Author: Veeda_admin

Bioassays are involved in each stage of drug discovery, starting from Target Identification until discovering the Lead compound.

Bioassays provide valuable information that displays the therapeutic potency of a drug under investigation.

The data generated during bioassay also plays a vital role in drug development and quality control of finished biological products.

Properly designed bioassays help assess the biological effect, activity, signal transduction process, and receptor binding ability of drug products or biologics on biological targets (proteins) when compared to a reference or standard for a suitable biological system.

The pharmaceutical and biotech companies involved in drug discovery and development are continuously challenged with developing biologically relevant assays for the analysis of multiple potential mechanisms.

The process involves the use of quality critical reagents, use of specific cell lines, and purified test drug and reference drug products, which at times may become a constraint.

Most of these activities require sufficient time, which may become a limiting factor for biopharma manufacturers.

It is worth outsourcing activities to reputed CRO service providers to save time in developmental efforts and also to have an unbiased opinion on the functional activities of the drug product.

Veeda Group has qualified and experienced scientists to design, develop, execute, and validate bioassays for companies, providing premier bioassay services (in vitro and in vivo) that generate meaningful data to support pharmaceutical and biotech companies in their drug discovery and development journeys.

Veeda Group’s Experience in Development and Execution of Bioassays includes:

• Plaque Reduction Neutralization Test (PRNT assay)
• In Vitro Skin Sensitization Human Cell Line Activation test (h-CLAT assay)
• Nab Assay
• Assay Development (Pharmacodynamics, Pharmacokinetics, Immunogenicity, and Biomarker Assessment)
• In Vivo Bioassays for drug molecules like Luteinizing Hormone, Epoetin, HCG, Recombinant FSH, β-HCG, and Insulin.
• ADCC assay for biosimilars and other different assays like Ex Vivo assay, Cell-based assay, Receptor Binding Assay, Cytokine Release Assay, and ADA assay.

Veeda Group provides Integrated Discovery, Development & Regulatory Services with its multiple technology platforms:

• Exploratory toxicology studies
• Regulatory toxicology studies
• In vitro Bioassays
• Ex vivo Bioassays

The group also has the experience to handle a diverse range of Biotherapeutics like Therapeutic Monoclonal Antibodies, Insulin & Insulin Analogues, Cytokines, Low Molecular Weight Heparins, Biosimilars, Hormones & Biomarkers.

Veeda group has demonstrated capabilities to develop recombinant proteins such as non-glycosylated proteins and glycoproteins derived from either bacterial or mammalian host expression systems.

Bioassays in Preclinical Drug Development

Biological assays or bioassays are essential tools in preclinical drug development.

Preclinical bioassays can be in vivo, ex vivo, and in vitro.

In vivo, bioassays provide a more realistic and predictive measure of the functional effects of tests with reference drug products or standard material of defined potency, along with the application of statistical tools, study-specific lab techniques, and adherence to a well-designed study protocol.

These assays capture the complexity of target engagement, metabolism, and pharmacokinetics of novel drugs better than in vitro bioassays.

The most commonly used experimental mammals in in vivo efficacy assays are mice and rats.

Occasionally, other species may be used depending on the sensitivity & suitability of the assays.

Development and Validation of Bioassays

Bioassays are used as a screening method to identify the signals that indicate desired biological activity from a set of compounds.

In general, two different types of signals can be generated by a bioassay: a linear dose-response and a sigmoidal (S-shaped) dose-response.

Since one solution does not fit all bioassays, it is good to evaluate and analyze the data to develop a precise approach to carry out each bioassay.

The life cycle stages of a bioassay are divided into:

• Stage 1: Method design, development, and optimization
• Stage 2: Procedure performance qualification
• Stage 3: Procedure performance verification (fit for purpose)

Developing a bioassay that meets regulatory requirements and gets a drug product registered is a very complex process.

Developing a bioassay includes many strategies and tactical designs like selecting the correct in vivo platform, proper method or plate design, data analysis, system/ sample sustainability strategy, method implementation, method performance, and monitoring.

There are several steps to be followed for the development and validation of bioassays, such as dose-response and curve-fitting selection, development of reference, calculation of potency, bioassay characterization, design of bioassay calculator, standardization and automation of bioassay, and finally, evaluation.

Both method development and validation of bioassays include three fundamental areas:

1. Pre-study (Identification and Design Phase) validation
2. In-study (Development and Production Phase) validation
3. Cross-validation or method-transfer validation

During method development, assay conditions and procedures are selected to minimize the impact of potential sources of invalidity.

Coming to the statistical validation for an in vivo assay, it involves four major components:

1. Adequate study design and data analysis method
2. Proper randomization of animals
3. Appropriate statistical power and sample size
4. Adequate reproducibility across assay runs.

Parallel group design, randomized block design, repeated measures design, and crossover design are the basic types of experimental designs used in in vivo assay.

The following are the key factors that should be kept in mind while designing an in vivo assay:

• All meaningful biological effects (pharmacologically) should be statistically significant.
• If biologically relevant assays are not present, then a range of plausible effects can be considered.
• The key endpoints should be well-defined before the beginning of the assay.
• Animals should be allocated randomly in an appropriate manner to the treatment groups.
• The dose levels should be selected appropriately. Dose and curve-fitting selection is among the most critical aspects of bioassay development. The dose is determined depending on the type of model used to fit the signal to the data. For Sigmoidal designs, a four- or five-parameter logistics (4PL or 5PL) model fits the data, whereas for linear designs, a parallel line analysis (PLA) model fits the data.

For a 4PL model, nine doses are recommended:

1. Three doses in the lower asymptote
2. Three doses in the upper asymptote
3. Three doses in the linear range

In contrast, for a PLA model, a minimum of four doses is recommended. A minimum of three consecutive doses is required to plot the dose curve.

• The selection of control groups and time points to collect samples should be optimal.
• The design strategies should minimize variability and maximize information.

To understand the design, developments, and statistical validation of in vivo bioassay in more detail, reach out to us at https://veedalifesciences.com/.

