Formulation of biologics

Question 1

What are the most common forms of biopharmaceuticals

Answer 1

Therapeutic monoclonal antibodies(mAbs)
Insulin
Monoclonal antibodies 
Vaccines 
Peptides used in hospitals such as teicoplanin and vancomycin

Question 2

What are biopharmaceuticals

Answer 2

Medicines comprised of:

monoclonal antibodies (often based on the subtype IgG1),

antibody drug conjugatesor ADC (a toxic small molecule is attached to the antibody which specifically binds cells),

interleukins,

peptides and

virus like particles (e.g. AZ covid vaccine)

Question 3

How do we derive molecules to be used in biologics

Answer 3

Unless were using peptides which can be synthesised, mAbs, ADC, virus like particles are produced usingbiological processes (bioprocess).

Question 4

Which host cells do we use to produce biologics

Answer 4

The current host cells for the production are E. Coli, yeastsor Chinese Hamster Ovary (CHO) and Human Ambryonic Kindney (HEK) cells. As the process are complex, long and repetitive this is now very often done by robots

Question 5

Which 3 ways can we utilise monoclonal antibodies in the treatment of disease and which sort of things can they treat in each instance

Answer 5

1. Protein therapeutics which have an enzymatic or regulatory activity: In this category will be found proteins that replace another protein which is deficient or abnormal, augment an existing cellular pathway (upregulates) or provide a new function or activity to the target cell.
2. Proteins with special targeting as they can interfere with a molecular pathway or the organisms’ physiology, or, deliver other compounds or protein (case of ADCs).
3. Protein vaccines which can be used to protect against a deleterious foreign agent, treat an immune disease or cancer.

Question 6

What is unusual about mab molecules

Answer 6

mAbs are massive molecules compared to small drug molecules

Question 7

How are mAbs named

Answer 7

Each mAb name has four parts.

The first one is a unique prefix chosen by the company, the second prefix letters are related to the type of target (e. g. tumour colon, mammary or non-tumour cardiovascular neurologic), the 3rdpart is a prefix that reflects the source of the variable chain (mAbs have evolved over the years we’ll see this next year and are becoming more and more human or humanised) and the final part is simply mAb.

Question 8

Heparins correct name is Trastuzumab. What does each part of the name refer to

Answer 8

Tras is the unique prefix given by the company, tu indicates it was meant to target many different tumours, zu indicates it is a humanized antibody

Question 9

What factors make a biologic unique

Answer 9

The IgG isotype, their target, how these are delivered i.e. IV, SC, the concentration of the mAb formulation, the buffer and its pH and the main excipients of the formulation

Question 10

How do we formulate MAbs and proteins

Answer 10

Biologics are either formulated as a liquid formulation (often at concentrations higher than 10mg/mL) or lyophilised (a solid to be reconstituted before use, a lower concentration between 1 and 10 mg/mL).
Administration has been mainly subcutaneous or intra-vascular but it can also be intra muscular.

Question 11

What issues do we face when using mAbs

Answer 11

mAbs are mainly used for long term conditions. Although they require less frequent doses than small drug molecules (their half-life is on average 3 week). The necessity of injecting yourself or being in hospital may lead to compliance issues.
mAbs and other biologics have the potential to elicit immune responses

Question 12

What are the benefits and drawbacks of formulating mAbs as solid (lypholised)/liquid forms

Answer 12

For the solid or lyophilised form: Advantages - dose and injection volume is adjustable (can be adapted to the patient)

Disadvantages - can be more expensive to couple a solid form to a delivery device such as the dual chamber.

For the liquid form: Advantage - more convenient to the end user (e.g. no preparation, no shaking) potentially better compliance. The dose is also more accurate.

Disadvantage - storage in aqueous media increases the probability of chemical degradation by hydrolysis.

Less stable than the lyophilised form over time which results in a shorter shelf life.

Their physical stability is more difficult to control - shaking and air/water interface are likely to lead to unfolding of the mAb and ultimately its aggregation.

Question 13

Why do we use buffers in the liquid formulation of mAbs and what are some examples of the ones used

Answer 13

mAbs need to have a pH that guarantees their solubility (ionisation) and stability. Thus a buffer will be used in the formulation. Common buffer used in formulations are acetate, citrate, histidine or phosphate; the reason is that their pKa is close to the pH of interest.

Question 14

What pH range of buffer do we use to ensure good solution stability of mAbs

Answer 14

As mAb (IgGs) have an isoelectric point of about 8, having solutions at pH between 5.5 and 6.5-7 guarantees a good solubility. Importantly the buffer concentration is kept low to adapt to physiological pH upon administration to the patient

Question 15

Why do we include salts in the formulation of mAbs

Answer 15

As with any injectable, these product need to be isotonic or isosmotic, and, salts are needed to control the stability/conformation of the protein.

