A Strategic Roadmap for Peptide Preclinical Studies: 3 Key Stages

Peptide therapeutics have proven to be a reliable pharmaceutical platform, with a growing record of FDA approvals and commercial success. Yet not every promising candidate makes it to the clinic. The difference? A strategic, three-stage preclinical approach that transforms individual studies into a cohesive regulatory roadmap.

A deliberate, three-stage peptide development strategy turns disconnected studies into a coordinated plan that anticipates regulatory expectations at every step. Each stage builds on the last to create the data foundation required for IND approval, while keeping timelines and budgets on track through focused execution.

READ MORE: The 4 Most Promising Therapeutic Applications for Peptide Drug Development

Stage #1: Early Screening Studies

What This Stage Addresses

Early screening determines whether a peptide candidate is worth further investment. It establishes initial characterization, including target binding affinity, proof-of-concept efficacy in biological systems, and basic stability profiling to identify potential red flags before significant resources are committed.

Why It’s Critical

Skipping this stage can lead to costly failures later. A peptide that looks promising in silico may show weak target affinity in practice. One with strong in vitro activity might degrade quickly in plasma. Discovering these issues during GLP toxicology turns a manageable setback into a major delay.

Key Study Components

In Vitro Target Engagement Studies

Surface plasmon resonance provides quantitative binding kinetics to confirm that the peptide achieves nanomolar to low-micromolar affinity for therapeutic efficacy. Selectivity profiling against related targets helps prevent off-target effects that complicate clinical development.

Cell-based functional assays define dose-response relationships and effective concentration ranges, while duration-of-effect studies clarify whether standard dosing intervals are feasible or if formulation adjustments will be needed.

Preliminary Stability Assessment

Peptides degrade through enzymatic cleavage, hydrolysis, oxidation, and aggregation. Early stability screening identifies vulnerabilities that can guide chemical modification and formulation decisions before a significant investment is made.

Enzymatic degradation studies determine whether the peptide remains stable long enough in circulation to reach its target, while formulation compatibility testing ensures that excipients do not accelerate degradation or promote aggregation.

Initial ADME Screening

Permeability testing with Caco-2 or PAMPA systems provides early insight into oral bioavailability or confirms that injectable delivery will be required. Plasma protein binding studies across species help predict interspecies pharmacokinetic differences that could complicate regulatory submissions.

Metabolic stability testing in liver microsomes and plasma identifies key enzymatic breakdown pathways. Preliminary tissue distribution studies using fluorescently labeled analogs show how effectively the peptide reaches target tissues and whether it accumulates in areas that may raise safety concerns.

Stage #2: Preclinical Candidate (PCC) Stage Studies

What This Stage Addresses

PCC studies answer a defining question: Is this peptide ready for regulated toxicology studies? This stage delivers deeper characterization through disease-relevant efficacy models, detailed pharmacokinetic and pharmacodynamic profiling, and preliminary safety assessments that determine a therapeutic window.

Why It’s Critical

This is where the development plan solidifies. PCC studies generate the data needed to justify candidate selection to both regulators and internal teams. They establish dosing ranges, support early clinical trial design, and identify formulation needs before manufacturing scale-up.

When programs progress without a comprehensive PCC package, regulators frequently challenge the rationale for candidate selection or dose justification, leading to delays and additional study requirements.

READ MORE: Five Pointers to Successfully Navigate the Challenges of Peptide Therapeutic Development

Key Study Components

Advanced Efficacy Studies

Disease-relevant animal models extend proof-of-concept into predictive validation. The best models use clinical endpoints that closely mirror human outcomes, such as tumor reduction in oncology, improved glycemic control in metabolic diseases, or measurable symptom improvement.

Mechanism of action studies confirm that the peptide produces the expected biological response under physiological conditions. Biomarker analysis supports target engagement and provides pharmacodynamic data for dose selection.

Comprehensive PK/PD Characterization

Multi-species pharmacokinetic studies establish exposure profiles and show whether interspecies scaling will be straightforward or complex. Route-specific data clarifies whether subcutaneous dosing provides sufficient exposure or if intravenous delivery is required.

