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Applied Biological Chemistry > 7. Therapeutic Proteins > Flashcards

7. Therapeutic Proteins Flashcards

1
Q

Give examples of therapeutic proteins and diseases & disorders.

A
  1. Avastin (monoclonal antibody)
    • Cancers
  2. Humulin (peptide)
    • Diabetes
  3. Remicade (monoclonal antibody)
    • Rheumatoid arthritis, Crohn’s disease, etc.
  4. Betaseron, Rebif, Avonex (interferon-b)
    • Multiple sclerosis
  5. Humira (monoclonal antibody)
    • Crohn’s disease, rheumatoid arthritis, coilitis, etc.
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2
Q

Describe the differences between protein and small molecule drugs regarding the following:

  • Size
  • Synthesis
  • Delivery
  • Regulation
  • Availability
A
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3
Q

What are the various dosage forms of therapeutic proteins?

A
  • Aqueous solution formulation in a vial
  • Lyophilized (freeze-dried) formulation in a vial
  • Prefilled syringe with aqueous solution formulation
  • Dual-chambered syringe with lyophilized formulation and diluent
  • Cartridge with aqueous solution formulation; used with injector system
  • Multidose vial or cartridge
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4
Q

What is the dosage form of BetaSeron?

A
  • BetaSeron is supplied as a freeze-dried formulation in a vial
  • The kit also contains a reconstitution system and a syringe containing diluent which is also used for the injection.
  • Can be stored at room temperature, but has limited stability after reconstitution
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5
Q

What are the instructions for preparing BetaSeron?

A
  1. Preparing for your Betaseron injection
  2. Mixing Betaseron
  3. Preparing the injection
  4. Choosing injection site
  5. Injecting Betaseron
  6. Disposing used syringes and vials
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6
Q

What is the dosage form of Herceptin?

A
  • Herceptin is supplied as a freeze-dried formulation in a vial
  • Also supplied is a diluent containing 1.1% benzyl alcohol as an antimicrobial preservative.
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7
Q

What are the steps of preparing Herceptin?

A
  1. Using a sterile syringe, slowly inject 20 mL of the diluent into the vial containing the lyophilized cake of Trastuzumab. The stream of diluent should be directed into the lyophilized cake.
  2. Swirl the vial gently to aid reconstitution. Trastuzumab may be sensitive to shear-induced stress, e.g., agitation or rapid expulsion from a syringe. DO NOT SHAKE.
  3. Slight foaming of the product upon reconstitution is not unusual. Allow the vial to stand undisturbed for approximately 5 minutes. The solution should be essentially free of visible particulates, clear to slightly opalescent, and colorless to pale yellow.
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8
Q

What solution should not be used to reconstitute Herceptin?

A

Dextrose 5% should NOT be used.

Herceptin should only be reconstituted with the supplied BWFI, USP, and 1.1% benzyl alcohol preserved. However, if the pt is hypersensitivity to benzene, the pt can use Sterile Water for Injection.

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9
Q

What is the dosage form of Avonex?

A
  • One version of Avonex is supplied as a aqueous solution in a pre-filled syringe
  • Convenient for patients or care givers.
  • Must keep refrigerated and protect from freezing, high temperature, light, etc. (more about this later)
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10
Q

What are the steps of preparing Avonex?

A
  1. Hold the AVONEX prefilled syringe with the cap pointing down and with the 0.5 mL mark at eye level
  2. With 1 hand, hold the AVONEX prefilled syringe right under the cap and with the cap pointing up
  3. With the other hand, grasp the cap and bend it at a 90º angle until the cap snaps off
  4. Open the sterile needle package and take out the covered needle. Hold the AVONEX prefilled syringe with the glass syringe tip pointing up. Press the needle on the AVONEX prefilled syringe glass tip
  5. Gently turn the needle forward until it is tight and firmly attached
  6. Choose the right AVOSTARTGRIP titration device for your weekly dose.
  7. Put the right AVOSTARTGRIP device on a flat surface with the door open
  8. ine up the AVONEX prefilled syringe over the AVOSTARTGRIPdevice with the plunger pointing to the left and the needle pointing to the right
  9. Push the AVONEX prefilled syringe down into the AVOSTARTGRIP device until both ends “snap” into place
  10. Using two fingers, push the door down until it closes over the AVONEX prefilled syringe
  11. Check to make sure that the AVONEX prefilled syringe is in the AVOSTARTGRIP device the right way and that the door is tightly closed
  12. Inject the medication
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11
Q

A small fraction of patients using therapeutic proteins is initially effective, but the patient may develop neutralizing antibodies to a given product.

