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We'll kick off with Joseph Denby from CPC Scientific.
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He's a Senior Manager of Business Development.
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He has the background in biochemistry and nearly a decade of experience in commercial sector.
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And CPC Scientific is supporting their clients end to end from discovery to commercialization in advancing peptides and oligonucleotides and new chemical entities.
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Please go ahead, you will be great.
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Thank you very much.
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So thank you all for bearing with me before the lunch break.
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Hope you're all thoroughly caffeinated as I am.
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So my name is Joseph Denby from CPC Scientific and thank you for joining our talk today on green approaches to peptide manufacturing from minimal protection strategies in API synthesis to macro cyclisation and peptide oligonucleotide conjugation in ecofriendly solutions.
1:00
So peptide oligo conjugation or as I'll refer to it more throughout the presentation as POC consists of a targeting peptide linked to a cargo oligo.
1:11
These peptides are highly effective at targeting cell surface receptors such as integrins and G protein coupled receptors, which as we all know are often overexpressed in cancer cells.
1:23
POC's can target tumour specific antigens, viral antigens and various tissues including tumour cells, endothelial cells, the liver and the brain.
1:33
So the therapeutic component is the oligo moiety which can encompass single or double stranded DNA, modified DNA like phosphothioated modified DNA and custom RNA oligos including siRNA, ASOs, miRNA, locked nucleic acids, and more.
1:50
Beyond the customisation capabilities of POCs, they offer exceptional therapeutic potential due to their enhanced tissue potential penetration, including the blood brain barrier, high specificity and selectivity, reduced off target effects, improved pharmacokinetics such as extended half-lives and bioavailability and lower immune responses.
2:10
Additionally, POCs exhibit reduced toxicity compared to small molecules with a more focused distribution to target tissues.
2:23
So POCs contain both a targeting moiety as we discussed the peptide and an oligo moiety and that's designed to be released in the target cell.
2:30
So the linkage or conjugation chemistry plays a key role in where and how the oligo is released.
2:37
Premature POC cleavage by systemic enzymes such as neutrophil elastase or carboxylesterase 1C will affect dosing and could reduce off target toxicity.
2:47
Cathepsins are lysosomal proteases that play crucial roles in protein degradation, extracellular matrix remodelling, and immune response regulation.
2:57
They are also heavily implicated in cancer progression, invasion, metastasis, and drug resistance.
3:04
Therefore, developing POCs that undergo intracellular cleavage within lysosomes is crucial to the advancement of new POC drugs.
3:12
So, the 12 principles of green chemistry, and many of you be familiar with, provides a framework for creating safer, more sustainable chemical processes.
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These principles focus on reducing waste, minimising the use of hazardous materials, improving energy efficiency, and incorporating renewable resources.
3:29
The goal is to prevent pollution at source rather than dealing with it after it's created.
3:34
These principles are critical because they allow industries to reduce their environmental impact, improve safety and achieve long term sustainability.
3:43
So those 12 principles were formulated by Paul T. Anastis and John C. Warner in their seminal 1998 book, Green Chemistry: Theory and Practice.
3:52
Of these 12 principles, this talk will focus more on the prevention and reduction of hazardous chemicals and solvents, reduced derivatives, i.e., minimal protection, and selective and recyclable catalysts.
4:05
So the goal of developing greener manufacturing processes for solid phase peptide synthesis, or SPPS, is to enhance atom economy while reducing or eliminating the use of environmentally hazardous solvents and reagents.
4:20
In alignment with the 12 principles of green chemistry, we aim to use less hazardous chemical synthesis whenever possible, select safer solvents by avoiding unnecessary reactions and choosing those compatible with ecofriendly solvents and avoid unnecessary derivatization.
4:36
So the use of blocking groups, protection deprotection steps or temporary modifications of physical chemical processes which generate waste and decrease atom economy.
4:49
So, despite the advantages of SPPS, it still requires the side chains of certain amino acids to be protected from undesired reactivity during synthesis.
4:59
The installation and removal of these protection groups will also result in a lower atom economy in the overall production process.
5:07
Removal of the protection groups often requires large volumes of TFA, which can result in lower yields and pose a significant risk to the environment.
