The FFA method is more rapid than the other two methods and measures host cell infection before a plaque is formed. It lends itself to high throughput applications since it can be applied to 96 well fluorescence technology and relies on host cell infection that may be evaluated within 24–72 h. Thus, there is a more rapid turnaround in acquiring results expressed as fluorescent forming units (FFU). However, this assay does not measure functional biology. In contrast, the endpoint dilution or TCID50 assay quantifies the amount of virus needed to kill 50% of the infected host cells (produce a cytopathic effect). This method can take as long as 7 days due to allowing for sufficient cell infectivity time. Finally, plaque assays are generally the referee or standard methods for determining active virus concentration and measured infection, replication as well as killing properties in terms of plaque forming units (pfu) [48]. This process can take up to 14 days to complete depending on the virus type being analyzed. Since the FFA method does not produce an equivalent response to the plaque forming method, it is strongly recommended to carry out sufficient correlation between the FFA and plaque assays to note differences, and to ultimately correlate with pfus. A variety of factors have been noted in the literature that can influence the response of these assays and should be consulted pertaining to process, formulation and decomposition [49].

371

E

t3:12 t3:13

Potential resolution

Deamidation, cyclic imide Aggregation, precipitation

T

t3:11

C

t3:9 t3:10

R

t3:8

Instability

R

t3:7

N C O

t3:6

Formulation compositions

Arginine, dibasic K-phosphate, egg protein, EDTA, gentamicin sulfate, hydrolyzed porcine gelatin, monobasic K-phosphate, monosodium glutamate, sucrose RotaTeq (rotavirus) Fetal bovine serum, sodium citrate, sodium phosphate monobasic monohydrate, sodium hydroxide, sucrose, polysorbate 80 DryVax (smallpox) Glycerin, phenol, polymyxin B, neomycin sulfate, chlorotetracycline hydrochloride, and streptomycin sulfate ACAM2000 Glycerin, HSA, mannitol, neomycin, phenol, polymyxin B Zostavax (shingles) Bovine calf serum, dibasic sodium phosphate, hydrolyzed porcine gelatin, monosodium glutamate, MRC-5 DNA and cellular protein, monobasic K-phosphate, neomycin, KCl, sucrose YF-VAX (yellow fever) Gelatin + sorbitol (stabilizers) and egg protein Attenuvax (measles) Gelatin, sorbitol, sodium phosphate, NaCl, sucrose, fetal bovine serum, neomycin, human serum albumin Meruvax II (rubella) Neomycin, human serum albumin, sorbitol, sodium discontinued phosphate, sucrose, NaCl, hydrolyzed gelatin, fetal bovine serum ProQuad (measles/mumps/ Serum albumin, hydrolyzed gelatin, monobasic rubella/varicella) K-phosphate, monosodium glutamate, MRC-5 cellular protein, neomycin, sodium bicarbonate, sorbitol, sucrose, KCl Varivax (vericella) Dibasic & monobasic sodium phosphate, EDTA, fetal bovine serum, gelatin, glutamate, monobasic K-phosphate, monosodium glutamate, MRC-5 DNA and cellular protein, neomycin, KCl, sucrose MMR-II (measles/mumps/ Amino acids, fetal bovine serum, glutamate, rubella) hydrolyzed gelatin, neomycin, recombinant HAS, sodium phosphate, sorbitol, sucrose, vitamins Rotarix (rotavirus) Amino acids, calcium carbonate, dextran, sorbitol, sucrose, vitamins, xanthan a Vivotif (typhoid) Amino acids, ascorbic acid, casein, dextrose, galactose, lactose, sucrose, yeast extract a OncoTICE (tuberculosis) Lactose, Sauton medium RotaShield (rotavirus) Stabilizer: Sucrose, monosodium glutamate, K-monophosphate, K-diphosphate, Residuals: Fetal bovine serum, neomycin sulfate, and amphotericin B

U

t3:5

t4:1 t4:2

Table 4 Resolving stability challenges.

E

t3:1 t3:2

9


372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407

433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455

459 460 461 462 463 464 465 466 467 468 469 470 471

C

431 432

E

429 430

R

427 428

R

425 426

O

423 424

C

421 422

N

419 420

U

417 418

Liquid dosage forms, whenever practical, can be more easily administered to patients and therefore are typically the most desirable dosage form. They also lend themselves to simpler device configurations. A strategy that has been implemented for FluMist is the freezing of the liquid at very low temperatures (below the glass transition temperature) to sustain longevity in a frozen state and yet permit ease of administration as a thawed liquid. Although such a strategy may be appropriate in a seasonal flu setting, it does become more of a challenge pertaining to cold chain management for non-seasonal vaccine cases. There are other cases where clear liquid dosage forms have been developed and are on the market. For example, the stable liquid suspension formulation of the GSK Rotarix vaccine (Table 1) is orally administered and can be stored in the refrigerator (not to be frozen) with a 3-year shelf-life. Although this is suitable for ease of administration, it can still pose cold-chain management challenges. This achievement tends to be the exception as many of the live attenuated vaccines are solid dosage forms. Finally, a strategy that can be effective is to reconstitute a solid dosage form and administer immediately in liquid form. In this latter scenario, the longevity afforded as a solid is leveraged against the immediate reconstitution followed by ease of administration as a liquid.

475 476

3.2. Lyophilized

495

F

Live attenuated and inactivated viral vaccines can be developed as stable liquids (Tables 1 and 2), or as lyophilized, spray-dried, and foam dried dosage forms. Liquid stabilization is perhaps the most challenging as degradation kinetics and dynamic processes are typically more favorable (greater physico-chemical reactivity) in such cases. Nevertheless, it has already been noted above that there are cases where liquid stabilized dosage forms can be practical with relatively short expiry (seasonal vaccines like influenza). A solid dosage form strategy on the other hand has advantages in that it lends itself to greater flexibility in acquiring long-term refrigerated stability, potential room temperature stability, and resilience to transportation induced instability (e.g., jarring, or interfacial modes of degradation). These types of dosage forms lend themselves to be robust against external stresses like temperature excursions, physical and chemical decomposition. Thus dry solid dosage

415 416

474

O

458

414

3.1. Liquid

R O

3. Dosage forms

412 413

P

457

410 411

forms should be part of the development strategy for live attenuated 472 and inactivated vaccines. 473

Lyophilization involves several stresses like freezing (e.g., coconcentration and crystallization of solutes), sublimation and dehydration. In terms of reducing molecular motion that may be required in destabilizing reactions, the freezing process should be examined (rate of freezing) and the influence of added agents (e.g., glassy state stabilizers like the disaccharides) on the glass transition temperature (Tg′) [58]. It is not just the attainment of keeping the Tg′ high at subzero temperatures that matters, but it also is the state of the API in that glassy medium that also has import to achieving the greatest stability of the dosage form. For example, surfaces (ice, air) in the frozen matrix may contribute to destabilization for proteins [59,60]. More recently a good case has been reported to show that β-relaxation governs protein stability in sugar-glass matrices and that increasing antiplasticizing additives correlates with increased protein stability [61]. With complex viral vaccines, protein, phospholipid and nucleic acid destabilization need to be taken into account as well during freezing, primary and secondary drying. The influence of freezing on a viral preparation can be assessed by conducting freeze/thaw experiments. There is not much reported data to examine sublimation stress (during primary drying) on live viral vaccines as it is virtually unexplored. However, given that it involves the transition of the ice phase to vapor without going through a liquid phase state, if the ice surface imparts an interfacial instability, then perhaps the removal of the ice surface could contribute some destabilization. Such mechanical distortion induced by sublimation of biofilm surface features has been attributed to increased strain on the viscoelastic matrix [62]. It is of some concern here, because live viral vaccines can have both protein and membranous structures that could be impacted differently (Fig. 1). Additionally, vaccine preparations are typically quite dilute and so surface mediated alterations are presumed to be more impactful. After primary drying, phospholipid bilayers can be potentially altered physically (e.g., fusing, forming liquid crystals to gel phase transitions) resulting in leaky porous structures. It has been reported that some sugars (like sucrose and trehalose) are capable of preventing damage from dehydration while maintaining lipids in a fluid-like phase [63]. In this context there have been studies that have examined the compositions of different stabilizers pertaining to lyophilized live attenuated vaccines with phospholipid envelopes. Both trehalose and