One can also read the guidelines mentioned by NIH by visiting the link:

https://www.ncbi.nlm.nih.gov/books/NBK92013/pdf/Bookshelf_NBK92013.pdf

References

1. A. Little, “Essentials in Bioassay Development,” BioPharm International 32 (11) 2019
2. Padmalayam, Ph.D., Assay development in drug discovery
3. Zwierzyna M, Overington JP (2017) Classification and analysis of a large collection of in vivo bioassay descriptions. PLoS Comput Biol13(7): e1005641. https://doi.org/10.1371/journal.pcbi.1005641
4. White JR, Abodeely M, Ahmed S, Debauve G, Johnson E, Meyer DM, Mozier NM, Naumer M, Pepe A, Qahwash I, Rocnik E, Smith JG, Stokes ES, Talbot JJ, Wong PY. Best practices in bioassay development to support registration of biopharmaceuticals. Biotechniques. 2019 Sep;67(3):126-137. doi: 10.2144/btn-2019-0031. Epub 2019 Aug 5. PMID: 31379198.
5. F Chana and Hursh D. Bioassays through the Product lifecycle: Perspectives of CDER and CBER reviews.
6. Haas J, Manro J, Shannon H, et al. In Vivo Assay Guidelines. 2012 May 1 [Updated 2012 Oct 1]. In: Markossian S, Grossman A, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Bookshelf URL: https://www.ncbi.nlm.nih.gov/books/

Lab Informatics: Revolutionising Pharma R&D

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Cancer is a deadly disease leading to the death of many individuals across the globe.

Biotech and Pharmaceutical researchers are carrying out extensive studies to develop drugs to treat cancer.

However, the current medications used in cancer treatment have many loopholes.

They are toxic, lack specificity, and have short half-lives.

The difficulty in administering complex oncology molecules, along with the above hurdles, has led to side effects, non-compliance, and patient inconvenience with many current treatments for cancer.

Liposomes are nano-sized drug delivery systems that have been shown to be quite effective in improving the selectivity of cancer chemotherapeutic agents.

However, clinical trial experts face many challenges when designing a Bioequivalence (BE) study for generic oncology drugs.

It includes selecting the study population, selecting the individual dose for patients, selecting the required study design (cross-over vs. steady-state design), and processing samples at investigator sites due to sampling uncertainty, high patient dropout rates, and stringent regulatory guidelines.

A bioequivalence study is generally conducted in healthy volunteers if the drug has shown a safety profile in a healthy population and is not a narrow therapeutic index drug.

However, the same is not ethically and medically acceptable in most anticancer drugs because of cytotoxicity in a healthy population.

Moreover, the regulatory requirements also vary from region to region.

How to Design a Study for a Generic Oncology Product on Liposome Injection Involving Cancer Patients?

Study Overview

Veeda Clinical Research completed an open-label, randomized, two-treatment, two-period, two-sequence, single-dose, multicentric, fasting, cross-over bioequivalence study of Doxorubicin Hydrochloride Liposome Injection 2 mg/mL in ovarian cancer patients for an Indian based Sponsor Company towards submission to the USFDA.

The study was completed within the stipulated timeframe with meticulous project management.

In both periods, the subjects received a 50 mg/m² single dose (intravenous infusion) of Doxorubicin Hydrochloride Liposome Injection 2 mg/mL (either the test or reference product), according to the randomization schedule created before the trial, on the first day of the chemotherapy cycle.

The washout period was at least 28 days between each consecutive dosing period.

Each cycle began with the collection of serial blood samples; a total of 25 blood samples were collected, with the last blood sample collected at 360.00 hours in each period.

Blood samples starting from 72.00 hours to 360.00 hours were collected on an ambulatory basis in each period to determine free and liposomal encapsulated doxorubicin plasma concentrations for PK analysis.

Subjects Inclusion and Exclusion Criteria

The study involved female patients between the ages of 18 and 65 who had ovarian cancer (confirmed through cytological and histopathological tests) and who were already receiving or scheduled to start therapy with the reference listed drug (RLD) or the reference standard product.

The four major inclusion criteria in this study were:

• Subjects with Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2
• Subject with Left Ventricular Ejection Fraction ≥ 50%
• Subjects with a life expectancy of at least three months are determined by checking liver, kidney, and bone marrow function.
• Subjects who had recovered from minor (at least one week) and major (at least four weeks) surgery.

Women who were pregnant, lactating, or planning for a family were excluded from the study.

A total of 18 parameters were judged under exclusion criteria.

Some of the major exclusion criteria were:

• Impaired cardiac function with the occurrence of unstable angina/arrhythmia/ myocardial infarction/ Qtc prolongation/ coronary artery bypass graft surgery/ heart failure/ symptomatic peripheral vascular disease within the last six months.
• Known history of brain metastasis.
• Pre-existing motor or sensory neurotoxicity of a severity ≥ grade 2 according to NCI criteria.
• Positive test results for hepatitis and HIV.

Reporting and Handling of Adverse Events

The investigators reported six serious adverse events (SAEs) during the entire study.

Fever with diarrhea was reported in two subjects.

Another two subjects were observed with Nausea, Vomiting, and weakness.

Fever due to hospitalization and Non-neutropenic fever with acute gastroenteritis were also found in one subject each.

At the time of writing this article, all SAEs have been resolved after constant follow-ups with the patient.

During the study, hypersensitivity reactions due to Doxorubicin Hydrochloride Liposome Injection were avoided by administering Prophylactic Antiemetic and Dexamethasone Injection 8mg.

Conclusion

The study was successful, as the test product showed bioequivalence with the reference product.

The pharmacokinetic parameters like Cmax, AUC_0-t, and AUC_0-∞ were within the range of 80.00 to 125.00%.

Veeda Clinical Research provided end-to-end services in identifying and selecting the clinical trial sites, preparing and submitting regulatory documents like protocol, ICF, CRF, and Clinical Study Report to the drug regulatory authority on behalf of the sponsor company.

Trained and experienced nurses and investigators regularly monitored the oncology patients who participated in the study.

