Protein and Insulin

Question 1

How does denaturation of a protein occur?

Answer 1

Can be caused by thermal stress, extremes of pH, or denaturing chemicals

Question 2

When is denaturation likely to be greater?

Answer 2

Denaturation is likely to be greater at the air–water interface as compared to a solid–water interface because of the mobility

• For example, shaking allows a continuous creation of a new interface, thus providing a massive surface area that can lead to large-scale denaturation

Question 3

Why does the aggregation of a protein occur?

Answer 3

Aggregation could result because a protein adsorbs and then unfolds at the air–water interfaces generated by shaking or shear, thereby exposing the hydrophobic amino acids, which are normally located in the interior

• The exposed hydrophobic amino acid side chains of one molecule interact with those of another to form aggregates

Question 4

What are the considerations for a dry powder or solution (protein formulation considerations)?

Answer 4

Dry powder

Cryoprotection and lyoprotection

Solution

Solubility

> Preservation

> Some excipients may also be added to the reconstituting solution rather than to the formulation directly

Question 5

What are the formulation excipients for protein?

Answer 5

• Bulking agents
• Tonicity modifiers
• Cryoprotectants & lyoprotectants
• Buffer system
• Preservative
• ‘Albumin, Amino acids
• Carbohydrates, polyhydric alcohol, propylene glycol, cyclodextrins
• Chelating agents
• Surfactants

Question 6

What is the role of bulking agents in protein products? What is an example?

Answer 6

protein drugs are very potent, very small quantities are required in the product. Bulking agents, therefore, allow pharmaceutical processing and the production of a presentable lyophilised product –> make the product look full

• Mannitol is a commonly used bulking agent. Other possible bulking agents include glycine or hydroxyethyl starch

Question 7

What happens if non-isotonic solutions given larger than 100mL are given? What is used to adjust tonicity?

Answer 7

Nonisotonic solutions, particularly if given in quantities larger than 100 mL, can cause hemolysis or crenation of RBC.

• Mannitol, in addition to use as a bulking agent, can adjust tonicity if present in the right concentration
• Dextrose or sodium chloride can also be used to control tonicity

Question 8

What are cryoprotectants and lyoprotectants? Give an example of each.

Answer 8

some excipients may stabilise the protein during freezing, and others may stabilise the protein during drying

• excipients that help against freezing are called cryoprotectants –> polyethylene glycol
• excipients that help against drying are called lyoprotectants –> sucrose or trehalose

> sucrose or trehalose also cryoprotectant

Question 9

What effects does buffer have on a protein formulation>

Answer 9

Besides pH control, the buffer system used can also affect the solubility of the protein.

• Generally inorganic buffers are more commonly used

Question 10

What is susceptible to microbial contamination and growth? What is needed?

Answer 10

Peptide and protein drugs

• Therefore, preservatives can be an important formulation component
• This is especially true for multiple-dose vials

Question 11

The reconstituted solution of lyophilised powders is required to be stable for about 2 weeks. When can the preservatives be added?

Answer 11

The preservative can be added to the powder formulation, or it can be a part of the solution used for reconstitution

Question 12

What are examples of preservatives used in protein formulations?

Answer 12

Eg benzyl alcohol, methylparaben, phenolic antimicrobial preservatives

• The choice of an appropriate preservative is critical because several preservatives may cause precipitation or turbidity in the reconstituted solutions

Question 13

Why is albumin used in protein formulations? How does it work?

Answer 13

HSA (human serum albumin) is often used to stabilise and to prevent adsorption of therapeutic peptides and proteins to various surface

• Albumin prevents surface adsorption of proteins by preferentially adsorbing to surfaces. Indirectly, this may stabilise the protein molecule because less adsorption would lead to higher recovery of the protein drug

Question 14

What role do amino acids have in protein formulations? What is the most commonly used one?

Answer 14

• Amino acids have been used to reduce surface adsorption, inhibit aggregate formation, and stabilise proteins against heat denaturation
• Amino acids may also increase protein solubility –> small neutral amino acids or those containing charged side chains are most effective

The most commonly used amino acid in marketed products is glycine

Question 15

What role do carbohydrates have in protein formulations? What are some examples?

Answer 15

Carbohydrates will usually not disrupt protein structure or function

• Improve stability in solution
• carbohydrate excipients have also been found to reduce the moisture-induced aggregation of proteins in the solid state
• Carbohydrates may also increase the solubility of proteins

Nonreducing sugars such as sucrose or trehalose should be used when possible

Question 16

What do polyhydric alcohols include? What do they do?

Answer 16

Polyhydric alcohols include molecules that are trihydric or higher, such as glycerol (glycerin), erythritol, arabitol, xylitol, sorbitol, or mannitol

• stabilise the native structure

> mannitol is often used as a bulking agent in lyophilisation

> glycerol has also been reported to suppress the aggregation of proteins

Question 17

What role do cyclodextrins have in protein formulation?

