1. Stages of Drug Development
Any drug development process must proceed through several stages in order to
produce a product that is safe, efficacious, and has passed all regulatory requirements.
Pacific BioLabs can assist you through all stages of drug developoment. Our scientists
can help you to determine your testing needs, and our experienced staff can perform
the critical tests and studies that are necessary to win FDA approval.
To get you started, below we have provided an in-depth overview of many stages in the
drug development process and necessary studies. Keep in mind this is just a guide; if
you have any specific questions call Pacific BioLabs at 510-964-9000 to speak to a
knowledgeable resource who can help you identify what testing you may need to
Detailed Stages of Drug Development
2. Product Characterization
3. Formulation, Delivery, Packaging Development
4. Pharmacokinetics And Drug Disposition
5. Preclinical Toxicology Testing And IND Application
6. Bioanalytical Testing
7. Clinical Trials
Discovery often begins with target identification – choosing a biochemical mechanism
involved in a disease condition. Drug candidates, discovered in academic and
pharmaceutical/biotech research labs, are tested for their interaction with the drug
target. Up to 5,000 to 10,000 molecules for each potential drug candidate are subjected
to a rigorous screening process which can include functional genomics and/or
proteomics as well as other screening methods. Once scientists confirm interaction with
the drug target, they typically validate that target by checking for activity versus the
disease condition for which the drug is being developed. After careful review, one or
more lead compounds are chosen.
When the candidate molecule shows promise as a therapeutic, it must be
characterized—the molecule’s size, shape, strengths and weaknesses, preferred
conditions for maintaining function, toxicity, bioactivity, and bioavailability must be
2. determined. Characterization studies will undergo analytical method development and
validation. Early stage pharmacology studies help to characterize the underlying
mechanism of action of the compound.
Formulation, Delivery, Packaging Development
Drug developers must devise a formulation that ensures the proper drug delivery
parameters. It is critical to begin looking ahead to clinical trials at this phase of the drug
development process. Drug formulation and delivery may be refined continuously until,
and even after, the drug’s final approval. Scientists determine the drug’s stability—in the
formulation itself, and for all the parameters involved with storage and shipment, such
as heat, light, and time. The formulation must remain potent and sterile; and it must also
remain safe (nontoxic). It may also be necessary to perform leachables and
extractables studies on containers or packaging.
Pharmacokinetics And Drug Disposition
Pharmacokinetic (PK) and ADME (Absorption/Distribution/Metabolism/Excretion)
studies provide useful feedback for formulation scientists. PK studies yield parameters
such as AUC (area under the curve), Cmax (maximum concentration of the drug in
blood), and Tmax (time at which Cmax is reached). Later on, this data from animal PK
studies is compared to data from early stage clinical trials to check the predictive power
of animal models.
Preclinical Toxicology Testing and IND Application
Preclinical testing analyzes the bioactivity, safety, and efficacy of the formulated drug
product. This testing is critical to a drug’s eventual success and, as such, is scrutinized
by many regulatory entities. During the preclinical stage of the development process,
plans for clinical trials and an Investigative New Drug (IND) application are prepared.
Studies taking place during the preclinical stage should be designed to support the
clinical studies that will follow.
The main stages of preclinical toxicology testing are:
Acute tox studies look at the effects of one or more doses administered over a period of
up to 24 hours. The goal is to determine toxic dose levels and observe clinical
indications of toxicity. Usually, at least two mammalian species are tested. Data from
acute tox studies helps determine doses for repeated dose studies in animals and
Phase I studies in humans.
Repeated Dose Studies
Depending on the duration of the studies, repeated dose studies may be referred to as
subacute, subchronic, or chronic. The specific duration should anticipate the length of
3. the clinical trial that will be conducted on the new drug. Again, two species are typically
Genetic Toxicity Studies
These studies assess the likelihood that the drug compound is mutagenic or
carcinogenic. Procedures such as the Ames test (conducted in bacteria) detect genetic
changes. DNA damage is assessed in tests using mammalian cells such as the Mouse
Micronucleus Test. The Chromosomal Aberration Test and similar procedures detect
damage at the chromosomal level.
Reproductive Toxicity Studies
Segment I reproductive tox studies look at the effects of the drug on fertility. Segment II
and III studies detect effects on embryonic and post-natal development. In general,
reproductive tox studies must be completed before a drug can be administered to
women of child-bearing age.
Carcinogenicity studies are usually needed only for drugs intended for chronic or
recurring conditions. They are time consuming and expensive, and must be planned for
early in the preclinical testing process.