Tissue distribution studies confirm whether the peptide reaches target tissues at effective concentrations. PK/PD modeling defines the exposure-response relationship that supports dose selection for first-in-human trials.

Metabolite identification determines whether degradation products contribute to efficacy or toxicity, which becomes important when regulators compare human and animal metabolite data.

Preliminary Safety and Toxicology

Early safety testing helps prevent setbacks in GLP studies. Acute toxicity studies define safe starting doses, while multiple-dose tolerance work identifies any accumulation or dose-limiting effects.

Safety pharmacology assessment evaluates potential cardiovascular, respiratory, or neurological risks that could require monitoring in clinical trials. Detecting these issues early allows for formulation or candidate adjustments before investment.

Immunogenicity assessment is essential for peptides intended for chronic dosing. Epitope prediction and in vitro T-cell assays help identify immune responses that could neutralize therapeutic activity or cause hypersensitivity, guiding formulation strategy and peptide design.

Stage #3: IND-Enabling Studies

What This Stage Addresses

IND-enabling studies deliver the safety and toxicology data required for clinical trial authorization. Conducted under Good Laboratory Practice (GLP) standards, these studies meet FDA and EMA expectations and provide the risk assessment that supports first-in-human dosing. They also ensure manufacturing and analytical readiness for the production of clinical materials.

Why It’s Critical

These studies form the foundation of every regulatory submission. They define the safety margins that determine the starting clinical dose and guide the monitoring strategy for early-phase trials.

Programs that move too quickly through this stage often face clinical holds, as agencies closely examine study design, execution, and interpretation. Any gaps or inconsistencies can trigger requests for additional studies, delaying clinical progress and increasing costs.

Key Study Components

GLP Toxicology Studies

GLP toxicology work provides the foundation of the IND safety package. Single and repeat-dose toxicity studies in two relevant species establish how the peptide is tolerated and define the exposure limits that support first-in-human dosing. Study duration should match or exceed the intended clinical schedule to ensure adequate coverage.

A complete toxicology program typically includes:

• Clinical pathology: hematology, clinical chemistry, and urinalysis
• Histopathology: examination of all major organs for microscopic changes
• Toxicokinetics: confirmation that adequate exposure occurred at all dose levels

These studies define the no-observed-adverse-effect level (NOAEL) and provide the safety margin regulators use to justify starting doses in clinical trials.

For programs involving women of childbearing potential, reproductive and developmental toxicity studies further evaluate fertility, embryonic development, and pre-/postnatal outcomes.

Safety Pharmacology Package

Cardiovascular safety evaluation extends beyond hERG channel screening. Agencies expect a comprehensive package that includes in vitro hERG testing, in vivo cardiovascular assessment in conscious animals, and evaluation of QT interval effects.

Additional studies assess respiratory and central nervous system function to identify any effects that may require monitoring during clinical trials. Early identification of these risks helps shape the clinical protocol and patient safety strategy.

Regulatory-Grade PK and Bioanalytical Validation

GLP-compliant bioanalytical method development must meet FDA and EMA standards for accuracy, precision, selectivity, and stability. Cross-species validation ensures consistent toxicokinetic data across studies.

Definitive tissue distribution work defines peptide biodistribution throughout the body, identifying potential accumulation in sensitive organs. Mass balance and elimination studies confirm clearance routes, while predictive human ADME modeling supports a scientifically justified first-in-human dose.

READ MORE: Bioanalytical Strategies for Peptide-Drug Conjugates (PDCs)

Conclusion

The difference between peptide programs that reach clinical trials and those that stall in preclinical development often comes down to execution. Each stage builds on the last, transforming individual studies into a connected, data-driven program that anticipates regulatory requirements and reduces risk.

Rushing through or omitting key studies consistently results in additional regulatory questions and extended timelines, a pattern seen across the industry when development teams attempt to accelerate progress without adequate foundational data.WuXi AppTec offers integrated peptide development platforms that encompass all three study types under one roof. Our GLP-compliant study designs, global regulatory experience, and proven track record of successful peptide IND submissions give development teams the clarity and confidence needed to move forward efficiently.