T

456

An example using the TCID50 method for a thermal stability test comparing unheated to heated sample at 37 °C for 2 days has been applied to a polio virus trivalent vaccine [50]. A vaccine batch is deemed to pass the test if the loss of titer on exposure is not greater than 0.5 log10 TCID50 per human dose [51]. Potency of live attenuated polio virus has been determined in an in vitro TCID50 assay with statistical significance [50]. The assay is a complex biological reaction between virus, cells, antisera and culture media. The recommended storage temperature for live attenuated poliovirus vaccine is 2–8 °C, so the test at 37 °C is an accelerated degradation test measuring the vaccine response to thermal stress. For inactivated vaccines, since they lack the ability to infect, the FFA, TCID50, and plaque assays can be used as negative controls. Thus, these assays may be used as screens to establish that the antigen cannot replicate and infect, and is indeed inactive. However, these assays do not measure the ability of the inactive vaccine to impact immunity. In this case, animal (in vivo) immunogenicity studies typically are carried out to assess potency (immunogenic response) and minimal efficacious dose levels (e.g., vaccination challenge/protection assay) [52]. Alternatively, in vitro assays that target proteins that make up the inactivated virus envelope have been developed (e.g., haemagglutinin-neuraminidase (HN)), such as the case for Newcastle disease virus (NDV) [53]. In this assay, the antigen (HN) is extracted from an oil emulsion (containing isopropylmyristate) and analyzed by an in vitro ELISA method specific to quantitate HN-protein of NDV. Another example is the single-radial-immunodiffusion (SRID) in vitro method commonly used for inactivated viral vaccines (e.g., influenza, polio, and rabies) [54]. The method is based on the agglutination of erythrocytes to assay the haemagglutinin (HA) antigen content of inactivated viral vaccines (e.g., influenza). The method can be extended to test whole virus and subunit vaccines [55]. Emphasis here requires attention to the acquisition of in vitro/in vivo correlates to validate responses of activity for inactivated as well as viral-like particles (VLPs). Thus animal correlates become the standard, although this can sometimes be difficult to achieve due to complexities involving CD4+ and CD8+ pathways of CMI that may not correlate exclusively with humoral responses. The approach to establish a reliable activity assay is to carry out an in vitro relative potency (IVRP) assay and correlate the assay response with an in vivo animal study [56]. In brief the method involves dosing animals (e.g., mice) within a suitable range (from high to low dose) and evaluating post-immunized sera against a pre-immunized serum control. The sera are statistically tested using an ELISA based method to assess the neutralizing antibody response to the antigen in vivo [57]. An IVRP assay is then used; a sandwich immunoassay with separate monoclonal antibodies is used for capture and detection. Antigenicity of the vaccine (e.g., antibodies raised against epitopes that neutralize the antigen) is measured by the IVRP assay. The IVRP response is then evaluated to examine the correlation with the in vivo animal (e.g., mouse) response.

D

408 409


E

10


477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494

496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533

555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570

575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597

C

553 554

E

551 552

R

549 550

R

547 548

N C O

545 546

U

543 544

O

A potential means of achieving higher temperature stability (i.e., ambient or common household refrigerator temperatures) of a vaccine is through formulating the drug product as a solid, wherein the active ingredient is immobilized in an excipient matrix. Although this is commonly performed by lyophilization, some vaccine candidates may exhibit poor process-related stability due to various degradation mechanisms as described above. Spray drying represents an alternative means of producing an immobilizing matrix, and has been previously explored for stabilization of live attenuated enveloped virus vaccines such as measles [25] and tuberculosis [67] as well as for other vaccine types such as influenza subunit vaccines [68]. Spray drying is a process wherein a liquid solution or suspension is atomized into fine droplets, which are then dried to form solid particles using a high temperature gas and collected. The solid particles generated by spray drying may be further processed, e.g., in tableting or capsule filling, or used directly, e.g., in an inhalation dosage form. High temperatures (e.g., 100 °C or higher) are commonly used to rapidly dry droplets; however, due to wet bulb kinetics, the drug product itself does not actually experience these high temperatures but is instead exposed to a downstream temperature below its glass transition temperature (Tg) that is more amenable to its physical and/or chemical stability [69]. By the time drying is essentially complete and the drug is exposed to higher processing temperatures, the formulation will have been designed such that its Tg is much higher than these temperatures (e.g., Tg N 80 °C) and stability is maintained until the spray dried product is removed from the processing

541 542

R O

573 574

540

P

3.3. Spray-dried

538 539

D

572

536 537

T

571

sucrose sugars were used in such a study for 17D yellow fever virus vaccine with an RNA genome [64]. While comparing 6 preparations, it was found that a composition with 10% sucrose alone was more favorable than 15% trehalose (w/v). Furthermore, a preparation containing 2% sorbitol + 4% inositol and a more complicated preparation with 10% sucrose, 5% lactalbumin, 0.1 g/L CaCl2 + 0.076 g/L MgSO4 also gave superior performance among the six preparations tested on stability. One surprising result from these stable compositions was that although the preparation containing the divalent salts yielded a promising stability profile, it showed lower infectivity titer. Thus, the divalent salts had an inhibitory effect that might be due to the established fact that many metal ions can accelerate the spontaneous degradation of RNA [65]. From the above example, it is clear that different disaccharides can have distinguishing effects on stability, and stability of the vaccine must be linked to infection (a point noted above with regard to assays that are intended to measure potency). The integrity of the genome is important for infectivity and replication. That is perhaps why chelators are typically used in some formulations of live viral vaccines. Different strains of enveloped viruses can behave differently subjected to lyophilization in the same formulation buffer system. In a case study where several strains of respiratory syncytial virus (RSV) were freezedried in SPGA (sucrose, phosphate, glutamate, albumin) [66], different strain specific behaviors in stabilization were observed (Fig. 3). In the figure, there are three strains represented, RSV-11657, RSV-R5059, and RSV-M1016. The strain RSV11657 shows a commonly observed biphasic stability degradation pattern (sharp initial decrease followed by a more gradual loss in infectivity) depicted in Fig. 3A. The stability profile for this particular strain appears more resilient than the others comparing the 4, 25, and 37 °C data with that of strain RSV-R5059 that shows a more dramatic drop in infectivity at the same temperatures (Fig. 3B). Another point to be recognized is the afforded stability retained by the solid dosage form at 37 °C for RSV-M-1016 in contrast to that for the non-freeze-dried (liquid suspension) sample (Fig. 3C). In the nonfreeze-dried case, rapid losses in infectivity can be seen to occur within a 24 h period. The solid lyophilized sample retains a significantly greater proportion of infectivity by comparison. These findings suggest an effective development strategy to screen particular strains for those with best responses toward a stable profile as a requisite for developability.

