Therapeutic proteins

What are the downsides to therapeutic proteins

Unstable in solution

High molecular weight

Microbial contamination

Low production yield

Sterility in cell culture and purification

Prone to enzymatic degradation eg proteolysis

Expensive

High viscosity and low solubility in high concentrations

Can end up forming aggregates that can cause loss of activity and end up unfolded and adsorbed

Need care during storage and shipping - cold chain

Complex purification steps

List strength, route of delivery, storage temp shelf life and dosing frequency

Strength micrograms to 100mg

Route of delivery IM IV SC

Storage temp - 2-8 degrees of plasma derived proteins

Shelf life - 12-36 months

Dosing frequency - weekly quarterly monthly

What can increase susceptibility to oxidation

Metals and peroxides in delivery devices and excipients can catalyse oxidation

Amino acids like cys tyr trp met are prone to oxidation

Proteins can be oxidised can make them inactive and unfolding can make them susceptible to proteolytic degradation

What can oxidation affect

Can affect stability, safety, protein degradation, structure activity , purities

What causes aggregation

Protein plus air water interphase causes aggregation

When can protein aggregation happen

Can happen in storage, manufacture and transport

When can protein aggregation arise

In high temperatures , ph mechanical stress

Name some types of aggregates

Soluble/unsoluble, covalent/non covalent , reversible/irreversible native/denatured

What effect can aggregates have

Can cause harmful side effects , can activate the immune system

What should you assess for proteins in production (bug) phase to the clinic

Look at pyrogen removal, sterility, purity , viral contamination

What should the final dosage form look like

Solids which are freeze dried

Solutions

What excipients can be used for proteins

Adsorption agents

Anti aggregation

Solubility enhancers

Osmotic agents

Preservatives

Buffers

In the body how are proteins eliminated

By T and b cells

Can be eliminated by proteolysis - endopeptidases and exopeotidases

Small proteins eliminated by kidney less than 30kda

Side effects of proteins

Loss of adsorption

Unwanted allergic reactions

And in storage - can be easily destroyed by mild storage conditions can be unstable, and they're held by non covalent forces

How can proteins fold

Can fold as globular structures

What kind of interactions can occur in secondary and tertiary structures

Non covalent structures such as hydrophobic , hydration, electrostatic, van der waals

How are water molecules bound in proteins

Can be bound internally and externally

What do water molecules bound internally to proteins what are they bound to

Bound to hydrogen bond donors and acceptors and binding whether loose or tight can affect protein structure and function

Compare dried enzymes and cooling a protein

Dried enzymes - add water then becomes active

Cool under 40 degrees - some water doesn't freeze since tightly bound

Describe interactions that can cause protein aggregation

Protein and container can interact and cause aggregation and precipitation

Chaperones - can prevent misfolding and aggregation

Denaturation - unfolding causes loss of function of protein

Adsorption - can adsorb to container cause aggregation or precipation

List some degradation processes that can lead to change in primary structure

Degradation can cause change in stereochemistry - degradation

Oxidation - methionine and cysteine - can be oxidised by reactive oxygen species, normally hydrophobic then get oxidised and becomes hydrophilic and moves on to the surface causing degradation

Disulphide exchange - exchanged with other disulfide causes change in protein function and stucture

Proteolysis - degradation by enzymes - can change protein structure and function

Deamidation- cleave amide bond changes structure and function

What locations does chymotrypsin cleave at

Phe,tyr and trp

What locations do tyrosine cleave at

Lysine and arginine

How can you change equipment to stabilise proteins

Have smooth glass walls - reduce adsorption and precipitation

Avoid polystyrene and silanol or plasters

Dark opaque walls prevent oxidation

Air tight container and argon atmosphere to reduce air oxidation

What factors can you do to improve protein stability

Refrigeration - use low temperature to prevent denaturation and microbial growth and metabolism

Freeze - to store long term

Degradation due to presence of water can have physical and chemical degradation even if it's refrigerated

Freeze drying - can improve stability

First freeze product

Then primary drying - remove ice under vacuum

Then secondary drying - remove bound water

Lyprotectants and cryoprotectants - what does PEG do

PEG - coats protein

But not good stabiliser

What is sucrose used for

Preservative above 60%

Freezes water molecules around protein

What can the presence of water promote

Can promote chemical and physical degradation

What can be used to prevent degradation in presence of water

Can do freeze drying to improve stability - done by sublimation not evaporation

What excipients can be used for proteins

Cryoprotectants - for reducing sugars

Polyols - to improve solubility eg PEGV

Surfactants - to reduce adsorption and aggregation

Solubility enhances like polyols amino acids, surfactants - especially for non glycosylated proteins

Anti aggregation agents - reduce hydrophobic interactions at interphase eg albumin surfactants phospholipids

Buffers - to maintain ph - eg use phospholipid citrate of acetate

Preservatives MET TRY HIS TRP - prone to oxidation use ascorbic acid - use preservatives for multiple injections phenol paraben