Answer 17

The mechanism by which cyclodextrins are likely to minimise protein aggregation is molecular encapsulation of the hydrophobic amino acid side chains that will undergo hydrophobic interaction

Question 18

For surfactants;

A) What is one possible mechanism of protein stabilisation?

B) What reduces protein self-association and interaction with hydrophobic surfaces?

Answer 18

A)

One possible mechanism of protein stabilisation by surfactants is the preferential adsorption of the surfactant at the interface

• Thus, the protein cannot adsorb at the interface to unfold and undergo aggregation

B)

Surfactant binding to the protein and reduce its available hydrophobic surface area

• the protein-surfactant complex is more hydrophilic than either the surfactant or the protein, thereby increasing the protein solubility and reducing its tendency to aggregate

Question 19

What do chelating and reducing agent do in a protein formulation? What are examples of these agents?

Answer 19

Some anions and cations will directly bind to the protein

• For example, divalent cations will often bind to certain proteins and lower their solubility
• Removal of these ions by a chelating agent such as EDTA may help to maintain protein solubility
• EDTA will inhibit the metal catalysed oxidation of sulfhydryl groups

Question 20

Examples of handling proteins in a hospital setting?

Answer 20

• For example, the protein may aggregate if reconstituted with vigorous shaking or may adsorb to the huge surface area of an intravenous administration set during delivery.
• Incompatibilities may develop during preparation of admixtures, or a simple step such as dilution may cause precipitation of the protein

Question 21

About half of the parenteral products are marketed as lyophilised powders, which need to be reconstituted just before use. What is the diluent used?

Answer 21

The diluent is usually WFI USP and may be provided with the dry powder as a convenience

• Some products are to be reconstituted with bacteriostatic water for injection, which contains benzyl alcohol as a preservative
• These products can generally be stored for about 14 days after reconstitution.

Question 22

What is the self-assembly behaviour of insulin?

Answer 22

• Insulin is a quaternary structure protein (i.e., it normally exists in a selfassociated form rather than as a monomer).
• Insulin exists as a monomer only at a very low concentration (<0.1 μM, ~0.6 μg/ml). At higher concentrations, insulin exists as a dimer.
• The dimers are believed to result from the hydrophobic association of the B23 to B28 regions on insulin monomers.
• In the presence of zinc ions and in the pH range 4.0 to 8.0, three dimers come together to form a hexamer.
• At concentrations of 2 mM and above, the hexamer is formed at neutral pH without the assistance of zinc ions

Question 23

What are some formulation factors affecting the self-assembly of insulin?

Answer 23

• Electrostatic repulsion between insulin monomers occurs at high pH: the monomer carries two negative charges at pH 7.5 and six negative charges at pH 10.5. As the pH is reduced, protein charge–charge repulsions are reduced, shifting the equilibrium toward oligomers.
• Increased ionic strength screens the charge repulsions, favoring the formation of oligomers
• Chelation of zinc ions by EDTA has been reported to cause hexamers to deaggregate to dimers
• Sodium glycocholate, a bile salt, may be capable of dissociating insulin oligomers to monomers

Question 24

What are the goals of diabetes treatment?

Answer 24

• To keep the blood sugar as normal as possible without serious high or low blood sugars
• To prevent tissue damage caused by too much sugar in the blood stream

Question 25

What are the examples of regular insulin?

Answer 25

• REGULAR human insulin injection [rDNA origin]) is a short-acting insulin that has a relatively short duration of activity as compared with other insulins
• Humulin R [Regular insulin human injection, USP (rDNA origin)] consists of zinc-insulin crystals dissolved in a clear fluid.

Question 26

For Hexameric Insulins;

A) What does insulin tend to form hexamers in contact with?

B) What does hexamer have to equililbrate back to, to be biollogically useful?

C) When is it not available

Answer 26

A)

Tend to form hexamers in contact with zinc in the bloodstream

B)

Back into monomers for insulin to bind to its receptors and to be biologically useful

C)

Not readily available for the body when insulin is needed in large doses, such as after a meal

Question 27

What is NPH insulin? Why type of suspension is it?

Answer 27

NPH human insulin injection [rDNA origin]) is an intermediate-acting insulin with a slower onset of action and a longer duration of activity than Humulin R.

• Humulin N [Human insulin (rDNA origin) isophane suspension] is a crystalline suspension of human insulin with protamine and zinc

Question 28

What is a mixrure of NPH nd Regular Insulin?

Answer 28

(70% human insulin isophane suspension, 30% human insulin injection [rDNA origin]) is a mixture insulin. It is an intermediate-acting insulin combined with the onset of action of Humulin R

Question 29

What is the idea behind recombinant insulin analogues?

Answer 29

• One that is faster acting and more bioavailable than natural insulin, to supply the level of insulin needed after a meal
• The second is one that needs to be less bioavailable, and released more slowly over a 24-hour period to supply the basal level of insulin for the day

Question 30

What is lispro insulin?