5:15
While side chain protection reduces some common side chain reactions in SPPS, removing the protection groups can result in their reattachment to the peptide by electrophilic addition.
5:24
If not sufficiently scavenged, protection group reattachment may become permanent and compromise the crude purity.
5:32
So the solvents commonly used in SPPS include diethyl ether, dimethylformamide, dichloromethane, acetonitrile, acetic acid, and water, and these vary significantly in their environmental toxicity.
5:46
Our goal is to eliminate protection groups that require highly hazardous solvents, hence minimal protection, such as diethyl ether and TFA for their removal.
5:56
While the use of other hazardous solvents like DMF can be reduced, it cannot be completely eliminated.
6:02
Another objective of this work is to reintroduce recommended solvents whenever possible, particularly for reactions like click chemistry which are compatible with aqueous solvent mixtures such as water, T-Butyl alcohol.
6:16
So SPPS is a method in which this is a bit.
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I appreciate that some of you already know this, but there are multiple tracks for the conference.
6:22
It's a method in which amino acids are sequentially added to a growing peptide chain anchored to a solid resin support.
6:28
After each coupling and deep protection step, the resin bound peptide undergoes washing cycles to remove access reagents and byproducts.
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This approach offers key advantages over the liquid phase peptide synthesis, as the use of access reagents drives the reactions to completion and therefore ensures more efficient couplings.
6:48
All synthesis steps are carried out in the same reaction vessel, eliminating the need for material transfer.
6:55
As a result, intermediates do not require purification, isolation, or characterization until the final product is cleaved from the resin and all temporary protection groups are removed.
7:06
Despite these advantages, during the elongation phase, as shown in the diagram, repeated washing with DMF, i.e., filling and draining cycles is required to remove excess reagents and prevent contamination.
7:20
Effective washing is crucial for maintaining high yields and minimising side reactions, particularly during longer synthesis, despite efforts to minimise these steps.
7:32
So let's proceed with the first case study of our talk on sustainable peptide manufacturing, which involves the minimal protection strategies we're using SPPS.
7:40
So as mentioned earlier, the side chains of certain amino acids require temporary protection groups in order to stop undesired reactions from occurring at these sites during the process.
7:50
However, some amino acids are more reactive than others, so different levels of protection are needed.
7:56
So in the Minimal Protection Strategy and SPPS section of our talk, we showcase two case studies where peptide elongation is achieved using unprotected side chains of serine, threonine, tyrosine, hydroxyproline, tryptophan, histidine and arginine.
8:12
So these strategies are applied to two specific target peptides, a novel peptide small molecule conjugate we developed incorporating an acid sensitive small molecule and a goserelin peptide API derivative.
8:24
The goal is to demonstrate how minimal protection approaches can simplify synthesis while maintaining efficiency of preserving sensitive functional groups.
8:34
So in traditional SPPS, hydroxyl groups on serine, threonine, tyrosine and hydroxyproline side chains are protected with T butyl groups which are removed during cleavage with 95 to 100% TFA with scavengers like water, phenol or thioanisole.
8:51
tBoc groups on tryptophan and trityl groups on histidine imidazole, rings are removed similarly.
8:55
Arginine's guanidine protection is more complex, so protection strategies have evolved to minimise cleavage times and reduce side reactions as prolonged exposure to strong acids can degrade the peptide.
9:11
In our first case study, which involves the synthesis of a peptide small molecule conjugate, we compare the solvent utilisation and atom economy of two methodologies.
9:20
So standard SPPS and minimal protection SPPS.
9:25
So the key distinction between these approaches is that minimal protection SPPS eliminates the need for TFA and diethyl ether, thereby avoiding the use of hazardous solvents that pose risk to both health and the environment.
9:42
In the conventional SPPS approach, hydroxyl bearing amino acids are protected with T butyl groups and the amino group of lysine is protected with a tBoc group.
9:51
Elongation is carried out on 2-Chlorotrityl or Wang resin solid support using appropriately Fmoc protected amino acid derivatives and a coupling reagent.
10:01
Cleavage is performed using a TFA cleavage cocktail, 95% TFA, 2.5% ETT and 2.5% water, followed by precipitation of the crude peptide with cold diethyl ether.
