Biologics

Question 1

What are biologics?

Answer 1

biologics are large, strucuturally complex molecules that have therapeutic effect. Usually drugs are small.

Question 1

What are some advantages and disadvantages of biologics as drugs?

Answer 1

+ Have high specificity for drug targets
- They aren’t accessible by chemical synthesis
- They are not membrane permeable unlike previous small drugs
- They are more likely to be antigenic- provoke an immune response
- More sensitive to handling and storage conditions

Question 2

Can biologics be chemically synthesised?

Answer 2

NO- We have to convince certain cells to make the desired proteins e.g. insulin

Question 3

What is the average size of a biologic (+ compare it to a ‘normal’ small drug)?

Answer 3

The average biologic molecular weight is >2 KD
e.g For a certain monoclonal antibody drug that is a biologic e.g. rituximab, its MW = 144,000 daltons but for a small molecule medicine e.g. Acetylsalicylic acid (Aspirin)= 180 daltons

OR
the anticancer agent paclitaxel has a size of 854 Daltons (Da), while the commercially available G-CSF (i.e. filgrastim), has a size of 18 000 Da.

Question 4

What are some examples of current biologics in use?

Answer 4

The Ebola vaccine
Monoclonal antibodies e.g. Rituximab & trastuzumab
Insulin
Erythropoietin

Question 5

What is recombinant production of therapeutic proteins?

Answer 5

The heterologous expression (heterologous as the protein of interest doesn’t occur naturally in the cell) of recombinant proteins =

‘Involves the introduction of a gene or cDNA coding for the protein of interest into a suitable producer organism’

Question 6

What are the steps for recombinant production of therapeutic proteins?

Answer 6

• Identify the target gene, then isolate and amplify it
• Integrate the target gene into a suitable cloning vector such as plasmid DNA
• Then introduce this vector into the host cell. Host cells e.g. E.coli, yeast, animal cell lines
• Grow this host cell in vitro (in the lab)
• Assess all the cells grown to identify which contain the target protein
• Once identified, Isolate e.g. by centrifugation or filtration and purify e.g. by chromatography the target protein for use

Question 7

What are the commonly used host organisms for producing recombinant proteins?

Answer 7

• Yeast, especially s.cerevisiae
• Microorganisms e.g. E.coli
• Animal cell lines
• Insect cells
• Plants

Question 8

What are the advantages and disadvantages of using E.coli as a host?

Answer 8

+ It is well understood as a micro-organism
+ Produces a high yield of heterologous proteins
+ Is quick and cheap- allows for rapid growth of proteins in inexpensive media
+ Is possibility to scale up production

• Heterologous proteins accumulate intracellulaly- meaning they have to lyse the cells and extract the proteins
• There is the presence of LPS on the surface of E.Coli which is pyrogenic and so would induce a fever
• There is inability to undertake post-translational modifications (MAIN DISADVANTAGE)

Question 9

What is a post-translational modification?

Answer 9

These are any changes via covalent modifications of the peptide sequence (of chosen amino acids) once the peptide chain has been synthesised.
e.g These can be glycosylation, phosphorylation, sulphation, glycation, deamidation, and deimination to amino acids

Question 10

Using E.Coli as a host has many advantages, so why isn’t is always used?

Answer 10

Because E.coli is unable to undertake any post-translational modifications as it is a prokaryotic cell

Question 11

How are proteins made?

Answer 11

• mRNA sequence is translated into an amino acid sequence in a ribosome
• The completed polypeptide chain must then be folded correctly into its 3D conformation. This is NOT covalent
• But any post-translational modifications (PTM) are covalent e.g. Glycosylation, phosphorylation, acylation, hydroxylation, Acetylation

Question 12

What are some examples of post-translational modifications?

Answer 12

Glycosylation- Attachment of carbohydrates
phosphorylation- Attachment of a phosphate group
acylation- Attachment of an acyl group-
hydroxylation- Attachment of a hydroxyl group
Acetylation - attatchment of an acetic acid group

Question 13

What is glycosylation?

Answer 13

The enzymatic process that attaches polysaccharides to proteins/lipids/other organic molecules. There are 2 types- N-linked (most common) and O-linked glycosylation.

Question 14

What is an ogliosaccharide?

Answer 14

An oligosaccharide is a saccharide polymer containing a small number (typically three to ten) of monosaccharides (simple sugars)

Question 15

What is the difference between N-linked and O-linked glycosylation?

Answer 15

In N-linked glycosylation, a protein can exhibit multiple glycan linkages ranging from one to many. In contrast, O-linked glycosylation typically includes only a single sugar residue added to a serine or threonine residue.