Clinical Analysis Strategies for Complement Biomarkers

Complement (C) is a group of heat-resistant proteins that exist in human and animal serum or tissue fluids, have enzymatic activity after activation, and can mediate immune and inflammatory responses. It is named “complement” because it can assist and supplement the specific antibody-mediated lytic activity that is heat-resistant. Currently, more than 50 soluble proteins and membrane-bound proteins have been identified, collectively forming the complement system and playing crucial roles in both innate immune defense and adaptive immune responses. These complement components can be further divided into intrinsic complement components (such as C1q/C1s/c1r, C2~C9, MASP-1, MASP-2, factor B (FB), and factor D, etc.), complement regulatory proteins (such as factor H, FH), factor I (FI), factor P, C1 inhibitory factor (C1-INH), decay accelerating factor (DAF), membrane accessory protein (CD46 or MCP), and membrane reactive lysis inhibitory factor (CD59)), as well as complement receptors (such as CR1~CR5, etc.) based on their roles and functions.

Under normal circumstances, most complement in serum exists in the form of inactive zymogens. In pathological conditions, the complement system can be activated through three independent and intersecting pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). Specifically, AP uses antigen-antibody complexes as the primary activators, which bind to C1q to initiate the activation process. This process sequentially involves C1r C1s, C4, C2, and C3, ultimately forming the C3 convertase (C4b2a) and the C5 convertase (C4b2a3b). Unlike CP activation, the activation of AP does not rely on antigen-antibody complexes. It crosses C1, C4, and C2 to activate C3 directly, and forms C3 convertase (C3bBb) with the participation of factors B, D, and P. Through the C3 positive feedback amplification loop, more C3 convertase and C3b are generated, and multiple C3b and C3 convertase combine to form C5 convertase (C3bB3b). In addition, unlike CP and AP, the LP pathway is mediated by mannose-binding lectin (MBL), which activates complement MASP-1 and MASP-2, as well as C4, C2, and C3, to form the C3 convertase (C4b2a or C3bRb) and the C5 convertase (C4b2a3b or C3bB3b). The above three pathways converge at the activation of C3 to form C5 convertase (C4b2a3b or C3bB3b), which then enters a common terminal pathway to cleave C5 into C5a and C5b. C5b then binds to C6-C9 to form the Terminal Complex C5b-9 (TCC). When a target membrane is present, TCC mediates irreversible damage to the target cell membrane, associated with complement activation in the form of the membrane attack complex (MAC). If there is a lack of target membrane, the formed C5b-9 complex can bind to complement regulatory proteins (such as S protein) to form stable sC5b-9. The common terminal effect of complement activation is the dissolution of target cells.

The timely and moderate activation of the complement system can mediate various biological effects, including cell lysis, bacterial and viral effects, exerting regulatory effects to enhance the phagocytic ability of phagocytic cells, clearing immune complexes, causing inflammatory reactions, and participating in adaptive immune responses. Under normal circumstances, the content of various components in the body’s complement system is relatively stable, and their activation is closely regulated to prevent self-damage. However, when there is a genetic complement deficiency or abnormal complement activation, it may cause various diseases, such as pneumonia caused by various bacterial infections, systemic lupus erythematosus, atypical hemolytic uremic syndrome, C3 glomerulopathy, paroxysmal nocturnal hemoglobinuria, and Alzheimer’s disease. Accurate detection of complement activity and component content is crucial in the diagnosis and treatment of these diseases, as well as in the development of corresponding drugs.