10:14
The crude material is then extensively washed with cold diethyl ether with a total wash volume of 350 litres per mole.
10:22
In the minimally protected SPPS approach, elongation is carried out on a 2-Chlorotrityl resin with all hydroxyl bearing amino acids left unprotected.
10:32
Since hydroxyl groups are weak nucleophiles, they do not interfere with the coupling of other amino acids.
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However, protection is required during cleavage with strong acids to prevent dehydration.
10:43
The epsilon amine of lysine has protected the labile DDe group, which can be removed with dilute hydrazine.
10:50
Peptide cleavage from the resin is achieved using 30% HFIP in dichloromethane, but acetic acid can be used as an alternative.
10:57
This approach avoids the use of both TFA and diethyl ether.
11:05
In a large scale preparation, the quantities of TFA and diethyl ether are considerable, totalling a combined volume of 380 litres per mole for the traditional approach.
11:12
Crude purity can be misleading as it's mainly influenced by the acid sensitivity of the small molecule in the conjugate.
11:23
In Part 2 of our minimal protection case study, we expand our approach to include unprotected side chains of histidine, tryptophan, and arginine.
11:32
We demonstrate the synthesis of a goserelin peptide API impurity, showcasing how a convergent peptide fragment strategy can be used to eliminate the need for TFA and diethyl ether eliminate side chain protection of arginine, histidine and tryptophan.
11:47
Similar to other LHRH peptide APIs such as buserelin, T-butyl protected D-serine is incorporated into the product and remains protected throughout the synthesis.
11:58
So this cartoon depiction represents a convergent synthetic approach where we combine 7 amino acid fragments containing a T-butyl protected serine with the resin bound dimer fragment of the C terminus modified hydrazine.
12:12
After this coupling, the peptide is cleaved from the resin to provide goserelin impurity. To prevent arginine side reactions during the activation step.
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Fmoc Arg is available as the chloride salt form i.e., Arg(HCl).
12:26
In this form, arginine can be activated without the risk of delta lactam formation.
12:32
Once incorporated into the peptide chain during the elongation phase, the guanidinium salt is converted to the freebase guanidine group in the presence of a base.
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So the synthesis of the heptamer fragment was achieved by elongating on 2-Chlorotrityl resin using standard solid phase peptide synthesis methods.
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Fmoc glue was directly coupled to the resin under anhydrous basic conditions, i.e., Diisopropylethylamine.
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Fmoc deprotection was carried out by treating the resin with 20% piperidine in DMF for 20 minutes.
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Fmoc-D-Ser(tBu) was then coupled to the resin using Diisopropylcarbodiimide and HOBt in DMF deal.
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Fmoc-D-Ser(tBu)-Leu-CTC resin.
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That's a lot of acronyms.
13:20
Following this, tyrosine, serine and tryptophan were coupled sequentially to the resin, all without side chain protection, using DIC and HOBt
13:29
To minimise racemisation of histidine, particularly at the alpha carbon, HOOBt was used instead of HOBt, which reduced the racemisation from approximately 2% to 0.56%.
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After coupling pyroglutamate, the fragment was cleaved from the resin and isolated.
13:50
With the heptamer fragment prepared, we proceed to the second phase of the synthesis, beginning with Fmoc hydrazine Chlorotrityl resin, Fmoc-NH-NH-CTC resin.
14:02
The first step involved coupling Fmoc proline using carbodiimide and HOBt chemistry, followed by Fmoc deprotection.
14:10
At this point, we introduced arginine in its HCl salt form to prevent delta lactam formation.
14:16
So Fmoc-Arg(HCl) was then coupled using carbodiimide chemistry with Oxyma as a coupling additive and Oxyma stabilises the activated ester, reducing unwanted side reactions and improving coupling efficiency.
14:31
After deprotecting the Fmoc group from the arginine side chain, the guanidinium chloride group was free based.
14:37
While this approach avoids delta lactam formation, it doesn't fully prevent side reactions from nucleophilic attack during future coupling.
14:46
So to minimise these side reactions, we reduce the frequency of couplings following the step.
