Webinar: Benefits of Monodisperse Activated PEGs in ADC Development
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Watch the webinar here: http://bit.ly/PEGsWebinar As the field of antibody-drug conjugation chemistry has advanced, the use of linkers to impart "drugability" has also been growing. Recent literature shows a variety of linear and branched monodisperse PEGs being called on to modify the PK/PD profile of some of the most active but lipophilic payloads. In this webinar, we will survey the types of PEGs that are used in ADCs and give examples of successful implementation to change the biophysical properties of the construct. In addition, we will focus on PEGs and why activated PEGs are widely used to improve the pharmacokinetics of drugs (such as pegylated proteins, peptides etc.). Raw materials used in this field may have a variety of polydispersity. However, when it comes to the use of activated PEGs as linkers for ADCs, the requirements are significantly higher. Therefore, the choice and control of the PEG linker is crucial for the successful development and accelerated time to market for your ADC. This webinar addresses critical success factors such as: • Survey of current PEG enhanced ADCs • Why PEGs are used designing ADCs • Critical parameters for aPEGs used as linkers • Requirements in terms of analytical capabilities
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Webinar: Benefits of Monodisperse Activated PEGs in ADC Development
- 1. Merck KGaA Darmstadt, Germany Lisa L. McDermott Director, Process and Analytical Development Olaf Bartram Global Product Manager, PEGs and Innovative Technologies Benefits of monodisperse & Activated PEGs to Accelerate ADC Development
- 2. The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada.
- 3. Monodisperse PEGs in ADCs Presentation Overview 2 Executive Summary 3 1 4 Hydrophobicity Challenges Summary Critical PEG Attributes
- 4. • Aggregation of Final ADC • Drug Load Limitations • High loaded species need to be removed • Limit R&D to low DAR Analogues • PK/PD impact • Fast clearance rate • Rate increased with increasing DAR • increased lipophilicity • Decreased therapeutic window Monodisperse PEGs in ADCs Challenges Encountered with Hydrophobic Toxins Impact of toxin solubility
- 5. • Alternative scaffolds • Optimize linker to offset lipophilicity of payload • Site specific payload attachment • Payload bearing site modification • SAR mAb, payload, linker and conjugation method • Alternative Formulation or Additives Monodisperse PEGs in ADCs Challenges Encountered with Hydrophobic Toxins Tools
- 6. Monodisperse PEGs in ADCs Impact of Drug Loading on Efficacy Higher DARs - Accelerates ADC clearance Higher DARs - Non-linear impact on tumor growth Kevin J. Hamblett et al. Clin Cancer Res 2004;10:7063-7070 Pharmacokinetics of cAC10 and cAC10– antibody-drug conjugates. SCID mice were injected via the tail vein with 10 mg/kg cAC10, E2, E4, and E8. Plasma samples were analyzed by ELISA to determine the antibody or antibody-drug conjugate concentration. DAR2 DAR8 DAR4 DAR0
- 7. Why PEGs can help? • Improved pharmacokinetics • Enhanced solubility • Improved stability • Increased circulation time • Decreased amount of protein required for therapeutic efficacy • Decreased dosing frequency due to optimized biodistribution • Reduced renal clearance increases circulation time • Decreased toxicity • Improved safety profile • Reduced immunogenicity • Reduced antigenicity • Reduced proteolysis • Generally recognized as safe (GRAS status by FDA) Dumb-Bell like PEG-drug structure Monodisperse PEGs in ADCs
- 8. Monodisperse PEGs in ADCs Transfer of the protein pegylation concept to ADCs with DAR8: what is the desired PEG size? Maleimide-PEG-Glucuronide-MMAE with variable PEG lengths Burke et al., Mol Cancer Ther 16(1) 116
- 9. Monodisperse PEGs in ADCs Pharmacokinetic and clearance is a function of PEG length, demonstrated for a DAR8 ADC Burke et al., Mol Cancer Ther 16(1) 116
- 10. Monodisperse PEGs in ADCs Increasing PEG length shows a positive effect on Pharmacodynamic and increased tolerability and mouse survival rate Burke et al., Mol Cancer Ther 16(1) 116
- 11. Monodisperse PEGs in ADCs Selected Microtubule Targeting Drug Linkers that contain PEG Groups DBCO-PEG4-linker MMAF payload (http://www.globalengage.co.uk/biologics/docs/Steiner.pdf , Sutro) PEG4, Mar. Drugs 2017, 15, 99 https://pdfs.semanticscholar.org/40fe/c075fbef09881fd87402276267645af078a7.pdf Mol Cancer Ther; 16(1) January 2017, 116-123 SGN-CD48a, PEG12, SeattleGenetics
- 12. Monodisperse PEGs in ADCs Selected DNA Targeting Drug Linkers that Contain PEG Groups Nature Reviews, 2017 (16) 315 PEG3, Immunogen ACS Med Chem Lett 2016, 7, 983 PEG8, Spirogen Mol Cancer Ther 2018, 17(10), 2176 , Immunogen Oncotarget Vol.6, No.26, 22496 PEG8, Immunomedics
- 13. Monodisperse PEGs in ADCs Linear and branched PEG in ADC examples CD-70, PEG2+2*4 , Ambrex US20150141624A1 HU2H11, PEG5, Sanofi Molecules 2017, 22(8), 1281
- 14. Monodisperse PEGs in ADCs Presentation Overview 2 Executive Summary 3 1 4 Hydrophobicity Challenges Summary Critical PEG Attributes
- 15. 1. PEGs are well known substances, generally recognized as safe (GRAS), biologically inert with hydrophilic properties 2. Widely used as non-toxic auxiliary materials in multiple technical applications and amongst others in cosmetics and food and pharmaceuticals (such as medical treatment of constipation) 3. After synthetic activation PEGs can be conjugated (PEGylated) with proteins, peptides, small molecules or can act as linkers in ADCs. 4. PEGylated products show improved pharmacokinetics, increased circulation time and decreased toxicity. Introduction Polyethylene Glycol (PEG) Monodisperse PEGs in ADCs
- 16. 16 Monodisperse PEGs in ADCs Polydisperse PEGs • Statistical distribution results from equal probabilities of ethylene oxide polymerizations. There is only a neglectable difference between PEG fractions of different chain lengths. • Thus even under the most ideal conditions a distribution will be obtained. If the conditions are not ideal, the distribution will deteriorate even more. Polymerizations will result always in a molecular weight distribution.
