Drug substance for early clinical phase initiation – the underestimated risk of unexpected N‑nitrosamine impurities

The chemical synthesis and supply of a drug substance is a time critical step in the process of defining and selecting a lead drug candidate for progress into a toxicology program and subsequently into first-in-human clinical trials. The presence of impurities can hinder toxicology results and delay entry into the clinic therefore it is important to identify and mitigate this risk. There are multiple potential causes of the formation or introduction of impurities regardless of the synthesis route used, including the materials used, the process conditions, potential cross-contamination, etc., and these can lead to several types of impurities.

The recent recalls of sartans and other medicinal products due to the presence of N‑nitrosamine impurities, increased the awareness of the impact of material selection, process changes, and cross-contamination, as well as the limitations of the existing analytical methods used. Many N‑nitrosamines are known to be carcinogenic to animals and are reasonably anticipated to be human carcinogens.

Authors: Lieven Van Vooren – Scientific Director, Arno Vermote – Senior CMC Writer

Table 1: Types of impurities in drug substance

The case of N-nitrosamine impurities

N‑nitrosamines can be formed by a nitrosating reaction between an amine (secondary, tertiary, or quaternary amines) and a nitrosating agent (e.g., nitrite salts) under acidic reaction conditions. Nitrosating reactions can potentially occur during production under certain conditions and when nitrogen-containing solvents, reagents, and other raw materials are used. In addition impurities can be carried over during the manufacturing process when using already contaminated equipment or reagents. In cases where N-nitrosamines can form or are carried over during production, the impurities should be controlled and removed during the manufacturing process.

Regulatory requirements and documentation

Since this discovery of N‑nitrosamines in pharmaceuticals, regulatory authorities in Europe and the U.S. have undertaken numerous activities to assess the extent, understand the root cause, establish expert working groups, and develop appropriate N‑nitrosamine impurity guidelines.

One of the essential parts of Health Authority regulations is a risk assessment of the chemical synthesis. Based on a solid understanding of the root causes of formation of N‑nitrosamines and relevant experience in chemical synthesis, the occurrence of N‑nitrosamines in the drug substance can be avoided and/or controlled sufficiently. Ardena ensures compliance with these guidelines through coordinated action among its Centers of Excellence. The Ardena Dossier Development team has a critical role to play in all this and has written over 100 nitrosamine risk assessment reports for multiple customers.

These risk assessments consist of 3 steps.

The first step is to perform a comprehensive risk evaluation to identify if there is a risk of N-nitrosamines being present in the API and/or finished product. As illustrated in Figure 1, both the potential formation of N‑nitrosamines and the cross-contamination risk are scrutinized.

Figure 1: N-nitrosamine investigation – Step-by-step plan, Step 1

The presence of amines and nitrosating agents in combination with the reaction conditions and potential inducers or inhibitors of nitrosamine formation are examined. Potential cross-contamination is evaluated by looking at recycled materials, multipurpose equipment, packaging, etc.

As illustrated in figure 2, if a risk is identified, confirmatory analytical testing (i.e., Step 2) is required in order to confirm or refute the presence of nitrosamines. If the presence of nitrosamines is confirmed, effective risk mitigating measures should be implemented (i.e., Step 3).

Figure 2: N-nitrosamine investigation – Step-by-step plan, Step 2 & 3

Proactive risk mitigation – Case study

N-nitrosamine risk assessments are necessary not only for commercial products but are also highly recommended during the development phase to investigate potential contamination.

The importance of assessing potential N‑nitrosamine contamination early in development can be illustrated by a typical customer request that Ardena addressed successfully In a drug substance synthesis project, one of the raw materials was a piperidine derivative. The manufacturer of this raw material indicated that during the synthesis piperidine-like structure secondary and/or tertiary amines were used in the same and subsequent steps as nitrosating agents. As the raw material was introduced in one of the final steps of the drug substance synthesis, it could not be excluded that N‑nitrosamines would be present in the eventual drug substance. In addition, theoretical purging calculations did not conclude that the potential nitrosamines were sufficiently purged. As a proactive mitigation plan in order to avoid analytical testing (i.e., Step 2 in Figure 2), the synthesis route was modified with the introduction of the piperidine derivative at the start of the synthesis as well as the addition of an extra crystallization step in the end.

By monitoring this potential nitrosamine formation early in development, we were able to react quickly and immediately choose the most suitable synthesis route for further development, without the risk of having to change the synthesis later in development.

To ensure the rapid initiation of toxicology studies and entry into the clinical phase, it is not sufficient to rely only on controlling the impurities below phase-appropriate specification limits. The synthesis of the drug substance must also be well understood, controlled, and accompanied by phase-appropriate analytical methods. To meet the regulatory requirements comprehensive documentation is essential as well as confidence in the scalability of the chemical synthesis to support the later clinical phases and commercialization. More information about this in our whitepaper titled ‘Phase-appropriate analytical method development’. To avoid undesirable and unexpected impurities during later phases, these should be considered during synthesis, analytical method development, and regulatory documentation.

The relevant CMC documentation and if necessary direct support in FDA, pre-IND, or EMA scientific advice meetings is provided by Ardena’s regulatory experts. More information about this in our whitepaper titled ‘Leverage the power of CMC in early drug development’.

N-nitrosamines – Acceptable limits in medicinal products

A specific challenge of N‑nitrosamine impurities is their very low acceptable levels, as defined by their acceptable daily intake (ADI; Table 2). Consequently they depend on the targeted daily clinical dose which can quickly exceed the ADI.

Table 2: Some specific N‑nitrosamines and their acceptable daily intake (ADI)

If multiple different N‑nitrosamines are present, the ADI limit must be calculated on the sum of all N‑nitrosamines and their specific ADIs. Such low thresholds imply that even traces from chemical precursors or non-optimized process conditions can lead to limits being exceeded. Since the FDA guidelines require analytical methods for N‑nitrosamines which have limits of quantitation (LOQ) in the parts-per-billion (ppb) range to ensure that the ADI is not exceeded, the management of N‑nitrosamine impurities must be part of the risk assessment and mitigation process.

An integrated approach to manage N‑nitrosamine impurities in clinical drug supplies

As illustrated above, by carrying out a risk assessment before deciding on the synthesis route of a new drug substance Ardena ensures that impurities are reduced to a minimum and that a targeted analysis is carried out regarding the possible impurities. This risk assessment is later formalized to support the regulatory documentation according to the ICH Q11 guideline.

By their nature N‑nitrosamines are impurities that occur in the ppm range and can be present in various analytical artifacts. In addition to the regulatory need to determine the presence of N-nitrosamines in marketed pharmaceutical products, there is a demand for appropriately sensitive and specific analytical methods. The challenge is that the methods must be developed and qualified specifically for each drug substance and/or drug product. Analytical method development includes appropriate sample preparation, separation, and detection of individual N‑nitrosamines. In the case of N‑nitrosamines, thermal and pH stability must be taken into account during sample preparation. Due to the extremely low concentration of the N‑nitrosamine impurities extraction methods are also usually used, mostly solid phase extraction (SPE), liquid-liquid extraction (LLC), or direct liquid extraction (DLE). The resulting sample is then further separated by gas chromatography (GC) or liquid chromatography (LC) and the individual N‑nitrosamine artifacts determined.

The limit of quantification (LoQ) plays an important role as it provides the minimum level at which an analyte can be quantified with acceptable accuracy, and it should be used for N-nitrosamine testing and decision-making

To cover the full range of possible N‑nitrosamines, Ardena develops a product-specific approach from one or several analytical methods that provide the highest possible resolution while being cost- and time-efficient.

Conclusion

Ardena’s integrated science-driven and concerted approach in drug substance synthesis, analytical method development, and regulatory documentation assures compliance with the latest N‑nitrosamine impurity guidance and requirements.

References

Alsante et al (2014). Recent trends in product development and regulatory issues on impurities in active pharmaceutical ingredient (API) and drug products. Part 1: Predicting degradation related impurities and impurity considerations for pharmaceutical dosage forms. AAPS PharmSciTech 15(1): 198-212.
Bharate SS (2021). Critical analysis of drug product recalls due to nitrosamine
impurities. J Med Chem 64(6): 2923-2936.
Brown et al (2016). Analysis of past and present synthetic methodologies on medicinal chemistry: Where have all the new reactions gone? J Med Chem 59(10): 4443-4458.
Charoo et al (2019). Lesson learnt from recall of valsartan and other angiotensin II receptor blocker drugs containing NDMA and NDEA impurities. AAPS PharmSciTech 20(5): 166.
Chidella KS et al (2021). Ultra-sensitive LC-MS/MS method for the trace level quantification of six potential genotoxic nitrosamine impurities in telmisartan. Am J Anal Chem 12: 227-240.
EMA (2020). Assessment Report. Nitrosamine impurities in human medicinal products. EMA/369136/2020. https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-assessment-report_en.pdf
EMA (2020). Meeting highlights from the Committee for Medicinal Products for Human Use (CHMP) 12-15 October 2020 https://www.ema.europa.eu/en/news/meeting-highlights-committee-medicinal-products-human-use-chmp-12-15-october-2020
EMA (2021). European Medicines Regulatory Network approach for the implementation of the CHMP Opinion pursuant to Article 5(3) of Regulation (EC) No 726/2004 for nitrosamine impurities in human medicines. EMA/425645/2020. https://www.ema.europa.eu/en/documents/referral/european-medicines-regulatory-network-approach-implementation-chmp-opinion-pursuant-article-53/2004-nitrosamine-impurities-human-medicines_en.pdf
EMA (2022). Fifth Nitrosamine Implementation Oversight Group (NIOG) meeting. https://www.ema.europa.eu/en/events/fifth-nitrosamine-implementation-oversight-group-niog-meeting
EMA (2022). Questions and answers for marketing authorisation holders/applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No 726/2004 referral on nitrosamine impurities in human medicinal products. EMA/409815/2020. https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-questions-answers-marketing-authorisation-holders/applicants-chmp-opinion-article-53-regulation-ec-no-726/2004-referral-nitrosamine-impurities-human-medicinal-products_en.pdf
FDA (2019). GC/MS headspace method for detection of NDMA in valsartan drug substance and drug products. https://www.fda.gov/media/115965/download
FDA (2021). Control of nitrosamine impurities in human drugs. Guidance for industry. February 2021. https://www.fda.gov/media/141720/download
FDA (2021). Updates on possible mitigation strategies to reduce the risk of nitrosamine drug substance-related impurities in drug products. https://www.fda.gov/drugs/drug-safety-and-availability/updates-possible-mitigation-strategies-reduce-risk-nitrosamine-drug-substance-related-impurities
Fritzsche M et al (2022). NDMA analytics in metformin products: Comparison of methods and pitfalls. Eur J Pharm Sci 168: 106026.
ICH Q11 (2012). Development and manufacture of drug substances (chemical entities and biotechnological/biological entities): https://database.ich.org/sites/default/files/Q11%20Guideline.pdf
King et al (2020). Ranitidine – investigations into the root cause for the presence of N‑nitroso‑N,N‑dimethylamine in ranitidine hydrochloride drug substances and associated drug products. Org Process Res Dev 24(12): 2915−2926.
Leistner et al (2020). Risk assessment report of potential impurities in cetirizine dihydrochl J Pharm Biomed Anal 189: 113425.
Parr MK & Joseph JF (2019). NDMA impurity in valsartan and other pharmaceutical products:
Analytical methods for the determination of N-nitrosamines. J Pharm Biomed Anal 164: 536–549.

Bioanalytical characterization and monitoring of ADCs supporting safety and efficacy from pre-clinical to clinical

Antibody-Drug-Conjugates (ADC) have emerged as an efficient technology to deliver cytotoxic drugs into cancerogenic cells. The mechanism is based on an antigen-mediated uptake of a cytotoxic drug conjugated antigen via clathrin-mediated endocytosis and release of the drug after lysosomal cleavage to its intracellular target.

With 14 ADCs currently approved worldwide, ADCs have proven their therapeutic value in increasing efficacy and reducing toxicity especially in oncology. Recent research however has shown that the cytotoxic drug mechanisms, drug-antibody ratio, antibody penetration and processing, resistance and off-target drug effects of ADCs can be further enhanced.

This continues to drive high investments into ADC research and development increasing the demand for external partnerships and expert support.

Translating the drug design concept of an ADC into a final product remains a major challenge. The cytotoxic small molecule drug can covalently bind to the monoclonal antibody at multiple sites during the synthesis leading to a heterogeneous mixture of unfavorable ADC products. In order to move into the preclinical and early clinical phase the exposure-response relationship for efficacy and safety must be established. For IND/IMPD filing, a bioanalytical strategy tailored to each of the ADC components is required by the regulatory authorities.

