2013

6th Annual Lyophilization and Emerging

Drying Technologies Conference Review Pep Talk 2013 Protein Science Week, Renaissance Hotel & Palm Springs Convention

Centre in Palm Springs, California USA

21st-25th of January, 2013.

E. Ekenlebie, A. Ingham

Aston University

Document release date: 05/04/13

http://www1.aston.ac.uk/lhs/staff/az-

index/ingham/

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Contents

1 Summary ......................................................................................................................................... 3

2 Buzz Sessions .................................................................................................................................. 4

2.1 BuzZ Session A: Improving Lyophilization by Annealing .................................................... 4

2.2 BuzZ Session B: QbD in Freeze Drying and Design of the Freezing Process ........................ 5

3 Presentations from Main Conference Days 1 and 2 ........................................................................ 5

3.1 Advances in Controlled Nucleation ........................................................................................ 5

3.2 Lyophilization Cycle Development: Navigating the Path to a Biological Animal Health

Vaccine Product .................................................................................................................................. 7

3.3 Beyond Controlled Nucleation ................................................................................................ 8

3.4 Risk Mitigation in Lyophilization Process Development and Tech Transfer ......................... 8

3.5 Formulation Strategies for Successful Spray Drying and Case Studies .................................. 9

3.6 Phase behaviour of Excipients in Lyophilized Formulations: Potential Implications on

Product Performance ......................................................................................................................... 10

3.7 Mannitol Crystallinity and Stability of IgG in Lyophilized Formulations ............................ 11

3.8 Scale-Up of Controlled Nucleation to a Production Environment ........................................ 12

3.9 Challenges and Considerations for Developing Lyophilized Biopharmaceuticals ............... 12

3.10 New Process Technologies for Fast, Continuous Freeze Drying .......................................... 14

3.11 Spray-Dried Biopharmaceutical Powders for Drug Substance Bulk Storage Application ... 15

3.12 Lyophilization of Saccharide and Salt Containing Systems in Bottles: Collapse,

Crystallization, and Implications ...................................................................................................... 16

3.13 Controlled Nucleation: The Impact of Nucleation Method and Freezing Temperature on the

Process and Properties of Freeze Dried Sample ............................................................................... 17

5 Poster............................................................................................................................................. 18

5.1 Submitted Poster Abstract ..................................................................................................... 18

5.2 Poster Award ......................................................................................................................... 19

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1 Summary

A report capturing personal notes from the 6th

Annual Lyophilization and Emerging Drying

Technologies Conference held from 22nd

-24th

January, 2013. This conferences was part of the

Pep Talk 2013 Protein Science Week held at the Renaissance Hotel & Palm Springs

Convention Centre in Palm Springs, California USA from 21st-25

th of January, 2013.

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2 Buzz Sessions

Buzz sessions were forty five minutes round table discussions held on Tuesday, January

22nd, 2013 prior to the main conference event. Attendees were free to participate in any of

the 42 discussion sets. The timing of these sessions meant one could only attend 2 of 4

closely related to lyophilisation.

2.1 BuzZ Session A: Improving Lyophilization by Annealing

This session was chaired by Charlie (Xiaolin) Tang, Ph.D., Associate Director, Formulation

Development, Regeneron Pharmaceuticals.

Issues discussed included reducing drying time, improving cake structure, annealing in an

amorphous system and maximizing crystallinity using an optimal annealing step. Annealing

is performed between the Tg’ and Teu to encourage full crystallisation. Crystallinity can be

measured using Differential scanning calorimetry (DSC) or X-ray Powder diffractometry

(XRD) insitu. The use of DSC can be challenging and is easier investigating changes in glass

transition (Tg) of a pure compound by measuring heat flow over time. Chair admitted his

attempts via this route had not been encouraging.

Benefits of annealing include a resulting increase in sublimation rate and decrease in primary

drying time. Lower product temperatures can be attained because of lower product resistance.

It causes crystallisation of bulking agent at the freezing stage instead of during primary

drying. This prevents the vial breakage phenomenon as seen in mannitol. Mannitol and

glycine containing formulations are commonly annealed. Thicker product walls may result in

pure amorphous systems when annealed.