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Fig. 3. Freeze-dried RSV:11657 (A), freeze-dried RSV:R-5059 (B) and kinetic of inactivation of freeze-dried and non-freeze-dried preparations of RSV: M-1016 (C) at 4 °C (□), 25 °C ( ) and 37 °C ( , ). All are formulated in SPGA (sucrose, phosphate, glutamate, albumin). Note all data plotted from [66].

equipment. In the case of a vaccine formulation, the drug concentration will be extremely low and the formulation Tg will be exclusively a function of the excipient composition and residual moisture content. The high achievable Tg is the main driver of long-term physical stability; however, the excipient composition may still negatively impact the chemical stability and potency of the vaccine as already described. Depending on the target droplet size, significant shear stress may be introduced to the drug solution stream during atomization as the solution expands at the end of a pressure nozzle, passes through a high-pressure gas stream, or sheets across a rotating disc. The magnitude of stress typically increases with decreasing particle target size, as more energy is required to break the liquid stream into smaller droplets. For a given solution feed rate, the shear stress and potential for potency loss will increase with decreasing nozzle orifice diameter, increasing atomization gas pressure, or increasing rotating disc speed. The potential negative impact of


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4. RSV live attenuated vaccine dosage form stabilization

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In the example that follows, a live attenuated vaccine against RSV was evaluated as it pertains to stability in lyophilized and spray-dried dosage form scenarios. Protruding through the lipid bilayer membrane (envelope) are the G and F glycoproteins responsible for viral attachment and fusion to host cells. There is also the SH membrane protein that has been shown to be responsible for decreased Th1 responses (inhibits host antiviral response). Encapsulated is the RNA genome and accompanying phosphoprotein, Large L, nucleocapsid (N), and M2-1 (or matrix) proteins that make up the RNA dependent RNA polymerase composition of the virus. Thus, these components with some variation make up the general features described in Fig. 1A. There have been a variety of different attenuated RSV vaccines brought forward for clinical testing. Among them are the attenuated gene deletion vaccines that include ΔNS1, ΔM2-2, ΔM2-2NS2 and rRSVA2 cpts248/404/1030/ΔSH (MEDI-559; MedImmune and National Institute of Allergy and Infectious Diseases) [76]. The latter (MEDI-559) is a recombinant temperature-sensitive RSV with a deletion of the SH membrane protein gene. Stabilization will thus be dependent upon the differences in protein compositions of the membrane as well as the proteins and genome housed within the envelope of this particular vaccine subjected to a variety of stresses involved in the preparation of the formulated drug product.

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A pH profile study was carried out to determine the optimal range of potency for MEDI-559 and it was determined optimal within the range extending from pH 5.5 to 7.5. Examining the loss of potency based on various stages of freeze-drying (Fig. 5), it is apparent that the differential loss for the trehalose containing formulation is less than that for the sucrose containing formulation during freezing. Subsequent primary drying losses are comparable during and after primary drying. Hence, trehalose was a preferred excipient to use in achieving a stable lyophilized

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A vacuum foam dried (VFD) product is generally regarded as mechanically stable, manufactured by boiling biological solutions or suspensions under vacuum at temperatures above their freezing point. Stresses commonly encountered during vacuum foam drying include boiling, foaming, and drying under collapse conditions (well above the Tg′). During the boiling phase, live attenuated vaccine particles can experience high shear forces, short durations of subzero temperatures (due to evaporative cooling) and cavitation. Foaming can introduce interfacial and rapid repeated change of local environment. Drying is usually carried out at collapse conditions and involves desiccation and temperature stresses. Controlling the foam structure is a key process parameter. Bubble inflation is arrested by increasing viscosity that occurs simultaneously during the drying phase. Bubbles and foam cells rupture and collapse repeatedly until foam is stabilized. The process is scalable and relies on control of pressure and temperature, container geometry, surface tension, viscosity, bubble nucleation and growth rate. The stability advantage gained in some vaccine applications is not always found to be universal or well characterized. The foam drying process can be carried out in a lyophilizer and involves aspects of optimization to achieve appropriate viscosity and surface tension properties of the solution. This is carried out using appropriate excipient compositions to modify the properties of the suspension medium. The process requires biological solutions to be filled to no more than 20–25% of the container volume prior to boiling above their freezing point to produce a foam scaffold (referred to as the primary drying step) [72]. A succeeding step is carried out at elevated temperature to further dry the sample and increase the Tg (lower residual moisture). Although in principal the process is rather straightforward, some common problems encountered include violent boiling, splattering and inconsistent onset of bubble nucleation. Some potential resolutions may involve mixing, chamber rotation, seed crystals, use of ultrasound, and stabilizing excipients. The resulting foam dried product characteristics are low specific surface area (much lower than lyophilization, or spray dried dosage forms) and slow water release and uptake. Potential solid dosage form applications to viral vaccine products have been investigated using VFD and found to offer an alternative to lyophilization practices where freeze-labile biologics are generally vulnerable. For example, VFD optimization of a thermolabile Newcastle disease (paramyxovirus) vaccine was found to be a suitable alternative to lyophilization where the virus was prone to denature during freezing and dehydration stresses [73]. Common formulation practices involve pH profiling, sugar screens (e.g., sucrose, trehalose) and surface tension characterization (e.g., using pluronic polymers, and non-ionic surfactants) to optimize foam and stability properties. It has been reported that increased surface viscosity correlates with increased foaming and afforded foam [74]. The solution properties must then be optimized with VFD cycle parameters, involving shelf temperature equilibration and appropriate vacuum control.

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In another example, a vector vaccine candidate, recombinant bovine/human parainfluenza virus type 3 (PIV3)/RSV F2 (MEDI-534), was evaluated for stability in lyophilized, spray-dried, and foam dried dosage forms [75]. In this particular case, the VFD vaccine turned out to offer superior storage stability to either lyophilized or spray-dried products. The reason cited was due to low specific surface area, low vaccine surface coverage and high enthalpy recovery. It was noted that the presence of surfactant did not play a role nor did annealing above the Tg′ (in the lyophilized case) have an impact on the stabilization of this vaccine type. In Fig. 4, a room temperature stable VFD MEDI534 product has been shown to be attainable extending out beyond 45 months at 25 °C.

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shear on the integrity of macromolecules in the solution state during atomization has been reported [70]. Degradation mechanisms driven by shear include denaturation and aggregation [71]. Formulation design to ensure integrity in the solution state and during atomization will depend on the particular properties of each macromolecule – in the case of vaccine formulations, due to the low concentration of the antigen itself the preferred stabilization approaches to date have involved use of very mild atomization conditions [28] and/or high solution disaccharide contents [67]. Both options tend to drive particle size upwards, and as such are useful for generating powders for reconstitution but less useful for generating small particles for direct nasal or pulmonary delivery. The specific form of the antigen (i.e., enveloped or non-enveloped) will also influence the chosen stabilization strategy, as non-enveloped viruses will be more amenable to spray drying due to absence of the lipid bilayer and associated membrane proteins.

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Fig. 4. Foam dried MEDI-534 formulation at 4 °C ( ) and 25 °C (□).


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Fig. 6. Stability results for MEDI-559 lyophilized formulations with 10% ( ) and 30% (□) trehalose at 4 °C (A) and 25 °C (B).

