8. DIFFERENCES BETWEEN SMALL MOLECULES AND BIOLOGICALS
8.1 Core di+erence: size and complexity
Small molecule drugs are usually small, chemically simple and well-defined compounds,
whereas biologicals are much larger and structurally more complex. Examples:
• Acetylsalicylic acid (aspirin): small molecule, ~21 atoms
• Human growth hormone: protein biological, ~3000 atoms
• IgG antibody: antibody biological, ~25,000 atoms
→ Biologicals are not just “big chemicals”:
Feature Small molecule drugs Biologicals
Structure simple, well-defined complex, heterogeneous
Characterization easy complete characterization not possible
Manufacturing defined chemical synthesis produced in living cells, challenging process
control
Identical copy possible? yes → true generics no → biosimilars
Stability relatively stable sensitive to external conditions: heat, light,
agitation (f.e., COVID-19 vaccines)
8.2 What counts as a biological?
There is no single straightforward definition of a biological:
• FDA: biologicals are generally large, complex products produced through biotechnology in
a living system such as microorganisms, plant cells, or animal cells, and are often harder to
characterize than small molecule drugs.
• EMA: a biological is a medicine whose active substance is made by a living organism.
• ICH S6: biologicals are products derived from characterized cells using expression systems
such as bacteria, yeast, insect, plant, or mammalian cells. Their active substances include
proteins, peptides, derivatives, and products containing these components, and may be
produced from cell cultures or recombinant DNA technology, including transgenic plants and
animals.
→ Biologicals are complex medicinal products made by or derived from living systems
DiXerent classes of biologicals include:
• Proteins with a specific regulatory/enzymatic function: human insulin
• Peptides: leuprorelin, which is a GnRH analogue suppressing sex hormones
• Vaccines with a prophylactic/therapeutic function: APOBEC3A
• Antibodies: adalimumab, which is an anti-TNF mAb for inflammation
• Nucleic acid therapeutics: siRNA/miRNA-based approaches that lead to decreased protein
production by inhibiting translation or degrading mRNA
COVID-19 examples:
• DNA/RNA vaccines and viral vector vaccines belong to the biological-type category
• Remdesivir is a small molecule antiviral agent
• Anti-spike antibodies are biologicals / antibodies
1
,8.3 Antibodies = immunoglobulins
Antibodies are large proteins of about 150 kDa and play an important role in the immune
response. Humans have five antibody isotypes:
• IgD: B-cell signaling
• IgE: allergens/parasites
• IgA: mucosal protection
• IgM: early immune response
• IgG: main circulatory antibody
Therapeutic antibodies are mainly IgG-type antibodies, because IgGs have useful therapeutic
properties, especially a long half-life.
An antibody has two main functional parts:
• Fab: variable region, binds the antigen/target → gives target specificity
• Fc: constant region, can interact with Fc receptors on immune cells, the complement
system, and FcRn (neonatal), which protects IgG from degradation and helps prolong
half-life
Antibody fragments are smaller antibody-derived structures, such as:
• Fab fragments
• single-chain variable fragments / scFv
As they lack the Fc region, they usually lose Fc-related eXects such as long IgG half-life and Fc-
mediated immune eXector functions, unless engineered otherwise.
• faster clearance
• lower background
• less immunogenicity
• better penetration
8.4 What’s di+erent about therapeutic antibodies?
1. Mode of action of biologicals / antibodies
Antibodies are close to the “magic bullet” concept because they can bind their targets with high
selectivity and aGinity.
Main mechanisms by which antibodies can act:
• Blocking soluble ligand/receptor → antibody prevents ligand-receptor interaction
o Example: CGRP/CGRP receptor antibodies to treat migraines
• Activating antibodies → antibody activates a receptor instead of blocking it
o Example: REGN538, NPR1 agonist antibody
• Antibody-dependent cellular cytotoxicity (ADCC) → antibody binds antigen on target cell →
Fc recruits immune eXector cells → release cytotoxic granules → target cell dies
o Example: Rituximab binds to CD20 on B-cells → cell becomes “tagged”
• Complement-dependent cytotoxicity (CDC) → antibody binds target cells → complement is
activated → membrane attack complex → pore formation → target cell dies due to water
entering the cell
o Example: Rituximab
2
, • Conjugated antibodies → antibody carries a toxic/radioactive/drug payload to a target cell
o Example: humanized anti-CD33 mAb linked to calicheamicin, which is a highly toxic
anticancer compound → antibody brings the payload to the target cell, limiting oX-
target toxicity compared with conventional chemotherapy.