Annealing can also be performed on dry powders. The approach is to anneal a pure

amorphous system below the Tg. This causes the redistribution of water. However, water

redistributed homogenously is optimal when compared to water clusters which have less

interaction with the API (increase stability). Annealing reduces free volume in a formulation.

The higher the free volume, the lesser the stability due to increased molecular mobility.

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2.2 BuzZ Session B: QbD in Freeze Drying and Design of the Freezing Process

Received apologies for chairman's absence. Discussions were lead by Sajal Patel of

Medimmune with input from all on various aspects of the subject area. Sajal stressed on a

need for formulators to know the critical parameters of materials for cycle development.

Process validation was discussed briefly. There was no one defined practice across the

various companies represented. However it was common to design high and low energy

demand cycles (for example 2 large cycles and 4 cycles at laboratory scale) as well as

demonstrating with data that transfer to laboratory scale indeed worked.

Using a typical influenza platform as an example, only lyophilisation robustness is of interest

initially. Design space is looked into after phase 2. Phase 1 and 2 were more about pace

without wasting time.

Smart lyo technology received praise across the table.

Shelf mapping is a common requirement in industry usually every 2 years. There are

differences in performance between brand new and aging dryers requiring periodic

assessment. Yearly comparison of same cycle from a dryer may show slight differences.

Historical data of production freeze dryers is important. Modifications may include fluid and

condenser changes.

Ramp rate and aggressive cycles are typical areas of caution. Knowledge of final dryer units,

geometry and product limits are necessary.

3 Presentations from Main Conference Days 1 and 2

3.1 Advances in Controlled Nucleation

Michael Pikal, Ph.D., Pfizer Distinguished Endowed Chair in Pharmaceutical Technology &

Professor of Pharmaceutics, University of Connecticut

Addressed the absence of proper control of the nucleation during freezing as compared to other stages

in freeze drying which are directly controllable.

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Surface area of the cake is dictated by the surface area of the ice which in turn causes intra and inter

batch variation as a result of interaction between protein and ice. Although it may not apply to all

systems this explains the observed variations in aggregation across batches.

Bias is commonly seen in thermocouple and non thermo coupled vials with collapse predominant in

the latter. Non thermo coupled vials experience about 10 of supercooling resulting in smaller ice

crystals and about 10% longer drying time.

Laboratory and clean room data showed similarities in nucleation temperature. Small temperature

variations are seen in nucleation temperature of BSA-20% sucrose and 5% dextran systems.

Dr Pikal emphasised supercooling was a big challenge in manufacturing and went on to explain the

current available techniques for controlled nucleation.

Ice fog Technique by IMA LIFE: Super cools product without freezing causing the chamber to be

humid. Pressure is reduced with introduction of nitrogen which forms ice when it hits the humid air

and pushes the ice into the vials to trigger crystal growth.

Freeze Booster by Millrock: Also an Ice fog technique. It can be cheap to retrofit to existing dryers

since it does not require a high pressure capability.

Control Lyo by Praxair:Technique uses a depressurisation mechanism. Drying chamber is pressurised

with an inert gas and shelves are cooled to desired nucleation temperature. Chamber is subsequently

depressurised to induce nucleation. There may be issues with aseptic manufacturing standards.

Further discussing depressurisation, Dr Pikal mentioned it was not clear what the exact mechanism of

action for this technique was. He outlined cavitation, bubble formation, ice fog or adiabatic cooling

possible explanations. Using Human growth hormone which is a good model for investigating

aggregation at gas-liquid interfaces revealed no evidence of bubble formation using an Hplc assay pre,

post and 14 days at 5 . Not used yet in production although it’s proven to work in laboratory.

Investigations revealed the temperature of the air in the drying chamber dropped suggesting the air

temperature in the chamber may be the mechanism. Using Argon is also more effective than nitrogen

and this is explained by the Joule-Thompson coefficient.

Higher super cooling yields larger Specific surface area (SSA) and hence faster secondary drying.

Work on air borne particulate count and its relation to ice nucleation temperature showed no

significant differences but more data is required.

Controlled nucleation is not yet FDA GMP but is good freeze drying practice.

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3.2 Lyophilization Cycle Development: Navigating the Path to a Biological

Animal Health Vaccine Product

Wanda Isaacson, Senior Principle Scientist, Veterinary Medicine Research and Development,

Biological Formulations, Pfizer

Therapeutic targets are usually disease complexes requiring a combination of viruses,

bacteria and protein fractions. Maintenance of potency after freeze drying is a priority.