Authors: Melloney Dröge, PhD, Francesco Bonardi, PhD
This whitepaper has been written with the contribution of Byondis B.V.

With 14 ADCs currently approved worldwide, ADCs have proven their therapeutic value in increasing efficacy and reducing toxicity especially in oncology [1]. Recent research however has shown that the cytotoxic drug mechanisms, drug-antibody ratio, antibody penetration and processing, resistance and off-target drug effects of ADCs can be further enhanced [2].

This continues to drive high investments into ADC research and development increasing the demand for external partnerships and expert support.

ADC are complex molecules consisting of a target-specific antibody, a cytotoxic drug (payload) and a covalent linker connecting the drug to the antibody. ADCs have a narrow therapeutic window, and their effectiveness may be compromised by undesired toxicity based on metabolic instability, poor pharmacokinetics, or off-target effects [3]. This risk can be mitigated by careful characterization, as well as in-vitro and in-vivo assessment prior to first in human studies, to predict the impact of their inherent heterogeneity and potential anti-drug antibodies on their pharmacokinetics (PK), pharmacodynamics (PD), safety, and efficacy [4]. To ensure efficacy and minimise toxicity outside the targeted tissue, the linkage and amount of the drug molecules bound to the antibody and the drug-antibody ratio (DAR) must be optimized and reproducibly manufactured. Robust analytical characterization and monitoring methods during the entire drug development process are essential in order to achieve the targeted product performance and the required regulatory documentation for IND/IMPD and NDA filing.

The R&D challenge

In contrast to a typical small molecule, ADCs consist of multiple elements which need to be identified during regulated bioanalysis: the total antibody content, the antibody conjugated with at least one payload, the free payload, and the total conjugated drug [5]. ADCs are also known to induce anti-drug antibodies (ADA) against structural parts of the ADC. ADAs can lead to neutralization and impact the efficacy of the ADC. It is therefore recommended that their presence is investigated early on in development [6]. The evaluation of immunogenic responses and the formation of ADAs in the preclinical and clinical phases remains a major challenge that requires a strategic and experience-based approach [7].

Developing the ADC specific bioanalytical strategy

The design of the bioanalytical approach must take into account the determination of the ADC with regard to the antibody, fully and partially conjugated antibody, off-target conjugated antibody, unconjugated drug, drug-conjugate metabolic stability, pharmacokinetics, and potential immunogenicity. In particular, detection of ADCs and their components in biological fluids may be challenging due to the complexity of the matrix.

Standard analytical methods do not exist for the bioanalytical characterization of the proteinic, chemical, and immunogenic components. The bioanalytical strategy of choice should therefore be unbiased by the bioanalytical platform and determined by a case-by-case scientific approach. During the design of the bioanalytical method the availability of specific reagents and equipment should be considered. (Figure 1).

Figure 1: Implementation of an analytical strategy during the transition from the pre-clinical to the clinical stage

Major principles of ADC assay methods

Selecting the right technology for the right target requires a high degree of expertise and in-house capabilities.

Liquid Chromatography (LC) is a versatile separation technology platform that can be operated in different ways to separate chemical and/or biological components from a variety of samples. Due to its versatility LC has emerged as the separation method of choice for ADCs in the growing field of biotechnological therapeutics. In particular when coupled to a Mass Spectrometer (LC-MS/MS), the separated components can be detected qualitatively and quantitatively with high precision and selectivity[8]. LC-MS/MS has proven capable of discriminating between two different co-administrated therapeutic antibodies in human serum [9].

The Ligand Binding Assay (LBA) platform is a detection method based on the formation of a complex between a receptor and a ligand. The receptor and/or one or more ligands are tagged or labeled to qualify and quantify the substrate of interest by measuring the intensity of the signal emitted by the complex. The most common LBAs are the enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence (ECL) or AlphaLisa, which can also be used for evaluation of immunogenicity and the formation of antidrug antibodies (ADA)[10].

When the use of LBAs is hampered by the lack of critical reagents or selectivity issues hybrid LC-MS/MS can be used as an alternative approach. The combination of immunoaffinity purification and LC-MS/MS (also referred to as immunocapture-liquid chromatography) allows the detection of ADCs in both in-vitro and in-vivo samples [11]. The approach consists in separating the targeted antibodies by pulldown followed by a proteolytic step and quantification of antibody specific protein sequences by LC-MS/MS. Hybrid LC-MS/MS provides quantitative information on the total antibody, total ADC, or total payload depending on whether the reagents used in the purification step target the antibody or the payload.

The role of reagents for ADC analytics

Analytical procedures for ADCs require highly specific and highly sensitive reagents compared to standard analysis. These reagents range from method specific e.g., biotin or ruthenium of the conjugated and unconjugated anti-body to the generation of anti-drug antibodies and anti-idiotype antibodies. The anti-idiotype antibodies which target the variable regions of antibodies must be identified and produced in sufficient quantity for each development program. Anti-idiotype antibodies represent the antigenic region specific for the antibody and are required for ADC selective PK and immunogenicity (ADA) analysis. These can be produced or in-vitro, for example by phage display of antibody libraries[6], or by animal immunisation and amplification in-vitro. Consequently, reagent manufacturing is a time critical step in bioanalytical development for ADC development and characterization.

The importance of regulatory compliance

Following selection of an ADC candidate for the preclinical phase all investigations and data must take into account the requirements for IND/IMPD submission, approval, and the first clinical study. The European Medicines Agency (EMA) and the Food and Drug Administration (FDA) have provided regulatory expectations for antibody therapeutic entities and recently additional guidelines have been drafted emphasising the importance of PK and immunogenicity assessments [16]. These requirements are part of the IND/IMPD application therefore the bioanalytical methods used must be sufficiently specific and validated for the scientific questions and fully documented according to these guidelines.

Case study

An innovative approach in ADC development to achieve better homogeneity and target tissue delivery is being pursued by Byondis. One of their lead compounds is a novel ADC developed by their unique platform technology utilizing a highly potent duocarmycin linker-drug (vc-seco-DUBA) for site specific conjugation, and hydrophobic linker-drug shielding to maintain its cytotoxic potency. After confirming the site-specific ADC conjugation versus random conjugation with their platform and the expected increase in efficacy in a xenograft mouse model[17], Ardena was requested to provide timely bioanalytical services to establish the pharmacokinetic profile of the ADC in the phase 1 clinical trial. A strategic plan was developed, coordinated by a dedicated project manager, for the bioanalysis in blood to quantify the total antibody, the conjugated antibody, and the payload concentration, and to determine potential ADAs and their impact on PK, PD, and immunogenic reactions. Due to the very low concentration of the ADC and the payload, the sensitivity and selectivity of the method needed to be able to determine qualitatively and quantitatively at pg/mL concentrations. Each assay format and analytical platform also required product specific reagents.

For the determination of the antibody and conjugated antibody, reagents for the LBA with high specificity to the antibody of the ADC were required. Heavy chain only antibodies (HcAbs) derived from camelids were used due to their immune specificity of the distinctive variable domain (VHH)[18]. Llamas were immunized with the naked ADC antibody and the VHH mRNA was collected from peripheral whole blood to obtain high quality RNA using Reverse Transcriptase Polymer Chain Reaction (RT-PCR). The RNA was then incorporated into phage display vectors in order to express the antibody and amplify the VHH of interest in E. coli [19]. After purification of the anti-idiotype VHH antibody (AIDA) against the ADC, the AIDA was labeled with biotin and Horseradish Peroxidase (HRP) for LBAs. In addition, magnetic beads covered with immune-capturing moiety for the ADC were also manufactured. These reagents were used to determine the total and conjugated antibody concentration by a sandwich ELISA streptavidin coated microtiter plate pre-coated with the AIDA and ADC respectively. The determination of the payload in pharmacokinetic studies is challenging due to the very low concentration, metabolites, and cleavage site (e.g. payload-linker-amino acid artefacts).

A highly sensitive LC-MS/MS method was developed to determine the payload and validated based on 8 calibration points in the range of 1.00 – 100 pg/mL. The payload and internal standard were isolated by liquid-liquid extraction from K2-EDTA plasma and separated by ultra-performance-liquid-chromatography (UPLC) followed by tandem mass spectroscopy (MS/MS). The calibration curve obtained for the payload showed a linear correlation across the expected in-vivo concentration in human plasma providing the required sensitivity, precision, and recovery (Figure 2).

Figure 2: Calibration curve for the payload in K2-EDTA plasma across the plasma concentration of 1.00 – 100 pg/mL

A sandwich ELISA was used to validate the conjugated antibody in human K2-EDTA plasma across 12 calibration points to cover the in-vivo concentration of 2.00 – 1250 ng/mL. The concentration of the total antibody in human K2-EDTA plasma was validated across 12 calibration points ranging from 10 – 1500 ng/mL using a sequential sandwich ELISA. The ELISA methods developed used different biotinylated capture antibodies coated on the surface of a streptavidin coated microtiter plate. After adding the substrate sample for a specific time under optimized conditions the specific anti-idiotype antibodies labeled with HRP were added to produce the concentration dependent signal. The bioassays were qualified for their selectivity, specificity, precision, and accuracy. In table 1 the validation results of the conjugated antibody determination are shown.

Table 1: Validation of the conjugated antibody concentration measurement in human K2-EDTA plasma across the expected in-vivo concentration for the pharmacokinetic study in humans

Alpha technology was used for the immunogenicity assessment due to its sensitivity when measuring ADAs in complex biologic fluids like plasma. Acceptor beads coated with the ADC and biotinylated ADC were produced for the assay. These reagents form specific complexes with the ADA and through excitation of the donor beads the emitted light signal of 615 nm is proportional to the amount of ADA present, and is quantified by running a standard curve[20].

Methods were developed and validated with ELISA, Alpha Technology and LC-MS/MS assays in the required ng/ml range according to the defined bioanalytical strategy and agreed timelines. The validation according to GLP and GCP requirements included regression type, selectivity, accuracy and precision, stability, dilution linearity, and robustness. A comprehensive documentation of the (bio)analytical methods relevant for the IND/IMPD filing was provided.

Conclusion

Targeted delivery of cytotoxic drugs by ADCs have fulfilled their potential and become part of the clinical repertoire for the treatment of cancer. Considering the potential of ADC application to the numerous unmet clinical needs, in-vitro and in-vivo bioanalytical methods are required to characterize the ADCs as well as to predict and finally prove the safety and efficacy during the translation from the pre-clinical to the clinical phase. The complexity and intrinsic heterogeneity of ADCs, their complex in-vivo distribution, metabolism and drug release patterns present challenges in bioanalytical characterisation, in-vivo prediction, and monitoring. Ardena’s multidisciplinary experts have designed a development program specific bioanalytical strategy which considers the required performance criteria for the individual analytical targets within the overall characterisation requirements and bioanalytical procedural framework. The broad services platform and scientific expertise at Ardena allows for the development of a bioanalytical strategy tailored to the properties of the therapeutic drug and therefore of the Sponsor. Ardena involves the Sponsor in every step of the process so that the methods can be customised to the smallest detail.

References

[1] Z. Fu, S. Li, S. Han, C. Shi, and Y. Zhang, “Antibody drug conjugate: the ‘biological missile’ for targeted cancer therapy,” Signal Transduction and Targeted Therapy, vol. 7, no. 1. Springer Nature, Dec. 01, 2022. doi: 10.1038/s41392-022-00947-7.

[2] L. N. Tumey, “An Overview of the Current ADC Discovery Landscape,” in Antibody-Drug Conjugates, 2078th ed., vol. 2078, Tumey LN, Ed. Humana Press Inc, 2020, pp. 1–22. doi: 10.1007/978-1-4939-9929-3_1.

[3] H. Donaghy, “Effects of antibody, drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates,” mAbs, vol. 8, no. 4. Taylor and Francis Inc., pp. 659–671, May 18, 2016. doi: 10.1080/19420862.2016.1156829.

[4] Y. Anami et al., “Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors,” Cell Rep, vol. 39, no. 8, May 2022, doi: 10.1016/j.celrep.2022.110839.

[5] B. Gorovits et al., “Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group position paper,” Bioanalysis, vol. 5, no. 9, pp. 997–1006, May 2013, doi: 10.4155/bio.13.38.

[6] S. Harth and C. Frisch, “Recombinant Anti-idiotypic Antibodies in Ligand Binding Assays for Antibody Drug Development,” in Methods in Molecular Biology, vol. 2261, Humana Press Inc., 2021, pp. 291–306. doi: 10.1007/978-1-0716-1186-9_18.

[7] G. Shankar et al., “Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products,” Journal of Pharmaceutical and Biomedical Analysis, vol. 48, no. 5. pp. 1267–1281, Dec. 15, 2008. doi: 10.1016/j.jpba.2008.09.020.