Question 16

What is the difference in tonicity between iv and im injections

Answer 16

IV injections require isotonic preparations, whilst IM or SC formulations may be able to handle hypertonic or hypotonic conditions (as they are not directly administered to the blood).

Question 17

Why dont wetend to use NaCl in the formulation of mAbs

Answer 17

NaCl may contribute to the corrosion of steel during bioprocessing.
Additionally the amount of NaCl may affect the viscosity of the solution a

Question 18

Why do we include surfactants (surface active agents) such as polysorbate 80 or 20 in the formulation of mAbs

Answer 18

mAbs can unfold –by doing so they expose their hydrophobic core. Then there are two possibilities: either they refold and it is fine or they form aggregates with other unfolded mAbs in order to minimise the contact of the hydrophobic core with water.

These aggregates are not acceptable as they reduce the therapeutic index and can result in an immunogenic response in the patient.

Now most unfolding happens at interfaces especially the air/water interface.

To reduce unfolding, surfactants are used as these will migrate to the interface and occupy the interface more efficiently than the mAbs.

Question 19

How do we ensure all interfaces of the antibody are covered by the surfactants

Answer 19

To guarantee that all interfaces are covered by the surfactants, the concentration of surfactant used in the formulation is above the cmc of the surfactant.

Question 20

What is a drawback of using surfactants in the formulation of mAbs

Answer 20

Polysorbates may oxidise if care is not taken. When this happen, the free radical will lead to aggregation of the mAbs.

Question 21

Why do we include antioxidants in the formulation of mAbs

Answer 21

Antioxidants such as EDTA are also often added to formulations in order to limit the chemical degradation of mAbs via oxidation mechanisms fuelled by the presence of metal ions in solution.

Metal ions may have different origins such as metallic parts in the different steps of bioprocessing or the container in theformulation steps.

Reducing agents such as glutathione which can reverse oxidation are also used in formulations

Question 22

Why do we include protein stabilisers in the liquid formulation of mAbs

Answer 22

They limit the unfolding of the proteins. The way they stabilise protein is by preferential exclusion (based on the concept that some compounds, e.g. Arginine, sucrose can remain out of the sphere of hydration of the protein and be able to stabilise the protein by attracting water molecules and thus resulting in a more compact protein)

Question 23

Unstable proteins are also an issue in lypholised proteins, How do we address this

Answer 23

Excipients are used as cryoprotectants i.e. protect the molecule during freezing.

Typical examples of cryoprotectantsare PEG, sucrose or trehalose. These protect as water crystals may induce aggregation. They do work in a similar manner as the previously mentioned excipients i.e. preferential exclusion.

Question 24

What are the issues with using sugars as cryoprotectants

Answer 24

Disaccharides are susceptible to hydrolysis at low pH. Indeed hydrolysis of sucrose results in the formation of glucose and fructose at pH5 which ultimately leads to the degradation of IgGs

Question 25

What are the most common routes of chemical degradation

Answer 25

The most common routes for chemical degradation are oxidation, de-amidation and aspartic acid (Asp) isomerisation and cross-linking (and be aware that chemical degradation may lead to physical degradation; however physical degradation does need chemical degradation to occur)

Question 26

Which regions are present in an iGe

Answer 26

IgG has an Fc region (CH2 and CH3 regions), Fab region, the heavy chains and the light chains, and, importantly the Complementary Determining Region or CDR which is where most of amino acid (AA) sequences are modified as this region binds to the antigen.

Question 27

What happens to mAbs binding ability during modification and why does this occur

Answer 27

When modifying, altering the amino acid residue sequence, the conformation or binding ability of the mAb may change. This may be due to the fact that the novel AA may have a different polarity or charge thus will change the repulsion or attractive forces of the molecule; it may be also the result of the bio synthesis process.

Question 28

What is de-amination

Answer 28

De-amidation is a common chemical degradation route and is dependent on the AA

Question 29

Which are the most suceptible amino acids for chemical degradation via oxidation

Answer 29

Methionine, Tyrosine, Histidine,Tryptophan and Cysteine

Question 30

How are each of the following amino acids oxidised:

Methionine, Histidine,Tryptophan and Cysteine

Answer 30

Methionine oxidation is frequent in mAbs (Herceptin again when exposed to light at 27°C and above).

Histidine oxidation often happens via metal catalysed reactions (which can happen during the bioprocess or in the final formulation (release of metal ions from the container).

Tryptophan oxidation occursalso via metal catalysed reactions (e.g. Trp in CDR of palivisumab when exposed to UV light) however this is rare in mAbs.

Cysteine has been shown in several mAbs to result in the cross-linking of intermolecular disulphide bridges.

Question 31

What does the term physical degradation refer to

Answer 31

Physical degradation refers to the conformation changes, aggregation or surface adsorption due to physical stresses such as temperature, shaking, pressure.

Question 32

What do conformational changes in proteins lead to and via what means do they occur

Answer 32

Conformation changes in proteins lead to denaturation or unfolding.
Sources of conformational changes are temperature changes, ice formation due to freeze thaw, shear forces, changes in ions in solution, changes in protein-protein interactions.

