ADME of Macromolecules

Question 1

Key differences between biotechnology-
based products and chemical products

Answer 1

Biologics/Biopharmaceutical Products
* Molecular weight: larger, typically in
kilodaltons (kDa)
* Derived from living sources –human &
animal tissues, cells & microorganisms
* Not easily characterized and refined to
high degree of purity.
* Often called by the same name despite
modifications in one or more amino acid
residues.
* Behave more predictably and lesser
side effects.

Traditional Chemical-based drugs
* Molecular weight: typically < 1000 Da
* Can be chemically synthesized and
purified to homogeneity.
* Chemical modification usually leads
to drastic changes in activity and new
drugs for new uses.
* May have off-target effects (side
effects).

Question 2

what does Biopharmaceuticals consist of?

Answer 2

Recombinant proteins, monoclonal antibodies, nucleic acid-based products, enz, Ab, etc

Question 3

Challenges of using Biopharmaceuticals

Answer 3

Stability (denaturation and degradation)
immunogenicity if impure and from non-human origin
Poor distribution due to permeability of vasculatures

Question 4

why does P have poor oral F?

Answer 4

poor Stability, poor permeability

Question 5

Name 2 transport mechanism for Absorption of protein drugs after SC or IM administration

Answer 5

1. Diffusion - Inversely related to MW/size of
   proteins → limited for large proteins
2. Convection - not limited by MW unless the
   protein molecules are enormously large and
   gets entrapped in the ECM. affected by steric hinderance and charge interactions

Question 6

For larger proteins (e.g. > 16-20 kDa), movement across capillary membrane
_______; absorption mostly occur via the _______________, drain into ______________
and _____________, then finally into ___________

Answer 6

slow, lymphatic system, lymph nodes, larger lymphatic vessels, circulatory system

Question 7

For smaller proteins (e.g. < 16-20 kDa), absorption can be via both ___________
and _______________. ____________ (i.e. blood flow throughout tissue) can
influence capillary absorption

Answer 7

circulatory, lymphatic systems, Perfusion

Question 8

Name 2 rate limiting factors that can cause changes to absorption rates of proteins
drugs after SC/IM administration

Answer 8

1. Interstitial fluid transport rate
2. Lymphatic transport rate

Question 9

________________ of protein drugs via ______________ pathway of trans-capillary transportation of proteins accounts mainly
for protein drug distribution.

Answer 9

Passive movement; convective or diffusive

Question 10

What are the key points of the Two pore model to characterize tissue level protein disposition

Answer 10

Key points of two pore model:
1. Endosomal space represents the porous tissue microvascular endothelium.
2. Two types of pores exist in the endosomal layer: small and large pores.
3. Fluid passes through both small and large pores and can re-circulate.
4. Passive movement of protein molecules from vascular (plasma) space to interstitial space using both small and large pores via diffusion (PS) or fluid phase convection (J). Extent of movement and distribution related to MW/size.
5. Larger proteins: have more limited distribution and display slower distribution into and out of tissues.
6. Mathematical equation utilizing a number of derived parameters to predict PK
profile of proteins of different sizes.

Question 11

Metabolism of protein drugs is via _________ by _______________ in _________, ___________ and ____________

Answer 11

proteolysis; proteolytic enzymes (e.g. activated proteases); interstitial fluid (extracellular fluid)
present in tissues/organs; On cell surfaces; Intracellularly once protein drugs are
taken up into cells

Question 12

The ____________ is a ___________________ sharing atructural similarities with _________________

Answer 12

Neonatal Fc receptor (FcRn), IgG Fc receptor; MHC Class I molecule

Question 13

FcRn binds to _____________

Answer 13

IgG and serum albumin

Question 14

Role of FcRn

Answer 14

Plays critical role in IgG homeostasis by mediating antibody recycling by
allowing intracellular trafficking of antibodies resulting in antibodies escaping
degradation in lysosomes.
* Plays a role in recycling of serum albumin (same intracellular trafficking
mechanism as antibodies) –> increase half-life of IgG and albumin

Question 15

slide 18/19 ic7

Question 16

Protein drugs can be eliminated via:

Answer 16

1. Proteolytic degradation (extracellular and intracellular)
2. Renal (glomerular) filtration (dominates renal excretion of protein
   drugs)

Question 17

Factors affecting renal excretion of proteins:

Answer 17

• Cut-off molecular weight of protein (proteins > ~50 kDa are not renal
  eliminated due to them being too big to pass through renal glomerular
  barrier).
• Charge of protein (positively charged proteins have higher renal
  filtration than negatively charged proteins of same size due to
  negative charges on glomerular basement membrane).
• Shape and rigidity of protein (affect how well proteins undergo
  glomerular filtration)
• Tubular reabsorption (tubular epithelium have net negative charge →
  positively charged proteins get more reabsorbed)

Question 18

Name 3 Common strategies to improve PK profile of protein therapeutics

Answer 18

• Glycosylation of proteins
• PEGylation of proteins
• Increase in size (MW) by means of fusion proteins

Question 19

Glycosylation involves addition of _________ (carbohydrates) to specific amino acids in a protein

Answer 19

glycans

Question 20

One universal strategy: _____________ of proteins increase circulation half-time by increasing size of protein or modifying binding
to glycoprotein receptors

Answer 20

N-linked glycosylation

Question 21

Polyethylene glycol (PEG) have 2 types :

Answer 21

PEG with free hydroxyl at both ends and methoxylated PEG (mPEG)
with hydroxyl at one or both ends methoxylated

Question 22

PEG conjugation involves Reactive functional groups of activated PEG/mPEG attached to ________

Answer 22

an amino group on amino acids like lysine, sulfhydryl -SH group in
cysteine or other nucleophilic groups on amino acids

Question 23

2 types of configurations:

Answer 23

Linear or branched PEG/mPEG polymers conjugated to protein drugs →
give rise to PEGylated proteins
of different extended half-lives

Question 24

PEGylation increases circulation half-lives of proteins drugs through:

Answer 24

• Increase in the size of conjugated protein
• Glomerular filtration of small proteins is retarded if PEG molecules with
  MW 40-50 kDa is conjugated to the proteins.
• Decrease elimination by proteolysis
• PEG molecules form a protective layer on surface of protein molecules,
  decreasing chance for proteolytic enzymes to interact with and break down
  proteins.
• Decrease elimination by action of antibodies and activated immune cells
• PEG molecules form a protective layer on surface of protein molecules,
  decreasing recognition by antibodies or activated immune cells (e.g.
  macrophages, dendritic cells, NK cells, etc).
• This is also the property that make PEGylated proteins have reduced
  immunogenicity/antigenicity

Question 25

______________________ fused to a therapeutic protein to utilize FcRn-mediated recycling (so as to increase half-lives of the therapeutic protein) to enhance circulation half-lives.

Answer 25

Fusion proteins with Fc domain of antibody or albumin used to a
therapeutic protein

Question 26

Name 4 Impact of recombinant DNA (rDNA)
technology on Biopharmaceutical products

Answer 26

cheaper, safer, and abundant supply of
protein-based biopharmaceuticals.

1. Overcomes limitations regarding source availability.
   Natural sources often rare and expensive. In addition, yield can be low due
   to limited amounts present in natural sources.
2. Allows production of safer biopharmaceuticals. E.g. eliminates transmission
   of blood-borne pathogens such as HIV, Hepatitis B virus if product is
   directly isolated from infected sources.
3. Provides an alternative way to obtain protein-based products other than
   direct extraction from inappropriate source material (e.g. urine, placenta).
4. Since gene of interest can be synthetic, offers opportunities for scientists to
   design desirable mutations to produce engineered protein-based
   biopharmaceutical products that possess advantages (such as greater
   clinical efficacy, greater protein stability for longer self-life, as well as
   shorter/longer circulation half-life)
   Recom

Question 27

In recombinant protein making,
upstream processes include selection
of the _____ transfected cell that possesses
the best cell growth properties and
highest protein yield to develop a master cell line.

Answer 27

1

Question 28

ic8 slide 9-11

Question 29

In pharmaceutical industry, QC is performed to confirm _______________ of final product to pre-determined specifications.

Answer 29

conformance

Question 30

____________ is a biologic that is almost an identical alternative version of the original biologic (called __________________) that is manufactured by a different
company.

Answer 30

biosimilar; innovator biologic, reference biologic

Question 31

Can a biosimilar biologic be entirely
identical to the innovator biologic?

Answer 31

Each biological product displays variability even between different
batches of the same product due to the variability of the biological
expression system and manufacturing process. → Process of
manufacturing biopharmaceuticals (upstream and downstream
processing) has crucial influence on the nature of the final biological
product

Mabs are larger proteins with post-translational modifications →
impossible to engineer a biosimilar 100% identical to its innovator
biologic; produce a highly similar biosimilar instead