The study was completed successfully with fewer patient dropouts, abiding by the principles of Good Clinical Practice.

Finally, the product was approved by the USFDA.

Experienced personnel, including the Principal and Clinical investigators team at different sites, the project management team, CRAs, phlebotomists, nurses, the medical writing team, and the bioanalytical team of Veeda Clinical Research, are responsible for successfully completing this clinical trial.

Hi, I am Mansi Shah, a clinical research nurse with over 9 years of experience. I started my practice in 2013 at Sterling Hospital after completing my GNM nursing course.

I have been working with Veeda since 2015.

As a clinical research associate, my day mainly constitutes of assisting in research activities and ensuring volunteer safety, protection, and that volunteers are well supported throughout the research study.

Even though every research is unique and every day is varied, I’m a seasoned Senior Research Associate, and my duty is to be right alongside the research participants in the journey, from the day of admission to their dosing and till the time they get discharged.

However, the most important task of a clinical research nurse involves determining the consent of suitable volunteers who consent to proceed with the study.

I have to ensure that the volunteer understands what the research seeks to accomplish and the protocols associated with it.

After educating the volunteers, I have to check her/his eligibility through OVIS and double-check it through routine screenings like blood and urine tests.

A usual day starts with a doctor assigning me my duty as per the study slots.

I go to my designated location, mind the volunteers, and check their vitals.

I hope and work to minimize the risk of adverse events during the research, but the risk is always there.

Identifying adverse events at the earliest possible time requires disciplined training and an in-depth understanding, thereby minimizing risk to research participants.

With the nature of conducting novel research, the risk of adverse events is always there, and the way we counteract it is by having ICU wards with doctors and nurses on standby so that we can treat any complication with haste.

Volunteer Safety is of the utmost importance to me and to Veeda, and we take all the necessary measures, in terms of personnel skills and our infrastructure, to ensure the same.

Besides taking care of research participants, documenting and recording information during clinical trials is the most important responsibility that a research nurse has.

And we at Veeda ensure the Quality and reproducibility of data by taking a meticulous approach and following the highest level of integrity.

I am extremely passionate about my job as I feel I am a part of something that is larger than us & larger than my role.

I wanted to be a part of it, as I get to be a part of the research that aims to test an experimental practice on willing volunteers and see it become a part of standard practice, therefore, saving many lives in the future.

Hi, I am Gangichatti Laxman Kumar & I work as a Clinical Research Associate with Veeda, and this is how a day in my life looks like

Although I’m based out of Hyderabad, I might be visiting a site that’s in a completely different part of the country by the time you read it.

This blog is supposed to walk you through a typical day in the life of a CRA.

A Clinical Research Associate plays a crucial role within the pharmaceutical business.

A CRA is responsible for pre-study qualification visits, reviewing the study progress, checking the quality & accuracy of data collection, ensuring compliance of patients with trial visits, and ensuring good clinical practices are maintained throughout the trial.

After successfully completing Pharma-D, I started working as a Safety Associate to the regulatory bodies; after that, I switched to clinical research operations and started working as a CRA in Oncology, Neurology, Endocrinology, Cardiology, and General Medicine.

I have also worked in the department of BA/BE trials, where I experienced a multi-functional team, and finally moved to Veeda Clinical Research, where I got the opportunity to work in BA/BE studies as well as Late Phase Trials in the field of Oncology.

Being a CRA, I have to spend a significant amount of time traveling to and from all the research sites that I have been assigned, which are spread throughout the country, and I visit 4 to 5 sites in a day.

The very fact that I have to be constantly on the move, which happens to be a part of my job, adds a travel aspect to the mix that always remains fresh.

I believe that Social interaction plays an important role in learning, and through this role, I have the opportunity to interact with a wide range of people, from site coordinators to doctors to project managers, which has proven to be quite effective in my cognitive development.

My standard operating day comprises of monitoring and supervising data files as a part of the source data verification process to ensure that the site is entering data accurately and in a timely manner.

The safety of a patient is of the utmost importance at Veeda, and I, along with my staff, regularly assess patient notes to ensure the safe undertaking of procedures as per the protocol.

Every role comes with its own set of challenges, and the role of a CRA is no different. Veeda offers workplace flexibility, which helps me deal with challenges calmly & efficiently.

Being a CRA, I practice a fast-paced lifestyle, but for me, the sense of accomplishment I get from tackling all those challenges is what makes me choose this line of profession every time.

In our last blog on Master protocols, we discussed the definition of master protocol, the types, and the advantages of using Master Protocol in clinical trials.

In today’s article, we would like to present to you the parameters that are kept in consideration while designing a master protocol for oncology drugs and biologics.

During the preparation of master protocols, different parameters are kept in consideration, such as:

• Specific Design Considerations
• Biomarker Development Considerations
• Statistical Considerations
• Safety Considerations
• Regulatory Considerations

Specific Design Considerations in Master Protocols

1. Use of a Single Common Control Arm

The FDA recommends using a single control arm with the current System Organ Class (SOC) when developing a master protocol that assesses multiple drugs in a single disease.

The SOC for the target population can be revised during the trial if a new drug is approved or if scientific evidence emerges that renders it unethical to randomize patients based on the previous SOC.

In such a situation, the FDA recommends that the sponsor suspend patient enrollment until the protocol, the SAP, and the protocol-informed consent document are modified to include the new SOC as the control.

2. Novel Combination of Two or More Investigational Drugs

When writing a master protocol involving two or more investigational drugs as a combination product, the sponsor should summarize the following.

• Safety of the combinational product
• Pharmacology of the combinational product
• Preliminary efficacy data for each investigational drug
• Rationale for the use of the drugs as a combination product
• Evidence of any synergistic effect (if any) of the two or more investigational drugs when given in combination.

The FDA strongly recommends that sponsors ensure the identification of the Recommended Phase II Dose (RP2D) for each drug with antitumor activity in all cases.

3. Studies With Drugs Targeting Multiple Biomarkers

The FDA highly encourages early discussion of biomarker research strategies when a sponsor plans to use one or more biomarkers to guide patient selection for trials.