Complement Related Testing and Sample Processing

1. Complement activity or function testing

The determination of total complement activity mainly reflects the activity of classical pathway complement components (C1 to C9). In clinical practice, the CH50 (50% Complex Hemolytic Activity) test, which measures the amount of complement required to cause 50% lysis of red blood cells, is commonly used as an indicator to evaluate total complement activity. Because complement can activate classical pathways to cause hemolysis in sheep red blood cells sensitized with antibodies (such as hemolysin), when the concentration of sensitized sheep red blood cells is constant, the hemolysis rate is positively proportional to complement content, which can be used to detect complement activity through classical pathways. Additionally, for the detection of complement activity in the alternative pathway, EGTA chelation of calcium ions can be used to block the action of C1 and prevent classical pathway activation of complement. Add rabbit red blood cells that can activate factor B, activate the complement pathway, cause hemolysis of rabbit red blood cells, and the complement activity of the pathway (AH50) that causes 50% hemolysis of rabbit red blood cells. The aforementioned 50% red blood cell hemolysis spectrophotometric method for evaluating complement activity is relatively complex to operate, and the individual animal source of the blood sample easily influences the results. It requires a highly robust detection method and consistent detection results. Currently, commercial ELISA kits can also be used to evaluate the complement activity of the classical, alternative, and lectin pathways, which are relatively easy to operate; however, the precision of the results is relatively poor. In addition to CH50 and ELISA detection, liposome-based methods are also used to evaluate classical pathway complement activity, which can meet high-throughput requirements. In practical applications, appropriate detection methods can be selected according to clinical needs for complement activity and function testing.

2. Complement component detection

Due to C3 being a core component of the three complement activation pathways, and C4 being a shared component of the classical pathway and lectin pathway, complement levels of C3 and C4 are commonly used in clinical diagnosis of autoimmune and renal diseases. In addition, C1q is an important component that initiates the activation of the classical complement pathway, and measuring its level can further clarify the activated complement pathway during disease occurrence. On the other hand, given the critical roles played by C3 and C5 convertases in the activation of the complement system, it is of great clinical significance to detect the content of complement components such as target substrates (C3 and C5), components (such as Bb), regulatory proteins (such as FH, FI, and C1-INH), and activation products (including C3a, C3b, C3dg, C5a, and sC5b-9) around these two active enzymes. Currently, multiple detection platforms and commercial ELISA kits are available to achieve quantitative detection of various complement components, including C3, C5, Bb, FH, C3a, C5a, and sC5b-9.

3. Selection and storage of test samples

It is advisable to choose serum samples for the relative quantification of complement activity through various activation pathways; however, plasma samples with added ethylenediaminetetraacetic acid (EDTA) are often used for quantifying complement activation products in vivo. EDTA can chelate calcium and magnesium ions, block the classical and alternative pathways of complement, and effectively reduce complement reactivation in vitro after sample collection. If the test sample needs to retain complement function while being anticoagulated, specific thrombin inhibitors such as piludine can be added. In addition to anticoagulants, the temperature and time during sample collection, storage, and transportation can also affect complement activation in vitro. If samples used for complement analysis need to be stored for an extended period, they should be frozen at -70 ℃ or lower. The requirements for sample storage may vary depending on different detection indicators, but in general, samples need to be frozen within 24 hours after collection and preparation; otherwise, it will affect the results of all types of complement detection. Meanwhile, it is not recommended to freeze complement samples at -20 ℃ as it requires a longer freezing time compared to -70 ℃, during which complement activation continues. Frozen samples should be transported in dry ice and subjected to minimal freezing and thawing during testing whenever possible.

Complement Related Testing and Sample Processing

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired pluripotent hematopoietic stem cell disease characterized by chronic intravascular hemolysis due to acquired defects in red blood cells that are abnormally sensitive to complement activation. Clinically, it is characterized by intermittent episodes of sleep-related hemoglobinuria, which a decrease in whole blood cells or recurrent thrombosis may accompany. The various inhibitors developed for complement C5 can inhibit the activity of related complement, thereby efficiently treating terminal pathway-mediated diseases caused or amplified by abnormal complement activation.