14:52
We then coupled the heptamer to arginine before cleaving the fully assembled goserelin impurity E with 1% TFA in dichloromethane, yielding a crude purity of 69%.
15:04
Following reverse phase chromatography purification, we obtained a final yield of 10.8% and then epimerisation purity of 98.9%.
15:15
So you can see the crude HPLC here shows a product peak with a retention time of approximately 16 min with baseline separation from other minor impurities, which makes purification theoretically simple and efficient.
15:30
So in conclusion, for this portion of the talk, the principles of green chemistry has enabled for the efficient and sustainable synthesis of both our peptide drug conjugate and goserelin API impurity.
15:43
We should see from this an increase in atom economy by the reduction of unnecessary protection groups, specifically T-butyl, PBF, trityl and tBoc.
15:53
We utilised safer solvents by the exclusion and replacement of diethyl ether and trifluoroacetic acid.
15:59
So now we're going to talk about the green approaches for conjugating the peptide to the oligo.
16:06
So peptide oligonucleotide conjugates are the hybrid molecules I explained earlier that combine the two moieties through covalent linkages, enhancing the delivery and stability of therapeutic nucleic acids.
16:18
POCs, as I will refer to them throughout the presentation, can improve cell targeting, uptake, intracellular trafficking that makes them very valuable in drug development and gene therapy applications.
16:30
So linker design may involve a broad range of chemistries tailored to achieve specific release mechanisms.
16:36
Some linkers incorporate reversible functional groups, such as disulfide bonds, which remain stable in oxidative environments but cleave under the reductive conditions inside cells.
16:48
Other linkers, though irreversible, are cleaved by intracellular enzymes.
16:52
For instance, sequence specific substrates like the VC-PABC are cleaved by lysosomal cathepsin B, facilitating targeted drug release.
17:02
In contrast, non-reversible linkers such as triazoles formed through click reactions or Thioetha bonds remain stable under the most biological conditions, making them useful for applications where prolonged stability is required.
17:18
So click chemistry, specifically the copper catalysed azide-alkyne cycloaddition, offers significant advantages over the traditional Huisgen’s 1,3-dipolar cycloaddition.
17:30
While the original Huisgen reaction forms a mixture of 1, 4 and 1, 5 triazoles under thermal conditions, click chemistry provides a highly selective, fast, and efficient synthesis of 1, 4 disubstituted triazoles under mild conditions.
17:45
The use of copper catalysts in click chemistry drastically reduces reaction time and enhances product yields, making the process more practical and versatile for a wide range of applications, including drug development, biomolecule labelling, and material science.
18:02
Additionally, click chemistry is compatible with aqueous conditions and a variety of functional groups, offering greater flexibility and biocompatibility compared to that uncatalyzed weakened reaction.
18:13
And crucially, click chemistry enables the use of green chemistry methodologies.
18:18
So macrocyclic peptides, especially head to tail lactams, offer several advantages.
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They're very big right now, including enhanced systemic stability such as resistance to proteolysis, improved binding affinity and specificity, and more features as well.
18:33
A wide range of modalities exist for the macro-cyclisation of peptides and can generally be characterised into either side chain to side chain, i.e., disulfide bridges, head to tail in the case of peptide lactams, back to bone to backbone and any variation of the three.
18:50
The chemistry and functionality of cyclisation may involve amide linkage, IL actin bridge, disulfide bridge, thioether sulphide bridge, alkene bridge, hydrocarbon stabled, and others.
19:02
For POC assembly, the ISI group is typically installed on the peptide moiety due to the chemical stability and compatibility with SPPS. So we typically prepare POCs with alkynes on the oligo and azides on the peptide, though the functionalities can be swapped if needed.
19:20
Standard alkyne groups require copper catalysis to facilitate the click reaction.
19:25
However, by using strained alkynes such as DBCO or BCN, strain promoted azide alkyne cycloaddition or SPAC allows for copper free conjugation.
19:36
The absence of copper can be compensated for by the kinetic advantages of ring strain.
19:41
Whether we use traditional copper catalysed click chemistry or copper free conditions.
19:45
The reaction can be performed in environmentally friendly solvents such as water and ethanol.
