Nejadnik Material - Lectures 19, 20, 21

What does the current status of biologics look like?

A steady increase/takeover in recent years

Most are for cancer, derm, hematology, neurology, I.D, rheumatology, pulmonology

What are some examples of biologic drugs?

Proteins, Vaccines, Oligonucleotides, Polysaccharides

What is included in the protein category of biologics?

monoclonal antibodies, mAbs

What is a mAb?

A type of antibody prepared via cloning cells to produce antibodies for specific antigens

Whats the size of mAbs/large proteins?

150-300 kDa/ 10 nm

Whats the size and structure of biologic drugs?

Very large complex molecules/mixtures of molecules

Difficult to characterize a complex biologic, so some components of a finished biologic may be unknown

How do biologics compare to small molecule drugs?

When compared biologics have:

High target affinity

Exquisite specificity

SUperior safety

Longer half lives

What can changes in a biologics structure do?

Can result in:

Efficacy, stability, safety, elegance, and manufacturability issues

Secondary non-responders?

Are individuals who responded to the initial treatment effectively, but then the treatment goes on to fail for them

Caused my immunogenicity, antidrug antibodies formed

Stirring?

Puts much more mechanical stress on the protein formulation than shaking

Basically degrads the drug, pushes proteins to the surface and the surface forces act on them denaturing them

CTSDs?

Closed system drug transfer devices

Nothing goes in nothing goes out

Pneumatic Tube transport

Compressed air tubes, bank teller tubes

Stress yet to be analyzed, but stress may be at maximum when the contain of a IV bag/syringe hits the receptacle in the tube system

What are the characteristics of biologics size?

Large size, with critical tertiary (sometimes quaternary) structure

What are the characteristics of small molecule drugs size?

Pretty small, often without any higher order structure

What are the characteristics of biologics structure?

Exact structure may not be known, and a small change (difference in amino acids) may not lead to a new product

What are the characteristics of small molecule drug structure?

Well defined, and a small modification can lead to a new molecule with a different therapeutic or toxicological outcome

Where are biologics produced?

Recombinantly in living cells, like E. coli or chinses hamster ovary cells, with complex purification and characterization 

Where are small molecule drugs produced?

Normally synthesized using organic chemistry methods, relatively straight forward characterization

How are biologics delivered?

Delivered parenterally by injection or infusion, usually not stable at room temperature except for dired dosages

How are small molecule drugs delivered?

Orally with a tablet or capsule, usually stable at room temperature

How are biologics regulated?

At all steps from initial fermentation via purification and dosage form production to delivery to patients 

How are small molecule drugs regulated?

From the bulk drug substance, characterization at this stage is usually all that is needed for generic

What are generics like for biologics?

Biosimilars, and they are just starting to get approved, much more extensive and lengthy regulatory process

What are generics like for small molecule drugs?

Tons of generics available, much more simple and rapid regulatory process

How does a single target mAb work?

Blocks one things, typically can interaction with a ligand

Like receptors on a cell, target protein on a virus/bacteria, or proteins in plasma

How does a antibody drug conjugate (ADC) mAb work?

Attaches a small molecule anticancer drug/other therapeutic agent to an antibody

How do multispecific mAbs work?

Blocks/other actions

Can bridge immune cells/target cells

Simultaneous inhibition of receptors/cytokines

Target multiple immune checkpoints increasing immune response 

What are nanobodies?

A new modality

Produced from fragments of camelid heavy-chain only antibodies

Have enhanced multi-targeting capabilities and stability

What happens during manufacturing?

Producing the drug substance

• Host cell development, producing protein, purifying, freezing

Producing drug product

• compounding, filtering, filling, inspect

What are the challenges with biologic administration?

Non-invasive routes are basically impossible

Only really formatted as injectables 

Safety of mAbs?

Generally safe

Have high specificites, precise action, long half lives (infrequent dosing)

Potential adverse effects of mAbs?

Target related toxicity and modality related toxcitiy

Target related toxicity

Mechanism based

Immunosuppression and mAb interaction with target antigen in other tissues

Modality related toxicity

Component of the immune system

Acute response at time of injection and development of antidrug antibodies 

What are non-specific interactions?

Formed via different types of atoms/molecules/molecular groups/surfaces described in generic terms

What are specific interactions?

Unique combinations of physical forces/bonds between two macromolecules acting together in space to give a strong/non-covalent

What are some other ways to refer to specific interactions?

Complementary, lock and key, ligand-receptor, and recognition interactions

How are physical properties influenced by intermolecular forces?

M.P/B.P increase as forces become stronger

Capillary action, H bonding between tube and water allows water to move up tube

Viscosity, as theres more interactions there is less flow 

What is surface potential?

The electrical potential on the surface of a molecule

So if a surface has negative charges it will attract positive ions

What is the stern layer?

A tightly bound layer of solvent and counter ions on the solid surface of a molecule

The counter ions will partially neutralize the surface’s charge

What does the stern layer mean?

The layer will partially block of the electrical field, and will not extend as strongly into the solution anymore

As you move farther from the particle surface the electric potential decreases

What is the shear plane/slipping plane?

A layer beyond the stern layer 

What is zeta potential?

The difference between the shear plane and the electrically neutral bulk medium

What does zeta potential decide?