Answer 30

Humalog® (insulin lispro, rDNA origin) is a human insulin analog that is a rapid-acting, parenteral blood glucose-lowering agent.

• Chemically, it is Lys(B28), Pro(B29) human insulin analog, created when the amino acids at positions 28 and 29 on the Cterminal end of the B-chain are reversed
• This modification did not alter receptor binding, but blocked the formation of insulin dimers and hexamers. This allowed larger amounts of active monomeric insulin to be available for postprandial, or after meal, injections

Question 31

What is humalog formulation? What does it consist of?

Answer 31

The vials, cartridges, and Pens contain a sterile solution of Humalog for use as an injection. Humalog injection consists of zinc-insulin lispro crystals dissolved in a clear aqueous fluid

• Each milliliter of Humalog injection contains insulin lispro 100 Units, 16 mg glycerin, 1.88 mg dibasic sodium phosphate, 3.15 mg Metacresol, zinc oxide content adjusted to provide 0.0197 mg zinc ion, trace amounts of phenol, and water for injection. Insulin lispro has a pH of 7.0 to 7.8. Hydrochloric acid 10% and/or sodium hydroxide 10% may be added to adjust pH

Question 32

For Insulin Aspart;

A) How was it created?

B) Results in less of a?

C) What does it prevent the formation of?

Novo Nordisk created “aspart” and marketed it as NovoLog® as a rapid acting insulin analogue

Answer 32

A)

• it was created through recombinant DNA technology so that the amino acid, B28, which is normally proline, is substituted with an aspartic acid residue

B)

• Results in less of a tendency to self-associate for insulin aspart than for human insulin

C)

• Analogue also prevents the formation of hexamers, to create a faster acting insulin

Question 33

What does NovoLog contain? (NovoRapid)

Answer 33

NovoLog is a sterile, aqueous, clear, and colorless solution, that contains insulin aspart (B28 asp regular human insulin analog) 100 Units/mL, glycerin 16 mg/mL, phenol 1.50 mg/mL, metacresol 1.72 mg/mL, zinc 19.6 μg/mL, disodium hydrogen phosphate dihydrate 1.25 mg/mL, and sodium chloride 0.58 mg/mL. NovoLog has a pH of 7.2-7.6. Hydrochloric acid 10% and/or sodium hydroxide 10% may be added to adjust pH.

Question 34

For APIDRA (recombinant insulin analog);

A) What does the structure of insulin glulisine affect?

B) when given IV

C) When given subcut, APIDRA has?

Answer 34

A)

• This structure of insulin glulisine affects not only self-association but also the isoelectric point, which is shifted lower (pH 5.1; human insulin, pH 5.5), which enhances its solubility at a physiologic pH

B)

• APIDRA has the same glucose-lowering effect as one unit of regular human insulin when given intravenously

C)

• When given subcutaneously, APIDRA has a more rapid onset of action and a shorter duration of action than regular human insulin

Question 35

FOR Glargine insulin Lantus;

A) How is LANTUS created? (clue: modifying AA)

B) What does it result in?

C) What does LANTUS consist of?

Answer 35

A)

LANTUS is created by modifying three amino acids

• Two positively charged arginine molecules were added to the Cterminus of the B-chain, and they shift the isoelectric point from a pH of 5.4 to 6.7, making glargine more soluble at a slightly acidic pH and less soluble at a physiological pH.
• Replacing the acid-sensitive asparagine at position 21 in the Achain by glycine is needed to avoid deamination and dimerisation of the arginine residue

B)

• these three structural changes and formulation with zinc result in a prolonged action when compared with regular human insulin

C)

LANTUS® [insulin glargine injection (rDNA origin)] consists of insulin glargine dissolved in a clear aqueous fluid

Question 36

Compare Lantus Vs NPH? Which onset of action is slower?

Answer 36

When the pH 4.0 solution is injected, most of the material precipitates and is not bioavailable.

• A small amount is immediately available for use, and the remainder is sequestered in the bloodstream.
• As the glargine is used, small amounts of the precipitated material will move into solution in the bloodstream, and the basal level of insulin will be maintained over a 24 hour period.
• The onset of action of subcutaneous insulin glargine is slower than NPH human insulin

Question 37

What is Levemir? How does it differ from human insulin? What is the duration of action mediated by?

Answer 37

LEVEMIR® (insulin detemir [rDNA origin] injection) is a sterile solution of insulin detemir for use as an injection. Insulin detemir is a long-acting basal insulin analog, with up to 24 hours duration of action.

• Insulin detemir differs from human insulin in that the amino acid threonine in position B30 has been omitted, and a C14 fatty acid chain has been attached to the amino acid B29.
• The duration of action of LEVEMIR is mediated by slowed systemic absorption of insulin detemir molecules from the injection site due to the self-association of the drug molecules. In addition, the distribution of insulin detemir to peripheral target tissues is slowed because of binding to albumin

Question 38

What are factors affecting dissolution?

Answer 38

• Particle size
• Surface area
• Morphology