Animal products are regulated by the US department of agriculture. Drying is governed by

the same four design space, formulation, freezing, primary and secondary drying.

It's important to know whether the formulation can be initially frozen or can be lyophilised

directly from fermentation. Osmolarity is of importance because high salt content and pH

shifts kill live organisms. The goal here is potency with other attributes such as appearance,

reconstitution time and stability secondary.

Apart from characterisation of excipients, it’s important to establish bulk antigen stability.

For example storing at various temperatures over time was shown to be important because

formulations may be in room temperature for a period of time before loading into lyophilizer.

If required, -80 storage can be very expensive.

Nucleation temperature, freezing temperature and time are all necessary considerations. It’s

important to know the limitations of the dryer being used. Lyophilisers available for

speaker’s work were typically 10 to 50 years old.

Occasionally it may be possible to dry above the collapse temperature due to high solute

content from debris and other cells components.

An ideal moisture content is less or equal to 4%w/w since too dry a sample is not ideal for

live organisms. Secondary drying of about 16 hours is common.

Dr Isaacson emphasized how no two formulation developments were the same because

vaccines differ and cycles also need to be designed to fit the freeze dryer.

Capacitance and pirani gauges were used in combination to determine end of primary drying.

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3.3 Beyond Controlled Nucleation

Matthew Ling, Manager, Research & Development, Millrock Technology, Inc.

Matthew spoke about recent work using Accu Flux lyo heat flux measurement system in

nucleation studies. This sensor system allows the continuous monitoring of the drying

process to inform heat flux, vial transfer coefficient, mass transfer, product temperature and

predict primary drying.

Problems associated with cake appearance and longer drying time were evident in all

uncontrolled nucleation samples treated by either controlled freezing or annealing.

Data showed annealing with or without controlled nucleation was not significantly different

in primary drying time but cake bottom shrinkage was seen with the uncontrolled nucleated

samples.

Matthew concluded with anticipated developments with this sensor system.

3.4 Risk Mitigation in Lyophilization Process Development and Tech Transfer

Mark Yang, Ph.D., Director, Fill Finish Development, Genzyme

Dr Mark Yang brought his presentation into perspective with production scenarios which

resulted in product losses and additional cost. Use of “not so dry stoppers” lead to the sudden

decline in product stability as a result of moisture uptake from stopper. Particles may also be

shed into final products from the vial or packaging. US vial-EU ISO vial formulation transfer

problems may also add to cost.

Referred to the FDA’s Code of Federal Regulations (21 CFR 211.94) for desired

characteristics of container closures. These include a need to be clean (with validated

depyrogenation), provision of adequate protection and not being reactive.

Types of glass vials (type 1 class, clear/amber/treated/untreated) and stoppers

(butyl/Neoprene/others) were mentioned. Attention was drawn to problems with differences

in EU and US vial blowback and stoppers. Warned moisture uptake took place during

autoclaving. A 20mm lyo stopper could hold up to 5mg of water and may be released

overtime into dried powder. The drying cycle in itself does not remove moisture.

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Igloo configuration for stoppers is most popular and the use of butyl and halo butyl rubber

dominates in industry. Stoppers may be coated with silicone, Teflon or fluorotec.

Washed glass vials are tested for particulates (spiking with polystyrene beads suspension),

contaminates ( NaCl challenge) and endotoxins (Lal gel clot).

Typical stopper preparations include washing, rising with water for injection, sicliconization,

sterilization and drying.

Design of experiments (DOE) may be employed developing a robust lyo process due to

limited recourses and time.

Dr Yang further discussed the various process development stages clearly highlighting

concerns were appropriate.

Freezing, radiation effects, shelf temperature differences, sample load, heat transfer and mass

flow resistance are some key variations between laboratory and commercial production. Key

strategy is to keep the product temperature the same in research and in production. Others

include knowledge of critical product parameters, robustness studies and use of a scale down

model are others.

Fill finish development was finally discussed along the lines of excipients, thawing of frozen

bulk, compounding process (pH range, sequence and buffer), studies (compatibility and

agitation), Isolator decontamination (1ppm of hydrogen peroxide may be too much for some

products), lyo appearance and characterisation of finished product.