A defined plan for the allocation of eligible patients should be present.

Patient selection studies must be analytically checked with well-defined parameters for master protocols involving drugs that target multiple biomarkers.

4. Adding and Stopping Treatment Arms

Before beginning the clincial trial, the sponsor should make sure that the master protocol and its corresponding SAP identify conditions that would contribute to adaptations, such as introducing a new experimental arm or arms to the study, re-estimating the sample size based on the interim analysis results, or discontinuing the experimental arm on the rules of futility.

5. Independent Data Monitoring Committee (IDMC)

The master protocol should provide details of the IDMC involved in monitoring efficacy results and details of the Independent Safety Assessment Committee (ISAC) involved in monitoring safety results.

However, the IDMC can perform both the functions of safety and efficacy.

For marketing an oncology drug, if the basis of the marketing application involves one or more sub-studies, the FDA recommends the inclusion of an independent radiologic review committee to perform blinded tumor-based assessments.

Biomarker Development Considerations

Master protocols assessing biomarker-defined populations should explain the rationale behind the use of that particular biomarker.

The sponsor should employ in vitro diagnostic (IVD) tests that are analytically validated, establish procedures for sample acquisition, handling, and the testing and analysis plans as early as possible.

The sponsor may need to submit the IVD’s analytical validation data to the FDA (CDRH or CBER) to determine whether the clinical results will be interpretable.

Statistical Considerations

If a sponsor introduces randomization into the design of an umbrella trial, the FDA recommends using a standard control arm whenever possible.

Bayesian statistical methods or other methods for dropping an arm, modifying sample size, or implementing other adaptive strategies can be used in the preparation of master protocols.

The SAP should include details on the implementation of Bayesian or other methods as described in the FDA guidance for industry, Adaptive Design Clinical Trials for Drugs and Biologics, and the guidance on Enrichment Strategies for Clinical Trials to Support Approval of Human Drugs and Biological Products.

Statistical considerations for master protocols can be strategized in four different ways:

1. Nonrandomized, Activity-Estimating Design
2. Randomized Designs
3. Master Protocols Employing Adaptive/Bayesian Design
4. Master Protocols With Biomarker-Defined Subgroups

Safety Considerations

The sponsor should implement a structured team of ISAC or an IDMC to assess the safety as well as the efficacy of all master protocols.

The constitution of this committee and the definition of its responsibilities should be well-defined in the IND.

A sponsor should not begin a clinical trial until the master protocol has been reviewed and approved by an IRB or IEC.

The FDA encourages the use of a central IRB to promote the IRB analysis of master protocols.

The sponsor is required to conduct a safety review of master protocols more frequently than annually and provide the investigator with the details.

If the master protocol contains proposals to include pediatric patients in the study, the FDA advises that the IRB include a pediatric oncology expert in its team who has expertise with the regulatory criteria for the enrollment of pediatric patients in clinical investigations, including parental approval and consent.

The informed consent document should be submitted to the Institutional Review Board (IRB) for review.

Additional Regulatory Considerations

Each master protocol should be submitted as a new IND to the FDA.

This is done to avoid miscommunication, owing to the sophistication of master protocols that may hamper patient safety.

If the sponsor is conducting a clinical trial on more than one indication for oncology drugs or biologics, the IND should then be forwarded to the Office of Hematology and Oncology Products at the Center for Drug Evaluation and Research (CDER) or the Center for Biologics Evaluation and Research (CBER) for approval.

REFERENCE

Master Protocols: Efficient Clinical Trial Design Strategies to Expedite Development of Oncology Drugs and Biologics, Guidance for Industry, Draft Guidance. U.S. Department of Health and Human Services, Food and Drug Administration, September 2018.

With many new therapeutics approved annually, the demand for biologics has seen exponential growth in the pharmaceutical market.

In the bioanalytical community, the study of large molecules is now a hot topic of discussion.

The snowballing importance of peptides and proteins as therapeutic agents, combined with the colossal opportunities offered by new MS-based technology, has unlocked a new world for bioanalytical scientists.

Ligand-binding assays (LBAs) such as enzyme-linked immunosorbent assays (ELISA) or UV identification of individual peptides using high-performance liquid chromatography (HPLC) are the standard methods for the quantification of biologic drugs.

However, these methods are typically expensive, are time-consuming to develop, and have limited selectivity and antibody cross-reactivity.

This results in a lack of interference specificity and high background levels that are not appropriate for fulfilling the specifications of the biopharmaceutical industry to identify different proteins and peptides with increasing sensitivity and reproducibility.

Liquid chromatography combined with tandem mass spectrometry (LC-MS-MS) has been widely used for small molecule bioanalysis in pharmaceutical laboratories since the 1980s.

Like smaller molecules, LC-MS-MS also carry advantages for biologics:

• It is not susceptible to cross-reactivity of the antibody because LC-MS-MS involves direct assessment of the analyte’s chemical properties.
• It provides excellent selectivity, being able to discern and quantify extremely homologous isoforms with precision and accuracy over a large linear dynamic range, even at low levels.
• Due to its high analytical sensitivity and selectivity, in addition to its high-throughput capability, LC-MS/MS has been considered the primary technique to measure the concentrations of first-generation and second-generation antipsychotics in schizophrenia patients.

Mass spectroscopy has gained increased interest for peptide and protein analysis over LBA because:

• LBA detects molecules based on binding affinity and 3D conformational structure, but it may not be able to distinguish between a protein and its metabolites.
• In contrast with LBA, MS-based approaches have the potential and would be able to produce more precise data on unchanged peptide/protein levels in situations where metabolism hampers reliable LBA data.
• MS techniques usually offer absolute concentrations of medications. This can depend on the form of an assay for LBA methods, and they may provide either an absolute or a free concentration of drugs.

However, LC-MS-MS-based bioanalysis for large molecule drugs poses a range of new obstacles, like difficulties in sample processing and extraction measures for the quantification of large molecules.