What are these types of patients called?

A

Secondary non-responders.

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12
Q

What occurs to the drug in a patient considered to be a secondary non-responder?

A

Patients often develop immunogenicity of the product because the “drug acts like vaccine.” That is, a patient develops anti-drug antibodies. As a result, the drug can be neutralized and/or cleared rapidly.

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13
Q

What can occur when a patient develops immunogenicity to a therapeutic protein?

A
  • Loss of efficacy
  • Uncontrolled disorder (e.g., Crohn’s)
  • Death (e.g., with replacement enzyme)
  • In rare cases, anti-drug antibodies can react with patients’ endogenous proteins (e.g., Eprex cases)
  • In some cases, patients can be switched to alternative therapy when one drug fails (e.g., Crohn’s patient switched to Humira from Remicade)
    • Patients may become immune to all approved medications
    • However, for many patients, there is not an alternative
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14
Q

What patient and product factors affect immunogenicity of therapeutic drugs?

A
  • Patient factors include genetics, co-administration of other drugs, infections, etc.
  • Product factors include type of protein, duration of treatment, route of administration, etc.
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15
Q

What is an adjuvant?

Describe its relationship to the efficacy of a protein therapeutic.

A
  • An adjuvant is a substance which enhances the body’s immune response to an antigen
  • Numerous studies using purified protein antigens have shown that without “adjuvant,” there is weak immune response
  • Many adjuvants are particulate (e.g., aluminum salt microparticles and nano- and microparticles of other materials)
  • Typically, antigen protein is adsorbed onto the particles
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16
Q

What is the correlation between aggregates/particulates the probability of a patient becoming a secondary non-responder?

A
  • Clinical studies with early human therapeutic protein products (e.g., growth hormone, interferon-alpha) showed immuogenicity rates correlated directly with levels of aggregates/particles
  • Even older human studies (60’s and 70’s) showed particles and aggregates stimulate immunogenicity
  • Numerous studies from 60’s and 70’s in human patients and in animals documented that removal of aggregates/particles eliminated immunogenicity and sometimes invoked toleranceto foreign proteins.

There is a direct correlation between aggregate/particle content and clinical rates of immunogenicity for the IFN-β products.

17
Q

What is the pathway of protein aggregation?

A

M → M* → Soluble Aggregates → Insoluble aggregates

  • M* is partially unfolded molecule which can be part of the native state ensemble.
  • Thus, even under conditions greatly favoring the native state, aggregation can proceed at unacceptable rates.
  • M* may be promoted at interfaces
18
Q

What stresses can cause protein aggregation?

A
  • Agitation, freeze-thawing, high temperature, light, mechanical shock, incompatible diluent, bag or ”tubing” IV bags
  • Often, aggregation occurs because of exposure to air-liquid or liquid-solid interfaces.
  • Adsorption to foreign particles
  • Mishandling by patients, pharmacies and/or clinics
19
Q

How does the interface of vials and syringes cause protein aggregation?

A
  • Protein molecules adsorb to most interfaces.
  • Layers of adsorbed protein form gels or films.
  • Rupture or sloughing off of film can lead to particles in the bulk solution phase
20
Q

What molecules have been added to protein therapeutics to prevent protein aggregation? Were they effective?

A
  • Sucrose and methionine did not Inhibit aggregation of soluble TNF inhibitor.
  • Trp has also been utilized, but led to the formation of radicals in solution.

This study indicated that a small fraction of protein molecules may receive “sublethal” damage due to light exposure.