- 17. 17 Monodisperse PEGs in ADCs Monodisperse PEGs • Monodisperse PEGs do have an impact on the pharmacokinetic and pharmacodynamics of your ADC • Production of monodisperse PEGs by purification of polydisperse PEGs or synthesis of defined PEG units with the same molecular weight • Typically used for ADCs are PEG4 – PEG24 units • Maleimide as functional group is targeting cysteine at the antibody • Functional azide group connects to payload • High purity and defined impurity profile are beneficial for registration • High GMP level required for successful commercialisation of your ADC ADCs require monodisperse PEGs as linkers with a uniform molecular mass.
- 18. 18 Monodisperse PEGs in ADCs Monodisperse PEGs • Target purity of 90-95% or higher - depends on analytical method • Sensitive to oxidation and hydrolysis - determined via derivatization • Adsorption of elemental impurities (heavy metals), salts, polar compounds Control elemental impurities via ICP-MS Control residual solvents and reagents via GC, IC, KF and HPLC Purity of monodisperse PEGs is the main quality parameter.
- 19. Separation of single PEG units in relation to PEG size Figure: Separation of different PEGs using HPLC-CAD. Sample concentration for all: 0.1 mg/mL. Source: C. Theiss, U. Holzgrabe / Journal of Pharmaceutical and Biomedical Analysis 160 (2018) 212–221 Monodisperse PEGs in ADCs Lower number of PEG units are easier to separate. Approx. 7-23 PEG units
- 20. Comparison of detector responses CAD ELSD UV Mass on detector Relativedetectorresponse Both ELSD and CAD have non-linear response curves compared to UV. ELSD tends to underestimate low level signals. LoQ is typically lower for CAD compared to ELSD. CAD overestimates small impurities. For both - CAD and ELSD - small, semi- volatile compounds might be evaporated and therefore not visible or underestimated. Understanding the characteristics of the different detectors is key for the correct interpretation of analytical results. CAD = Charged Aerosol Detector ELSD = Evaporative Light Scattering Detector UV = UV Detector Monodisperse PEGs in ADCs CAD-ELSD-UV Widely used UV detectors are not applicable for PEGs as such.
- 21. ELSD Purity: 98.9 % area Largest impurity: 1.0 % area Number of impurities: 5 Monodisperse PEGs in ADCs Case study: Same sample measured with Evaporative Light Scattering Detector (ELSD) and Charged Aerosol Detector (CAD)
- 22. ELSD Purity: 98.9 % area Largest impurity: 1.0 % area Number of impurities: 5 Monodisperse PEGs in ADCs Case study: Same sample measured with Evaporative Light Scattering Detector (ELSD) and Charged Aerosol Detector (CAD) CAD Purity: 89.0 % area Largest impurity: 5.8 % area Number of impurities: 18 Interpretation of CAD and ELSD derived results need solid analytical knowledge
- 23. Penta ethylene glycol (PEG 5) Hexa ethylene glycol (PEG 6) Monodisperse PEGs in ADCs Gas Chromatography (GC) for volatile compounds PEGs up to approx. six ethylene glycol units are volatile enough to be analyzed by GC.
- 24. Monodisperse PEGs in ADCs Presentation Overview 2 Executive Summary 3 1 4 Hydrophobicity Challenges Summary Critical PEG Attributes
- 25. • Low solubility of lipophilic payloads limits high DAR ADCs • PEGs in ADC linker enables higher soluble ADCs with higher DARs • Quality, especially purity of PEGs in ADC is a key parameter for regulatory success • Determination of PEG purity depends significantly on analytical setup Monodisperse PEGs in ADCs Summary Overcoming ADC Solubility Issues
- 26. Merck and the vibrant M are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources. © 2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.
