Protein Pharmaceuticals (V)“Production Consideration”

Dr. Aws AlshamsanDepartment of Pharmaceutics

Office: AA87Tel: 4677363

aalshamsan@ksu.edu.sa

Objectives of this lecture

By the end of this lecture you will be able to:1. Describe the problems associated with

protein formulations2. Numerate strategies to improve protein

formulations3. Understand the difficulty of scaling up

pharmaceutical protein industry

Problems with proteins

• Large molecules– Very hard to be synthesized chemically

• Unstable:– Held by weak forces

– Easily destroyed in vitro and in vivo

• Hard to obtain in large quantities by extraction– Loss or denaturation of many proteins during the process

• Easy to contaminate– Most proteins are given parenterally

• Difficult to formulate for large scale purposes– Reproducibility is a challenge

Solving the problems

Solving the problems

Microbial consedirations

• Sterilization:– It is impossible to sterilize the end product– All equipments must be sterilized– Assembled in aseptic conditions “BSL and Cleanroom”

• Quality control:– Viral testing– Bacterial testing– Pyrogen testing

Cleanroom

• An environment of controlled level of contamination that is specified by:1. The number of particles/volume

2. Particles size

• Air entering is HEPA filtered to exclude dust, airborne microbes, and aerosol particles

• Working staff must wear personal protective equipments (PPE)

Cleanroom

Turbulent Cleanroom Laminar Flow Cleanroom

Cleanroom

ClassMaximum Particles/ft³

ISOequivalent≥0.1 µm ≥0.2 µm ≥0.3 µm ≥0.5 µm ≥5 µm

1 35 7.5 3 1 0.007 ISO 3

10 350 75 30 10 0.07 ISO 4

100 3,500 750 300 100 0.7 ISO 5

1,000 35,000 7,500 3000 1,000 7 ISO 6

10,000 350,000 75,000 30,000 10,000 70 ISO 7

100,000 3.5×106 750,000 300,000 100,000 700 ISO 8

US FED STD 209E cleanroom standards

In November 2001, US FED STD 209E was cancelled

Cleanroom

Class

Maximum Particles/m³FED STD

209Eequivalent≥0.1 µm ≥0.2 µm ≥0.3 µm ≥0.5 µm ≥1 µm ≥5 µm

ISO 1 10 2.37 1.02 0.35 0.083 0.0029

ISO 2 100 23.7 10.2 3.5 0.83 0.029

ISO 3 1,000 237 102 35 8.3 0.29 Class 1

ISO 4 10,000 2,370 1,020 352 83 2.9 Class 10

ISO 5 100,000 23,700 10,200 3,520 832 29 Class 100

ISO 6 1.0×106 237,000 102,000 35,200 8,320 293 Class 1,000

ISO 7 1.0×107 2.37×106 1,020,000

352,000 83,200 2,930Class

10,000

ISO 8 1.0×108 2.37×107 1.02×107 3,520,000

832,000 29,300Class

100,000

ISO 9 1.0×109 2.37×108 1.02×108 35,200,0008,320,00

0293,000 Room air

ISO 14644-1 cleanroom standards

Viral Decontamination

• There is no well-determined mean to detect viruses in the cell culture

• Each lab has a level of biocontaminents AKA Biosafety Level (BSL):

1. BSL1:Well-characterized agents not know to cause disease to a healthy adult human being

2. BSL2: BSL1+ agents of moderate potential hazard to personnel and environment (e.g. HBV and Salmonella)

3. BSL3: Agents which may cause serious or potentially lethal disease after inhalation but to which treatment is available (e.g. TB, Anthrax, and SARS)

4. BSL4: High individual risk of aerosol-transmitted lab infection that cause severe or fatal diseases to which no treatment or vaccine is available (e.g. Ebola and Marburg)

Viral Decontamination

• Viral contamination can be from the host cell line or nutrients present in the growth media (e.g. FCS)

Category Type Example

Inactivation

Heat Pasteurization

Radiation UV-light

Dehydration Lyophilization

Cross linking Formaldehyde

Neutralization Antibodies

Removal

Chromatography Affinity chromatography

Filtration Ultrafiltration

Precipitation Cryoprecipitation

but these processes may be harmful to the product

Bacterial Decontamination

• Filtration sterilization of the final product by bacterial filter “0.22 m membrane filter”

• Antibiotics must be added to the cell culture to inhibit bacterial contamination– What if the expression system is bacterial?

• Complete removal of antibiotic residues from the final product is very difficult

Pyrogens

• The process of pyrogen removal AKA depyrogenation refers to the removal of pyrogens such a “endotoxins” from solutions.