[8] X. Zhu, S. Huo, C. Xue, B. An, and J. Qu, “Current LC-MS-based strategies for characterization and quantification of antibody-drug conjugates,” Journal of Pharmaceutical Analysis, vol. 10, no. 3. Xi’an Jiaotong University, pp. 209–220, Jun. 01, 2020. doi: 10.1016/j.jpha.2020.05.008.

[9] S. Schokker et al., “Development and validation of an LC-MS/MS method for simultaneous quantification of co-administered trastuzumab and pertuzumab” MAbs, 2020 Jan-Dec;12(1):1795492. doi: 10.1080/19420862.2020.1795492

[10] T. Wyant, L. Yang, and M. Rosario, “Comparison of the ELISA and ECL Assay for Vedolizumab Anti-drug Antibodies: Assessing the Impact on Pharmacokinetics and Safety Outcomes of the Phase 3 GEMINI Trials,” AAPS Journal, vol. 23, no. 1, Jan. 2021, doi: 10.1208/s12248-020-00518-0.

[11] Y. Huang, S. Mou, Y. Wang, R. Mu, M. Liang, and A. I. Rosenbaum, “Characterization of Antibody-Drug Conjugate Pharmacokinetics and in Vivo Biotransformation Using Quantitative Intact LC-HRMS and Surrogate Analyte LC-MRM,” Anal Chem, vol. 93, no. 15, pp. 6135–6144, Apr. 2021, doi: 10.1021/acs.analchem.0c05376.

[12] Fda and Cder, “Bioanalytical Method Validation Guidance for Industry Biopharmaceutics Bioanalytical Method Validation Guidance for Industry Biopharmaceutics Contains Nonbinding Recommendations,” 2018. [Online]. Available: http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htmand/orhttp://www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/default.htm

[13] EMA, “Committee for Medicinal Products for Human Use (CHMP) Guideline on bioanalytical method validation,” 2011. [Online]. Available: www.ema.europa.eu/contact

[14] Fda and Cder, “Immunogenicity Testing of Therapeutic Protein Products —Developing and Validating Assays for Anti-Drug Antibody Detection Guidance for Industry,” 2019. [Online]. Available: https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htmand/orhttps://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm

[15] EMA, “Committee for Medicinal Products for Human Use (CHMP) Guideline on Immunogenicity assessment of therapeutic proteins,” 2017. [Online]. Available: www.ema.europa.eu/contact

[16] FDA, “Clinical Pharmacology Considerations for Antibody-Drug Conjugates Guidance for Industry DRAFT GUIDANCE,” 2022. [Online]. Available: https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-guidances

[17] R. G. E. Coumans et al., “A Platform for the Generation of Site-Specific Antibody-Drug Conjugates That Allows for Selective Reduction of Engineered Cysteines,” Bioconjug Chem, vol. 31, no. 9, pp. 2136–2146, Sep. 2020, doi: 10.1021/acs.bioconjchem.0c00337.

[18] G. Gonzalez-Sapienza, M. A. Rossotti, and S. Tabares-da Rosa, “Single-domain antibodies as versatile affinity reagents for analytical and diagnostic applications,” Frontiers in Immunology, vol. 8, no. AUG. Frontiers Media S.A., Aug. 21, 2017. doi: 10.3389/fimmu.2017.00977.

[19] C. M. Hammers and J. R. Stanley, “Antibody Phage Display: Technique and Applications,” 2014, doi: 10.1038/jid.

[20] Z. T. F. Yu et al., “Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA,” Sci Rep, vol. 5, Jun. 2015, doi: 10.1038/srep11339.

View or download the PDF version of this whitepaper.

Deciphering the complex characteristics of nanoparticles by asymmetric flow field-flow fractionation

The focus on drug discovery has been shifted from the traditional small molecules to biologics and new modalities as a result of increasing research to address unmet medical needs. This evolution requires drug delivery technologies capable of carrying the drug intact to specific sites in the body and across cellular membranes to reach their targets. Engineered nanoparticles have emerged over the past decades as effective and customizable drug delivery platforms.

Due to the versatility of nanoparticulate systems they are defined broadly as materials engineered with at least one dimension or structure in the range of 1–100 nm or as particles with properties related to their nanoscale dimension, even if this dimension is up to 1 μm. In Ardena we define nanoparticles as having a size up to 200 nm. While the concept of entrapping the drug in nanoparticles has been known for many years, the increasing mechanistic understanding and constant advances in optimizing nanoparticulate drug delivery technologies for specific applications continues to accelerate the progress and use in drug product development .

Specific regulatory pathways to stimulate nanoparticle-based drug delivery systems have been drafted by regulatory authorities providing meaningful guidance for research, development, and manufacturing. With the outbreak of the pandemic, the successful development and emergency use of mRNA lipid nanoparticulate vaccines in record time was a quantum leap forward for nanoparticle drug delivery technology. This encouraged drug developers to intensify their investments in engineered nanoparticle delivery for innovative therapeutics approaches.

Introduction

The focus on drug discovery has been shifted from the traditional small molecules to biologics and new modalities as a result of increasing research to address unmet medical needs. This evolution requires drug delivery technologies capable of carrying the drug intact to specific sites in the body and across cellular membranes to reach their targets [1]. Engineered nanoparticles have emerged over the past decades as effective and customizable drug delivery platforms.

Due to the versatility of nanoparticulate systems they are defined broadly as materials engineered with at least one dimension or structure in the range of 1–100 nm or as particles with properties related to their nanoscale dimension, even if this dimension is up to 1 μm [2]. In Ardena we define nanoparticles as having a size up to 200 nm. While the concept of entrapping the drug in nanoparticles has been known for many years, the increasing mechanistic understanding and constant advances in optimizing nanoparticulate drug delivery technologies for specific applications continues to accelerate the progress and use in drug product development [3].

Specific regulatory pathways to stimulate nanoparticle-based drug delivery systems have been drafted by regulatory authorities providing meaningful guidance for research, development, and manufacturing [4]. With the outbreak of the pandemic, the successful development and emergency use of mRNA lipid nanoparticulate vaccines in record time was a quantum leap forward for nanoparticle drug delivery technology [5]. This encouraged drug developers to intensify their investments in engineered nanoparticle delivery for innovative therapeutics approaches.

Characterization of nanoparticle drug delivery systems for pre-clinical and clinical testing

Nanoparticles is an emerging field in biomedical research and drug development. They are developed to overcome the barriers and limitations of traditional drug delivery systems and they can be designed to target the delivery of a drug compound to certain tissues, cells, or intracellular targets, to have reduced toxicity, to prolong systemic exposure, to increase bioavailability, or to overcome other barriers of traditional formulation approaches. Various nanoparticles exist with a broad range of chemistries (e.g., based on lipids, peptides, polymers, metal oxides), architectures (e.g., micelles, vesicles such as liposomes, solid nanoparticles, core-shell like structures), and surface properties.

Their uniqueness is based on their physicochemical properties, especially particle size distribution (PSD) (i.e., average particle size and size polydispersity), surface charge, and structure. These properties can be optimized to enable the entrapped or conjugated drug to be delivered to a specific target or to improve physicochemical stability in the biologic fluids. Consequently nanoparticles are complex by nature because of their multiple characteristics. In addition some specific requirements result from the potential association of nanoparticles with specific adverse reactions like immunological responses such as complement activation-related pseudoallergy (CARPA) and induction of cytokines related to the particle size, zeta potential, and formulation components, which must be carefully considered throughout development [6, 7].
Establishing the critical quality attributes (CQAs) and associated specifications is not trivial. Neither is setting the proper excipient specifications, process and manufacturing conditions to be reproducible for clinical and commercial GMP manufacturing [8]. Me-too generic versions of iron sugar nanoparticles were developed and considered similar to the originator, nevertheless later clinical studies revealed significant differences in efficacy and clinical performance in comparative trials [9].

In drug development a similar challenge exists as the formulation and process are not completely understood and validated in the early phases of drug development. To mitigate the risk a comprehensive, in-depth characterization of engineered particles is essential from early drug development onwards to ensure consistency and an accurate interpretation of the preclinical and clinical results. Such characterization requires a whole range of different analytical methods and experience in selecting the right analytical method for the specific formulation characteristic. The CQAs and the methods to measure them can be divided in 5 categories (Figure 1). The PSD and shape in particular are considered to be CQAs for any nanoparticle as they effect the pharmacokinetics, distribution, interaction with cells, and cellular uptake [10].

Figure 1. The CQAs of nano-formulations can be categorized in five groups.

From the various analytical methods applied by Ardena for the in-depth characterization of nanoparticles, asymmetric flow field-flow fractionation (AF4) coupled with multi-angle light scattering (MALS) and dynamic light scattering (DLS) has been established for qualitative and quantitative analysis of PSD, molar mass distribution (MMD), shape, and free drug compound analysis for a broad range of different types of nanoparticles.

Asymmetric flow field-flow Fractionation (AF4)

Field-flow fractionation (FFF) is a flow-assisted separation methodology similar to liquid chromatography where the separation takes place inside an open channel under mild conditions [11, 12]. FFF encompasses a family of techniques based on the principle of a thin channel geometry creating a laminar channel flow with a parabolic flow profile and an applied field perpendicular to the channel flow. Over the years FFF systems with different applied force fields (such as thermal, centrifugal, flow, electric or magnetic forces) have been developed to separate biomolecules and nanoparticles according to their specific properties such as size, mass, or charge. In biopharmaceutical sciences the asymmetric flow field-flow fractionation (AF4) has become the major FFF technology used where the applied field is an additional flow, and the separation is solely based on the hydrodynamic size. The AF4 channel consists of a solid top plate with fluid connectors and a bottom plate with a semipermeable membrane placed above a frit (Figure 2). The carrier liquid is allowed to pass through (cross-flow) while the particles are retained, accumulating close to the membrane. Due to their upwards diffusion, the particles are entering different flow streams of the parabolic flow profile and they separate based on their diffusion coefficients and, therefore, based on their hydrodynamic size.

AF4 can fractionate the different subpopulations of the nanoparticles and unbound components based on size for qualitative and quantitative analysis. In contrast to high-performance size-exclusion chromatography (HPSEC), AF4 does not use packing material or chromatographic support exposing the samples to very low pressure and shear forces, which enables labile nanoparticles to be analysed. Other advantages compared to HPSEC are higher sample recovery, operation with nearly all biorelevant buffer media, and a higher upper size limit. AF4 is ISO standardized acknowledged by regulatory authorities as a method for nanoparticle size characterization [13, 14]. It is recognized as a very versatile separation method especially for colloidal systems, such as macromolecules (e.g., proteins, polymers), nanoparticles (based on polymers/lipids/metal oxides/biomolecules) and other complex systems (e.g., blood plasma, viruses). Its application ranges from the early formulation development and characterization, including forced and real time stability testing through to the clinical, and if required to the commercial GMP release testing.

Figure 2. Schematic illustration of the separation mechanism in AF4.

Due to the simultaneous actions of cross flow and diffusion, particles are migrating along the channel with different velocities and separate according to their size; smaller particles move faster because they diffuse into the higher flow velocity region due to their higher diffusion coefficients. [Courtesy from Wyatt https://www.wyatt.com/library/theory/flow-field-flow-fractionation-theory.html]

When coupled online with various detectors AF4 can evaluate multiple CQAs of a complex product sample simultaneously without the need of sample pre-treatment. To determine PSD, MMD, aggregation, or morphology, AF4 coupled with DLS and/or MALS are methods of choice during the lead formulation, process development, and the (pre-)clinical studies. The AF4-MALS/DLS system allows particle size characterization with high resolution even in very complex systems [15]. The PSD obtained by DLS is based on the hydrodynamic size (Rh) and by MALS on the radius of gyration (Rg), which is the root mean square distance of the particles from its center of gravity. This principle favors MALS as a technology to study surface interactions of nanoparticles with endogenous proteins, monitor the change of the particle size and analyse the structure of secondary nanoparticles with adsorbed proteins. In addition indirect information regarding the shape can be achieved from the ratio Rg/Rh [15]: for compact spheres the ratio is 0.77, for empty liposomes 1, and for elongated particles >1.

Application of AF4 in drug development

During the nanoparticle pre-formulation preliminary characterization of the PSD by (batch mode) DLS or nanoparticle tracking analysis (NTA) can serve the purpose of selecting the lead formulation candidate (Figure 3). Although DLS and NTA are suitable to measure samples with a very narrow size distribution, the results are less accurate for samples with broader size distributions and should serve only for batch-to-batch comparison purposes. In addition DLS has low resolution (cannot discriminate between monomer and dimer) and NTA measures only a very small fraction of the sample. Transmission electron microscopy (TEM) is a great tool to visualize the nanoparticles but it requires sample preparation that may alter the size, the images are only two dimensional, and it can estimate PSD based in a very small number of particles. For the preclinical studies an in-depth orthogonal, high resolution nanoparticle analysis, including the evaluation of their fate in relevant biologic fluids is essential to develop a critical understanding of the pharmacology and toxicology of the designed nanoparticles [15].