The reasons include the following:

• The background peptides and proteins in the biological matrices compete with the biotherapeutic molecule of interest, creating interference problems and impacting accuracy.
• The lack of significant evidence during quantification arises from being unable to catch free drugs that may circulate in serum.

Recently, many LC-MS-MS technological advancements have been made that can help solve all of these concerns.

In particular, the increase of ionization efficiency and ion transmission in recent triple quadrupole instruments has greatly enhanced sensitivity, allowing biologics to be detected at picogram or sub-femtogram levels.

Advances in technologies inside the LC-MS-MS include improved ion collision focusing, which brings more ions to the detector, as well as upgrades to the dynamic range of the detector to increase bioanalysis sensitivity and efficiency.

Recently, there has been a growing interest in integrating LBA immunoaffinity enrichment with LC-MS-MS quantification to integrate LBAs with the sensitivity and selectivity of LC-MS-MS technologies with greater precision and wider immune capture capabilities.

Automated Column-switching LC–MS/MS, Microextraction packed sorbent (MEPS)/LC-MS/MS, and Disposable Pipette extraction (DPX)/LC-MS/MS are some of the recent techniques that have been used to quantify large molecules.

Two major methods are widely used when using LC-MS/MS-based technologies for the bioanalysis of large molecules:

1. Intact analyte LC–MS(/MS) approach

This approach is predominantly used for peptides, small proteins, and oligonucleotides with a molecular weight typically below 4–8 kDa.

2. LC–MS/MS approach using a digestion step

This approach is more complex and mainly used for proteins or larger peptides.

This approach involves an (enzymatic) digestion step in addition to the intact analyte approach, where the protein/peptide is digested into smaller peptides.

Today, it is most common to use traditional LC-MS/MS triple quadrupole instruments for quantification for both the intact and the digested analyte approaches.

According to the existing standards, 4-6-15 (four out of six QC samples should be within 15% of the nominal value) is used as an approval criterion for large molecular LC-MS/MS assays. 4-6-20 approval requirements are proposed for larger intact analytes, in particular, if a hybrid LC-MS/MS approach is used.

A labeled peptide for peptide analysis, or either a labeled intact protein or a labeled signature peptide, can be used as an Internal Standard (IS) to establish a successful LC-MS/MS method.

Several guideline documents have been issued by the ICH and FDA to help standardize large-molecule bioanalysis studies. These recommendations can be found on the website of the appropriate regulatory agency.

While LC-MS-MS technologies have progressed to be more appropriate for biological bioanalysis, for non-experts who need to create and measure new biologics, the variety of mass spectrometry technologies and techniques, sample preparation methods, and reagents could be overwhelming.

The new advances in instrumentation and software will bring substantial changes in the consistency and efficiency of bioanalysis tests, providing more accurate and compliant results with significant patient safety consequences.

REFERENCES

1. Suma Ramagiri, Trends in Bioanalysis Using LC–MS–MS. The Column, The Column-12-07-2015, Volume 11, Issue 22.
2. Magnus Knutsson, Ronald Schmidt & Philip Timmerman, LC–MS/MS of large molecules in a regulated bioanalytical environment – which acceptance criteria to apply? Future Science, BIOANALYSIS VOL. 5, NO. 18, https://doi.org/10.4155/bio.13.193

The United Kingdom comprises England, Scotland, Wales, and Northern Ireland. It is an island nation in northwestern Europe.

The exit of the United Kingdom from the European Union to become a “third country” on January 31, 2020, is termed as Brexit.

The withdrawal agreement, which provided a transition period of one year, came to an end on December 31, 2020.

Thus, the Medicines and Healthcare Products Regulatory Agency (MHRA) has been the UK’s independent authority for medicines and medical devices since 1 January 2021.

Brexit will have both direct and indirect effects on the future of UK and EU clinical trials.

The impact of Brexit on pharmaceutical companies will be seen at the levels of regulatory alignment with respect to the forthcoming implementation of the EU Clinical Trial Regulation (EU CTR).

As the best universities for research in the study of clinical and pre-clinical medicine are present in the UK with strong regulatory and IP safety structures, the United Kingdom has become a major global centre for the pharmaceutical industry.

In addition, most generic pharmaceutical companies are registered with a UK address.

The departure from the EU would thus lead to hectic structural shifts, with a huge amount of time and investment on both sides.

Impact of Brexit on Outsourcing of Clinical Trials

Till now, many pharmaceutical companies based out of Europe were outsourcing their projects to contract research organizations (CROs) and contract manufacturing organizations (CMOs) based in the UK.

Post-Brexit, these scenarios may change.

As of now, the European Commission has given its decision that the UK authorities will have partial access to Article 57 and will also have partial access to the EudraVigilance database.

Because of Brexit, CROs and CMOs located in the United Kingdom are no longer members of the EU, and this will have a dramatic impact on the European portion of the clinical trials for the delivery of investigational medicinal products (IMPs).

The effect of clinical trials on the supply chain post-Brexit will totally disrupt the new drug development process due to major negative financial and economic effects.

Brexit can influence the clinical trial and drug discovery scenario that may involve access to drugs and Investigational Medicinal Products (IMPs), results, financing, and the workforce of clinical trials.

For BE studies carried out in the EU, the reference product can be made to a RefMP (UK Reference product) that has been granted in the Union in accordance with Articles 8(3), 10a, 10b, or 10c of Directive 2001/83/EC.

It is important to understand for the sponsor and the CRO that bioequivalence studies conducted with a medicinal product sourced in the UK can be used by EMA if the new MA using those BE studies has been granted before January 31, 2020.

Conclusion

United Kingdom is the 2^nd destination of Indian Pharmaceutical exports after the USA. Some CROs have an internal Brexit Task Force comprised of talented individuals who very well know their roles and responsibilities.

CROs are preparing themselves to engage and capitalize on the new regulatory process in the UK and EU so as to avoid costly delays and disruptions of clinical trials.

However, many questions still remain unanswered.

One of the biggest issues refers to complaints regarding the shipping of materials from the UK to the EU for clinical trials.