3.5 Formulation Strategies for Successful Spray Drying and Case Studies

Tarun Mandal, Ph.D., McCaffrey/Norwood Endowed Professor of Pharmacy & Director, Center for

Nanomedicine & Drug Delivery, College of Pharmacy, Xavier University of Louisiana

Flowability, production time and particle size were some spray drying advantages highlighted. Other

applications include granulation of poorly compressible API, solubility enhancement as tackled with

co spray drying, microencapsulation and pulmonary delivery.

Spray dried excipients are generally between 40-400microns after granulation and exhibit excellent

flow behaviour. Aero dynamic diameter is of prime importance in pulmonary delivery. Particle sizes

of 2-6micron or greater than 10 microns are desired for deep lung and upper respiratory tract

deposition respectively.

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Professor Tarun further discussed formulation development strategies. This process is driven by the

intended purpose of the formulation as well as physical properties of the API and excipient. Spray

drying is not ideal for an API not stable at 40-50 . Choice of solvents need careful thought and must

be at final FDA residual levels. Fine particles (hard to collect) and large particles (ideal for

granulation) are produced from low and high viscous feed solution respectively.

Delved into control of process parameters namely atomizer nozzle size, liquid flow rate, inlet and

outlet temperature. Particle size is inversely proportional to atomization flow rate. Unlike outlet

temperature, Inlet temperature can be controlled. To avoid high residual solvent content, the outlet

temperature must exceed the boiling point of the solvent.

Discussing particle formation mechanisms, Professor Tarun spoke on stress induced deformation

caused by changes in material and pressure. This often results in wrinkled, hollow or dimpled particle

shapes.

Session ended with numerous case studies showing work in spray drying including chitosan granules,

micro encapsulation and enhancement of solubility using PVP solid dispersions.

From key questions

In the case of low Tg formulations, a secondary drying method may be adopted e.g. tray drying in the

freeze dryer for 24 hours.

Responding to a common concern about thermal damage to an API, the speaker mentioned the rate of

evaporation was high thus making temperature not the main mechanism of destruction for proteins

but the air/water interface.

3.6 Phase behaviour of Excipients in Lyophilized Formulations: Potential

Implications on Product Performance

Raj Suryanarayanan, Ph.D., Professor, Peters Endowed Chair, Department of Pharmaceutics,

University of Minnesota

Implications and prediction of crystallisation of formulation components were discussed in depth.

Formulation components are desired in the solid state as crystalline (small molecules, bulking agents)

or amorphous (buffers, lyoprotectants).

Buffers crystallisation can be worrying in formulation development. Whereas the sodium dihydrogen

phosphate buffer component will not crystallise, disodium hydrogen phosphate does into the

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dodecahydrate resulting in a pH shift from 7.4 to 3.5. Low concentration buffers are the best choice

for low crystallization.

Examining work from synchrotron radiation, the speaker showed how even low buffer concentrations

stood the risk of crystallisation. High sensitivity techniques are required to show crystallisation events

in 1mM sodium phosphate.

PH swings are seen in succinate buffers as a result of sequential buffer component crystallization.

Beta succinic acid and monosodium succinate were products detected by synchrotron XRD. DSC

confirmed melt endotherms of these components. High lyoprotectant concentrations were shown to

inhibit buffer crystallisation.

Trehalose content in avastin, herceptin and lucentis drug products are 6%w/v,2%w/v and 10%w/v

respectively.

No report cites trehalose crystallisation during lyophilisation. However, a look at 4% trehalose in

frozen solution crystallised to its dihydrate after annealing for 24 hours. It then undergoes phase

transition during drying to the amorphous form. In the presence of mannitol, trehalose crystallisation

was accelerated while sucrose completely inhibited trehalose crystallization. A high API

concentration of an antibody will also prevent trehalose crystallisation. A relation also exists between

inhibition of mannitol crystallisation and increasing protein concentration.

Retaining mannitol in amorphous form can be detrimental to storage stability due to moisture release.

Buffers should be used only when indeed necessary.

.