Figure 3. Particle size characterization during nanoparticle formulation and process development.

Phase appropriate scaling from comparative sizing with DLS/NTA to full scale characterization of size and aggregation/interaction in standard media and biorelevant media after separation from interacting protein.

Multiple analytical methods can be applied to the fractionated subpopulations. Online MALS and DLS detectors are used for the determination of PSD, MMD, morphology, free components, shape, nanoparticle stability (aggregation), and protein binding in biologic fluids [16, 17]. Online UV detection can be used for the determination of a single component concentration along with the particle size measurement [18]. Furthermore many other analytical technologies can be coupled offline with AF4 by collecting fractions for further analysis to investigate if there are size-dependent differences in their zeta potential, drug content, purity, stability, or surface structure [19].

At Ardena we have an AF4 system coupled online with RI, UV, MALS and DLS detectors. The system is also connected to a fraction collector which enables offline coupling with various other techniques. We understand the versatility of AF4 as a separation technology and leverage the strength of each analytical method for the in-depth nanoparticle characterization by selecting the most meaningful scientific product-specific approach for each nanoparticle according and appropriate to the stage of the development.

References

McGoron AJ (2020) Perspectives on the Future of Nanomedicine to Impact Patients: An Analysis of US Federal Funding and Interventional Clinical Trials. Bioconjugate Chem. 2020, 31(3): 436-447
Mühlebach S (2018) Regulatory challenges of nanomedicines and their follow-on versions: A generic or similar approach? Adv Drug Del Rev 131: 122–131
Mitchell et al (2021) Engineering precision nanoparticles for drug delivery. Nature Rev Drug Discov 20: 101-124
Halamoda-Kenzaoui et al (2019) Anticipation of regulatory needs for nanotechnology enabled health products. Publications Office of the European Union, Luxembourg, doi:10.2760/596822, JRC118190
Schoenmaker et al (2021) mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int J Pharm 601: 120586
Clogston JD (2021) The importance of nanoparticle physicochemical characterization for immunology research: What we learned and what we still need to understand. Adv Drug Del Rev 176: 113897
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Giddings et al (1993) Field-flow fractionation: Analysis of macromolecular, colloidal, and Particulate Materials. Science 260(5113): 1456-1465
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Caputo et al (2019) Measuring particle size distribution by asymmetric flow field-flow fractionation: A powerful method for the preclinical characterization of lipid-based nanoparticles. Mol. Pharmaceutics 16: 756-767
Caputo et al (2019) Measuring particle size distribution of nanoparticle enabled medicinal products, the joint view of EUNCL and NCI-NCL. A step-by-step approach combining orthogonal measurements with increasing complexity. J Contr Rel 299: 31-43
Ventouri et al (2022) Field-flow fractionation for molecular-interaction studies of labile and complex systems: A critical review. Anal Chim Acta 1193: 339396
Klein et al (2020) Advanced nanomedicine characterization by DLS and AF4-UV-MALS: Application to a HIV nanovaccine. J Pharm Biomed Anal 179: 113017
Ansar et al (2020) Characterization of doxorubicin liposomal formulations for size-based distribution of drug and excipients using asymmetric-flow field-flow fractionation (AF4) and liquid chromatography-mass spectrometry (LC-MS) Int J Pharm 574: 118906

Phase-appropriate development of a phase 1 oral dosage form

During the drug development process there are many critical milestones and many more potential pitfalls which could seriously impact the timely progression of a new compound into the next phase. One of the major milestones is the entry into clinical phase 1, where a new compound is administered to healthy subjects for the first time. The greatest challenges arise from the fact that both the synthesis and characterization of the drug substance is still limited and yet clinical material must be produced and released for use in humans. For clinical trials the drug must be converted into a form that has sufficient stability and bioavailability, and at the same time can be provided in an administrable form of different dose strengths. However there are multiple drug characteristics (table 1) which might prevent or at least challenge the provision of clinical materials of an orally administered drug compound for the first in human trials.

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Critical attributes of drug substances

Poor or pH dependent aqueous solubility
Poor permeability
Narrow window of absorption
High pre-systemic metabolism
High first pass effect
Poor crystal formation
Poor compactability
Polymorphic changes
Degradation at gastric pH
Narrow therapeutic window

List 1: Some of the major drug characteristics which can cause issues in clinical material provision

Consequently the development of a suitable phase 1 clinical formulation and its cGMP manufacturing is a critical milestone and essential for planning and ensuring the first in human clinical trial starts on time. The objective of the phase 1 clinical trials is to establish a pharmacokinetic and safety profile of the drug in healthy volunteers. A dosage form is required that isable to secure sufficient plasma levels and exposure to the systemic circulation as well as to show drug stability during the course of the study.

Development of a phase 1 formulation
Supporting a phase 1 formulation development program should be a balance between different elements such as the performance and quality requirements for a phase 1 formulation, and the necessary pharmaceutical technology approach to achieve this. Ardena uses a rational approach which starts with a thorough review of the available physicochemical characteristics of the drug substance complemented by additional analysis if required. These data support a risk assessment leading to [Figure 1]. In the ideal case the drug compounds can be metered accurately in two-piece hard capsule in the dose strengths required to support the phase 1 clinical trials, however very few drug compounds have suitable characteristics for this and instead require specific formulation development work and drug delivery technologies.

Figure 1: Rational approach for phase 1 clinical drug product development and manufacturing
For the majority of drug substances more extensive formulation development work is required to obtain a clinical product with the desired quality and clinical performance to avoid an inconclusive phase 1 clinical trial or the program having to be put on hold and subsequently repeated with a new, more adequate phase 1 product [1].

Poor aqueous or pH dependent solubility and poor permeability are common properties of new chemical entities [2, 3]. Drug compounds with these characteristics do not achieve sufficient plasma concentrations without the application of solubility enhancing drug delivery technologies. Generally the development of bioenhancing drug delivery formulation and processing is complex and requires resource intensive development programs for the commercial formulation. For clinical phase 1 drug delivery formulations, platform technologies and scientific expertise have evolved enabling drug compounds to reach clinical trials quickly and effectively..

Unfavourable characteristics such as high lipophilicity or high lattice energy can cause molecules to have poor aqueous solubility For drug compounds with high lipophilicity determined by logP, clogP or logD7.4 lipid-based formulation can be considered. Such formulations need to be developed on scientific grounds and should employ a rational selection of a lipophilic phase with or without surfactants or cosolvents such as polyethylene glycol. In such systems the drug compound is dissolved or dispersed, enhancing the bioavailability for example through the formation of supersaturation during lipid dispersion or digestion [4].

For drug compounds with poor aqueous solubility, for example due to a high lattice energy or molecular weight beyond 500 Da, the bioavailability can be influenced by increasing the rate of dissolution which is dependent on the surface area and the physical form of the drug.

A substantial increase of the drug surface area exposed to the aqueous phase and the dissolution rate can be achieved by reducing the particle size e.g., by micronization or nanotization. Since particle size reduction is accompanied by particle charging and agglomeration, the milling process must be carefully selected and the micronized or nanosized drug generally requires the support of formulation with functional excipients like wetting agents, disintegrants and glidants [5].

By changing the physical state from crystalline to amorphous, the drug is transferred into a thermodynamic state where the intra-molecular forces in its crystalline state are reduced and inter molecular hydrogen bonds with water increase. When stabilized into their amorphous states, drug compounds show an increase in kinetic solubility and can form metastable supersaturated solutions which translates into an increased in-vivo rate of absorption [6]. To convert a crystalline compound into its amorphous form two major pharmaceutical technologies able to generate a solid dispersion are used: spray drying and hot melt extrusion.

In the spray drying process the compound is dissolved together with polymers in solvent and then atomized by spraying into small droplets, which dry instantaneously to form distinct solid microparticles of the amorphous drug stabilized by the polymer [8].

In a hot melt extrusion process (HME), the compound is mixed with polymeric excipients and heated above the glass transition temperature until molecular mixing is achieved where the compound is embedded in the polymer matrix in its amorphous form and solidifies by cooling [7].

Over the past decades we have gained a good understanding of the mechanisms of HME and spray drying process technology along with several successful product launches using each. Spray drying provides some advantages over HME including better particle size reduction, higher porosity and better wettability, which are favorable from the early development and phase 1 clinical supply perspective [9].

Development of a phase 1 dosage form
To orally administer the drug and its formulation in the phase 1 clinical program, different dose strengths in a single dosage form unit are required. Additional excipients (e.g., binders, diluents, disintegrants, wetting agents, glidants) and processing steps (e.g., wet/dry granulation, compression, coating) are often required for oral dosage forms which can add unnecessary complexity and potential risks for stability or performance issues to the phase 1 manufacturing and supply [10]. While tablets are the most common oral dosage form with established manufacturing processes, there are some important factors that are not favorable for the tablet dosage form in the early clinical trials. Tablet manufacturing requires a drug powder blend with excellent flow properties on the machines to achieve content uniformity for which in most cases an additional granulation process (e.g., dry or wet granulation) is needed. Drug compounds generally have an unpalatable flavour, requiring a taste masking coat on the tablet. The disintegration and dissolution of the tablet are driven by its porosity which is dependent on the powder properties, the compression forces and profile applied, and the coating. To keep the phase 1 formulation as simple as possible, two-piece capsule technology has emerged as the dosage form of choice due to their ability to dose drug compounds or microparticles with poor flow properties, their provision of effective taste masking and retention of the dissolution characteristics of drug compound formulation in the absence of a strong powder compression step [11, 12]. In addition the application of capsule technology for complex compounds and delivery forms has been greatly expanded by the introduction of new capsule polymers (e.g. HPMC) and filling technologies.

Analytical characterization and in vitro-dissolution
Analytical characterization is an essential part of clinical product development and manufacturing as this builds the basis of the product release specification which is mandatory for the IND or IMPD filing. Accurate analytical methods for drug compound stability, impurities, content uniformity, drug dissolution and other quality criteria are needed to fulfill these regulatory requirements. From a phase 1 product development perspective in-vitro dissolution tools play a particularly important role in the formulation and dosage form selection process. The in-vitro tools may also include predictive methods with biorelevant or discriminatory dissolution media or different dissolution apparatus in order to confirm the assumptions made during the drug compound assessment and definition of the most suitable clinical product development approach [13].

Beyond oral small molecules
The emerging clinical investigations of life biotherapeutics create distinct challenges for entry into phase 1 clinical trials. For example, probiotics are live microorganisms which are very sensitive to water activity, heat and oxygen and need to be stabilized for pharmaceutical applications. Two major techniques are being used: micro-encapsulation into polymeric systems and controlled removal of water. Micro-encapsulation of a probiotic requires intense investigations into suitable techniques, biopolymers and micro-encapsulation processes, which is similar to freeze drying or lyophilization technologies operating at freezing temperature and low pressure (vacuum). In contrast to these techniques, spray drying has emerged as the technology of choice due to the low energy consumption, lower operational costs, high yield, industrial feasibility, and continuous manufacturing. Experienced scientists can easily adjust the spray drying process using temperature, feed rate, drying time and drying medium to obtain the desired probiotic stability [14]. While tableting of probiotics has become a feasible option, the selection of the necessary excipients is of utmost importance for the compression of probiotics into tablets. The excipients need to be screened for each strain of probiotics regarding their suitability and stabilizing effects as no predictions can be made [14]. While this might be justifiable for the phase 3 and commercial formulation, encapsulation of the conditioned probiotics or spray dried probiotics in moisture reduced HPMC capsule avoids the intensive development and formulation work for the phase 1 clinical trials.

Pulmonary drug delivery is gaining increasing importance for local and systemic application of small molecules and biotherapeutics. They present another challenge for phase 1 clinical trials due to the need for a performance critical device component. Particles for pulmonary delivery must have a particle size < 5µm and need to be metered at very low doses into administration dose units. Such formulations typically consist of micronized drug attached to carriers (e.g., lactose) or engineered particles derived from spray drying technologies. Capsule-based inhalation devices are considered to be the technology of choice for phase 1 and beyond due to the existing expertise in inhalation powder development and processing, and availability of encapsulation equipment able to handle even cohesive powders. [15].

There is growing evidence for the benefit of fixed dose combination products, combining 2 or more different drugs or release profiles into a single unit. Clinical studies for combination products are more complex as they need to identify the contributions of each drug to the overall clinical benefit of the combination [16]. The individual drugs used are established, therefore the program will start with phase 2 trials to provide the clinical proof of concept (cPoC). The challenge for clinical manufacturing is the need for a variety of different combinations and dose strengths to be prepared and blinded. To avoid unnecessary formulation and product development work for this initial trial, distinct product units (e.g., powder mixtures, tablets, etc) are filled into a single capsule to support the variety of fixed dose combinations required for the cPoC [17].