Will the volunteers involved be at any risk? Or will international boundaries lead to delays in clinical trials and difficulty in site management?

Or will there be any imposition of tariffs that could lead to disinterest among pharmaceutical sponsors in the UK in carrying out clinical research?

Thus, it will be interesting to see what is in store for the CROs post-BREXIT.

However, because the UK and the EU account for less than 15-18 percent of total Indian pharmaceutical revenues, BREXIT is expected to have little impact on Indian pharmaceutical firms.

References

1. The Landscape for CROs post-Brexit: An Update. Accessed at https://dwlanguages.com/2018/02/22/cros-post-brexit/
2. Brexit Solutions, Clinigen Clinical Supplies, and Management. Accessed at https://www.clinigencsm.com/brexit-solutions
3. Questions and answers to Stakeholders on the implementation of the Protocol on Ireland/Northern Ireland, 11 December 2020. European Medicine Agency (EMA/520875/2020)
4. Future of clinical trials after Brexit. Cancer Research UK, School of International Futures (SOIF).

The drug development process for pharmaceuticals and biologics is strictly regulated across the globe by different regulatory authorities.

The process of drug development comprises the following stages:

• Stage 1: Target and lead identification, in vitro testing of tissues, plasma, etc., for bench testing of the product.
• Stage 2: Non-clinical testing in live animals (in vivo testing).
• Stage 3: Filing of IND (Investigational New Drug) to get approval to test on humans. If approved, the clinical trial begins with Phase I clinical trials, which are also known as first-in-human studies.
• Stage 4: Filing of NDA (New Drug Application) after successful Phase II trial completion. If approved, the clinical trial will begin for Phase III.
• Stage 5: Submission of the document to request approval to market the product after a successful Phase III clinical trial.

Phase I Clinical Trials

Phase I studies are designed to investigate the safety and tolerability, i.e., identifying the Maximum Tolerable Dose (MTD), as well as the pharmacokinetics and pharmacodynamics of an investigational drug in humans.

The Right Drug to the Right Patient with the Right Dose at the Right Time is the ultimate goal or objective of Phase 1 Clinical Trials.

To achieve the objective of Phase I studies, scientists carry out studies in the following sections:-

Clinical Pharmacology of the Drug

• It involves studies such as First-in-Human, SAD and MAD PK studies, Healthy vs. Patient Population, ADME (Mass Balance), Specific population, Drug Interaction, Population PK, Biomarkers, Pharmacogenomics, and other specialized safety studies.

Exposure-response (PK/PD) of the drug

• It involves dose selection and optimization, efficacy vs. safety, and clinical trial simulation.

Biopharmaceutics of the Drug

• It involves BA/BE and Food Effect studies

In vitro studies carried out with the drug

• It involves protein binding, blood-to-plasma partitioning, in vitro drug metabolism, transport, and drug interactions.

Bio-analytical methods

• It involves validating assays and generating performance reports.

For Biologics, scientists carry out immunogenicity and comparability studies.

This article focuses on the ADME studies involved in Phase 1 Clinical Trials.

What is Pharmacokinetics?

Pharmacokinetics is the study of the action of the human body on medicines.

Absorption, Distribution, Metabolism, and Excretion are the major steps involved when a drug enters the human body.

The physicochemical properties of the drug, the administration route, and intrinsic and extrinsic factors of the subject, such as diseases, organ dysfunction, concomitant medications, and food, are factors that affect the pharmacokinetic (PK) profile of an investigational drug.

Efficacy, Toxicity, C_max, and T_max are some of the important terms that we generally come across in PK studies.

ADME study is also known as a mass balance study.

ADME studies are important because they help to determine other clinical investigations that might need to be conducted in support of regulatory approval for a new drug.

The ADME is determined by attaching a radioactive isotope (radiolabel), such as carbon-14 (14C) or tritium (3H), to an investigational new drug and following the radiolabel in human subjects.

Human ADME studies are carried out by the sponsor to obtain valuable information about the investigational new drug, which includes:

• Determining the routes of elimination and clearance mechanisms of the drug.
• Identifying metabolites and determining the relative exposure of parent drug and metabolites.
• Confirming that the human metabolite profile is covered by the metabolite profile in animals from toxicology studies.

What Is the Type of Study Design Carried Out in ADME Studies?

ADME studies are typically single-dose studies with healthy males (4-6 in number) at the intended route of administration.

What are the Primary and Secondary Outcome Measures Considered in an ADME Study?

The primary outcome measures of ADME studies in Phase 1 trials include:

• 1) PK Parameters

Maximum observed concentration (Cmax), time to reach maximum observed concentration (Tmax), area under the concentration-time curve from hour 0 to the last measurable concentration (AUC_0-t), area under the concentration-time curve extrapolated to infinity (AUC_0-inf), apparent terminal elimination rate constant apparent terminal elimination half-life (t_1/2), apparent clearance, and apparent volume of distribution.

• 2) Urine and Feces PK Parameters

Amount excreted in urine over the sampling interval, renal clearance (CLR), and the percent excreted in the urine, amount excreted in feces over the sampling interval, and the percent excreted in feces

• 3) Metabolites

Metabolites of [14C]-DRUG MOLECULE and their PK parameters will be identified and calculated as deemed appropriate, based on plasma and urine concentration levels.

The secondary outcome measures involved in Phase 1 Clinical Trials include:

• Signs, symptoms, incidence, and severity of adverse events (AE).
• Abnormalities in clinical laboratory assessments, vital signs, electrocardiograms (ECGs), and physical examinations.

How Are ADME Studies Conducted in Phase 1 Trials?

Mass Balance or ADME studies are carried out with healthy male volunteers by administering them a single dose of the investigational drug labeled with Carbon-14.

The cumulative radiolabeled dosage used in these experiments is approximately 50-100 μCi.

It is based on the predictions of real tissue exposures from tissue distribution studies performed during preclinical trials involving animals.