4.2. Spray-drying RSV

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Spray drying was evaluated as a means of producing formulated MEDI-559 particles that may either be reconstituted for delivery as a liquid spray or administered directly as a dry powder to the nose for immunization. Previous evaluations of this virus indicated its low tolerance to freezing processes common to other stabilization methods (e.g., lyophilization); thus, the spray drying approach presents a potential opportunity to improve drug product stability by avoiding a freezing step. Spray drying development at MedImmune was conducted at the bench scale using a modified Büchi B-190 Mini Spray Dryer (Büchi Labortechnik AG, Flawil, Switzerland) and at the pilot scale using an Anhydro MicraSpray® 75 spray drying system (SPX Flow Technology, Søborg, Denmark). Excipient screening studies were conducted at the Büchi scale, followed by transfer of the lead formulation candidate to the Anhydro scale for evaluation of commercial-scale processing capability and long-term stability. Feed solutions for spray drying were prepared by thawing the drug substance formulation under refrigerated conditions, then performing any buffer exchanges, dilutions, and/or excipient additions required by the formulation recipe. Feed solutions were held at 2–8 °C for not more than 48 h before use. In most cases, solution titer was assessed prior to use; for studies summarized here, titer typically declined less than 0.2 log10 FFU/mL during this hold period. Most titer loss in the solution state was due to the thawing process, with little to no titer loss observed during buffer exchange, dilution, or excipient addition. Solutions were exposed to room temperature conditions over the 30 min to 2 h period typically required for spray drying. Supporting data obtained for a baseline formulation exposed to 25 °C for 24 h indicated no significant titer loss would be expected during spray drying. Formulations studied at the Büchi scale consisted of a standard buffer for maintenance of pH and ionic strength, a monovalent salt for tonicity, and a non-reducing disaccharide for bulking and virus immobilization during drying. Other additives were evaluated for their potential to assist with bulk matrix formation (e.g., mannitol, dextran) or to interact with and stabilize the viral lipid membrane (e.g., phospholipids and surfactants). Spray drying of most Büchi-scale lots was performed using process conditions intended to generate powders with residual moisture

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dosage form. It is, however, unknown as to why the trehalose offers improved stability with regard to the stresses encountered during the freeze-drying process. Further investigation into this area is needed, and perhaps with a deeper look into the glassy state property differences between the two sugars. Additionally, at the molecular level of understanding, differences in the way the two sugars interact with the phospholipid bilayer may play a role [63]. A comparison of stability performance with regard to trehalose compositions (high and low) at refrigerated and at room temperature is shown in Fig. 6. Although the Tg of both lyophilized products are approximately comparable (40.7 °C for 10% trehalose vs. 36.6 °C for the 30% trehalose formulated sample), at 4 °C there is a substantial drop-off in potency for the low trehalose containing sample providing

evidence that trehalose composition is important to the overall stabilization of this vaccine. Furthermore, the moisture content of 2.6% for the 10% trehalose containing formulation may suggest that too much dehydration is impacting overall stabilization of the vaccine (an area of further study). Evaluating performance at 25 °C depicts a gradual drop-off of the high trehalose containing formulation (Fig. 6B), but as experienced at refrigerated temperatures the low trehalose sample rapidly drops below the detection limit (as noted earlier for the refrigerated case by the first timepoint). The glassy state influence is likely to be a minor contributor to the overall stabilization in this case because of the dramatic differences at refrigerated conditions well below the Tg and thus, local influences about moisture content and molecular interactions of trehalose surrounding the live attenuated vaccine particles could provide a deeper understanding of the stabilizing mechanism. Clearly, a high trehalose containing formulation is still inadequate to achieve a room temperature stable lyophilized drug product for this vaccine. With the addition of a bulking agent (mannitol), the typical biphasic behavior was observed showing a rapid decline within the first month of stability followed by tapering off beyond 3 months (Fig. 7A and B). The longevity at 25 °C is insufficient to attain a room temperature stable product. The biphasic stability response observed is unknown and the subject of speculation within the literature. It has been noted as possibly arising from the presence of more than one population of infectious particles [66].

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Fig. 5. Titer losses during processing for MEDI-559 formulations with trehalose (□), Δ = 0.34 log10 FFU/mL, and sucrose ( ), Δ = 0.61 log10 FFU/mL.

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0.2 log10 FFU/mL. Qualitatively, the titer loss due to spray drying appeared to decrease slightly (i.e., by approximately 0.5 log10 FFU/mL) with increasing disaccharide content from 10% w/v to 25% w/v trehalose. The additional trehalose may provide more protection to the virus during the spray drying process. Certain additives such as dextran and myoinositol appeared to increase processing titer loss relative to a baseline TPK (trehalose, phosphate, and KCl) formulation (Fig. 8). Anhydro scale spray drying conditions were selected to achieve residual moisture contents of 3–4%. Powders were generated at outlet temperatures of both 50 and 60 °C for comparison to Büchi process results, with a drying gas flow rate of 80 kg/h. Powders generated at this scale were not secondarily dried. The two 10% w/v trehalose lots made at these two outlet temperatures exhibited a mean process titer loss of 0.8 log10 FFU/mL, which was consistent with the decrease in titer observed in spray drying at similar Büchi outlet temperatures. For the Anhydro-scale lots, solution feed rates ranged from approximately 7 to 11 mL/min, with powder yields exceeding 80%. Powders generated at this scale were not secondarily dried, as their moisture contents were less than 4% as targeted by the spray drying process design. Both formulation moisture content and Tg were influenced by composition. The TPK baseline formulation typically expressed a Tg of approximately 86 °C at the standard Büchi operating conditions. Variations in buffer composition (e.g., to citrate or Tris buffers) did not significantly change Tg or moisture within the ranges studied. In contrast, formulations with higher disaccharide contents tended to display higher Tgs and slightly lower residual moisture contents. Addition of other excipients had variable effect depending on the properties of the excipient. For example, addition of 5% mannitol to the baseline formulation resulted in a significantly lower Tg and somewhat higher moisture content, while addition of 0.1% leucine to the baseline formulation resulted in a higher Tg and lower moisture content. Addition of 10% myo-inositol to the baseline formulation resulted in the lowest Tg obtained in the study, while the moisture content was not reduced to the same extent. Formulation composition did not appear to systematically affect process-related titer loss. Midpoint Tgs obtained for the two Anhydro lots (i.e., 66–69 °C) were consistent with those seen for Büchi lots of equivalent compositions and residual moisture (i.e., not secondarily dried), indicating no influence of dryer scale on Tg. Büchi and Anhydro-scale lots were placed on stability at 4, 25, and 40 °C and monitored for up to 12 months. Titer data were obtained using both manual and IsoCyte plate reading methods. Of Büchi formulations, the TPK formulation provided one of the best and most consistent stability profiles. This formulation demonstrated an initial decline in titer of approximately 0.8 log10 FFU/mL after 1 month at 4 °C, but then declined a slight additional amount and stabilized after 3 months. At 25 °C, this lot declined by about 1 log10 FFU/mL after 1 month and then continued to decline more gradually up to 5.5 months (Fig. 8). Stability data suggest that this powder could be suitable for

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contents in the 3–5% range at an outlet temperature (i.e., 45 °C) low enough to minimize potential impact to viral titer. Additional lots were spray dried at higher outlet temperatures (i.e., 50 and 60 °C) to assess impact of temperature on viral titer. Solution feed rates were approximately 1 mL/min for all Büchi-scale lots. Process yields were typically 50% or less as gauged against total solids fed; however, yield is not a relevant process performance metric at the Büchi scale and as such was not routinely assessed. Powders generated at the Büchi scale were secondarily dried to achieve moisture contents of b 2% to minimize potential molecular mobility due to presence of residual water. Secondary drying was performed in open trays in a clean dry air-purged glove box at room temperature conditions for 24 h. At the Büchi scale, the mean titer loss due to spray drying was 0.6 log10 FFU/mL, while the additional mean titer loss due to secondary drying was

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Fig. 7. Stability results for MEDI-559 lyophilized formulations with sucrose (□) or trehalose ( ) and mannitol at 4 °C (A) and 25 °C (B). Frozen-liquid formulations with potassium phosphate (□) and citric acid ( ) excipients stored at −19 °C in droppers (C).