Conclusion
Understanding the various constraints of the early development phases, in particular the “first in human” study, the formulation, dosage form and analytical method development, Ardena consistently employs its “phase-appropriate” approach to efficiently provide the necessary evidence and confidence at each decision point. This applies to drug compounds beyond small molecules such as biotherapeutics and to dosage forms beyond oral delivery such as pulmonary drug delivery as well as complex drug products such as fixed dose combinations or drug compounds with special handling requirements (e.g., OEB 3 & 4 compounds, microdosing). Supported by the required documentation, the clinical batch is manufactured and supplied ahead of the start of the phase 1 trials.

References

Boyd, B.J. et al (2019) Successful oral delivery of poorly water-soluble drugs both depends on the intraluminal behavior of drugs and of appropriate advanced drug delivery systems. Eur J Pharm Sci 137:104967
Shulz, M.D. (2019) Two Decades under the Influence of the Rule of Five and the Changing Properties of Approved Oral Drugs. J Med Chem 62:1701-1714
Brown, D.G. & Wobst, H.J. (2021) A Decade of FDA-Approved Drugs (2010-2019): Trends and Future Directions. J Med Chem 64:2312-2338
Ditzinger, J. et al (2019) Lipophilicity and hydrophobicity considerations in bio-enabling oral formulations approaches – a PEARRL review. J Pharm Pharmacol 71:464–482
Wu, D. & Sarsfield, B.A. (2018) Particle Size Reduction: From Microsizing to Nanosizing. In: Early Drug Development: Bringing a Preclinical Candidate to the Clinic, Volume 1, Ed: Giordanetto, F. Wiley‐VCH Verlag GmbH & Co. KGaA
Iyer, R., et al (2021) Amorphous Solid Dispersions (ASDs): The Influence of Material Properties, Manufacturing Processes and Analytical Technologies in Drug Product Development. Pharmaceutics 13:1682
Simões, M.F., et al (2021) Hot-Melt Extrusion: a Roadmap for Product Development. AAPS PharmSciTech 22:184
Davis, M. & Walker, G. (2018) Recent strategies in spray drying for the enhanced bioavailability of poorly water-soluble drugs. J Contr Rel 269:110-127
Vasconcelos, T., et al (2016) Amorphous solid dispersions: Rational selection of a manufacturing process. Adv Drug Del Rev 100:85-101
Markl, D. & Zeitler, J.A. (2017) A Review of Disintegration Mechanisms and Measurement Techniques. Pharm Res 34(5): 890–917
Esseku, F. et al. (2010) The Effect of Overencapsulation on Disintegration and Dissolution. Pharm Technol 4:104-111
Wilding, I.R. et al (2005) In vivo disintegration profiles of encapsulated and non-encapsulated sumatriptan: gamma scintigraphy in healthy volunteers. J. Clin. Pharmacol. 45(1):101-105
Mann, J. et al. (2017) Validation of Dissolution Testing with Biorelevant Media: An OrBiTo Study. Mol Pharmaceutics 14:4192-4201
Gurram, S., et al. (2021) Insights on the Critical Parameters Affecting the Probiotic Viability During Stabilization Process and Formulation Development. AAPS PharmSciTech 22:156
Stegemann, S. et al. (2022) Focusing on powder processing in dry powder inhalation product development, manufacturing and performance. Int J Pharm 614:121445
EMA (2017) Guideline on clinical development of fixed combination medicinal products. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-clinical-development-fixed-combination-medicinal-products-revision-2_en.pdf
g., https://www.mg2.it/processing/capsule-fillers/

Leverage the power of CMC in early drug development

The drug development process, from drug discovery to the clinical proof of concept (PoC) is costly and high-risk. The high rewards of a successful development, both ethical and commercial, excites scientists, researchers, and investors . Starting from scientific endeavors, drug discovery is mainly driven by the pre-clinical pharmacology and toxicity as well as the fast entry into the clinical phase to establish the clinical PoC.

Once a pharmacological target is identified and lead chemical compounds emerge from the drug screening process, many activities around drug development take place in parallel, while at the same time depending on each other. Drug development is a multidisciplinary process by which data are generated in various scientific fields: the chemical synthesis of the drug candidate, its characterization, purity, stability, formulation into a finished drug product, the drug product performance, and biopharmaceutics in animal models. These data represent the critical learning curve for a drug and are essential for entry into clinical trials, risk mitigation, and ultimately, development into a marketable product. The systematic processing and documentation of these Chemistry Manufacturing and Control (CMC) data pose a major challenge, especially in expedited drug development and approval procedures, and for smaller companies.

Introduction

Once a pharmacological target is identified and lead chemical compounds emerge from the drug screening process, many activities around drug development take place in parallel, while at the same time depending on each other. Drug development is a multidisciplinary process by which data are generated in various scientific fields: the chemical synthesis of the drug
candidate, its characterization, purity, stability, formulation into a finished drug product, the drug product performance, and biopharmaceutics in animal models. These data represent the critical learning curve for a drug and are essential for entry into clinical trials, risk mitigation, and ultimately, development into a marketable product. The systematic processing and
documentation of these Chemistry Manufacturing and Control (CMC) data pose a major challenge, especially in expedited drug development and approval procedures, and for smaller companies [1].

Challenges in CMC and CMC regulations

The European Medicines Agency (EMA) CMC guidelines provide general guidance on the expected information to be presented in the Investigational Medicinal Product Dossier (IMPD). For investigational medicinal products (IMPs), the information required in the quality dossier should be phase-appropriate and focus on the risks, thereby taking into account the nature of the product, the patient population, and the underlying disease of the population being studied. [2]. Similar guidelines have been put forward for Investigational New Drug (IND) filings by the United States Food and Drug Administration (FDA) [3] and the Pharmaceutical and Medical Device Agency (PMDA) in Japan [4]. With the CMC guidelines, Health Authorities make clear that a minimum level of information and data is expected to ensure the safety of patients in the clinical trial. At the same time, they offer advance consultations to bring innovative drugs to patients quickly.

Recently, the FDA performed a survey on the success of IND submissions of oncology drugs. IND filings that were not successful primarily affected First in Human trials and were put on hold. Over 40% of these unsuccessful IND filings were due to CMC problems and were submitted predominantly by sponsors with limited regulatory experience. Resolving the quality issues took on average 114 days but, in some cases, up to more than two years. The review also demonstrated that sponsors with regulatory experience benefited most from the FDA consultations in the pre-IND meetings [5]. CMC is therefore a critical building block for product quality throughout the process, from product development to marketing, and will continue to progress dynamically in the future as also regulatory guidelines evolve. [6; 7].

Building up the right level of CMC at each stage

In the early development phase, the non-clinical studies (e.g., pharmacology, toxicology), have to take place in parallel with the manufacturing and characterization of the drug substance and its formulation development. Since both are interdependent, it is paramount to build a comprehensive and well coordinated project plan, which successively combines the most important data from both in order to obtain valid evidence on the efficacy, safety, and druggability of the compound. From a CMC perspective, the drug must be characterized in terms of solid state properties, impurities, stability, and solubility, and appropriate analytical methods must be validated. Thus, it is recommended to sufficiently investigate the solid state properties of the drug substance during lead compound selection in order to secure the non-clinical data and to determine the Critical Quality Attributes (CQA) [8; 9]. In addition,
impurities have always been an area of major concern, even more so since the revelation of high nitrosamine concentrations in blood pressure medicines known as ‘sartans’. It was shown how increased formation of nitrosamines occurred in the course of synthesis optimization, and GMP non-compliance resulting in stricter regulatory guidelines [10]. A multidisciplinary team can avoid such issues by building on the quality by design (QbD) principles for activ pharmaceutical ingredient (API) [11] and drug product [12] manufacturing as well as the early establishment of quality systems [13; 14]. This will ensure the generation of a stage-appropriate product, process understanding, and adequate CMC documentation.

Risk mitigation by utilizing external expertise

Ardena ensures the goal of moving from scientific conception to clinical PoC as quickly as possible through coordinated action among its Centers of Excellence (Figure 1).

Figure 1: Ardena’s Center of Excellence

Starting from chemical structure, synthesis, and physicochemical analysis, a “druggable” drug substance is developed followed by a scalable formulation based on the defined target product profile (TPP) . From the beginning, process parameters and control are evaluated and CMC documentation is prepared. Problems that occur in the course of development are therefore immediately identified and resolved. A common issue encountered during development is the solubility profile of a new compound resulting in low or highly variable bioavailability.

A very typical case that Ardena solved was a project with a drug substance with insufficient solubility. The analysis of the solid state properties showed that the cause was a low dissolution rate. Therefore, as a solution approach, the multiple enlargement of the crystal surface area by particle reduction to the nanoscale was developed. Different size reduction methods and stabilization of the nanoparticles were evaluated. During the nano-milling, the particle size change was monitored by laser diffraction allowing the selection of the best milling technology. As depicted in Table 2, the nano-milling led to a 20-fold decrease in the D90. Before selection of the final process parameter and performing the stability, the nanomaterial was analyzed with regard to potential physicochemical changes like the formation of an amorphous drug or different polymorphic forms. Subsequently, the nanoparticles in a suspension formulation were subjected to a stability study at 25°C/60% RH and 40°C/75% RH, confirming 2 weeks stability at accelerated conditions (table 1).

Table 1: Particle size reduction by milling (A) and particle size stability of the nanoparticles under accelerated conditions (B)

In preparation for the Phase 1 clinical trial and IND/IMPD documentation, a bioequivalence study of the nano-suspension against the reference suspension (i.e. with the unmilled API) was performed in Beagle dogs. The nanoparticles showed a 5-fold increase in bioavailability after oral administration (Figure 2).

Table 2: Preclinical study results

These data constituted an important part of the CMC documentation that Ardena’s regulatory experts addressed with the FDA in the pre-IND meeting and contributed to the success of the IND submission. In case of a positive clinical PoC, the collaboration will continue with the development of a solid oral dosage form based on the established nanoparticle technology.

Conclusion

Drug discovery is the source of pharmaceutical innovation driven by scientists from virtual companies through large pharmaceutical companies. The key objective and milestone of drug discovery is the clinical proof of concept. An important step towards this goal is the approval of the first clinical trial application. This requires not only the non-clinical pharmacology, toxicology, and kinetics studies, but also sufficient and phase appropriate CMC work an documentation, the relevance of which is often underestimated. As indicated in the FDA review of IND filings, the likelihood of success also depends heavily on sponsor experience and pre-IND meeting advice. However, throug timely consideration of CMC, appropriate expertise, and collaboration, CMC can also be instrumental for milestone decisions contributing to the overall success of a project. Combining the expertise from chemical synthesis and characterization, analytical method and formulation development, clinical supply, and regulatory expertise, partnering with Ardena can accelerate your drug development program, support science based milestone decisions, mitigate the risk, and prepare for a successful market entry from the early stages of development.

References

Dye et al (2016) Examining manufacturing readiness for breakthrough drug development. AAPS PharmSciTech 17(3): 529-38
EMA (2017) Guideline on the requirements for the chemical and pharmaceutical quality documentation concerning investigational medicinal products in clinical trials. EMA/CHMP/QWP/545525/2017
FDA (2015) IND application for clinical investigations require information and documentation of the chemistry, manufacturing, and control (CMC) (IND Applications for Clinical Investigations: Chemistry, Manufacturing, and Control (CMC) Information | FDA)
Singh S (2021) Insight on PMDA Regulatory Procedures, Key Stages, Timing, and CMC Requirements for Bio-Therapeutic Products in Japan. J Pharma Res Rep 2(1): 8-13
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Cauchon et al (2019) Innovation in Chemistry, Manufacturing, and Controls- A regulatory perspective from industry. J Pharm Sci 108: 2207-2237
Algorri et al (2020) Transitioning Chemistry, Manufacturing, and Controls content with a structured data management solution: Streamlining regulatory submissions. J Pharm Sci 109:1427-1438
Stofella et al (2019) Solid state characterization of different crystalline forms of sigagliptin. Material 12, 2351
Gyseghem et al (2009) Solid state characterization of the anti-HIV drug TMC114: Interconversion of amorphous TMC114, TMC114 ethanolate and hydrate. Eur J Pharm Sci 38:489-97
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ICH Q11 (2012) Development and manufacturing of drug substances (chemical entities and biotechnological/biological entities) (https://database.ich.org/sites/default/files/Q11%20Guideline.pdf)
ICH Q8 (R2) (2009) Pharmaceutical Development (https://database.ich.org/sites/default/files/Q8%28R2%29%20Guideline.pdf)
ICH Q9 (2005) Quality Risk Management (https://database.ich.org/sites/default/files/Q9%20Guideline.pdf)
ICH Q10 (2008) Pharmaceutical Quality Systems (https://database.ich.org/sites/default/files/Q10%20Guideline.pdf)

Formulation and Process Considerations for Optimising Spray-Dried Solid Dispersions

Although a technically challenging process, spray drying is a mature, well understood technique capable of transforming solutions or suspensions into solid particles. Although this process has been widely used in diverse industrial fields, it has become more and more demanded in pharmaceutical applications for the production of solid dispersions.