19:51
So in this example we use 5-Ethynyl-2'-deoxyuridine, a nucleoside analogue with the five positions of the uridine base modified with an ethynyl group, making it suitable for bioconjugation applications involving click chemistry.
20:06
The click reaction is carried out with a purified alkyne bearing oligonucleotide and an azide functionalized peptide.
20:13
The reaction is catalysed by copper sulphate with ascorbate as reducing agent and THPT, Tetramethyl-1,3-propanediamine serving as the ligand.
20:24
THPT acts as a chelating agent binding to copper ions and stabilising the copper catalyst, thereby enhancing the efficiency of the reactions by helping maintain copper in its active oxidation state.
20:36
The reaction is conducted in an eco-friendly aqueous phosphate buffer under mild temperature conditions with yields from 90% to quantitative.
20:51
Diode array detection or DAD is important for identifying impurities in peptide and oligo molecules.
20:58
Wavelengths at approximately 220 nanometres are used to detect the amide bond absorbents in peptides, while detection at 260 nanometres targets the aromatic side chains of the peptide and the base of the oligo moiety, so a range of strained alkynes can be used for click conjugations of POCs.
21:16
BCN is advantageous for faster reaction rates with additional ring strain of cyclopropane, while DBCO is more stable under physiological conditions, making it a common reagent in biorthogonal chemistry.
21:27
The scheme on the right illustrates our use of DBCO PEG NHS ester and its incorporation into the amino modified 5 oligo.
21:35
So the alkyne group of DBCO then reacts with the azide on that lysine side chain of the peptide.
21:41
Chiral BCN reacts with azides to form stereoisomers, which results in the generation of diastereomers.
21:47
This reaction is driven by the distinct spatial configurations of the chiral BCN structure, where the introduction of the azide group can occur at different positions relative to the chiral centres.
21:57
The resulting diastereomers exhibit different physical and chemical properties.
22:01
Unlike BCN, which forms stereoisomers, DBCO reacts with azides to produce regioisomers.
22:07
This reaction can lead to different structural isomers depending on the position where the azide group actually attaches, and that provides additional complexity and diversity in the conjugation products.
22:17
The selection of dyes for reported functionality will depend on the specific application or assay with the ability to convey covalently attach a broad variety of chemical dyes to either the peptide or oligonucleotide moiety, offering flexibility in tailoring conjugates to meet the needs of different experimental setups or detection methods.
22:36
Labelling POCs with fluorescent dyes is a widely used technique for tracking, detecting, and quantifying peptides in various biological applications.
22:43
The choice of dyes depends on several factors, including the intended application, the detection method, the chemical properties of the peptide and dye. Attachment sites for dyes can differ based on the design of the conjugate.
22:54
However, in this case, we attach FAM to the N terminus of the peptide, and key considerations for fluorescent labelling are always going to be brightness, water solubility, membrane permeability, contrast, quenching properties, and toxicity.
23:12
The VC-PABC cleavable linker can be easily incorporated into these conjugates and is independent of the conjugation site, so that allows for flexibility in designing POCs while maintaining functionality.
23:25
The cleavable valine-citrulline dipeptide sequence is specifically recognised by certain enzymes within the target cells, providing a controlled release mechanism for the peptide component.
23:35
Once the conjugate reaches the target site, these enzymes cleaves at the Val-Cit, releasing the oligo cargo inside the lysosome.
23:42
This linker is designed to be stable under physiological conditions, ensuring suitability for using complex biological systems without premature cleavage.
23:49
I just want to make sure I prioritise this next slide.
23:59
And if anybody would like to speak about this later, sorry, we are at booth 43.
24:03
Would love a conversation.
24:04
I apologise for running over parallel POC synthesis allows us to synthesise the peptide and oligo concurrently, significantly reducing turn around.
24:12
The oligo and peptide can be prepared and released within two to three weeks, well with conjugation occurring the third week.
24:17
So with parallel POC manufacturing release and delivery, this entire process can be completed in about four weeks.
24:22
So just an enormous thank you to the CPC Scientific R&D team who make these talks possible.
24:28
As I said, if you'd like a conversation regarding any of this, the post talk, myself and three of my colleagues, we will be at booth 43, would love for the discussion of anything pertaining to this.
24:36
Thank you very much.