Property Exotoxin Endotoxin

Chemistry Secreted proteins Shed lipopolysaccharide

Source Gram (+ve) or Gram (-ve) bacteria Gram (-ve) bacteria

Symptoms Specific action on target tissue Fever, diarrhea, vomiting, shock

Toxicity High / Fatal Weak / Rarely fatal

Immunogenicity Causes neutralizing Ab production Insufficient Ab production

Toxoid potential After formaldehyde treatment None

Fever potential Rarely Pyrogenic

Pyrogens• Lipopolysaccharide is a component of Gr (-ve)

bacteria cell wall

Pyrogenic part

Pyrogen Testing

• Rabbit Test:– Rabbits have similar endotoxin tolerance to humans

– Costly method and time consuming

– Inability to quantify the endotoxin level

• LAL Test:– Limulus Amebocyte Lysate (LAL) test

– FDA-approved for in vitro pyrogen testing

– High sensitivity 0.005 EU/mL

– Only detects LPS

– Gives false positives with Glucans

Pyrogen Removal

• Simple filtration sterilization and standard autoclaving conditions do not remove pyrogens

• Dry heat for 30 min at 250 oC would breakdown the endotoxin

• All equipments used in the production process must be endotoxin free

• The FDA’s maximum permissible endotoxin limit is 5 EU/kg/hr

• Intrathecal endotoxin limit is 0.2 EU/kg/hr

• Sterile water for injection is allowed to contain 0.25-0.5 EU/mL

Pyrogen Removal

• Ion Exchange Chromatography:– LPS is highly negative– Anion exchanger

• Ultrafiltration and Reverse Osmosis:– LPS has high molecular weight >10kDa

Ultrafiltration Reverse Osmosis

Pyrogen Inactivation

• Hydrolysis in order to cleave Lipid A from the polysaccharide component, Oxidation using hydrogen peroxide, and Heating at 250 oC for 30 minutes are commonly used methods inactivate endotoxin on solid surfaces. However, these methods would harm the therapeutic protein. Therefore, it is important to work with sterile endotoxin-free equipments under aseptic condition.

Cellular DNA

• Mammalian expression systems are immortalized cell lines by stable oncogene transfection

• Recombinant products may get contaminated with oncogen-bearing DNA fragments in the final product

• Purification process MUST remove cellular DNA and RNA

• DNA concentration in the final product should not exceed 10 pg/dose

Protein Contaminants• Source:

1. Growth media (FBS)

2. Host cells

3. Ligands from affinity chromatography columns

• Host version of the protein can be co-purified with the protein of interest

• Large-scale production prefers the use of serum-free media (e.g. in mAbs production) but this causes insufficient growth and lower yield of production

• Foreign protein contaminants can be hazardous and immunogenic. If not purified, they lead to miss-interpretation of the produced protein’s immunogenicity profile

Solving the problems

Solving the problems

Additives

1. Active ingredient

2. Solubility enhancer

3. Anti-adsorption/aggregation agent

4. Buffer components

5. Preservative/anti-oxidant

6. Lyoprotectant/cryoprotectant

7. Osmotic agents

8. Delivery systems

Solubility enhancer

• Problem:– Aggregation and precipitation especially with non-

glycosylated proteins

• Solution:– Proper pH and ionic strength– Cationic amino acids (Lys and Arg)– Surfactants (e.g. SDS)

Anti-adsorption Anti-aggregation

• Problem:– Hydrophobic sites causes adhesion and adsorption to

solid interfaces and leads to unfolding and aggregation

• Solution:– Proper pH and ionic strength– Surfactants (e.g. phospholipids and SDS)– Competitor protein (e.g. Albumin)

Buffer components

• Problem:– Protein solubility and stability depend to a great extent

on the pH of the surrounding environment. Temporary change in the pH can cause aggregation

• Solution:– Add buffer components– Citrate (pH 3-7), acetate (pH 3-7), and phosphate (pH

7-11) buffers– Choose buffer systems that do not crystallize during

freezing

Preservatives and Anti-oxidants

• Problem:– Oxidation occurs to (Met, Cys, Trp, Tyr, and His)– Contamination with microorganisms expecially in

multiple-dosing dosage forms

• Solution:– Replace oxygen in the vial with inert gas– Ascorbic acid– Preservatives at bacteriostatic concentrations (e.g. p-

hydroxybenzoic acid and thimerosal “thiomersal”)

Osmotic Agents

• Problem:– Most proteins are given parenterally. Therefore, they

must be administered as isotonic solutions. However, excipients used in this regard may influence protein structural stability

• Solution:– Sugars (e.g. sucrose) and polyhydric alcohols i.e.

sugar alcohol e.g. glycerol and PEG improves protein stability through preferential exclusion

Solving the problems

Storage1. Aqueous solutions:

• Stability of protein solutions depends on pH, ionic strength, temperature, and stabilizers

• Smooth walled glass

• Air-tight container

• Dark

2. Freeze-dried form (Lyophilized)

3. Dried form in compact state (pills)

Freeze Drying

• Presence of water in the protein solution promotes chemical and physical degradation, which reduces the expected shelf life

• Freeze drying removes water through sublimation and not evaporation

Freeze Drying

Freeze Drying

Freeze Drying Steps

• The freeze drying process consists of three steps:1. Freezing:

Crystallization of water molecules (bound and unbound to protein/excipients)

2. Primary drying:Removal of unbound water molecules by sublimation

3. Secondary dryingRemoval of protein/excipient bound water by sublimation

Freeze Drying Steps

In absence of proper excipients, irreversible damage to the protein

Lyoprotectant/Cryoprotectant

• PEG:– Coats the protein– Not a very good stabilizer

• Sucrose:– Freezes the water molecules around

the protein (preferential exclusion)– Also preservative above 60%

Lyoprotectants prevent over drying of proteins during freeze drying

You are now able to: Describe the problems associated with

protein formulations Numerate strategies to improve protein

formulations Understand the difficulty of scaling up

pharmaceutical protein industry

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