• Opsonization and phagocytosis → antibody coats target, making it easier for phagocytes to
engulf (= general antibody function)
Important: for cancer antibodies, Fc eXector functions such as ADCC/CDC may be useful. For
antibodies that are only supposed to block or activate a receptor, unwanted Fc-mediated killing
may be a safety issue.
2. Pharmacokinetics of biologicals / antibodies
Absorption
Biologicals are generally not given orally because they are large and unstable → poorly absorbed
and/or degraded. Therefore, they are usually administered:
• i.v. → immediate systemic exposure, with a rapid peak in blood
concentration
• s.c. / i.m. → slower absorption, mainly via the lymphatic system,
giving a delayed and broader concentration-time profile
Importantly, slower absorption does not mean ineXective absorption. In the alemtuzumab
example, s.c. administration reached blood concentrations similar to i.v. administration, but
required a higher cumulative dose. It was also more convenient and better tolerated, although
some patients developed anti-alemtuzumab antibodies.
Distribution
Biologicals distribute mainly through passive diGusion, convection and transcytosis, but
because they are large molecules, their tissue distribution is limited.
• Passive diGusion → movement from high to low concentration, but this is limited for
biologicals because of their large size and physicochemical properties.
• Convection → the main mechanism for many biologicals. Biologicals are
carried with pressure-driven fluid movement from the vascular space into
the interstitial tissue space through paracellular pores in the vessel wall.
This depends on vascular permeability, number/size of pores, tissue
pressure and lymphatic drainage.
• Transcytosis → transport across cells via vesicles; this can help biologicals cross cellular
barriers in some tissues.
After entering the interstitial space, a biological can either remain there, bind to its target, or drain
into the lymphatic system, which can return it to systemic circulation.
Because biologicals are large, they usually have a low volume of distribution (Vd): they do not
spread widely into tissues and mainly remain in blood and interstitial fluid.
Distribution can also be target-mediated: if the target is highly expressed in a tissue, the
biological can be “pulled” into that tissue by binding to the target, which can increase local
exposure and sometimes increase apparent Vd.
The blood-brain barrier is an important limitation for biologicals, because large antibodies
usually enter the CNS poorly. However, it is not completely black-and-white: CNS exposure may
depend on barrier integrity, disease state, transport mechanisms and antibody properties.
3
8.1 Core di+erence: size and complexity
Small molecule drugs are usually small, chemically simple and well-defined compounds,
whereas biologicals are much larger and structurally more complex. Examples:
• Acetylsalicylic acid (aspirin): small molecule, ~21 atoms
• Human growth hormone: protein biological, ~3000 atoms
• IgG antibody: antibody biological, ~25,000 atoms
→ Biologicals are not just “big chemicals”:
Feature Small molecule drugs Biologicals
Structure simple, well-defined complex, heterogeneous
Characterization easy complete characterization not possible
Manufacturing defined chemical synthesis produced in living cells, challenging process
control
Identical copy possible? yes → true generics no → biosimilars
Stability relatively stable sensitive to external conditions: heat, light,
agitation (f.e., COVID-19 vaccines)
8.2 What counts as a biological?
There is no single straightforward definition of a biological:
• FDA: biologicals are generally large, complex products produced through biotechnology in
a living system such as microorganisms, plant cells, or animal cells, and are often harder to
characterize than small molecule drugs.
• EMA: a biological is a medicine whose active substance is made by a living organism.
• ICH S6: biologicals are products derived from characterized cells using expression systems
such as bacteria, yeast, insect, plant, or mammalian cells. Their active substances include
proteins, peptides, derivatives, and products containing these components, and may be
produced from cell cultures or recombinant DNA technology, including transgenic plants and
animals.
→ Biologicals are complex medicinal products made by or derived from living systems
DiXerent classes of biologicals include:
• Proteins with a specific regulatory/enzymatic function: human insulin
• Peptides: leuprorelin, which is a GnRH analogue suppressing sex hormones
• Vaccines with a prophylactic/therapeutic function: APOBEC3A
• Antibodies: adalimumab, which is an anti-TNF mAb for inflammation
• Nucleic acid therapeutics: siRNA/miRNA-based approaches that lead to decreased protein
production by inhibiting translation or degrading mRNA
COVID-19 examples:
• DNA/RNA vaccines and viral vector vaccines belong to the biological-type category
• Remdesivir is a small molecule antiviral agent
• Anti-spike antibodies are biologicals / antibodies
1
,8.3 Antibodies = immunoglobulins
Antibodies are large proteins of about 150 kDa and play an important role in the immune
response. Humans have five antibody isotypes:
• IgD: B-cell signaling
• IgE: allergens/parasites
• IgA: mucosal protection
• IgM: early immune response
• IgG: main circulatory antibody
Therapeutic antibodies are mainly IgG-type antibodies, because IgGs have useful therapeutic
properties, especially a long half-life.