3.7 Mannitol Crystallinity and Stability of IgG in Lyophilized Formulations

Charlie (Xiaolin) Tang, Ph.D., Associate Director, Formulation Development, Regeneron

Pharmaceuticals, Inc.

Talk was a good follow up and verbally covered in buzz session previously chaired by Dr Tang.

Speaker focused on various aspects of mannitol crystallinity in formulations. Modulated differential

scanning calorimetry (MDSC) can be used to monitor mannitol crystallisation and optimise annealing.

Mannitol and glycine are well known to crystallise out resulting in vial breakage and increasing

moisture content on storage.

Annealing was introduced and discussed thoroughly to cover temperature and duration. MDSC, FDM,

XRD characterisation techniques were mentioned. Bulking agent concentrations need to be optimised.

Low protein concentrated formulations may require bulking.

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Tg' shifts are seen in mannitol formulations, increasing solute results in increasing shift without

annealing.

1:1 ratio of mannitol-sucrose showed greater crystallinity. Stability data showed a reasonable amount

of sucrose was enough to confer stability as demonstrated from FTIR data on native secondary

structure.

From key questions

Can one maintain mannitol amorphous during storage? Speaker in a response mentioned he didn’t

know but XRD could be used to test.

3.8 Scale-Up of Controlled Nucleation to a Production Environment

Mark Shon, Vice President, Technology Development, SP Scientific

Talk evaluated and demonstrated it was indeed possible to retrofit existing commercial scale

dryers with Control Lyo technology by Praxair. Spoke on the basics of controlled nucleation

and benefits through to the required mechanics for retrofitting.

Every 1 rise in nucleation temperature is accompanied with a 3% decrease in drying time

(Searls et al 2001).

Chamber must be able to withstand required pressurisation (including the isolation valve if

present), must have available ports for mechanical additions such as manifolds

(pressurization and depressurisation) and pressure transducers. A manual control box or

modifying the dryer’s controls is also required.

Demonstrated 100% controlled nucleation using 5%w/v mannitol (5-25ml fill volumes) and a

total of 8,071 vials using nitrogen as inert gas.

3.9 Challenges and Considerations for Developing Lyophilized

Biopharmaceuticals

Sajal Patel, Ph.D., Scientist, Biopharmaceutical Development, MedImmune

Introducing the talk, Dr Patel spoke on the lyophilisation formulation development process

and how critical it was to fully characterise formulation and dryer.

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Collapse temperature (Tc) is about 1-2 higher than Tg’. Higher protein content affords

higher drying temperatures. Data for a monoclonal antibody dried above 60mg/ml showed

significant difference between Tg’ and Tc. Every 1 rise in product temperature results in

about 13% reduction in drying time. Stable, elegant, scalable and a cost effective freeze

drying process are desired key objectives. To utilise heat and mass transfer models container

systems also require characterization.

Considerations and challenges are the freezing step, edge effects, determination of primary

drying end point and dryer load effects. Controlled ice nucleation and annealing can address

batch variation and also promote crystallisation. Vials not surrounded by about 6 other vials

may be described as experiencing edge effect vials. Such vials have higher product

temperature as well as shorter drying times due to radiative heat transfer. As shelf load

increases, drying time increases due to a reduction in product temperature.

Placing the talk into proper perspective, a needed 100% loading scenario based on previous

knowledge (40% dryer load of the same entity on the same dryer) and cycle transfer to a new

dryer were discussed. Primary drying time and chock flow predictions proved essential.

Critical to the final decision was the prediction that the lyophiliser’s pressure set point was

not attainable based on drying unit characterization.

Process Analytical Tools (PAT) for the stages of freeze drying was highlighted. A sharp drop

in pirani pressure indicates primary drying is near completion and was shown to be the best

compared to others. Smart freeze dryers (Lyostar II) uses theory of heat and mass transfer

together with user fed information from Manometric temperature measurements (MTM). A

target product temperature is then achievable by optimizing chamber pressure and shelf

temperatures. As a limitation, a 300cm2 minimum sublimation area is required.

Tunable diode Laser Absorption spectroscopy (TDLAS) measures gas flow velocity and

sublimation rate.

Other applications include product temperature, endpoint and residual water during

secondary drying. Duct length between chamber and condenser is a limiting factor.

Talk concluded with techniques for characterisation of frozen or finished product and how

operational, design and control space can contribute heavily to process development.