The volunteers are then kept in the clinical pharmacology unit (CPU) after administration until the radioactivity linked to the radiolabeled drug is quantitatively retrieved in the excreta (thresholds prescribed in the study protocol, normally in the range of 95% total and < 1 Bq/mL in the blood).

Blood samples collected during the study are analyzed for the PK properties of the parent medication.

The samples collected from this analysis are used in the circulation and excreted metabolite profiling.

Conclusion

The human mass balance study is an essential study of the drug development process.

ADME is also being carried out in the preclinical stage, but the safety and efficacy of the investigational drug can only be validated after determining the absorption, distribution, metabolism, and excretion (ADME) properties of the investigational drug on healthy human volunteers.

It can be rightly said that the human ADME (hADME) study provides a correlation between clinical observations and preclinical safety studies.

The key objective of the hADME study is to quantify, characterize, and identify drug metabolites present in the systemic circulation.

References

1. Clinical Pharmacology 1: Phase 1 Studies and Early Drug Development, US FDA.
2. What is a Human Mass Balance Study? Accessed at https://www.nuventra.com/resources/blog/what-is-human-mass-balance-study/
3. Why, When, and How to Conduct ¹⁴C Human Studies. Accessed at https://www.sgs.pt/~/media/Global/Documents/Technical%20Documents/SGS-Clinical-14C-ADME-Clinical-Trials-EN-09.pdf

Veeda, through its V-Konnect series, interacted with Dr. Susobhan Das and discussed “Current outlook of Biosimilar Development.”

About the V- Konnect

V-Konnect interview series is a program to get in touch with specialized industry experts to know their views on current relevant subject matters.

About Dr Susobhan Das – Founder & CEO at Amthera Life Sciences

Dr. Das is the Founder and CEO of Amthera Life Sciences Pvt. Ltd., a preclinical-stage biosimilar drug development company based in Bangalore.

Dr. Das has extensive techno-commercial experience in early-stage Biologics Development.

He has 20 years of experience in advanced biotechnology research and biopharmaceutical development.

He has served as a member of the USP Biologics and Biotechnology Expert Panel and also worked as a Director at the United States Pharmacopeia, India site.

Dr. Das has also worked at a senior management level at Intas Pharmaceuticals, developing biosimilars for global markets.

Dr. Das has worked as a member of the Expert Committee on Biologicals and rDNA Products: Indian Pharmacopeia Commission (IPC); Govt. of India.

He has authored research papers that are published in peer-reviewed National and International journals

Transcript.

1. What are the key international developments with respect to EU and USFDA biosimilar requirements?

A: One key development towards biosimilar acceptance has been the issuance of guidance on “interchangeability” by the US-FDA in May this year.

This will pave the way for the substitution of one product for the other without a prescriber’s involvement, as is the case for generic small-molecule pharmaceuticals.

This, I believe, is a significant action and will promote competition in the biologic market in the US.

Another development is the issuance of a revised guidance by the FDA titled “Development of Therapeutic Protein Biosimilars: Comparative Analytical Assessment and Other Quality Considerations,” also in May this year.

This is the revised version of an earlier guidance titled “Quality Considerations in Demonstrating Biosimilarity of a Therapeutic Protein Product to a Reference Product,” published on April 30, 2015.

FDA says this revision is to reflect on agency’s recommendations on the design and evaluation of comparative analytical studies intended to support a demonstration that a proposed therapeutic protein product is biosimilar to a reference product, and in anticipation that this will provide additional clarity and flexibility for product developers on analytical approaches to evaluating product structure and function.

For Europe, although the approval rate of Biosimilars is much higher than in the US, uptake of biosimilars is somewhat country-specific, with the large EU5 countries still not having interchangeability options.

However, payers have been significantly employing various tools, which may lead to higher biosimilar uptake.

For example, the introduction of prescribing targets, i.e., prescribing biosimilars to a predetermined percentage of patients.

The NHS of the UK introduced a biosimilar adoption framework with the idea that switching patients to a biosimilar may be inserted into clinical practice with incentive offerings for staff to offset switching costs.

This year in May, the NHS has published a document titled “What is a biosimilar medicine” for clinical and nonclinical stakeholders about the role of biosimilars in the healthcare system.

The document explains, among many other aspects, the overall savings from Biosimilars as well as suggests that a prescriber can switch from a reference to a biosimilar product.

However, switching at the pharmacy level is still not permitted without the consent of the prescriber as of now.

2. What are the main attributes for higher market approvals of Biosimilars in Europe compared to the US?

A: The first biosimilar, Zarxio, was approved in the United States only in 2015, whereas Omnitrope, another biosimilar, was approved by the European Medicines Agency (EMA) way back in 2006.

Since then, the EMA has approved more than 40 biosimilars as of 2019.

Essentially, this shows that EMA is the pioneering agency to advance biosimilars approval and uptake for the world.

To understand this, one may refer to the concept paper on the development of a guideline on the comparability of biotechnology-derived products published in 1998, which led to the introduction of a directive in EU legislation with the idea of “similar biological medicinal product” in 2001.

Therefore, a definition and a legal framework for market authorization for Biosimilars were first introduced in the world by the EU and are monitored and updated on an ongoing basis, which is key for a larger market approval rate of biosimilars in the EU.

By now, the EU has already gained experience of over a decade of Biosimilar use and has established the fact that biosimilars have similar efficacy and safety concerns to those of the reference products and can save a significant portion of healthcare costs.

Only three official biosimilars are in the market in the US, although around 15 are approved, and their uptake has been slower than anticipated.

For example, less than 15% for filgrastim biosimilar and 3% for the infliximab biosimilar hold as market share.

This is partly due to the lack of pricing incentives from biosimilars, as well as more attractive contract offers from the innovator product.

A host of other reasons for this slow approvals and uptake could be considerations on overall quality, safety, and clinical efficacy of the biosimilar, plus manufacturer reliability (supply without disruptions), reimbursement rates set by insurance companies or commercial payers, and support services for health care professionals and patients.

In other words, assurance on the efficacy and safety from the providers, as well as less out-of-pocket expenses, is key to most US patients.