Today’s APIs are increasingly insoluble and that is presenting new problems for formulators looking to manage the bioavailability and dosing of their formulas. As a result, a significant number of therapeutics gaining approval recently possessed poor biopharmaceutical properties that had to be managed through advanced processes and formulation strategies.

Improving the bioavailability of these new and existing drugs is turning out to be big business for contract development and manufacturing organizations (CDMOs) as pharma’s drug developers look to exploit both accelerated new chemical entity (NCE) and existing drug development pathways.

Introduction

Spray drying active pharmaceutical ingredients (APIs) in solution to overcome solubility hurdles requires part craft and great attention to process variables. In this article, Javier Gurrea, a spray drying manufacturing specialist at Ardena, explains how expertly applied spray drying technology offers drug innovators a faster route to higher-performing drugs.

Pharma leveraging SDDs more than ever

Several of the most popular drugs on the market today have had to manage poor solubility and low bioavailability. That trend isn’t slowing either. Pharma industry analysts estimate that as many as 40% of approved drugs and nearly 90% of the developmental pipeline drugs consist of poorly soluble molecules. (1)

For developers, changing formula chemistries and identifying different routes of administration are just a few of the ways the industry is seeking to profit from accelerated drug development routes including 505(b)(2) New Drug Applications (NDAs). The industry is finding that redeveloping existing formulations can quickly improve the therapeutic value of existing drugs to both payer and patient.

Regarding drug bioavailability enhancement, SDSDs have proven to be a highly controllable, flexible manufacturing strategy to improve the solubility of drugs – especially those with low aqueous solubility.

Because each product is unique, a deep knowledge of the key aspects of the formulation and the mechanistic understanding of spray drying process is required.

Understand your evaporation rate inside and out

During spray drying, the heat and mass transfer that takes place determines the characteristics of the particles being formed. This atomization of the solution is a crucial aspect of the process because it generates fine droplets in order to increase the surface area of the liquid exposed to the drying gas (2).

Initial mass transfer is characterized by a constant evaporation rate, equivalent to a pure solvent droplet—because it refers to the evaporation of the solvent on the surface of the droplet. This is followed by the diffusion of the solvent from the core to the particle surface (2). At this moment, the temperature of the particle suddenly increases and the particle formation rate diminishes due to the higher amount of solvent in the drying gas stream. Consequently, the evaporation rate undergoes a sudden decrease due to the droplet viscosity, which can solidify the surface first, hindering the solvent from leaving the interior of the droplet.

It is a key consideration not to be overlooked or dismissed lightly. The evaporation rate is crucial in stabilizing the amorphous form of the drug, as well as the time the particle is in contact with the hot gas may have an impact on the stability of the product obtained. In this sense, although the drying capacity of the gas can be increased by raising the process temperatures, it cannot rise indefinitely to avoid compromising the stability of the solid dispersion.

Tetris and the rush hour effects

It is important to not forget that the drying process takes place in milliseconds and during this brief point in time, different phenomena occur that can and will determine the characteristics of the particle being formed.

First, the droplet mass remains constant until the solvent begins to evaporate. Then, the amount of solvent of the droplet goes down and the solute content concentrates on the surface of the particle. As a result of this ‘concentration gradient’ there is a slight diffusion of solutes towards the nucleus of the particle as well. In this case, if the diffusion rate of solutes is not as fast as the decrease in droplet volume, a ‘crust’ can form on the surface of the particle (3).

As evaporation of the solvent continues, this outer skin can hinder the evaporation of the solvent from the core of the particle and, depending on the resistance and thickness of the crust formed, the particle can inflate or burst resulting in hollow or porous particle due to the internal pressure. This phenomenon is commonly known as ‘rush hour effect’ (4).

On the contrary, if the evaporation rate is low, the solute particles have enough time to migrate to the core of the particle during the solvent removal, resulting in denser and smaller particles (‘tetris effect’) (See Figure 1).

Figure 1. Effect of evaporation rate on transition from droplet to particle.

Fortunately, these phenomena can be explained by a simple equation referring to the Peclet number (Pe_i) equation which relates the variables that influence the characteristics of the resulting particles (5).

k is the evaporation rate
D_i is the diffusion coefficient of the solute

For a given solution composition, a low evaporation rate (low Pe_i) results in smaller and denser particles while a very fast evaporation rate (solvents with low boiling point, high Pe_i) provides particles of more volume, porous, less dense and with an enrichment of solutes on their surface.

Additionally, the solvent characteristics can also affect the distribution of the particle components, as can be seen in the equation, that includes the diffusion coefficient of the solute in the solvent.

When values of diffusion coefficient are low (corresponding to a higher Pe_i) there is an enrichment in solutes on the particle surface because the particle components diffuse more slowly than the particle size decrease during the solvent evaporation. In contrast when diffusion coefficients are high (lower Pe_i) the components will be uniformly distributed along the particle (3,6,7).

All things considered, by manipulating the key process inputs of spray drying, the properties impacting the dissolution behaviour such as morphology and particle size, as well as density and flowability (relevant to process quality and throughput goals in downstream processes like tableting or capsule filling) can be modulated.

Manage spray drying variables for best results

Manufacturers with experience have found that the better technicians are at leveraging a series of critical spray-drying process parameters, the more capable they are of generating desired particle morphologies. Breaking it down, precise particle formation control requires a thorough evaluation of both feed solution variables and process parameters:

Feed solution variables

Polymer content. The evaporation kinetics is influenced by the polymer concentration in the solution, which will result in a given solution viscosity. On the other hand, the miscibility of both components, and the potential to obtain a homogeneous system, is determined by the API-polymer ratio (8).

Solids content. Typical solids content used in amorphous solid dispersions are within the range of 10% to 30%. This solids content is inversely proportional to the evaporation rate (9). Regarding desired particle size, low-concentrated solutions generally produce small spherical particles (of high hygroscopicity and concentrated solutions often result in larger particles with a rough surface and high porosity (3,10).

Solution stability. This variable requires close examination, especially when large commercial volumes of solution are prepared, involving a large period of time between its preparation and its drying process, in order to avoid nucleation and crystalline growth (11).

Process parameters

Liquid feeding rate. This parameter dictates the time in which the particles are in the drying chamber, as well as the amount of solvent present in the gas stream and the subsequent outlet temperature observed. It is also directly proportional to the particle size and some authors have described that this feed rate could be inversely proportional to the solubility enhancement of the active ingredient (12).

Inlet temperature. Because inlet temperature has been postulated to be directly proportional to the obtained glass-transition temperature (T_g), it is inversely proportional to the crystallinity of the drug (13). High inlet temperatures can generate larger particles and may cause solvent entrapment in its core, resulting in the subsequent destruction of its outer skin while lower inlet temperatures, generate smaller denser particles with a rough surface (3,14).

Outlet temperature. Two aspects of outlet temperature should never be overlooked. If the outlet temperature is above the T_g of the product, it can adhere to the walls of the equipment due to the sticky characteristics of the compound, reducing process yield. Similarly, an outlet temperature too low will cause a high level of residual solvent in the product, compromising its stability (15).

Type of gas and flow rate. On the one hand, the type of gas used can influence the particle size. Gases with low density, such as nitrogen, result in smaller particle size (2,16). On the other hand, the higher the gas flow rate, the smaller the particle size obtained during the process and, in addition, it has been observed that working in open cycle produces higher yields than working in closed cycle (2,17).

Type of atomizers used. Depending on the design of the atomizing system, particles with different properties can be obtained using:

Rotary/centrifugal atomizers: These devices use a rotating disk to break the liquid stream into small droplets that are projected towards the walls of the drying chamber thanks to centrifugal force (18).

Bi-fluid nozzles: This is the most common type of atomizer employed in the pharmaceutical field. In these devices the liquid is put in contact with a gas stream resulting in a disintegration of the liquid into fine droplets. The characteristics of the atomization will be influenced by the characteristics of the solution or suspension and the gas used (density, viscosity, pressure, etc.) (19).

Pressure nozzles: This type employs hydraulic pressure to break the liquid stream through a nozzle, where a series of spiral-shaped inserts break the solution into small droplets. One advantage of these atomizers is that they allow the obtention of larger particles that facilitates the subsequent downstream process without needing to perform a dry granulation step to achieve the optimum flow and density features (20).

Programs run better with systematic Quality by Design

Although spray drying can be a challenging technology to master it has fast become the preferred way for drug developers to overcome the limitations of APIs with poor aqueous solubility due to its applicability to obtain amorphous solid dispersions or to dry nanosuspensions, for instance. In support of quality and reliability in process the industry is increasingly introducing spray drying in a more systematic and empirical way following ICH Q8 (21) guidelines and its primary Quality by Design (QbD) approach.

Having a deep knowledge of all the parameters that influence the process and their potential impact on particle formation is the initial and key step in starting a successful drug manufacturing program for tricky, insoluble formulations. The process is sophisticated and program planning requires a precision approach. Ardena has a long experience leveraging spray drying to meet highly potent drug products therapeutic and manufacturing goals. As a CDMO we offer access to all current GMP spray drying capabilities as well as complete manufacturing services in support of all clinical and commercial phase drug development.

References

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Much more than just smoke – from marijuana to medicinal cannabis derived therapeutics

The history of cannabis begins already in antiquity and its therapeutic use has been documented for 2700 years. In the year 1850, cannabis was included in the United States Pharmacopoeia (USP) before it was deleted in 1942, after a new legislation in 1937 classified cannabis as a narcotic and was subsequently prohibited. Since that time, the story of cannabis has been told by baby boomer generations as “the mind-expanding, peace-making smoke” that can land you in jail.

But beside the psychotropic effects, many patients and even doctors swore by the effectiveness of cannabinoids in important therapeutic areas like chronic pain, terminal cancer, and multiple sclerosis. Due to the illegal status these applications remained anecdotal until its legalization. Within a short period thereafter scientific interest took off at a rocket speed triggering a flood of clinical studies with parts of the cannabis plant, its resin or extracts investigating different indications. Until today only a few products were approved by regulatory authorities using either synthetic Δ⁹-tetrahydrocannabinol (THC) (Marinol ®, Syndros®, Cesamed®) or plant derived standardized cannabidiol (CBD) or CBD/THC extracts (Epididex®, Sativex®). Overconfidence in cannabis per se was soon challenged by many failed endpoints in clinical trials. These setbacks led to more basic research on the different cannabis varieties, the active components, their pharmacology and metabolomics, structure-activity relationships, synergistic effects of compounds (entourage effect) and targeted receptors. The more these research progresses the more we understand the complex nature of cannabis as well as the multitude of chemical compounds and structures with the potential of novel therapeutic application.

The metamorphosis of the “joint“ to a medicine

The history of cannabis begins already in antiquity and its therapeutic use has been documented for 2700 years [1]. In the year 1850, cannabis was included in the United States Pharmacopoeia (USP) before it was deleted in 1942, after a new legislation in 1937 classified cannabis as a narcotic and was subsequently prohibited [2]. Since that time, the story of cannabis has been told by baby boomer generations as “the mind-expanding, peace-making smoke” that can land you in jail.

But beside the psychotropic effects, many patients and even doctors swore by the effectiveness of cannabinoids in important therapeutic areas like chronic pain, terminal cancer, and multiple sclerosis. Due to the illegal status these applications remained anecdotal until its legalization. Within a short period thereafter scientific interest took off at a rocket speed triggering a flood of clinical studies with parts of the cannabis plant, its resin or extracts investigating different indications. Until today only a few products were approved by regulatory authorities using either synthetic Δ⁹-tetrahydrocannabinol (THC) (Marinol ®, Syndros®, Cesamed®) or plant derived standardized cannabidiol (CBD) or CBD/THC extracts (Epididex®, Sativex®). Overconfidence in cannabis per se was soon challenged by many failed endpoints in clinical trials. These setbacks led to more basic research on the different cannabis varieties, the active components, their pharmacology and metabolomics, structure-activity relationships, synergistic effects of compounds (entourage effect) and targeted receptors [3-5]. The more these research progresses the more we understand the complex nature of cannabis as well as the multitude of chemical compounds and structures with the potential of novel therapeutic application.