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3.10 New Process Technologies for Fast, Continuous Freeze Drying

Gerhard Winter, Ph.D., University Professor, Pharmacy, Ludwig Maximilian University of

Munich

Speaker highlighted road blocks in typical freeze drying process to include risk of collapse

and high diffusion lengths in large cakes. There is a need to address to economics and

increasing competition from spray drying techniques. Low cost bulk storage and mass

production solutions are needed.

Showed previous work on fluid bed freeze drying by Leuenberger et al, Basel and Pellet

freezing and drying (Gehrmann et al, Bayer TS, Leverkusen). Criticised bulk drying for

material handling issues.

Emerging technologies discussed were:

Stirred Freeze drying (Active Freeze drying) by Hosokawa micron.

Rotating drum Freeze drying (Lyo motion) by Meridion Technologies.

Two possible approaches for stabilization through storage to fill:

1. Freezing Frozen storage + Shipping Thawing + Liquid Dosing + Freeze drying

2. Bulk freeze drying Free flowing dry powder storage Redissolve (then

liquid dose) or powder fill.

Reviewing stirring method of freeze drying, sticking and flowability were clear concerns

from pictures. Dry free flowing powder is collectable.

Quoted the work of Abdul-Fattah et al 2008 linking high surface area to be a problem with

stability. Mentioned these powders have a high surface area. Increasing bulking agent also

increases SSA. The larger the SSA, the bigger the solid void interface and the faster

desorption however keeping SSA low is desirable for protein stability.

Stirred Freeze drying :

Freezing via dry ice or liquid nitrogen. Works and yields similar SSA as from vial freeze

drying however yields problematic with high sugar formulations.

Process for sucrose-Lysozyme system benefited from incorporation of mannitol and Amino

acids to increase yields of a less sticky character. Use of Liquid nitrogen freezing and small

amounts of trehalose also offered same benefit without bulking agent.

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High protein and low sugar formulations are good formulation models.

Meridion Technologies:

SprayCon: generates droplets into static cold gas atmosphere to form 100 micron frozen

droplets.

LyoMotion: has a rotating drum, vacuum and IR heater for sublimation.

Prof. Winter further showed previous work with atmospheric freeze Spray drying in fluidized

bed, spray freeze drying and dynamic bulk freeze drying.

Techniques used to access protein stability include HP-SEC and FT-IR used.

As opposed to low solid content (5%), 20% sucrose showed lower SSA and trend of lower

residual moisture.

3.11 Spray-Dried Biopharmaceutical Powders for Drug Substance Bulk Storage

Application

Mayumi Bowen, Ph.D., Senior Engineer, Pharmaceutical Processing Technology

Development, Genentech, Inc.

Defined the problem associated with storage of biologics in Genentech in the frozen form.

The very high cost involved lead to a need to explore areas to cut cost without compromising

drastically product stability and potency. Storage in 35-600Litres stainless steel tanks cost

$100-$150,000. Samples are frozen and as such require freeze-thaw skids costing about

$500,000. There are also additional cost of shipping, validation and maintenance of vessels.

Freeze thawing is time consuming coupled with mould growth in empty tanks. Complex

supply chains add to cost. Corrosion and safety are also concerns during handling (tanks are

very heavy and could be accidentally dropped).

Tackling this problem, disposable plastic freeze thaw bags and spray dried powders in

disposal plastic bags were explored. 2-8oC stored powders in bags could then be reconstituted

and filtered for further processing.

Touched on the basics of spray drying and stressed on desired physical properties such as

size, morphology, solubility and stability.

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Main objective was to cost effectively store monoclonal bulk powders with concerns for

yield, residual moisture and reconstitution time.

Finally various models of spray dryers were compared along the lines of maximum drying

output and problems that arise during use.

3.12 Lyophilization of Saccharide and Salt Containing Systems in Bottles:

Collapse, Crystallization, and Implications

Bakul Bhatnagar, Ph.D., Principal Scientist, Formulation & Process Development, PhRD,

Pfizer, Inc.

Describing the motivation for his work, Dr Bhatnagar mentioned the need for larger dried

product volumes for product development.

