Exam 3 Biologics: Solutions for Injection

Question 1

What is the indication and route of administration for Rituximab Rituxan?

Answer 1

indication: lymphoma, leukemia, arthritis
route: IV

Question 2

What is the indication and route of administration for Synagis?

Answer 2

indication: RSV in children
route: IM

Question 3

What is the indication and route of administration for Lucentis?

Answer 3

indication: macular degeneration
route: intra-ocular injection

Question 4

What is the indication and route of administration for Epogen?

Answer 4

indication: anemia in renal failure
route: IV or SC

Question 5

What is the indication and route of administration for NovoLog?

Answer 5

indication: diabetes
route: SC

Question 6

What is the indication and route of administration for Xgeva (a MAb)?

Answer 6

indication: bone metastases
route: SC

Question 7

What is the indication and route of administration for Avastin?

Answer 7

indication: cancers
route: IV

Question 8

Why are solution formulations popular for biologics?

Answer 8

1. are simplest and least expensive to manufacture
2. are convenient for patients and hospital personnel since they do not require reconstitution → given parenterally so having it in solution is convenient
3. can be inspected visually prior to administration

Question 9

What are some chemical concerns regarding biologics in solution?

Answer 9

1. efficacy
2. sterility
3. side effects → such as dose limiting immune response (for recombinant proteins) in which the body may recognize it as foreign and produce ADAs (anti drug antibodies) to limit the drug
4. pain on injection (IM or SC) → have to monitor the volume administered, pH (7.4), and tonicity/ionic strength

Question 10

What are some formulation concerns regarding biologics in solution?

Answer 10

1. all clinical concerns
2. stability → aggregation, chemical stability (deamidation or oxidation), shelf-life, and storage conditions
3. manufacturability → cost and manufacturing time

Question 11

What are some formulation variables for biologics in solution?

Answer 11

1. solution properties → pH, ionic strength/tonicity, drug concentration, volume, excipients (additives)
2. container, closure → materials
3. storage conditions → refrigerated v room temp or frozen

Question 12

What is the good thing about some formulation variables for biologics in solution?

Answer 12

we can control some of the variables

Question 13

What is the importance of pH as a formulation variable?

Answer 13

plays a role in the stability of peptides and proteins

Question 14

How is salmon calcitonin an example of how pH is a formulation variable?

Answer 14

1. salmon calcitonin (sCT) is a peptide used to treat osteoporosis
2. rate of sCT degradation rate depends on pH
3. pH of maximum stability is 3-4 → not a good pH for an injection since it is not physiological pH
4. additives make the stability worse (degradation rate constant is faster) → better without the excipients
5. if we want it to be given IM or SC, we want it to be around body pH (7.2-7.4) but sCT is more stable and has a slower degration rate at a lower pH

Question 15

How is beta lactoglobulin an example of how concentration is a formulation variable?

Answer 15

1. beta lactoglobulin is a milk protein and its structure is similar to MAbs → has beta sheet structure
2. aggregate content increases with increasing protein concentration
3. since SC delivery requires small volumes, MAb concentrations are being pushed to 100 mg/mL which increases aggregation risk

Question 16

What is the relationship between concentration and aggregation?

Answer 16

higher concentration = greater aggregation

Question 17

How is aggregation measured?

Answer 17

turbidity is greater when aggregates are present so turbidity is an indication for aggregation

Question 18

What are the three different methods in which proteins can aggregate?

Answer 18

1. chemical reaction → crosslink molecules
2. colloidal interaction → molecules stick together without any change to the structure
3. unfolding → partially unfolding the protein can expose hydrophobic domains that can stick together and make aggregates

Question 19

What is the simplest definition of protein aggregation?

Answer 19

proteins aggregate when one or more of the molecules of proteins come together → these aggregates tend to elicit an immune response and these responses are dose limiting

Question 20

What makes protein aggregation complicated?

Answer 20

you can have more than one of these reactions going on at a particular time → can have a molecule that is partially unfolded and then undergo chemical reactions to crosslink (can have other examples as well)

Question 21

What is the folding funnel?