Fig. 8. Examples of spray-dried formulation showing a biphasic response in titer over time stored at 4 °C ( ) and 25 °C (□).


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5. Administration route

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Complete immunity depends not only on the vaccine type but also on the route of administration. For example, non-mucosal injected vaccines elicit a systemic immune response [77,78], while immunization by mucosal routes not only generates a systemic response but also stimulates local immunity [79]. The vast majority of vaccines in use today are administered by intramuscular injection, even though the muscle is not a highly immunogenic organ [80].

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The stability of a frozen-liquid dosage form for MEDI-559 was tested at −19 °C at a pH of 6.5. The results are shown in Fig. 7C and depict the commonly observed biphasic behavior in the stability pattern out through 5 months of real-time storage. Stability in two different buffer systems is presented, and although the nonlinear fits do not seem to show much difference, the data points at around 5 months of stability seem to be significantly lower for the phosphate buffered case. Although the cause for the loss in stability is currently unknown, it may be due to changes in the phospholipid bilayer structure as a consequence of freezing and dehydration stresses for reasons previously mentioned. Additionally, the temperature for this set of preparations is slightly above the Tg′ of the samples in each case. Thus the additional mobility offered by viscous flow could also account for the slow decay of titer for this particular vaccine. Areas to focus on would be the role of disaccharide sugars on the stability of the vaccine (focus on the integrity of the lipid bilayer structure) and the use of polymers to raise the Tg′ so that storage at −19 °C is below and not above this assumed critical temperature parameter.

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The skin is an attractive site for vaccination because of the large number of dendritic cells and efficient drainage to the lymph nodes [81]. The epidermis is the outer layer of the skin enriched in melanocytes and Langerhans cells (a subtype of antigen-presenting DC). The outer dermis contains mast cells, dermal DCs, macrophages as well as Langerhans cells. Skin immunization can initiate a specific immune response by processing and presenting antigen fragments to naïve T cells in the lymph nodes (Fig. 9). Skin immunization approaches include liquid jet injections [82], particle-based methods such as epidermal powder immunization [83], and topical application methods such as electroporation, ultrasound, thermal or radio wave mediated ablation and microneedles [84–87]. Oily formulated excipients may aid induction into the lymphatic system. Out of these methods, microneedle technology is the only one that has attained a commercially viable setting. Fluzone intradermal (Sanofi Pasteur) is an inactivated microneedle product indicated for active immunization against influenza disease caused by influenza A and B. Scarification vaccination is another simple and convenient microneedle approach to deliver vaccines to the epidermis layer. For example, DryVax™ and ACAM200™ are commercially available for live-attenuated vaccines against smallpox delivered via scarification (Table 1). Mucosal vaccination can be achieved via oral, intranasal, pulmonary, rectal or vaginal routes [88]. The nasal route is the most straightforward and is suitable for vaccination, while still achieving both mucosal and systemic immune responses [89,90]. The nose is easily accessible and the nasal cavity is equipped with a high number of dendritic cells (DCs), T-cells and B-cells which are covered by an epithelial layer of cells containing some distinctive cells called microfold (M) cells [91]. The inductive tissue for the generation of an immune response in the respiratory tract is mediated by the nasal-associated lymphoid tissue (NALT), generally described as the Waldeyer's ring and consist of the nasopharyngeal tonsils, the bilateral pharyngeal lymphoid bands, the bilateral tubal and facial tonsils, and the bilateral lingual tonsils [92]. The M-cells, which are the gateways to the NALT, are the sites of antigen uptake that can mediate both strong local and systemic immune responses against pathogens that invade the body through the respiratory tract, producing Th1, Th2 and IgA-committed B cells (Fig. 10). In infants, lymphoid tissue is disseminated in the whole nasal mucosa. Thus, a broad distribution of the vaccine on the mucosal surfaces appears desirable for local action and systemic absorption [93]. A nasal inactivated vaccine against influenza virus (Nasalflu, Berna Biotech AG) was recalled in 2001 in Switzerland because of its associated incidence with Bell's palsy [94]. The gastrointestinal (GI) tract is a major route of entry for numerous pathogens. The intestinal immune system has to discriminate between harmful and harmless antigens. Initiation and generation of oral antigen-specific immune responses is mediated by the gut-associated lymphoid tissue (GALT) found in the intestinal tract. GALTs comprise several different organized lymphoid structures: Peyer's patches (PPs), mesenteric lymph nodes (MLN), as well isolated lymphoid follicles distributed throughout the walls of the small and large intestines [95]. PPs are well characterized sites for the initiation of intestinal IgA responses and are covered with a specialized epithelial region, termed follicle-associated epithelium (FAE), containing specialized antigen sampling M cells. Antigens delivered via the oral route are processed via M cells or through the epithelium [96], see Fig. 11. Oral vaccination can also induce both mucosal and systemic immunity. It offers several advantages: needle-free, easy and comfortable delivery and applicability to mass vaccination [97]. Live-attenuated vaccines have demonstrated good results, while oral delivery of non-living vaccines has proved to be extremely challenging, owing to poor stability of proteins, peptides and DNA in the acidic and enzyme-rich environment of the gastrointestinal tract [98]. Despite these environmental challenges, there are three successfully marketed oral live-attenuated vaccines against rotovirus (Rotarix [solid and liquid] and RotaTeQ) and typhoid (Vivotif) pathogens, see Table 1.

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refrigerated storage, but would not be sufficiently stable at room temperature. A different TPK formulation was also produced on the Büchi system to evaluate whether a higher disaccharide content could reduce process-related potency loss and/or improve stability. The titer decline for this formulation after 1 month was slightly less than that observed for the baseline formulation; in addition, the titer of this formulation at 25 °C stabilized by 2 months instead of 3 months. Preliminary data thus suggest that the changes adopted in this TPK formulation may improve viral titer stability; however, other factors may also play a role (e.g., residual moisture content) and remain to be fully characterized. The baseline TPK formulation was also spray dried on the larger scale Anhydro system at two different outlet temperatures (50 and 60 °C) and placed on long-term stability. Anhydro powders made at both outlet temperatures showed improved stability performance at both 4 and 25 °C relative to the Büchi powder. The Anhydro powder titers appeared to stabilize after 1 month for both storage conditions with no further decline. Relative powder moisture contents (i.e., between Büchi and Anhydro powders) may be influencing these results, and should be studied in more detail in the future. Results of initial evaluation of MEDI-559 formulation and spray drying process development indicated that, within the time frame studied, stabilization of powder viral titer at a storage temperature of 4 °C is possible with an initial loss of about 0.7 log10 FFU/mL after 1 month. Stabilization may also be possible at a storage temperature of 25 °C given additional optimization targeted at formulation composition and powder residual moisture content. Process-related losses of about 0.8 log10 FFU/mL appeared to be a result of the atomization process and will likely depend on the choice of atomization method. These initial results indicate that there is a possibility for development of a process at the Anhydro scale capable of generating powders suitable for nasal delivery, given selection of the appropriate atomizer. A change to a less aggressive method to increase particle size may result in reduced process-related titer loss.