Good explanation from online:
Glycosylation is an important modification to eukaryotic proteins because the added sugar residues are often used as molecular flags or recognition signals to other cells than come in contact with them.
- There are two types of protein glycosylation, both of which require import of the target polypeptide into the ER.
- N-linked glycosylation actually begins in the endoplasmic reticulum, but O-linked glycosylation does not occur until the polypeptide has been transported into the Golgi apparatus. Therefore, it is also the case that N-linked glycosylation can (and is) usually beginning as a co-translational mechanism, whereas O-linked glycosylation must be occurring post-translationally.
- Other major differences in the two types of glycosylation are (1) N-linked glycosylation occurs on asparagine (N) residues within an N-X-S or N-X-T sequence (X is any amino acid other than P or D) while O-linked glycosylation occurs on the side chain hydroxyl oxygen of either serine or threonine residues determined not by surrounding sequence, but by secondary and tertiary structure
- N-linked glycosylation begins with a “tree” of 14 specific sugar residues that is then pruned and remodeled, but remains fairly large, while O-linked glycosylation is based on sequential addition of individual sugars, and does not usually extend beyond a few residues

Question 16

What is a glycoform?

Answer 16

These are different forms of one protein caused by variation in glycosylation patterns.
They have the same protein sequence, but different sites of glycosylation
Can alter:
- Stability
- Solubility
- Serum half-life (bioavailability)
- Biological activity
- Immunogenecity

Question 17

Can prokaryotes perform post-translational modifications?

Answer 17

No because they don’t have the required machinery- as they don’t have membrane-bound organelles e.g. nuclei as they are prokaryotic cells
- This is where the use of E.coli becomes a limiting factor as it doesn’t have the necessary glycosylation, machinery to lead to these post-translational modifications.

Question 18

What cells are recommended for producing recombinant proteins?

Answer 18

Eukaryotic cells
Especially:
- Chinese hamster ovary cells
- Human embryonic kidney cells

Question 19

What is the HAMA response when trying to produce monoclonal antibodies?

Answer 19

HAMA = Human anti-mouse antibody response
This is a problem that occurs when using mouse cells to grow the monoclonal antibodies as when they are then injected into a human, it provokes an immune response leading to self-immunogenicity.

Question 20

How were the monoclonal antibodies produced using mouse cells made?

Answer 20

Via Hybridoma technology

Question 21

What is the process of Hybridoma technology to grow monoclonal antibodies in mouse cells?

Answer 21

• The mouse is immunised with specific antigens
• The immune system of the mouse will then produce antibodies to these antigens
• These antibodies are fused with immortal cell lines (long lasting) such as myeloma cells and fused with PEG and so they continue to replicate
• The cells are grown in a medium in large numbers = high yield
• The cells are then analysed and the most potent cells that produced the largest number of antibodies are selected.
• Then isolation and purification is needed- the process depends on whether the cell has secreted the desired protein or if it is still intracellular

Hybridoma = desired = mouse plasma cell + myeloma cell

Question 22

What factor determines which isolation and purification techniques are used and what are the 2 methods?

Answer 22

Depends on whether the cell has secreted the desired protein or if it is still intracellular.

1. Recovery of protein from producing organism-
   - Treat with chemicals e.g. detergents or alkaline conditions
   - sonification (Sonication is the act of applying sound energy to agitate particles in a sample) or homogenisation
   - Agitation in the presence of abrasives e.g. glass beads
2. Chromatographic purification

Question 23

What is a biosimilar?

Answer 23

Biosimilars are agents that are biologically and clinically comparable to the innovator product but not exactly the same
When a patent of a biologic expires (usually 20 years from submission), then different companies can try and make the drug- but it won’t be the exact same and wouldnt pass trials like small drugs would. The new drug may have the same protein sequence but has different properties due to post-translational modifications and so needs to be completely re-tested.

Question 24

Are biosimilars interchangeable in practice?

Answer 24

NO- they are not completely the same drug so can’t be switched without being prescribed. Also, if a physician prescribes a biosimilar and not the reference product, it is deemed a new therapeutic intervention.

Question 25

What do the EMA guidelines look at to deem whether a biosimilar is similar enough to the biologic drug?

Answer 25

• Manufacturing quality
• Non-clinical pharmacology and toxicology
• Pharmacokinetics
• Clinical considerations

Question 26

What is the ‘Minimally clinically important difference (MCID)?

Answer 26

This is the minimal difference between two clinical treatments beyond which regulatory bodies would consider the 2 drugs to be non-equivalent.

Question 27

What is the difference in the need for similarity in simple drugs and biologics and why?

Answer 27

With generic pharmaceuticals only the chemical structure of the active drug needs to be reproduced
- With biologicals both the protein structure and its folds need to be similar. Glycosylation patterns can affect protein folds and overall stability and may have a role in cell-cell adhesion
- To receive regulatory approval, generic drugs only need pharmacokinetic comparability to the original product. Pharmacodynamic and clinical equivalence do not need to be shown. With pharmacokinetic comparability, generic drugs are deemed to be fully interchangeable with the innovator product and the same therapeutic effects are assumed
- For biosimilars, even after regulatory approval they’re not interchangeable with the reference product.

• This is important as can lead to issues e.g. in the 1990s, cases of red cell aplasia (RCA) were reported in renal dialysis patients who were receiving treatment with subcutaneous epoetin alfa.
  After investigation, the most likely cause of the RCA was a formulation change, which led to antibody formation against all circulating erythropoietin. Removal of human serum albumin from the epoetin alfa formulation and its replacement with polysorbate 80 and glycine as stabilizers was suggested as the primary cause. This example illustrates how a small change in the development of a biological can lead to serious adverse events and patient complications

Question 28

What suffix do all monoclonal antibody drugs have?