The cannabis plants

Cannabis sativa is a polymorphic and complex plant growing under different environmental conditions, altitudes, and soils leading to a variety of genotypes and chemovars. For therapeutic applications more than 600 different varieties are available with substantial different cannabinoid and terpenoid compositions as well as other pharmacologic active compounds [4]. These differences lead to various and variable physiological and pharmacological responses [6]. Cannabis sativa L. contains about 565 chemical compounds from 23 different chemical classes of which around 100 are attributed to cannabinoids [7]. Yet, the chemical characterization of the plants and their extracts require specific analytical procedures to capture the multitude of components present. One of the challenges of cannabis analytics is the impact of the processing and extraction conditions, which can alter the chemical structures of the active compounds as well as the composition original present in the plant [8, 9].

The variety of different cannabinoids in the plant and their extracts display multiple often contrasting receptor interactions which might counteract desired effect. It is well established that an excessive central nervous system effect by some components is responsible for serious side effects including dizziness, dry mouth, nausea, fatigue, somnolence, euphoria, vomiting, disorientation, drowsiness, confusion, loss of balance, and even hallucination [10]. Therefore, purification technologies of cannabis are progressing for medical applications [11]. Identifying the chemical structures and compounds contributing to the desired but also undesired pharmacological effects provide a rich source of new active ingredients being it plant based compounds, semi-synthetic derivatives or newly created synthetic structures [12].

Cannabis analytical characterization

Characterizing botanical plants and their products can be very challenging as they are complex and dynamic systems. Metastable components in organs or cells of the plant can easily degrade or form artefacts based on the processing and analytical procedures. Some of the active chemical compounds might only be present in quantities which are close to the limit of detection. Suitable analytical procedures, which are required for medicinal use, are validated with regard to effective extraction and separation technology coupled with sensitive, selective as well as reproducible detection systems. Over the past years different approaches have been proposed coupling HPLC, RP-HPLC, GC, LC with HRMS/MS, MS/MS, FID or VUV [8, 13-15]. Most recent work also suggested that MIR spectroscopy can be used as a Process Analytical Technology (PAT) for the quantitative determination and monitoring of the major cannabinoids during product manufacturing [16]. In addition, the natural origin of cannabis makes it susceptible to microbiological, pesticide and heavy metal contamination which are subject to strict limitation in medical products [17]. The microbiology contamination can be determined for example by ICP-MS, MALDI-MS, APC or qPCR [13].

Cannabis regulations for medicinal use

The development of cannabis based medical products follow the established principles of evidence for efficacy, safety and quality as pharmaceuticals. The FDA considers Cannabis based products to be developed according to their guidance for botanical drug development [18] and the draft guidance on the quality considerations for clinical research of cannabis and cannabis-derived compounds [19]. One of the major concern specific to cannabis derived products remains the addiction potential of THC for which reason the FDA has set a maximum THC concentration of no more than 0.3 % by dry weight. Otherwise, it will be classified as Schedule I controlled substances under the Controlled Substance Act. Due to the multicomponent nature of cannabis, it is essential for new drug products to either standardize the cannabis-based product (e.g. extract) to specific quantities of the active components or extract and purify the active components into a single compound system or into fixed dose combination.

Drug development challenges of cannabis products

The physicochemical properties that are decisive for the formulation strategy are very different between the cannabis and cannabis derived actives. They depend on the provenance of the plants, the processing method and the properties of the individual components. The challenges related to cannabinoids like THC and CBD is their high lipophilicity (clogP > 6), low aqueous solubility (2 – 10µg/mL), low melting point (< 70°C), low oral bioavailability (< 20%), highly variable orally inhaled bioavailability, poor taste as well as their limited stability under standard storage conditions (< 6 months). After oral intake, cannabinoids are extensively metabolized by the cytochrome P450 system. Despite this high first pass metabolism, due to their lipophilicity they distribute substantially into lipid tissue leading to individual elimination half-lifes of > 24 h and in steady state of 2 – 50 days [20].

Pulmonary, nasal, topical, oral, mucosal and parenteral dosage forms have been investigated whereby each of the dosage forms has its own merits for specific clinical targets [21]. The highest bioavailability has been achieved through the pulmonary route with high inter- and intrasubject variability due to the individual inhalation performance. The bioavailability of oral delivery is dependent of the formulation. Lipophilic solutions or surfactant-based emulsion systems have demonstrated to achieve therapeutic plasma levels even though the bioavailability was low. Transmucosal delivery forms applied in the mouth or nasal cavity were able to achieve bioavailability of around 40 % as they circumvent the first pass effect, but might be limited in the achievable dose that can be delivered. Transdermal applications demonstrated their benefit based on their long term, constant delivery of small doses of cannabinoids for pain management. In the recent years engineered particles provided an interesting formulation approach that can be used for oral and parenteral delivery. Recently lipid, polymeric carriers or nanocarriers have been developed to achieve targeted delivery of cannabis derived products [22, 23].

Determination of cannabinoids and its metabolites in biological fluids

Bioanalysis of cannabis in biologic fluids and tissues has been of interest for many decades for forensic purposes. For medicinal purposes, bioanalytics are key for product development and regulatory clearance. Especially in the case of cannabis based medicinal products with multiple pharmacological active components, the existing lack of sufficient pharmacokinetic and pharmacodynamic understanding has to be seen as an opportunity rather than a threat [24]. Even though the determination of the pharmacokinetic profile of cannabinoids in blood and plasma remains a challenge [25] creative scientists have been successful in developing bioanalytical methods to support medicinal cannabis-based product development.

Within the development of the dosage forms bioanalysis is essential to quantify the desired concentration at the targeted site of action. This includes solid procedures to measure absorption, bioavailability and distribution of the drug and its metabolites in blood or serum. This is especially true for cannabinoids due to the high lipophilicity, instability at low pH and extensive hepatic first pass metabolism which require a targeted formulation approach. Chemical optimization of the cannabinoids to achieve better drug-like properties through structure-activity analysis can also be explored to improve the pharmacokinetic and pharmacodynamic properties [3, 26]. Consequently, the pharmacokinetic profiling of the different cannabinoids is a critical aspect to understand the complex kinetics of cannabinoids and their metabolites to improve efficacy and reduce adverse drug reactions.

The opportunity space for cannabis derived medicinal products

Cannabis and their derived products are being investigated for multiple clinical conditions [27]. A major challenge remains the complexity and variability of the cannabis plants and plant derived products that can explain a lot of disappointing results in clinical trials [28, 29]. This opportunity can be successfully seized if a systematic approach is followed. This approach includes a sequence of steps in purification, analytical, formulation, manufacturing, clinical study design, bioanalysis, and regulatory processes for which specific expertise is required.

Serving several medicinal cannabis projects successfully, some of the critical issues, their solutions and opportunities provided by Ardena will be explained with the help of the following examples.

As for any new drug development program, the cannabis derived product is considered as the active pharmaceutical ingredient (API), requiring an exhaustive characterization of its composition. An accurate analytical method must be able to separate all components qualitatively and capture them quantitatively. A method based on liquid chromatography (LC) coupled with ultraviolet spectra (UV) determination was used to qualify and quantify the major components (CBD and THC) in the cannabis derived product. The method was validated with regard to accuracy, precision, repeatability, intermediate precision, specificity detection limit, quantitation limit, linearity and range. The comparison of different batches of the API revealed sufficient compositional reproducibility so that purification and further standardization of the extract via column chromatography was not necessary for the formulation development.

Dependent on the intended therapeutic target the API has to be formulated into a dosage form which is capable to release the API at its site of absorption. To achieve fast and sufficiently high plasma concentration of purified CBD for pediatric use, a sublingual dosage form was considered. Due to its high lipophilicity, three different crystal engineering approaches were developed for CBD: nanocrystals, spray dried particles and amorphous solid dispersion by freeze drying. Different tablet formulations were screened and evaluated for stability and in-vitro dissolution. The lead formulation was than scaled up to assure processability as well as tablet quality and performance targets. While the tablet achieved all the targeted quality criteria, the initial palatability assessment raised concerns about acceptability by children. Based on data, literature and internal reviews, the potential root cause could be identified and the taste issue was resolved by formulation optimization.

Another case was the urgent request for the development and clinical supply of a stable oily oral formulation of THC and CBD at a defined ratio. Based on prior experience, the number of formulations in the screening could be kept at a minimum which also reduced the number of technical batches to be manufactured to serve initial stability testing. In parallel the analytical methods for CBD and THC in the oil formulation were developed and validated according to GMP standards. Clinical batches of the selected oily CBD & THC formulation as well as the respective placebo were manufactured and released for the clinical trials on time. To secure the program and its continuation, Ardena also qualified the supplier as well as put samples of the clinical batch in passive stability.

Measuring plasma concentrations of cannabis components and their metabolites is another specific challenge due to the very low plasma concentrations and required sample handling procedures.

During the course of many preclinical and clinical studies supported by Ardena, a set of fully validated cannabinoid bioanalytical methods based on LC-MS/MS analysis have been developed and validated according to the latest FDA and EMA guidelines for bioanalytical method validation.

For cannabinol (CBN), a sophisticated sample processing method has been designed and optimized. After addition of an internal standard (a stable isotope labeled variant of CBN) and sample derivatization combined with an extensive sample clean-up, a sensitive and quantitative bioanalytical assay has been validated in the range as low as 1.00 to 100 pg/mL. This method has been successfully applied in numerous regulated preclinical studies and in phase I and II clinical programs to provide detailed PK data in plasma and in tissue samples.

Another valuable example can be found in the combined assay for tetrahydrocannabinol (THC), 11-hydroxy tetrahydrocannabinol (11OH-THC) and cannabidiol (CBD). After liquid-liquid extraction of the samples, the compounds and their respective internal standards are derivatized and purified using solid phase extraction. The subsequent LC-MS/MS analysis then allows the accurate and precise determination of plasma levels as low as 0.1 ng/mL for each of these analytes. If necessary, the 11-nor-9 carboxy-THC metabolite of THC (i.e. THC-COOH) can be analysed by our laboratory as a separate assay. Due to the different physicochemical properties of THC and THC-COOH and the fact that THC-COOH levels in plasma remain relatively high over time, a combined assay is not optimal

Chemistry, Manufacturing and Controls (CMC) is yet another, often underestimated, requirement for the successful development of medicinal cannabis products. CMC is a mandatory procedure for the comprehensive documentation of the entire product development according to the respective guidelines to support any clinical trial and product approval.

Conclusion

After the legalization of cannabis, a dedicated science has emerged on the variety of therapeutic components and possible uses, from which new medicines are now emerging. The botanical origin of cannabis derived products poses special challenges to its development into a medical product and approved drug. Despite all the enthusiasm, it is therefore important from the outset to develop and document the product systematically in terms of quality, clinical efficacy and safety. In order to keep risks to a minimum during the course of development and to take advantage of new opportunities as they arise, cooperation with experienced experts has proven its worth. Especially in a highly competitive environment, they can be decisive for the market entry and success.