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Fig. 9. Skin can be divided into three regions: first, the outer most layer, epidermis, which contains stratum corneum; second, the middle layer, dermis; and third, the inner most layer, hypodermis. The Langerhans cells (LC), a subset of the dendritic cell family, reside in the basal and suprabasal layers of the epidermis; they transport and present antigens to CD4+ T cells in regional lymph nodes, initiating an adaptive immune response.

Vaccine antigen-virus Periciliary layer 7-10 µm

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Fig. 10. Schematic overview of the consecutive steps of successful nasal vaccine delivery: “1” mucoadhesion; “2” antigen uptake by Microfold (M)-cell transport that are present in the epithelium overlaying the nasal-associated lymphoid tissue (NALT); “3” delivery and activation/maturation of antigen-presenting cell (APC); “4” induction of B-cell responses.



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5.1. Nasal delivery

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Fig. 11. Antigen sampling in the gut-associated lymphoid tissue (GALT). Luminal antigens are transported to the Peyer's patches through M-cells that are present in the follicle-associated epithelium followed by the transfer to local APCs in the subepithelial dome (SED) and then into the thymus dependent area (TDA). Finally, transfer to the mesenteric lymph node (MLN) is where induction of B-cells takes place. Antigens may also enter through the follicle-associated epithelium directly (alternate pathway) without passage through Peyer's patches that migrate to the MLN where maturation/differentiation of B-cells occurs.

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Why nasal administration? Nasal delivery is an attractive route for 990 systemic drug delivery and also for needle-free vaccination. It addresses 991 hurdles commonly faced by other routes of delivery: avoids first-pass 992 metabolism by the liver, enzymatic activity in the gastrointestinal 993 tract, drug degradation, slow absorption and poor bioavailability. More994 over, it offers a promising opportunity for the delivery of vaccines due to 995 its non-invasive ease of administration, low antigenic dose required, 996 extent of high vascularization and large mucosal surface area for dose 997 absorption. 998 However, there are historical challenges that limit the potential of 999 nasal delivery which include dose volume restriction from 25 to 1000 200 μL, mucociliary clearance (half-life for clearance is of the order of 1001 15–30 min), small anterior anatomy and potential enzymatic degrada1002 tion [99–101].

5.2. Validating API

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5.2.1. Applying a proper model Even though nasal vaccination is a promising alternative to classical parenteral vaccination, efforts toward new effective vaccines have been slow, especially for killed-inactivated vaccines. Since it is expensive and time consuming to characterize particle deposition in the nasal cavity in vivo, one alternative approach is to use in vitro nasal models to evaluate nasal formulations and delivery technologies. There is no commonly accepted nasal model to examine the regional intranasal deposition. Typically, geometries are obtained from computed tomography (CT) scans or similar data of the human nasal cavity and from molds of cadavers. The nasal replica models from CT scans are built using rapid prototype techniques and frequently divided into subregions. Therefore, it is an accurate representation of the human nasal airways of particular age, gender or specific characteristic.

1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017

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The disadvantages of using cadavers are desiccation and shrinkage of the nasal cavity which translates into overestimation of the airways [102]. There is a reported study investigating intranasal delivery of dry powder using an anatomically correct nasal cavity model [102]. The models can serve to characterize and optimize nasal formulation properties for improved deposition and adsorption in a particular area of the nasal cavity. Evaluation of dispersal, uniform distribution and deposition rely on adjusting the formulation rheological properties. In addition, information pertaining to administration losses can be ascertained from mass balance associated with delivery efficiency. Thus, the model can not only serve as a valuable system in formulation development, but it can also provide useful data for device and delivery optimization.

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5.2.2. Pharmaceutical factors affecting nasal vaccine delivery Traditionally, nasal formulations were solutions or suspensions for administration of locally acting drugs with the nasal cavity. Currently, both aqueous solutions and suspensions are used to deliver drugs to treat allergic rhinitis, sinusitis, or related diseases [103]. An advantage of intranasal delivery is that it permits longer residence time at the site of delivery without being subjected to the degradation and clearance that would be encountered by the liver or gastrointestinal tract [104]. Vaccination via nasal mucosa has been widely studied in animal models and also in clinical investigations [105,106]. The commercially available intranasal delivery system of FluMist uses a liquid formulation that requires cold chain storage and transport to maintain vaccine potency. Liquid formulations have been commonly used for nasal delivery, but few using dry powder formulations. Solid formulations offer the potential to eliminate preservatives (commonly used in liquid formulations) and cold chain requirements, potentially offering long term stability for room temperature storage and shipping [107]. Despite the advantages, there are only a few marketed dry powder products for nasal administration (for example, Beclonetasone dipropionate delivered by Rhinocort Turbuhaler from

1030 1031


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18

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2

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V TS ¼ ρo do g=18η ¼ ρp dp g=18η

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Powder delivery device

Actuation

ST

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where ρo is the standard particle density (1000 kg/m3), ρp is the actual particle density, g is the acceleration of gravity and η is the air viscosity. Under the same atmospheric conditions, terminal velocity will depend on the particle density and size, which are determined by formulation manufacturing procedures (lyophilized and “milled,” spray-dried and foam “milled” as discussed above). As particle size increases so does the terminal velocity (Eq. (1)), leading to deposition most likely mediated by inertial impaction, an important factor in the deposition of drug in the nasal passages. In the spray-dried formulation, porous particles will have the same terminal settling velocity as a smaller particle with a higher density.

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It is important to discriminate between nasal and post-nasal fractions. For characterization of nasal powder sprays or aerosols, cascade impactors are employed for aerodynamic particle size characterization in the range of b5 to 25 μm [110]. Laser diffraction, a non-aerodynamic optical method, has become the industry standard for determining particle size distribution of powder plumes from the delivery device. Particle size distribution is reported as volume diameter defined by 10% (percent of sample below this particle size), 50% (volume median), and 90% of the cumulative volume undersize (d10, d50 and d90, respectively), span [(d90 − d10)/d50] and percentage of particles less than 10 μm. Laser diffraction is suitable for measuring both liquid and solid formulations, with sampling rates of up to 10 Khz and size distributions in the range of 0.1–3000 μm. From the immunologic perspective, antigenic nanoparticles can be rapidly taken up by the NALT M cells [111–113]. However, it increases the possibility of pulmonary deposition (an area to be avoided for delivery of live attenuated vaccines) rather that the upper respiratory tract. The nasal epithelium is covered by two layers: the gel (mucus) layer (0.5–2.0 μm thick) and the periciliary aqueous layer (7–10 μm thick) shown in Fig. 10. The mucus layer is propelled by the cilia toward the nasopharynx; it is replaced approximately every 12–15 min [114], that is an efficient clearance mechanism of inhaled particulates [115]. To trigger an immune response, the antigen must reach the NALT (see Fig. 12). Increasing the length of residence time of the antigen in the mucosa increases the impact on immunity. To increase residence time (facilitated by uptake and processing), viscosity-enhancing (mucoadhesive) agents (like chitosan, alginates, celluloses, thiolated polymers) have been incorporated in the antigenic formulation. Chitosan is a well-known polymer that has been used to boost the mucosal immune response after intranasal administration [105,116]. It has been shown to open tight junctions in the epithelia and thereby increase the permeability [117]. Using a chitosan solution, the residence time for 50% of the formulation to be cleared from the nasal cavity increased from 15 (without chitosan) to 40 min. More rapid clearance may not allow the formulation sufficient time for