The electrical repulsion between particles

What does increasing counter ions (salts/electrolytes) do?

The extra ions gather around the particle easier, and they neutralize the surface/shield and make the zeta potential smaller

What is DLVO theory?

Explains how Van der Waals and double layer forces act together in a liquid

What do van der waal forces do?

They are attractive forces, so the closer the particles the stronger the V.D. W

What do double layer repulsion forces do?

Act as repulsion forces

What is double layer repulsion?

Each particle has its own electrical double layer, (made up of the stern and slip plane), when another particle approaches their D.L.F overlap causing them to repel each other

What does it mean if zeta potential is large?

The repulsion is strong and the particles will stay apart

What does it mean if zeta potential is small?

Repulsion weakens, and van der waals forces dominate and the particles clump together

What does debye length measure?

How far the electrostatic effect persists

Large debye length means?

Farther electrostatic effect, so more repulsion

Primary

Sequence of amino acids connected via amine peptide bonds 

Secondary

Organizational structure introduced via specific hydrogen bonding interactions of different amino acids

Tertiary

Higher organization of protein folding that results from interaction with aqueous environment

Quaternary

Multi-protein organization into an organized complex

What can happen when changing a single amino acid in a protein chain?

Can change the:

Higher order structure

Post translational modifications

Solubility 

Chemical stability 

Function 

What can alterations of a sequence do?

Can make reactions faster or slower

Flasp

An insulin aspart injection

A rapid acting human insulin analog, the aspart is basically the same as regular human insulin, with the exception of a single substitution of the amino acid proline by aspartic acid

Murine antibodies

Produced entirely from mouse protein

Recognized as allogeneic proteins, leading to polyclonal human anti-mouse antibody (HAMA reactions)

HAMA had a neutralizing action leading to rapid murine antibody inactivation/affected their PK

Chimeric antibodies

Fc portion of the antibody, (part that dictates the function of the antibody) was chemically exchanged with human constant portion

First to market, Human + animal

Humanized antibodies 

90% human protein and 10% mouse protein

Fully Human antibodies

100% human mAbs 

Nomenclature of mAbs

Consists of a prefix, two substems, and a suffix

What does the prefix mean in mAb names?

Random to give a unique drug name

What are the substems used for in mAb names?

To designate the target and the source

What is the substem name for murine?

o

What is the substem name for chimeric?

xi

What is the substem name for humanized?

zu

What is the substem name for fully human?

nu

What is the suffix for mAbs?

For all its mAbs

Biosimilar mAbs are named as the reference drug followed by a four letter suffix made up of four random lower case letters separated from the reference name by a hyphen

What is a polyclonal antibody?

A collection of IgG molecules that bind to various epitopes on a target antigen

What is a monoclonal antibody?

A single IgG that binds to a specific epitope on a target antigen

Comes from a single B cell parent clone

What does fusing of proteins do?

Increase half-life of the antibody:

Increases size, slowing kidney filtration

Interaction with neonatal Fc receptor, helping recycle the antibody

What is Fc fusion?

When a Fc region of an antibody is fused to a biologically active protein/peptide

What is the point of Fc fusion?

It allows them making a single protein with the desired properties and eliminates the need for post production conjugation (PEGylation attaching chains to improve stability)

What is Alprolix?

A Fc Fusion protein, is a recombinant DNA derived coagulation factor IX concentrate for adults/children with hemophilia B

What can chemical instability result in?

Deamidation, oxidation, asp isomerization, disulfide shuffling, racemization, etc.

What can physical instability result in?

Conformation unfolding/misfolding, colloidal aggregation/precipitation, adsorption

What can occurs with multiple degradation processes?

Multiple processes can occur at different rates, creating multiple degradation products

What can chemical instability result in physically?

Add a ionizable proton (added charge), affecting H bonding and resulting in conformational changes

What can chemical instability result in biologically?

May/may not have impact on the activity of a protein, can increase immunogenicity

What two amino acids are most likely to deamidate?

asparagine and glutamine, with asn deamidating much faster than gln

What can results from either Asn or Gln deamidating?

An exposed carboxylic acid on the side chain, affecting hydrogen bonding

What influences rate of deamidation?

Greatly influenced by pH

What influences oxidation in amino acids?

Presence of oxidants, H2O2, metal ions, and light

How can the oxidant H2O2 get into a drug product?

Can be in the system due to surface sterilization, or as an impurity in a product

How can the oxidant metal ions get into a drug product?

From buffers, formulation components, or leachates from containers

What storage factors can greatly denature a product?

UV light and freezing

What happens on the company side during manufacturing?

Good control in both storage and transportation

What happens after a drug product leaves a company?

Less control over factors that affect product quality after the drug product is released/shipped 

What was noticed from patients storage their medications?

They do not store their biologics within the recommended temperature range

Measurable impact 50%

Biologics are what?

Big and fragile, more sensitive to pH than small molecule drugs

Why is there few biosimilars?

Harder to characterize and replicate 

What is denaturation and what can cause it?

Heat and pH changes

What is aggregation and what can cause it?

Unfolded proteins stick together, loss of activity or immunogenicity

What is adsorption?

Proteins stick to container walls, primarily IV bags and vials 

What does oxidation cause?

Loss of potency

What is an example of deamidation?

Asn → Asp / isoAsp conversion, alters structure and charge