To detect the complement activity of the total complement (classical pathway) and alternative pathways, we established analytical methods using CH50 and AH50 as core indicators, which can provide stable and reliable reference data for complement-related clinical disease research and treatment. And the AP activity detection was developed and validated using an ELISA kit. The validation parameters of these methods meet the predetermined acceptance criteria, which can increase the source of clinical research data for complement pathway-related diseases. For the detection of Free C5 in serum, we have established a corresponding detection method on the Gyrolab platform and successfully applied it to the content analysis of clinical research samples, which can be used for clinical drug research targeting C5. Additionally, we have a mature platform for determining C3 and C4 content in samples. We have established multiple specific methods for detecting complement activation products in blood samples, including allergen components C3a and C5a, pathway-specific component Bb, C3/C4 activation regulatory proteins FH/FI and C1-INH, as well as the terminal complement complex sC5b-9. In clinical research, the accurate and reliable quantitative detection of complement components involved in complement activation pathways at various stages can provide insight into the type and status of complement activation in diseases from multiple perspectives, and it can accelerate the development of clinical drugs targeting the complement system.

A Final Word

As our understanding of complement biology continues to expand, so too does the importance of precise, validated analytical strategies in clinical research. From detecting total complement activity to profiling key components and activation products, tailored assays play a crucial role in elucidating disease mechanisms and informing therapeutic development. With robust platforms and extensive methodological expertise, researchers can generate reliable data that supports the advancement of complement-targeted therapies and enhances patient outcomes across a broad range of immune and inflammatory conditions.

Five Keys to Success in Bispecific Antibody Development

Back in the 1990s, a cell phone performed one function. You dialed a number and placed a call. Now, smartphones have cameras, maps, music players, and more, offering multiple functions all rolled into one. In medicine, many treatments are still stuck in the brick phone era: one drug for one job. But the rise of bispecific antibodies (BsAbs) has ushered in the smartphone age for therapeutics, and the possibilities are exciting. 

However, as with all innovative leaps, the challenge lies in the details. Developing BsAbs requires comprehensive knowledge of their mechanisms of action, pharmacokinetic (PK) characteristics, and the best strategies to ensure a smooth path to market. Sponsors must overcome these hurdles to bring effective BsAbs to market in the most efficient manner possible.

Bispecific Antibodies: Promise and Challenges

Regulators approved the first BsAb for use in the United States in 2009, but their development has been the result of decades of steady progress. The concept was first introduced in the 1960s, and technological breakthroughs in the following decades pushed the idea towards reality. To date, regulators have approved around 20 BsAbs across the globe, most of which target cancers.

BsAbs have a unique design that allows them to perform two functions simultaneously. This enables novel therapeutic mechanisms such as redirecting immune cells to tumor cells, shutting down several disease signals, and focusing treatment precisely where it’s needed.

This dual-targeting capability offers promise in oncology, ophthalmology and immunology. However, BsAbs present complex development challenges that further intensify regulatory scrutiny due to their structural diversity, immunogenicity risks, and the need for precise target engagement. Overcoming these hurdles requires careful consideration and deliberate deployment of testing methodologies to ensure safety, efficacy, and consistency throughout the product lifecycle.

1. Master the Mechanisms of Action of Bispecific Antibodies

Bispecific antibodies have challenged conventional drug development paradigms throughout their four primary mechanisms of action have emerged. Understanding them is critical for us to better guide study design.

Bispecific T cell engagers
This type of BsAb acts like a matchmaker. It simultaneously binds to the T cell receptor and the cancer cell, forming a synapse between the two cell types. This activates T cells, which release the necessary molecules to kill tumor cells. 

Immune checkpoint (ICP) modulation 
Another type of BsAbs work by blocking a cancer’s ability to shut down the body’s immune system. These include some that dual-block ICP, and others that target ICP simultaneously with a target involved in other signaling pathways. 

Signaling pathway blockade 
These target faulty signaling inside cells. BsAbs can block two signaling pathways at once, or they can bind to two different parts of the same target. 

Functional mimicry
This type of BsAb is designed to act like a natural helper in the body, guiding molecules into the right spot to ensure a function occurs properly.