D

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AstraZeneca indicated for the prevention and reduction of severity of asthma symptoms) but none for immunization. In addition to the challenges associated with formulation stability, there are physiological hurdles encountered in intranasal vaccination: residence time, uptake through nasal epithelium, transport to DCs, DC uptake, and maturation-control of immune responses (Fig. 2). The physicochemical properties of a liquid droplet or dry powder (size, morphology, composition, surface charge, antigen release characteristics) as well as delivery system play a role in an immune response. The droplet (if liquid aerosol) or dry powder formulated particles will distribute spatially in the nasal cavity dependent upon size. Nasal formulations typically target droplets or particles larger than 10 μm diameter to ensure deposition in the upper respiratory tract [108]. It is estimated that approximately 50% of particles in the 2–4 μm size range pass the nasal cavity into the pulmonary mucosa [109]. The dimension used to represent airborne particle size and effects on deposition is the aerodynamic diameter. The aerodynamic diameter of a particle (dp) is the diameter of a spherical particle with a corresponding density of 1000 kg/m3 (do) that has the same terminal settling velocity in still air. The terminal settling velocity can be calculated by the following equation:

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Nasopharynx region

7 IT

Post-nasal filter

Y

8 Z X

Breathing Simulator

Fig. 12. Infant nasal cavity anatomical structures (outlined in red): nostrils (N), nasal valve area (NVA) and NALT [that includes the inferior turbinate (IT), middle turbinate (MT), superior turbinate (ST) and nasopharynx region]. The anatomically correct infant nasal cavity model was divided into six regions: “2” nasal valve area/nasal vestibule, “3” inferior turbinate, “4” middle turbinate, “5” superior turbinate regions and two nasopharynx sections noted as “6” and “7.” The nasal cavity assembly was mounted in a rotating platform about the z-axis (perpendicular to the plane of the page), mimicking infants in different positions (α2) as well as allowing multi-positions of the device relative to the face (α1). Breathing profiles were conducted through a post-nasal filter connected to a simulator apparatus.


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1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181

5.2.3. Deposition Airway channels direct nasal spray deposition to the anterior cavity rather than the NALT area (Fig. 12). More studies are needed using an anatomically correct model to accurately predict deposition in the nasal cavity. Predicting nasal deposition is important to assess technologies that target the turbinate region, where antigenic agents can potentially achieve a higher immunogenic response. This can be challenging because the airway channel can redirect droplets and powder particles away from the turbinate region (toward the anterior third of the nasal cavity). As proof of concept, a procedure was developed to quantify and predict nasal deposition using an anatomically correct nasal cavity model (for infants) based on formulation properties of a dried powder spray and different delivery trajectories. The anatomically correct geometry for the model was obtained from CT scans, and a replica was built using rapid prototyping (3-D printing technique). The cavity was divided into six regions: nasal valve area/nasal vestibule, inferior turbinate, middle turbinate, superior turbinate and two nasopharynx sections (Fig. 12). A mucosal adhesive polymer was spray-coated to the inner surfaces of the nasal cavity to mimic the mucus layer and minimize particle bounce. A breathing simulator was attached to the nasal cavity in flow trigger mode to maintain a constant flow rate of 6.1 L/min. One half of the breathing cycle rate was inhalation and one half was exhalation in the form of a square wave profile. The powder delivery device was actuated, via a pneumatic actuator from InnovaSystems (Moorestown, NJ), during the inhalation, exhalation or hold-on breath flow part of the cycle. Three commercially available inhalation grade lactose powders (DFE Pharma, the Netherlands) were used for this study with different size particles (d50 = 17 μm, d50 = 60 μm and d50 = 90 μm). The powder delivery device container was filled with approximately 25 mg of powder. The nasal cavity, pneumatic actuator and powder delivery device were mounted in a rotating platform stand, mimicking infants in different positions. A known mass was emitted from the delivery device, and powder deposition in each disassembled region of the nasal cavity was quantified by a colorimetric assay (MBL international Corporation, Woburn, MA) following the manufacturer's protocol. A mass balance approach was performed to determine regional deposition and losses (M: missing mass), M = total mass in delivery device container-Σ nasal cavity regions (Fig. 13). Nearly all particles N 50 μm were deposited in the nasal cavity [109]. The chosen lactase particle size fractions mainly represent particles which should not pass the nasal cavity, ≥10 μm, whereas particles in the 2–4 μm range generally pass through the human nasal cavity into

F O R O

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D

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T

1128 1129

C

1126 1127

E

1124 1125

R

1123

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N C O

1119 1120

efficient uptake/adsorption of antigen [99]. Since the mucus layer is an anionic polyelectrolyte at neutral pH [118], several studies have incorporated cationic-based compounds (i.e. chitosan, hyaluronic acid, N-trimethylchitosan chloride, Carbopol, cetyltrimethylammonium bromide, cationic maltodextrin or ceramide carbamoyl-spermine) to induce electrostatic interactions between the mucus layer and the vaccine formulation, which have resulted in reductions of clearance rates [119–121]. Mucoadhesion can also be promoted by hydrophilic polymers (i.e. sodium alginate, sodium carboxymethylcellulose, hydroxypropyl methylcellulose or Carbopol) which absorb to the mucus by forming hydrogen bonds. Nasal formulations are administered as nasal drops, powder sprays and as a metered dose through nebulization. To evaluate the performance of delivery devices, the FDA recommends characterization of spray pattern (ratio of the maximum to minimum cross section diameter of the plume), determination of plume geometry (identifies the angle and trajectory of spray), delivered dose (shot weight and emitted dose) and particle size distribution, factors that will depend on both the formulation and device. The FDA also recommends the use of automated actuators to eliminate variability between individual-specific measurements and to report spray distance between the device nozzle and the collection surface. The ultimate goal is to deliver a formulation with particles or droplets larger than 10 μm (to trap within the nasal cavity), with a uniform spray pattern and efficient intranasal deposition.