Compared plug and shell frozen cakes. Total drying time of sucrose was significantly shorter

(17 days) in shell frozen samples from 500ml 10%w/v sucrose. A shell freezer is required and

critical to the process are the height of the cooling fluid ( ethanol) ,sample fill and bottle

placement. A correlation exists between freezing angle, total drying time and final residual

moisture levels. Increasing freezing angle correlates with increasing cake height and thus

longer drying time.

Heat transfer is 9-10 times lower in bottles compared to vials.

Use of stoppers resulted in higher product temperatures, increased residual moisture, poor

reconstitution, collapse and protein aggregation. Aperture size influenced cake appearance

and the conclusion was to avoid stoppers.

Other vial work examined conservative and aggressive conditions for formulations (protein

,buffer, NaCl and sucrose (high or low). At lower sucrose-NaCl ratios, resulting cakes dried

at -20 good in appearance with short reconstitution times (30sec). Poorer reconstitution

time (90min with particles) was observed with +15 drying. Higher sucrose ratios at +15

however showed short reconstitution times (5mins) with poor cake appearance.

Characterised the frozen formulations using MDSC. Tg' is shown on reversible heat flow

curve and crystallization while melt events are on non reversible heat flow curves. NaCl

phase behaviour was evident since Nacl crystallization was inhibited and thus remained

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amorphous with lower Tg' (aprox -47 instead of aprox - 27 ) at high sucrose

concentrations and was confirmed with XRD.

From key questions

Product thickness in bottles was significantly high and as such stoppers should not impact.

Why is the stopper making a difference? (Dr Pikal)

Speaker did not know where this unexpected observation occurred.

3.13 Controlled Nucleation: The Impact of Nucleation Method and Freezing

Temperature on the Process and Properties of Freeze Dried Sample

Vamsi Mudhivarthi, Ph.D., Postdoctoral Fellow, Pharmaceutical Sciences, University of

Connecticut

A brief talk employing Beta galactosidase and sucrose systems to study controlled nucleation

using control lyo and freeze booster techniques. Also investigated vial and syringe drying in

aluminium and pexi glass moulds. It was evident that controlled nucleation at higher

temperatures resulted in smaller surface areas, lower drying resistances, bigger pore size

,higher mass transfer and thus shorter primary drying time.

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5 Poster

5.1 Submitted Poster Abstract

Process Optimization of Bulk Lyophilised Bio synthetics: Implications of Powder

Rheology.

E. Ekenlebie and A. Ingham*

School of Pharmacy, Life and Health Sciences, Aston University, Birmingham, UK, B4 7ET.

*Corresponding author- Inghamaj@aston.ac.uk

Synthetic biology is a research field combining biology and molecular engineering. Over the next

decade, it is expected to contribute to drug discovery and biosynthesis of pharmaceuticals. These

biologicals may require drying to maintain long term shelf life. Recovery of activity, an optimum

reconstitution time and an elegant appearance are priorities for many researchers.

Bulk freeze drying has been proposed to have a 30% cost savings when compared to conventional vial

systems during scale up. With aseptic continuous flow lyophilisers and a dosing system capable of

powder filling, this cost saving may be achieved. This study investigated the targeted dosing of

powder formulations of Immunoglobulin G (IgG), Lactate Dehydrogenase (LDH) and Beta

Galatosidase (Bgal) for compatibility with Accofil and Quantos powder dispensing systems. Mannitol

and sucrose were studied as carrier agents for the freeze dried proteins. Lyophilisation cycles

consisted of -37 primary drying (36hr), -20 secondary drying (9hr) with a constant -75

condenser and vacuum of 200mtorr (27 Pascal). Powder characterisation was performed with

compressibility index, Karl Fischer, helium pycnometry, analytical sieving and X-ray powder

diffraction.

Sucrose based formulations displayed higher levels of dosing errors with significance in lyophilised

sucrose from 1% w/v solution (p<0.05). A 10 fold improvement in RSD of dosed mannitol-IgG was

evident post milling. Select formulation batches from both dosing systems produced pharmacopeia

acceptance values of 0.16 and 0.43. A correlation between very poor flowability (p<0.05), increasing

solute concentration, dosing time and accuracy exists.

This study shows dosing lyophilised powders from bulk could be time efficient, economical and

meets regulatory requirements specified.

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5.2 Poster Award