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FDA (2020) Cannabis and Cannabis Derived Compounds: Quality Considerations for Clinical Research – Guidance for Industry (Draft Guidance) (https://www.fda.gov/media/140319/download)
Consroe et al. (1991) Controlled clinical trial of cannabidiol in Huntington’s disease. Pharmacol. Biochem Behav 40: 701–708
Bruni et al. (2018) Cannabinoid Delivery Systems for Pain and Inﬂammation Treatment. Molecules 23: 2478
Martin-Banderas et al. (2014) Engineering Δ⁹-tetrahydrocannabinol delivery systems based on surface modiﬁed-PLGA nanoplatforms. Coll and Surf B: Biointerface 123: 114-122
Hommoss et al. (2017) Mucoadhesive tetrahydrocannabinol-loaded NLC – Formulation optimization and long-term physicochemical stability. Eur J Pharm Biopharm 117: 408–417
Lucas et al. (2018) The pharmacokinetics and the pharmacodynamics of cannabinoids. Br J Clin Pharmacol 84: 2477–2482
Kraemer et al. (2019) Detectability of various cannabinoids in plasma samples of cannabis users: Indicators of recent cannabis use? Drug Test Anal 11:1498 – 1506
Braganca et al. (2020) Impact of conformational and solubility properties on psycho-activity of cannabidiol (CBD) and tetrahydrocannabinol (THC). Chem Data Collect 26: 100345
Gonsalvez et al. (2019) Cannabis and Its Secondary Metabolites: Their Use as Therapeutic Drugs, Toxicological Aspects, and Analytical Determination. Medicines 6: 31
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Rein JL. (2020) The nephrologist’s guide to cannabis and cannabinoids. Curr Opin Nephrol Hypertens 29(2):248-257

Prevent undesired immunogenicity from becoming the barrier for innovative biotherapeutics

Engineered therapeutic proteins, monoclonal antibodies (MAbs) as well as bispecific monoclonal antibodies (BsMAbs), genes or genetic sequences (e.g. mRNAs, oligonucleotides), and cell and engineered cell based therapeutics (e.g. stem cell, CAR-T-cells) have demonstrated the successful delivery of breakthrough therapies for many unmet medical needs. The enthusiasm continues to increase the pipelines of the pharmaceutical industry in their efforts to address the medical conditions for which no effective treatment currently exists. While immunological reactions are utilized in effective vaccine development as demonstrated recently by the introduction of a new generation of mRNA vaccines against SARS CoV2 in record time, undesired immunological reactions to engineered therapeutic biopharmaceutical compounds are a threat. Throughout the development of therapeutic biologic compounds, undesired immunogenicity remains one of the most important risk factors for patient safety. The FDA made it clear that it will not compromise on patient safety in clinical trials for the development of therapies even vaccines for the SARS-CoV2 virus, stating “Ensuring the safety of trial participants is paramount”

Recognizing the importance of immunogenicity

The increasing recognition of an imminent risk of immunogenic reactions of therapeutic proteins arose from various products in clinical development or early marketing. Vatreptacog alfa was a promising engineered factor VIIa derivative for the treatment of hemophilia patients. In the phase III clinical study (NCT01392547) neutralizing antidrug antibodies (ADAs) were observed in the vatreptacog alfa group in 10 % of the patients compared to less than 1% in the recombinant factor VIIa (rFVIIa) group. Even though the engineered factor VII product demonstrated superior efficacy, the higher immunogenicity led to an unfavorable risk-benefit profile and discontinuation of the program [2]. The mechanism of immunogenicity was elucidated by in vitro methods that could have predicted immunogenicity at the preclinical stage [3]. The knowledge generated by the in-vitro assays provided evidence for a neo-epitope generated by vatreptacog alfa and could guide the development of a deimmunized derivative without compromising on the superior efficacy [4]. Other examples are peginsenatide [5] and bococizumab [6], which despite clinical efficacy and benefits had to be discontinued due to the occurrence of unfavorable immunogenic reactions. Each of these withdrawals caused great financial damage, loss of reputation, and deprivation of a new therapeutic option for patients.

From science to regulatory guidance

To mitigate the risk of undesired immunogenicity leading to discontinuation or withdrawal of a biologic compound, substantial scientific efforts have been made over the past decade to better predict and assess immunogenicity of therapeutic biologics from early development onwards. This has also been strongly incorporated into regulatory guidance that expect in vitro predictions of the immunogenicity of a new biologic prior to first use in humans. The FDA guidance clearly states that the “development of valid, sensitive, specific, and selective assays to measure ADA responses is a key aspect of therapeutic protein product development.” Consequently, the FDA expects the sponsor to “provide an immunogenicity risk assessment as well as a rationale for the immunogenicity testing paradigm in the original investigational new drug application (IND)”. It is recommended that a multi-tiered testing approach is performed with sufficiently validated assay cut off points, sufficient sensitivity, specificity and selectivity, as well as precision, reproducibility, robustness, and sample stability in the assay. The guidance further considers the selection of the reagents used in the assay as a critical part of the assay development and expects the qualitative and quasi-quantitative assay results to be reported in an appropriate manner [7]. The EMA guidelines also emphasize the influence of patient and disease factors, as well as product factors, which may have an impact on clinical consequences of immunogenicity that need to be evaluated early. Therefore the EMA points out that “the evaluation of immunogenicity should be based on integrated analysis of immunological, pharmacokinetic, pharmacodynamic, as well as clinical efficacy and safety data.” It is considered that the recommendations will have to be adapted on a case-by-case basis to fit into an individual development program and that a Risk Management Plan (RMP) is provided [8].

Evaluating immunogenicity and mitigating the risk

Immunogenicity is induced by complex activation of cellular and/or humoral system response involving T cell activation and the formation of histocompatibility complex (MHC) molecules and subsequently antigen-presenting cells (APCs).There are three gene loci that encode the MHC class I proteins in humans (HLA-A, HLA-B and HLA-C) as well as the MHC class II proteins (HLA-DR, HLA-DQ and HLA-DP). Stable peptide-MHC-II complexes are transferred to APCs’ surface for presentation to CD4+ T cells, which can initiate, maintain and regulate immune responses, including the production of ADAs and neutralizing Antibodies (NABs). To assess immunogenicity a variety of technologies can be used covering binding affinity studies e.g. using surface plasmon resonance or ELISA, flow cytometry to determine MoDC and T cell activation, investigating cytokine release with ELISA or cell based assays using whole blood or purified specific subsets of cells like CD4+, CD8+ or T cells. One of the challenges of in vitro assays is how the HLA peptides and cells are obtained or processed and how the assays are performed. For example, due to the genetic variety, pooled samples from multiple donors are requested to cover at least 80 % of the human HLA-class II haplotypes [9]. To overcome some of the limitations of these systems, the profiling and identification of T cell epitope displayed by HLA Class II molecules after uptake of the full-length protein by monocyte derived dentritic cell APCs can be performed by LC-MS/MS systems. Additional assays are being considered to better understand the proliferation and activation of T cell effector functions by other stimulating signals such as CD80/86 on the APC and ligand CD28 on the T cell. Characterization and quantification of the immune complex (IC) formed by the ADA and the drug may help to predict the influence pharmacodynamics and pharmacokinetics to enable dedicated in vivo studies [10]. In addition to these assays, predictive models are progressing and have shown their potential to reduce the number of assays and allow, when parameters are well defined, weighted, and evaluated in an ex vivo setting, correlation with the clinical data [11-13]. However the evaluation of immunogenic responses remains specific to each protein and species and has to follow a product-specific system as well as rational scientific strategy and selection of the best suited analytical methods. Special skills, knowledge, and experience from different disciplinary areas are crucial for valid interpretation of the data and fast execution.

A case study on successful partnering with Ardena

A highly promising analogue of an endogenous protein was selected as a lead drug candidate for a first-in-human clinical study. An immunogenicity risk assessment classified the protein as one at high risk for immunogenic reactions. Ardena’s expertise was used to establish a valid immunogenicity profile of the protein prior to entering the clinical trial and to generate the immunogenicity assay and data necessary for IND filing. The development of the immunoassay for the analog protein required the development and production of specific antibodies against the protein, which are essential to serve as positive controls. The specific antibodies were obtained and purified from immunized rabbits in sufficient quantities for the entire immunogenicity study. Keeping assay development cost at a minimum, a bridging ELISA with an electrochemoluminescent read-out on the Mesoscale Discovery platform (MSD-ECL) was developed and validated in rat, monkey and human serum. The analogue protein was labelled with a biotin or sulfo-tag to serve as a capture or detection reagent respectively. The immunoassay was subsequently developed, qualified, and validated. Using a mastermix containing equal concentrations of biotinylated and sulfo-tag labelled drug, the complex of biotinylated drug – ADA – sulfo-tag labelled drug was detected on a blocked MSD Gold Streptavidin plate. A negative control pool was prepared per matrix by pooling at least 34 individual sources of rat or monkey sera or 51 individual sources of human serum, to which positive control antibodies of different concentrations were added. The minimal required dilution (MRD) was set at a 10-fold dilution of the samples containing ADA, as this dilution gave a response close to the response of non-specific binding. The assay sensitivity was between 1 and 10 ng/mL anti-drug antibodies, which is well below the regulatory required sensitivity of 100 ng/mL. The drug tolerance was between 10 and 100 µg/mL of drug at an anti-drug antibody concentration of 100 ng/mL. Using this established immunoassay, the immunogenicity risk of the protein can be ranked and the necessary data provided for the FDA. Early development of the immunogenicity assay laid the groundwork for further clinical evaluation of immunogenicity, which continues to be supported by Ardena for validation in humans.

Read the full case-study here.

Conclusion

The study of the immunogenicity of biological products remains a case-by-case consideration despite the growing number of in vitro and ex vivo assay options. It is always composed of preclinical and clinical data, for which specific experience and experimental setting are necessary. Prior to initiation of the first clinical studies in humans, a screening of immunogenic reactions in the form of potential ADA and, depending on the risk, neutralizing antibodies is required by the FDA and EMA. The immunogenicity assay must be adequately validated according to the guidelines e.g. with regard to sensitivity, specificity, selectivity, cut off points, minimal required dilution, etc. As a prerequisite for the first clinical trial and ongoing throughout development, a rationale for the chosen strategy of immunogenicity evaluation and a risk management plan is expected from the regulatory authorities. They strongly encourage to seek their scientific advice to ensure the desired trial participants safety and fast entry into the clinical trial program.

References

FDA (2020) Conduct of Clinical Trials of Medical Products during the COVID-19 Public Health Updated January 27, 2021
Lentz et al (2014) Recombinant factor VIIa analog in the management of hemophilia with inhibitors: results from a multicenter, randomized, controlled trial of vatreptacog alfa. J Thromb Haemost, 12: 1244–1253
Lamberth et al (2017) Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools. Sci Transl Med 9(372):eaag1286
Jankowski et al (2019) Mitigation of T-cell dependent immunogenicity by reengineering factor VIIa analogue. Blood Adv 3(17):2668-2678
Hermanson et al (2016) Peginasetide for the treatment of snemia due to chronic kidney disease –an unfulfilled promise. Expert Opin Drug Saf 15(10): 1421-1426
Ferri et al (2017) Bococizumab for the treatment of hypercholesterlaemia. Exp Opin Biol Ther 17(2):237-243
FDA (2019) Immunogenicity testing of therapeutic proteins products – developing and validating assays for Anti-Drug Antibody detection. (https://www.fda.gov/media/119788/download)
EMA (2017) Guideline on immunogenicity assessment of therapeutic proteins (https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-immunogenicity-assessment-therapeutic-proteins-revision-1_en.pdf)
Groell et al (2018) In-vitro models for immunogenicity prediction of therapeutic proteins. Eu J Pharm Biopharm 130: 128-142
Hoffman et al (2019) Generation, characterization, and quantitative bioanalysis of drug/anti-drug antibody immune complexes to facilitate dedicated in vivo studies. Pharm Res 36: 129
Rosenberg et al (2018) Immunogenicity assessment during the development of protein therapeutics. J Pharm Pharmacol 70: 584-594
Hamuro et al (2019) Evaluating a multiscale mechanistic model of the immune system to predict human immunogenicity for a biotherapeutic in phase 1. AAPS 21:94
Barra et al (2020) Immunopeptidomic data integration to Artiﬁcial Neural Networks enhances protein-drug immunogenicity Front. Immunol. 11:1304

Drug Product

Nanomedicine

Bioanalysis

Drug Substance

Drug Product

Nanomedicine

Bioanalysis

Drug Substance

Drug substance for early clinical phase initiation – the underestimated risk of unexpected N‑nitrosamine impurities

Table 1: Types of impurities in drug substance

Figure 1: N-nitrosamine investigation – Step-by-step plan, Step 1

Figure 2: N-nitrosamine investigation – Step-by-step plan, Step 2 & 3

Table 2: Some specific N‑nitrosamines and their acceptable daily intake (ADI)

Bioanalytical characterization and monitoring of ADCs supporting safety and efficacy from pre-clinical to clinical

The R&D challenge

Developing the ADC specific bioanalytical strategy

Figure 1: Implementation of an analytical strategy during the transition from the pre-clinical to the clinical stage

Major principles of ADC assay methods

The role of reagents for ADC analytics

The importance of regulatory compliance

Case study

Figure 2: Calibration curve for the payload in K2-EDTA plasma across the plasma concentration of 1.00 – 100 pg/mL

Table 1: Validation of the conjugated antibody concentration measurement in human K2-EDTA plasma across the expected in-vivo concentration for the pharmacokinetic study in humans

Conclusion

References

Deciphering the complex characteristics of nanoparticles by asymmetric flow field-flow fractionation

Introduction

Characterization of nanoparticle drug delivery systems for pre-clinical and clinical testing

Figure 1. The CQAs of nano-formulations can be categorized in five groups.

Asymmetric flow field-flow Fractionation (AF4)

Figure 2. Schematic illustration of the separation mechanism in AF4.

Application of AF4 in drug development

Figure 3. Particle size characterization during nanoparticle formulation and process development.

Phase appropriate scaling from comparative sizing with DLS/NTA to full scale characterization of size and aggregation/interaction in standard media and biorelevant media after separation from interacting protein.

References

Phase-appropriate development of a phase 1 oral dosage form

Leverage the power of CMC in early drug development

Formulation and Process Considerations for Optimising Spray-Dried Solid Dispersions

Much more than just smoke – from marijuana to medicinal cannabis derived therapeutics

Prevent undesired immunogenicity from becoming the barrier for innovative biotherapeutics