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Fig. 13. Deposition profiles in infant nasal cavity assembly with mean diameter powder particles of 17 μm (A), 60 μm (B) and 90 μm (C) with an airflow of 6.1 L/min (N: nostrils, NVA: nasal valve area, IF: inferior turbinate, MT: middle turbinate, ST: superior turbinate, S-1: nasopharynx section-1, S-2: nasopharynx section-2; F: filter and M: missing powder) during inhalation ( ), exhalation ( ) and hold-on breath ( ). The nasal cavity assembly was position at α2 = 45° from the horizontal plane, and the powder delivery device at α1 = 35° relative to the face.

the lower airways. With inhalation, exhalation or hold-on breathing conditions, a negligible amount of lactose was detected at the postnasal cavity (Fig. 13). In infants, 14%, 26% and 30% of the NALT is distributed in the inferior, middle and superior turbinates, respectively [122]. In general, N80% of the lactose deposition took place in the nostril, nasal valve/vestibule area, interior, middle and superior turbinates for the three breathing conditions. The nostril–nasal valve/vestibule accounts for ~50% and 40% for the inferior and turbinate regions of the total emitted dose. Both 2% and 5% of the total emitted powder was detected in the nasopharynx region for the d50 = 90 μm and d50 = 60 μm powders, respectively. In addition, 10% of the total was the result for the d50 = 17 μm (Fig. 13A–C). The deposition profile demonstrated that


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1243

The basic toxicity assessment of vaccines needs to be based on the best science available, and suitable animal for testing. For example, the susceptibility of mice to influenza viruses depends on both the strain of the mouse and on the strain of the influenza virus [124]. The subject animal should be considered in context with a meaningful study design (e.g., dosing schedule, device, route of administration). The route of administration should conform to the same for use in clinical trials. Thus, nasal delivered vaccine should be tested via nasal delivery in the animal and with the same device proposed for use in the clinical trials. Potential toxic effects of the product should be evaluated with regard to target organs, dose, route(s) of exposure, duration and frequency of exposure, and reversibility of observed toxic effects [125]. Histology of organs (brain, kidneys, liver, heart, and reproductive organs) and the site of vaccination administration should all be documented as part of the study. Full tissue examination will be required for novel vaccines without prior nonclinical or clinical knowledge and through consultation with the Regulatory Authority. The toxicity assessment of the formulated vaccine entity can be done as either a stand-alone toxicity assessment or as a combination safety/activity study with appropriate endpoints [125]. The potency of the administered vaccine material should be validated with an unused portion from the animal testing facility re-examined for activity to assure the material administered to the animals was still active (tested using a qualified suitable method described above).

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7. Device closure

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The drug product includes the vaccine active pharmaceutical ingredient, formulation, and container. Formulated drug products, whether, liquid, lyophilized, spray-dried or foam dried, need to be evaluated in context with the device or container closure that will be part of the product itself. That is susceptibility to leachables and extractables from the production of the vaccine to filling in the container or device would need to be assessed, and thus stability testing in the actual container over the shelf-life at recommended storage and handling conditions would be required. There is general regulatory guidance available on leachables and extractables [126,127].

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8. Conclusions

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A successful live or inactivated vaccine formulation strategy should consider the potential dosage form options, whether liquid, solid, suspension or other, in context with the delivery route intended and the stresses that impact stability. Identifying the primary instability pathways that affect potency can direct formulation design to the most impactful remedies. Moreover, the connection between in vitro correlation to the immunogenic response intended in vivo by a particular vaccine requires an improved understanding of the immune system than is currently known. Thus, in vitro potency must be correlated with suitable animal models. Additionally, characterization of neutralizing epitopes of antigens is important to elucidate properties specific to a deeper understanding of immunization. From the preceding information gathered, there has been little focus on specific instabilities pertaining to cause and effect of degradation relationships as they correlate to loss of potency by these rather complex molecular vaccines. There is a need to better understand freezedrying, spray-drying, and foam drying stresses on enveloped and nonenveloped viral vaccines. Furthermore it is important to understand how different viral envelope compositions respond to different stabilizing effects from disaccharide sugars and why differences exist among strains of the same virus. In addition, analytical tools that enable more detailed physico-chemical characterization of kinds of degradation processes involved for such molecules are fundamental toward grasping a firm scientific perspective applied to efficiently develop such vaccines. Finally, better characterization of these vaccine entities could lead to a more predictive understanding of their degradation behaviors and formulation conditions that confer robust stabilization. The potential to reduce

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O

6. Non-clinical toxicology considerations

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Extractables are chemical substances that can be liberated from the product contact surface through the action of an extracting solvent at specified conditions of temperature and time. Typical extractable compounds are fatty acids, antioxidants, rubber synthesis byproducts (e.g., nonylphenol isomers, 3′-oxybispropanitrile, stearic acid, 3,5 di-tert-butylphenol, docosanol, and dodecanol). All polymers contain extractables and extractable contaminants can also arise from stainless steel used in manufacturing. The polymerization process affects the levels and types of extractables. The sterilization process can contribute to the level or type of extractables. Leachables, on the other hand, are chemicals that migrate out of the product contact surfaces and into the product solution under conditions associated with the manufacturing process and from container closure. Contaminating components found in plastics that can be leached into the product are elastomeric monomeric agents, catalysts (e.g., dicumyl peroxide, cadmium), antioxidants (e.g., phenols, hydrochinon, and pyrogallol), softners (e.g., paraffin, tar, diethylhexyl phthalates, sebacates, organotin compounds), hardners (e.g., heavy metals, peroxides), fillers (chalk, sand, and zinc oxide) and releasing agents (serve to release the formed polymer from its mold). Suppliers cannot guarantee that no deleterious leachables will migrate into the drug product and so it is the responsibility of the drug product manufacturer to evaluate impact on safety and efficacy for the vaccine. Thus two strategies to evaluate potential of such contamination is to (1) follow stability in the container closure at real-time and accelerated conditions across suitable timeframe of shelf-life and (2) follow placebo (identical formulated product without API) in the same containment process over time at the same conditions. Analytical methods should also be appropriately tuned to the identification and separation of organic compounds that could be leached into the DP solution. Moreover, the influence of such compounds on the stability and safety of the vaccine should also be characterized. Information pertaining to safety and best practices for evaluating and managing extractables and leachables for orally or nasal inhaled drug products has been published by the Product Quality Research Institute [128].

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about 10% of d50 = 17 μm lactose powder was lost. Some powder was observed outside the cavity during the exhalation condition (Fig. 13A) which could be a potential exposure issue for healthcare professionals in a clinical setting. The experimental setup is a robust system that allowed quantification of lactose powder deposition in an infant cavity model with the ability to recover up to 99% of the actuated dose via a colorimetric assay. The system closely mimics some of the physiological nasal cavity conditions (viscoelastic properties of the mucus, breathing simulation and anatomically correct airways). Approximately 80–90% of the powder was distributed in the nostrils, nasal valve/vestibule area and inferior-superior turbinates for the mean diameter particles of 60 and 90 μm regardless of breathing condition. It is not surprising to find that most of the powder deposition taking place was in the posterior portion of the cavity, since the nasal valve has the smallest crosssectional area in the nasal passage [123]. Particles with a mean diameter of 17 μm might have undesired effects because some particles could reach beyond the turbinate region in the nasal cavity (Fig. 13A). The preliminary results presented here demonstrate that particle size modulates deposition site, but further testing will be needed to determine whether the device orientation has impact in powder deposition as well as impact on other breathing patterns. The lack of physiological mucus and mucociliary clearance are major limitations in the experimental setup. However, in vitro testing with a nasal cavity model can serve as a valuable system in formulation development or even for delivery technology evaluation despite its limitations.

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The authors would like to recognize the MedImmune Vaccine Formulation Group that participated in the acquisition of stability data for this work, specifically Shweta Hegde, Mengshu Zhao, Marcus Wong, Vu Tran, Dennie Magcase, Dr. Yi Ao, and Steve Heid, and also the MedImmune Vaccine Analytical Group, Lynne Williams, and Dr. Kuldip Sra. The authors thank Dr. Steven Bishop and Dr. Michael Washabaugh for critically reading the manuscript and their constructive suggestions. Finally, we thank Dr. Phillip Lovalenti for his efforts in development of an intranasal strategy for the live attenuated vaccine programs.

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