21
Q

What occured to protein therapies that underwent mechanical shock?

A
  • Recent work has documented that mechanical shock due to dropping a vial onto a surface causes violent fluid flows and cavitation (forming bubbles that release free radicals).
  • As a result, a model IgG formed visible and subvisible particles and was oxidized

Therefore, do not toss the box, drop the vial/syringe or “tube” the IV bag.

22
Q

Draw a picture describing the control of aggregation.

A
23
Q

Explain why pH can affect the aggregation rate of a protein therapeutic.

A

In this case, effects of pH an ionic strength on aggregation rate can be attributed at least in part to effects on thermodynamic stability of the native state.

24
Q

What is it important for researchers to quantify subvisible particles in protein therapeutics?

A
  • Subvisible particles are in every therapeutic protein product and many products cause non-response in fraction (e.g., 20-70%) of patients.
  • Beyond immunogenicity, particle sizes and levels are important product quality attributes.
  • Mass of protein (e.g., < 0.1%) in particles may not be detectable as loss of monomer.
  • Particle counts (MFI) provide highly sensitive measure of aggregation due to freeze-thawing
25
Q

What foreign particles can arise from glass containers, closures, filters, and pumps?

A
  • Glass particles from containers.
  • Glass cartridges and syringes are siliconized, and free silicone droplets can be generated.
  • In syringes, there may also be tungsten particles and tungsten salts from needle insertion process.
  • Rubber and/or silicone particles can come from stoppers.
  • Stainless steel and other particles from filling pumps.
  • Particles shed from filters during pre-filling sterile filtration and other filtration steps
  • Protein molecules can adsorb to these nano- and microparticles.

Unless they are filtered out during administration, these particles are delivered to patients.

26
Q

Why is filtration not a perfect method to prevent foreign particulate matter from being slouged off into a protein therapeutic?

A
  • Filters themselves can shed particles.
  • Protein molecules can adsorb/desorb to/from filter materials.

Both effects may cause protein aggregation. In fact, even when a filter is used, there was found to be > 20M nanoparticles/ml.

27
Q

During IV administration, aggregates and particles can form during mixing of protein formulation with IV diluent, storage in bag and/or infusion.

Therefore, what steps can pharmacists take to avoid protein aggregation?

A
  • Instructions with each product must be carefully followed, without any deviations
  • Proper diluent, mixing procedure and storage time and temperature must be used
28
Q

How can a pharmacist avoid accidental freeze-thawing of protein therapeutics?

A
  • “Blue ice packs” are at -20 C and can freeze products during shipping
  • Beware of cold spots in refrigerator
29
Q

What are the advantages and disadvantages of lyophilized formulations?

A
  • Advantages:
    • Cannot be frozen; exposure to subzero temperatures will not cause product damage
    • Exposure to high temperatures typically can be well-tolerated by lyophilized formulations
      • Many products are stable at ambient temperatures for days or weeks
    • May be less sensitive to light exposure
  • Disadvantage
    • Reconstitution needed
    • Risk of mishandling
30
Q

Stresses to protein therapeutics can occur even during “proper” IV preparation.

List a few examples.

A
  • Stabilizing excipients greatly diluted
  • Air-water and solid-water interfaces for protein adsorption, aggregation and particle formation
  • Particles from WFI, and IV bag, tubing, etc.
  • Silicone oil from syringes used in rehydration and for final saline flush of line
  • Peristaltic pump with silicone tubing
  • With 0.22 and 1.2 micron in-line filters, plenty of particles can still get through system; also filters and tubing can shed particles
  • Plasticizers leach into solutions
31
Q

Nanoparticles of Infliximab in IV saline stimulated:

A
  • T cell proliferation
  • IL-6 production in whole blood
32
Q

What were the conclusions of the Avastin Case Study?

A
  • Avastin repackaged in plastic syringes was contaminated by silicone oil micro-droplets.
  • Freeze-thawing, exposure to light and other improper handling can further degrade the product and increase levels of particles and protein aggregates.

Applied Biological Chemistry (7 decks)

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