2. Carefully Consider Dosing and Interactions of Bispecific Antibodies

Setting the right starting dose is critical when designing human trials of BsAbs. Some products already in the market behave predictably at higher doses, but at lower doses become less consistent.  Past clinical trials have shown that if a drug over activates T-cells, it can trigger a dangerous immune reaction called a cytokine storm. Researchers often use the Minimum Anticipated Biological Effect Level (MABEL) method to avoid this by setting very cautious first-in-human doses. 

To further mitigate the risk of a cytokine storm, developers might consider using pretreatments before administration of BsAbs. In addition, clinical recommendations for stepwise dose escalation can also potentially reduce the risk of cytokine storm. Despite these precautions, some BsAb drugs have been released with a boxed warning.

3. Establish Robust ADME Testing for Bispecific Antibodies

Drug developers must also overcome challenges caused by the complex properties of BsAbs. Appropriate Absorption, Distribution, Metabolism, and Excretion (ADME) testing can address these considerations by taking into account key lessons learned from previous BsAbs development. 

• Absorption: BsAbs have poor stability in the gastrointestinal tract and suffer from low permeability across the gut wall. This gives them extremely poor oral bioavailability. As a result, most are administered via injection. The bioavailability through the non-IV route is generally good. For example, the bioavailability of elranatamab is 56% after subcutaneous injection; The bioavailability of emicizumab is 80%-93% after subcutaneous injection.
• Distribution: Similar to monoclonal antibodies, BsAbs remain in the circulatory system and extracellular fluids due to their large molecular size, limiting their ability to enter cells and deep tissues. 
• Metabolism and Excretion: Because of their protein-based nature, bispecific antibodies don’t use the usual drug-metabolizing enzymes (like CYP450), so their metabolism and excretion are usually not a major concern in safety testing. Similar to the monoclonal antibody, BsAbs show different dosage linearity from small molecules. For example, when the dose of amivantamab is ≥20 mg/kg/week, the systemic exposure in cynomolgus monkeys is linear, but it presents non-linear PK when the dosage is < 20 mg/kg/week. which may be contributed by target-mediated drug disposal (TMDD).

4. Choose the Correct Bioanalysis Techniques 

Due to the structural complexity of BsAbs, researchers primarily use ligand-binding assay platform for their bioanalysis. This tool can detect the total amount of antibody, individual targets, or the fully intact antibody. 

Occasionally, more than one method is required because intact bispecifics and fragments are both active forms. U.S. regulators recommend using multiple approaches to get the complete picture, which is better to interpret the PK/PD relationship. 

For newer BsAb designs, such as probodies, ligand-binding assays may not be adequate, as they produce active forms after metabolism. In this case, liquid chromatography-mass spectrometry (LC-MS), may be more suitable as they can detect surrogate peptides. 5. Prepare for Immunogenicity Challenges
The artificial structure of BsAbs increases immunogenicity risks such as the formation of anti-drug antibodies (ADAs), which can induce severe drug-related toxicity. 

This is also a risk when developing BsAbs targeting solid tumors, and many immunotherapeutic applications for cancer have discontinued clinical development due to the formation of ADAs. 

Immunogenicity studies for BsAbs are best tailored to a specific research phase. In early preclinical studies, analyzing total ADAs helps interpret PK and TK data. During the IND stage, analysis of immunotoxicity and immune cell profiling is most helpful. And in clinical research phases, further assessments of immunotoxicity or neutralizing antibodies (Nabs) may be required.

A Final Word

BsAbs offer many advantages to researchers looking for new tools to fight debilitating and devastating diseases. Their unique nature allows for dual-targeting in a single molecule, and they can reduce off-target toxicity, enhance efficacy, and improve safety. 

Of course, there are challenges in their development. Developers must carefully monitor the risk of cytokine storms, monitor drug interactions, and establish robust testing to ensure these drugs are as effective as possible. But forward-looking sponsors, who engage with partners boasting a wealth of experience, expertise, and know-how, will be able to ensure each therapeutic candidate is rigorously characterized, safe, and ready for regulatory advancement.