Editor's Notes
- Non-denaturing water-soluble = chem properties for biological app, Non toxic, non-immunogenic How do they impact potency selectivity and PKPD
- Non-denaturing water-soluble = chem properties for biological app, Non toxic, non-immunogenic How do they impact potency selectivity and PKPD
- Thank you, Lisa and welcome from my side!
- Before we will talk about critical attribute of Polyethylene Glycol and the analytical challenges connected with that, I would like to give a short introduction. PEGs are used in many applications and industrial field, e.g. as humectant in food, dispersant in cosmetic, or even as ingredient of ink for inkjet printers. They are very water-soluble and generally recognized as safe (GRAS). The fact that they are also biologically inert make PEGs applicable for medical treatment. A well known application is the treatment of constipation. Via the addition of functional groups either on one or both ends of the polymer, PEGs can be conjugated to proteins, peptides or small molecules. Those –s so called- PEGylated products show improved pharmacokinetics, increased circulation time, decreased toxicity. Today we are focusing on PEGs as linker between an antibody and a payload molecule.
- The classical synthetic of PEGs shown here at the left side, is a polymerization of ethylene oxide that connects randomly and with an equal probability to the build polyethylene chains. The result is a statistical distribution of the molecular weight of this polydisperse PEG, shown in the MALDI-TOFF chromatogram on the right side. Each signal reflects the amount of PEG units of a polydisperse PEG. The level of distribution depends on the process conditions during polymerization. Needless to say that using such a PEG with multiple MW fractions as linker for ADCs would be not beneficial.
- With the use of so called monodisperse PEGs this issue can be solved. Since PEGs as linkers do impact significantly the pharmacokinetics and pharmacodynamics of ADCs, highly defined and pure PEGs are necessary. Monodisperse PEGs are produced either via purification of polydisperse PEGs or the synthesis of uniform PEG units leading to a defined molecular weight. Typical chain lengths used as linkers are 4 to 24 units of PEGs but also larger monodisperse PEGs can be manufactured. The right graph shows a PEG52 analyzed with MALDI-TOF with a clear separation of each PEG unit. A typical functional group is Maleimide to conjugate with the thiol containing amino acids of the anti body. At the other end of the PEG, an Azide group is used to connect to the payload via click chemistry. Beside the impact on pharmacokinetics and pharmacodynamics, the uniform molecular weight of the PEG makes it much easier to be analytically characterized. This can be beneficial the registration of the ADC during all clinical phases. Last but not least supply monodisperse PEG meeting c-GMP requirements, is crucial when it comes to commercialization.
- The target purity of monodisperse PEGs is usually in the range of 90-95%, but depends very much on the chosen analytical setup. I will show an example for that. Regarding sensitivity of oxidation and hydrolysis monodisperse PEGs don’t differ from polydisperse. To control this a chemical derivatization is needed in front of every analytical run. PEGs can easily adsorb elemental impurities, salt or polar compounds, which is analyzed by state of the art methods for ICP-MS, GC, IC, Karl-Fisher and HPLC.
- When it comes to PEG analytics we are facing some challenges. The separation of PEG units depends on the molecular weight, illustrated here by a graph from Theiss and Holzgrabe. The ADC relevant range of PEG 300-1000 Dalton shows already a significant difference in peak separation which has an impact on differentiation between purity and impurities. This has to be taken into account especially during the evaluation of your optimal PEG length and the respective analytical development.
- In addition the use of different detectors of HPLC systems for the determination of purity needs to be taken into consideration. With the absence of double bindings the most preferred UV light detector can not be used for monodisperse PEGs. Applicable detectors are ELSD and CAD, but both need a solid analytical expertise for the correct interpretation of results. ELSD is underestimating low level signals, on the other hand CAD overestimates them. Both detectors evaporates volatile compounds which are therefore not visible.
- Looking at an example of an monodisperse PEG measured with a HPLC-ELSD delivers a purity of almost 99%, 5 impurities, the largest with 1 aerea %.
- The same sample measured with a CAD leads to a drop in purity to 89% an increased number and content of impurities. This shows how diverse results can be using different analytical methods. Since there is no clear scenario whether using the one or the other technology, it is more about using both and to have the right partner with a broad expertise in analytical development to draw the right conclusion out of the results.
- On my last slide I would like to show that Gas chromatography is an option for small PEG units. Up to PEG6 units are volatile enough. Shown her is a PEG5 and PEG6 unit. Last one also multiple impurities have been detected. Now its time for two poll questions. How do you determine the purity of your monodisperse PEG? -read- And What do you or what would you request most from your PEG supplier? -read- Thank you your input is very valuable for us.
- Finally I would like to summarise Lisa explained that the low solubility of the lipophilic payloads is one of the main issues for ADCs PEGs used as linkers can help to overcome this to achieve higher DARs Especially the purity of monodisperse PEGs will determine the successful development & registration of your ADC The analytic of monodisperse PEGs is impacted by the used or available setup. ELSD CAD can be used but results need interpretation. GC is an option of smaller PEG units up to 6.