Answer 28

-mab

Question 29

What do the letters that precede the ‘-mab’ suffix in all monoclonal antibody drugs tell us?

Answer 29

The letters before tell us the cell origin of the monoclonal antibody (what cell line it was grown and produced in).

-u- : human
-o- : mouse
-xi- : Chimeric
-zu- : Humanised
-xi-zu- : chimeric and humanised hybrid

Question 30

Aside from the letters before ‘mab’ in monoclonal antibodies telling us the cell origin, what other factor can be found out via the drug name?

Answer 30

The disease target

-im- immune
-es- infectious disease
- vir- viral
-mel- melanoma
-col- colon

Question 31

What does the letter ‘u’ before ‘mab’ in the name of a monoclonal antibody mean for its cell origin?

Answer 31

Cell origin = human

Question 32

What does the letter ‘o’ before ‘mab’ in the name of a monoclonal antibody mean for its cell origin?

Answer 32

Cell origin = mouse

Question 33

What does the letters ‘xi’ before ‘mab’ in the name of a monoclonal antibody mean for its cell origin?

Answer 33

cell origin = chimeric (an organism whose cells are derived from two or more zygotes )

Question 34

What does the letters ‘zu’ before ‘mab’ in the name of a monoclonal antibody mean for its cell origin?

Answer 34

Cell origin = Humanised (monoclonal antibodies developed for administration to humans from non-human species e.g. mice)

Question 35

What does the letters ‘xi-zu’ before ‘mab’ in the name of a monoclonal antibody mean for its cell origin?

Answer 35

Cell origin= chimeric and humanised

Question 36

What can you tell about the monoclonal antibody ‘ Toclizumab’ and the ‘ Rituximab’ from their names?

Answer 36

Tocilizumab- has ‘zu’ before the mab suffix and so is originated from humanised cells
Rituximab- has ‘xi’ before the mab suffix and so is originated from chimeric cells.

Question 37

Discuss the European medicines agency (EMA) guidelines for approving biosimilars?

Answer 37

Manufacturers must prove to regulatory bodies e.g EMA that:
- Their medicine is highly similar to the reference medicine, not withstanding natural variability inherent to all biological medicines
- There are no clinically meaningful differences between the biosimilar and the reference medicine in terms of safety, quality and efficacy

Biosimilar development relies heavily on comparability studies to establish similarity to the reference product. This involves a comprehensive head-to-head comparison of the biosimilar and the reference medicine.
Is considered a step-wise process:
Step 1: Comparative quality studies = Looks at physical and chemical properties. Also, at the functionality- biologic and pharmacological properties
Step 2 : Comparative non-clinical studies = pharmacodynamic and toxicity
Step 3: Comparative clinical studies = pharmacokinetic/pharmacodynamic and efficacy, safety and immunogenicity

• Preclinical studies = Comparative non-clinical and toxicology
  studies
• Pharmacodynamics = Pharmacodynamic similarity markers should
  be based on clinical efficacy
• Pharmacokinetics = Single dose SC and IV in human volunteers.
  Criteria for acceptance based on clinical
  judgement
• Clinical trials = At least one equivalence trial relative to the
  reference product is required or a three-arm
  trial relative to the reference product and
  placebo. If both the SC and IV route of
  administration are possible, then two separate
  clinical trials must be undertaken
• Extrapolation to other disease sites. This may be permitted, but a final decision will usually be made on a case-by-case basis
• Drug safety = Safety must be demonstrated in at least one
  equivalence trial with the reference product
  in the control arm
• Immunogenicity All clinical trials must include antibody testing
• Post-approval requirements- Specific monitoring must be performed for efficacy in the extrapolated indications. A
  pharmacovigilance programme is required
  post-approval

Question 38

You are the scientist in charge of developing a new biosimilar for rituximab and are trialling
its production in a new cell line derived from Chinese Hamster Ovary cells. Your initial results
show differences in the glycosylation profiles between rituximab and your biosimilar, even
though the peptide sequences are identical.
Explain why you might expect to see some glycosylation changes in a biosimilar and give
three reasons why this might affect the performance of the biosimilar, and identify the main
step required to ensure no harmful side-effects have been induced by these changes. (example exam q - note down few ponts)

Answer 38

Since the originator drug’s production is proprietary, biosimilars are produced in a novel
cell line, and each eukaryotic cell line will induce different post-translational
modifications in the peptide sequence of the protein. The peptide sequence is generated
by a template driven synthesis within the cell based on the introduced rna sequence, but
post-translational modifications occur after peptide synthesis and are determined by
the available enzymes in the host organism, not the rna sequence.
Changes to the glycosylation can have significant effects on the biosimilar: they may
interfere with the mechanism of action of the drug; they may change the folding of the
protein, affecting its stability and/or its efficacy; and they can introduce novel
immunogenic responses in patients, or some population of patients.
Because of this, biosimilars are still required to undergo clinical trials before approval, even
if they have demonstrated comparable efficacy in preclinical testing