An antibody has two main functional parts:
• Fab: variable region, binds the antigen/target → gives target specificity
• Fc: constant region, can interact with Fc receptors on immune cells, the complement
system, and FcRn (neonatal), which protects IgG from degradation and helps prolong
half-life
Antibody fragments are smaller antibody-derived structures, such as:
• Fab fragments
• single-chain variable fragments / scFv
As they lack the Fc region, they usually lose Fc-related eXects such as long IgG half-life and Fc-
mediated immune eXector functions, unless engineered otherwise.
• faster clearance
• lower background
• less immunogenicity
• better penetration
8.4 What’s di+erent about therapeutic antibodies?
1. Mode of action of biologicals / antibodies
Antibodies are close to the “magic bullet” concept because they can bind their targets with high
selectivity and aGinity.
Main mechanisms by which antibodies can act:
• Blocking soluble ligand/receptor → antibody prevents ligand-receptor interaction
o Example: CGRP/CGRP receptor antibodies to treat migraines
• Activating antibodies → antibody activates a receptor instead of blocking it
o Example: REGN538, NPR1 agonist antibody
• Antibody-dependent cellular cytotoxicity (ADCC) → antibody binds antigen on target cell →
Fc recruits immune eXector cells → release cytotoxic granules → target cell dies
o Example: Rituximab binds to CD20 on B-cells → cell becomes “tagged”
• Complement-dependent cytotoxicity (CDC) → antibody binds target cells → complement is
activated → membrane attack complex → pore formation → target cell dies due to water
entering the cell
o Example: Rituximab
2
, • Conjugated antibodies → antibody carries a toxic/radioactive/drug payload to a target cell
o Example: humanized anti-CD33 mAb linked to calicheamicin, which is a highly toxic
anticancer compound → antibody brings the payload to the target cell, limiting oX-
target toxicity compared with conventional chemotherapy.
• Opsonization and phagocytosis → antibody coats target, making it easier for phagocytes to
engulf (= general antibody function)
Important: for cancer antibodies, Fc eXector functions such as ADCC/CDC may be useful. For
antibodies that are only supposed to block or activate a receptor, unwanted Fc-mediated killing
may be a safety issue.
2. Pharmacokinetics of biologicals / antibodies
Absorption
Biologicals are generally not given orally because they are large and unstable → poorly absorbed
and/or degraded. Therefore, they are usually administered:
• i.v. → immediate systemic exposure, with a rapid peak in blood
concentration
• s.c. / i.m. → slower absorption, mainly via the lymphatic system,
giving a delayed and broader concentration-time profile
Importantly, slower absorption does not mean ineXective absorption. In the alemtuzumab
example, s.c. administration reached blood concentrations similar to i.v. administration, but
required a higher cumulative dose. It was also more convenient and better tolerated, although
some patients developed anti-alemtuzumab antibodies.
Distribution
Biologicals distribute mainly through passive diGusion, convection and transcytosis, but
because they are large molecules, their tissue distribution is limited.
• Passive diGusion → movement from high to low concentration, but this is limited for
biologicals because of their large size and physicochemical properties.
• Convection → the main mechanism for many biologicals. Biologicals are
carried with pressure-driven fluid movement from the vascular space into
the interstitial tissue space through paracellular pores in the vessel wall.
This depends on vascular permeability, number/size of pores, tissue
pressure and lymphatic drainage.
• Transcytosis → transport across cells via vesicles; this can help biologicals cross cellular
barriers in some tissues.
After entering the interstitial space, a biological can either remain there, bind to its target, or drain
into the lymphatic system, which can return it to systemic circulation.
Because biologicals are large, they usually have a low volume of distribution (Vd): they do not
spread widely into tissues and mainly remain in blood and interstitial fluid.
Distribution can also be target-mediated: if the target is highly expressed in a tissue, the
biological can be “pulled” into that tissue by binding to the target, which can increase local
exposure and sometimes increase apparent Vd.
The blood-brain barrier is an important limitation for biologicals, because large antibodies
usually enter the CNS poorly. However, it is not completely black-and-white: CNS exposure may
depend on barrier integrity, disease state, transport mechanisms and antibody properties.
3