Currently, this has yet to happen in the US, although progress has been made to achieve these goals.

On the contrary, a range of different policies to generate pricing pressure, drive adoption, and ultimately yield cost-savings for their healthcare systems have been implemented in the EU countries, which has somewhat led to a higher uptake rate for the biosimilars.

3. What is the scenario of prescribers’ acceptance of biosimilars over the innovator biological products?

A: At the beginning of the biosimilar era, the differences between lots in quality characteristics were cited to be reason enough for great concerns on efficacy and safety of the product.

From this, we have come to a stage where regulatory agencies have formalized acceptable changes of quality characteristics in the “innovator products” with no impact on efficacy and safety.

We also have more than a decade of real-world experiences of biosimilar use with comparable efficacy and safety concerns in the EU.

Moreover, we now have the outcome of the NOR-SWITCH trial, which demonstrated that “switching from infliximab originator to CT-P13 [a biosimilar] was not inferior to continued treatment with infliximab originator”.

All of these experiences, I believe, have led to higher prescribers’ acceptance of biosimilars over the innovator product, given there are incentives attached all through the stakeholders chain (for example, for the provider, prescriber, payer, and insurer).

The EU is clearly way ahead in implementing policies with the above considerations and will reap huge benefits in healthcare cost savings.

Although slow, the US has finally initiated action that may eventually allow biosimilars to be interchangeable with the innovator product.

First, to this idea was the finalization of the guidelines on interchangeability this year in May.

4. What is your opinion on Indian biosimilar industry, whether it attained its potential or this is just the beginning of the journey?

A: Indian biosimilar industry has now been very firmly established with defined regulatory path and a number of large and medium manufacturers with more than 70 biosimilars approved.

India is also the first country to approve a biosimilar monoclonal antibody to Rituximab in 2007, and interestingly, without having a published guideline, which first appeared in the year 2012 and in a revised form in 2016.

This approval has tremendously helped the patients to have access to the product with almost half the cost of the innovator product.

Interestingly, another mAb, Trastuzumab, indicated for HER2-positive breast cancer, is now available at almost 65% less than the innovator price, due to the launch of an Indian biosimilar.

Moreover, 3 companies from India have biosimilar products registered in the US, the EU, and Japan.

This shows the maturation of the Indian biosimilar industry as a global player.

Although these facts are very positive, India still has huge gaps in filling up the affordability factor with its very low per capita income.

On the contrary, India has a very high number of incidences and disease burden in most therapeutic segments, such as Cancer, Diabetes, Infections, Arthritis, Blood factor disorders, etc.

Therefore, affordable and quality biosimilars are a big opportunity for India.

However, what is critically needed is a policy framework somewhat similar to that being followed in the EU, which incentivizes all the stakeholders involved with biosimilar use, including the insurance sector.

Unfortunately, medicine costs in India are largely an out-of-pocket expense, and this needs to change very rapidly.

Given that these policies are implemented, the Indian biosimilar industry has tremendous potential to impact healthcare in a significant way.

5. Where does China stand with biosimilar approvals and the regulatory requirements?

A: This year, in February, Chinese regulators approved their first biosimilar.

A biosimilar Rituximab indicated for non-Hodgkin Lymphoma.

Although biotherapeutics development in China continue to grow exponentially over the past decade, no biosimilar drug however was approved until 2019.

This is primarily because of the lack of a national regulatory guidance, which was first published in February 2015.

This guidance document followed the same principles and requirements consistent with those formalized by the FDA and EMA.

Some other changes also happened simultaneously to foster pharmaceutical approvals and market authorizations, such as the China Food and Drug Administration (CFDA), which is now the National Medical Product Administration (NMPA), which falls under the State Administration for Market Regulation (SAMR).

The Centre for Drug Evaluation (CDE), which reviews applications under NMPA, remains unchanged in function.

China currently has more than 200 biosimilars under clinical development.

Interestingly, two key recent developments in policy setting by NMPA can be seen either as a barrier to biosimilar growth or as bringing serious competition: One is the listing of foreign-made drugs for urgent unmet medical needs, which can be approved for registration without any clinical trials being conducted in China.

Forty-eight such drugs have been listed for public review, out of which 11 are biologic drugs.

The second one is reduced or no import cost of new cancer drugs or drugs for hard-to-treat cancer.

Another very interesting development is the Market Authorization Holder (MAH) program implemented by the Chinese regulatory agency as a pilot program, which allows holders of an NMPA biologics approval to have the option to manufacture the drugs on their own or use any contract manufacturer.

This policy has given a significant boost to the CMO industry inside China and will surely foster growth in the Chinese Biosimilar industry along with new drug development.

6. How switching and interchangeability affect biosimilars access and its market size?

A: EMA and the EU commission define 3 terms related to biosimilar switching: interchangeability, switching, and automatic substitution.

Interchangeability is a general term that includes both switching, when the prescriber decides to use one over another, and substitution, when this exchange happens at the pharmacy level without the consultation of the prescriber.

In the US, though, FDA-designated interchangeability may refer to automatic substitution at the pharmacy.

Europe has been at the forefront in terms of interchangeability and currently allows physician-guided transitions of biosimilars, restricting pharmacy-level substitution, and this is without any separate or additional
regulatory guideline or drug development criteria.

As a result, we see a very high uptake of Biosimilars in some select EU countries.

Therefore, we may envisage that interchangeability or substitution will surely bring competition as well as uptake and cost savings.

Indeed, a follow-on biologic to Lantus, like Basaglar, has gained a market share of around 30 percent, and the Neupogen market share is down by 20 percent from the competition of Zarxio, a biosimilar.

Disclaimer:

The opinions expressed in this publication are those of the Interviewee and are not intended to malign any ethic group, club, organization, company, individual, or anything.

Examples of analysis performed within this publication are only examples. They should not be utilized in real-world analytic products as they are based only on the personal views of the Interviewee.

They do not purport to reflect the opinions or views of the VEEDA CRO or its management.

Veeda CRO does not guarantee the accuracy or reliability of the information provided herein.