Answer 21

at the top of the funnel, it is higher energy state with lots of unstable protein conformations and more stable structures will have a lower energy state → at the top is unfolded protein and at the very bottom there is amyloid fibrils and amorphous aggregates

Question 22

What is the native state of the protein?

Answer 22

minimum amount of energy that the protein will form its native state

Question 23

What are important about amorphous aggregates and amyloid fibrils?

Answer 23

they are typically found in Alzheimer’s where it can happen in the body or even the vial → once these fibrils or aggregates form, they are hard to get rid of them since they are at a low energy state → essentially it can’t un-aggregate

Question 24

What are some important things to note about aggregation and surfaces?

Answer 24

1. aggregates are made worse by exposure of the proteins to surfaces such as the container surface or the air surface
2. container wall is more hydrophobic than the solution because almost everything is more hydrophobic than water so a partially unfolded protein that contains the hydrophobic domains will want to have contact with the container wall
3. air is also more hydrophobic than water/solution so the hydrophobic domains of the partially unfolded protein will want to have contact with the surface
4. will see partial unfolding at the air/liquid interface and at the container wall surface
5. folded native proteins have hydrophobic domains kept in the interior and hydrophilic domains that are exposed on the surface
6. the unfolded proteins won’t stay at the interfaces forever → will unfold at the interface and then come back into solution in its partially unfolded form which forms a dimer and then aggregates
7. if you shake a solution → the surface area for liquid air interface will increase and the three phase boundary is the worst place for aggregation (where solution, air, and the container wall meets)

Question 25

How are excipients a formulation variable?

Answer 25

1. excipients can be used to help stabilize the protein
2. excipients that are preferentially excluded from the protein surface promote interactions with water and stabilize native protein structure
3. excipients that bind to the protein can lead to denaturation and unfolding
4. exception: protein binding to native ligands can stabilize native structure

Question 26

What is preferential binding?

Answer 26

allows cosolvent molecules to come close to the protein → protein structure is de-stabilized by nonspecific binding to excipients

Question 27

What is preferential exclusion?

Answer 27

allows water molecules to come close to the protein to promote interactions with water → protein structure is stabilized (most proteins will be folded)

Question 28

What are the clinical effects of formulation using epoetin and PRCA as an example?

Answer 28

1. erythropoetin (EPO, Epoetin) is used to treat anemia in renal disease
2. formation of anti-EPO antibodies upon administration which binds to EPO (exogenous and endogenous) reduces drug effect and that of any naturally occurring EPO that remains
3. pure red cell aplasia (PRCA) can result and lead to sudden onset anemia and death
4. in Europe, increased incidence of PRCA has been associated with a change in container closure (stopper) → was traced to the change in the stopper but don’t know why PRCA occurred but it may be due to aggregation of EPO and greater incidence of production of anti-EPO antibodies (that resulted from aggregated form of EPO)

Question 29

Why do we want to avoid aggregated proteins?

Answer 29

they tend to be more immunogenic!

Question 30

What if solution formulation does not work (aka is not stable at room temp)?

Answer 30

1. store at refrigerated temperature
2. freeze
3. freeze dry (lyophilize) or spray dry to create a dry powder for reconstitution
4. re-engineer the protein molecule → redo the amino acids that may be causing problems
5. abandon drug candidate → look for a new drug candidate

Question 31

What are some practical considerations regarding solution formulations of biologics?

Answer 31

1. store at recommended temperature
2. protect from light if recommended
3. avoid agitation → no shaking unless it says on the label that you should!
4. examine vial for particulates prior to administration (looks clear or what it’s supposed to) → don’t want to be administering particulates for potential anaphylaxis or forming anti-drug antibodies that will limit the patient’s ability to receive the drug
5. be aware of the potential for adverse immune responses

Question 32

What is a good equivalent to not shaking biologics?

Answer 32

don’t shake a baby!

Question 33

What is an overall summary for solutions for injection?

Answer 33

1. solutions for injection are a common dosage form for biologics
2. solution properties desirable for clinical use (pH, volume) may not be optimal for stability → but can be manipulated
3. aggregation is a serious instability influenced by formulation, storage, and handling (example is agitation) → right at the hand of administration!
4. be aware of the potential for adverse immune response