Question 33

What causes aggregation of proteins

Answer 33

Aggregation may originate from conformational changes induced by covalent changes (chemical changes) but very often these are related to hydrophobic/hydrophilic issues (change of conformation due to stresses such as temperature, shaking, pressure).

Question 34

Why is aggregation undesirable

Answer 34

Aggregates are undesirable as they alter the pharmacokinetics, reduce the activity of the mAb and can lead to severe immune responses

Question 35

There are two categories of aggregates: reversible and non-reversible aggregates. What characterises each of these

Answer 35

Reversible aggregates resulting from the self-association of the proteins may be the consequences of the formulation or the delivery. This means that by altering the conditions e.g. pH, proteins may revert to the monomeric form from the aggregation form.Irreversible aggregates, however, cannot be reverted to the monomeric form.

Question 36

Define soluble aggregates

Answer 36

Soluble” aggregates are aggregates not visible in solution and these cannot be removed easily by filtration. As they are not visible in solution -they are below the size an eye can detect-, they are called subvisible aggregates and have a size between 100 nm and 10 m. These aggregates are thought to be immunogenic but this has not been established unequivocally.

Question 37

Which IG subtype is more prone to aggregation

Answer 37

Among thefour IgG subtypes, IgG2is more prone to aggregation than the 3 others (IgG1, IgG3andIgG4); this explains this subtype is less used for therapeutic proteins.

Question 38

How do companies determine whether their product will aggregate

Answer 38

Companies use accelerated stability testing to determine whether their candidate is prone to aggregation. This is a different concept than the one used for small molecules: protein candidates can be shaken, frozen and thawn or heated to determine the presence of aggregates. However,this is only indicative and compared against other previouscandidate resultsfor long term evaluation.

Question 39

Why is the air-water interface important in clinical practice

Answer 39

mAbs like other proteins are amphipathic macromolecules thus are likely to unfold at the air/water interface especially when the samples is shaken during transportation or mixed following reconstitution in the syringe. Thus particular attention should be given to the presence of air bubbles in the syringe.

Question 40

What are leachables

Answer 40

Leachablesare chemical compounds released from the container closure system when in stress conditions (e.g. temperature, shaking) in thesolvent. This often is related to pH temperature salt concentration of thesolution. Leachables include metals, organics, volatile compounds. As seen earlier about chemical degradation, it is obvious these may lead to degradation of the product whichis not acceptable.

Question 41

What are extractables

Answer 41

Extractables are a subset of chemical compounds eluting during normal storage or used conditions e.g. when using an infusion bag, or tubing. An obvious addition aspect to consider it the volume of the bags as having bags too large may result in large volume of air thus increased possibility of protein damage

Question 42

What is headspace and how does it relate to proteins

Answer 42

The head space is theair volume left in the syringe after filling thedesired volume of protein solution. This volume should be minimised was filling thesyringe as we have already seen that the air/water interface is believed to be the main source of unfolding of proteins and subsequently of aggregates formation.

Question 43

What are the challenges faced when administering protein solutions in infusions

Answer 43

infusion bags is that products may be reconstituted in larger volumes to lower the protein concentration. Care shouldbe taken in these cases to remember that diluting a concentration, not only the concentration of theprotein is decreased but also theexcipients concentrations. This will consequently affectthe concentration of surfactants and if this one is not sufficient to guarantee the presence of micelles (i.e. be above the cmc), proteins will be able toadsorb at interfaces, unfold and aggregate.

Question 44

WHat must we remember about the cmc

Answer 44

The cmc reflects an equilibrium and if there is not enough surfactants to cover all interfaces, micelles will disappear, thus the surfactants concentration will be below the cmc.

Question 45

How concentrated are SC injections of protein solutions

Answer 45

SC formulations are high protein concentrations (above 100 mg/mL), a small volume of solution is injected (1.5 mL or less) and proteins are slowly released in the blood compartment which in turn reduces the frequency of administrations.

Question 46

What drawbacks come with administering SC protein solutions

Answer 46

h increased concentrations, the distance between proteins decreases and protein protein interactions are more likely. This may mean more unfolding and ultimately more aggregation.

When the concentration of hydrophilic disperse systems increases, the viscosity of thesolution increases. This,in turn,maybe a problem for the patient (especially elderly patient) who would need to be very strong to inject themselvesas theinternal diameter of the needle is very small (0.2 mm) thus causes pressure build-up; this leads to the concept of syringeability.

Question 47

How viscous can a solution get before its too thick to syringe

Answer 47

Studies have shown that (related to the maximum glids force) injectables can have a maximum viscosity of 25 cP

Question 48

Which mAbs could be formulated as a SC injection

Answer 48

The viscosity is mAbs formulation varies greatly depending on the amino sequence of the mAb and the excipients used in the formulation.