These are typically similar in design to PK/ADME studies except that they use much
higher dose levels. They examine the effects of toxic doses of the drug and help
estimate the clinical margin of safety. There are numerous FDA and ICH guidelines that
give a wealth of detail on the different types of preclinical toxicology studies and the
appropriate timing for them relative to IND and NDA or BLA filings.
(See Regulatory/Animal Welfare and at FDA Guidances.)
Bioanalytical laboratory work and bioanalytical method development supports most of
the other activities in the drug development process. The bioanalytical work is key to
proper characterization of the molecule, assay development, developing optimal
methods for cell culture or fermentation, determining process yields, and providing
quality assurance and quality control for the entire development process. It is also
critical for supporting preclinical toxicology/pharmacology testing and clinical trials.
The Bioanalytical Team at PBL can support clinical trials. Clinical studies are grouped
according to their objective into three types or phases:
4. Phase I Clinical Development (Human Pharmacology)
Thirty days after a biopharmaceutical company has filed its IND, it may begin a small-
scale Phase I clinical trial unless the FDA places a hold on the study. Phase I studies
are used to evaluate pharmacokinetic parameters and tolerance, generally in healthy
volunteers. These studies include initial single-dose studies, dose escalation and short-
term repeated-dose studies.
Phase II Clinical Development (Therapeutic Exploratory)
Phase II clinical studies are small-scale trials to evaluate a drug’s preliminary efficacy
and side-effect profile in 100 to 250 patients. Additional safety and clinical
pharmacology studies are also included in this category.
Phase III Clinical Development (Therapeutic Confirmatory)
Phase III studies are large-scale clinical trials for safety and efficacy in large patient
populations. While phase III studies are in progress, preparations are made for
submitting the Biologics License Application (BLA) or the New Drug Application (NDA).
BLAs are currently reviewed by the FDA’s Center for Biologics Evaluation and Research
(CBER). NDAs are reviewed by the Center for Drug Evaluation and Research (CDER).
and Toxicokinetic Studies
Pharmacokinetic (PK) and toxicokinetic (TK) analyses are key
activities of early drug development. These studies can be
exploratory, or can be more extensive and formalized requiring GLP
compliance. Thoroughly understanding the DMPK of a potential
clinical candidate can have a huge impact on the success of a drug
PK and TK studies provide useful and required information that
informs no effect levels (NOEL), human equivalent doses (HED), and
pharmacokinetic/pharmacodynamic (PK/PD) drivers. Carrying out PK
studies enables the determination of PK parameters such as AUC,
clearance, volume of distribution, half-life, Cmax, and Cmin.
Pacific BioLabs’ integrated toxicology and bioanalytical services
can seamlessly perform in-vivo sample generation as well as
5. bioanalysis. PBL can conduct PK, PD and/or TK studies in most
rodent species, and has the instrumentation and expertise in its
bioanalytical lab to provide rapid and sensitive drug concentration
determinations from a variety of matrices.
PBL has experience working with many biomatrices, and extraction
conditions will be optimized to provide reproducible and robust
methods. Exploratory PK with bioanalysis can be implemented
simultaneously and completed rapidly in many cases. For GLP
compliant TK studies PBL provides the technical and regulatory (QA)
expertise to ensure method validation protocols, validation reports and
analytical standard operating procedures (SOPs) are all in compliance
with current GLP requirements.
PK, PD and TK Bioanalytical Lab Services
Pharmacokinetics (PK) Studies
PK Sample Analysis
Toxicokinetics (TK) Studies
Bioequivalence (formulation support)
MSD (Meso Scale Discovery) Assays
PK, PD and TK Available Species
PK, PD and TK Routes of Administration
Immunosorbent Assay –
ELISA is a technique that allows the identification and quantification of
known proteins to be ascertained by immunological means. The key
to ELISA is linking an antibody to an enzyme that is capable of
catalyzing a colorimetric or chemiluminescent reaction. ELISA was
first developed in the early seventies and replaced
radioimmunoassays which used radioactivity as the reporter
mechanism. For biological products, ELISA is a common method for
determining the presence and concentration of products. It is also
used in cell based assays, immunogenicity studies and as a screening
method. There are several common types of ELISA: Direct ELISA,
Indirect ELISA, Sandwhich ELISA, and Competitive ELISA.
In direct ELISA, an antigen is bound to the bottom of a 96 well plate
and a rest of the well is blocked with a blocking agent (usually BSA or
Milk). After washing, a primary antibody that is conjugated to an
enzyme (such as horseradish peroxidase) binds to the antigen at the
bottom of the well. The unbound primary antibody is washed away
and when the chemical substrate is added, the enzyme acts upon the
chemical substrate to produce a colorimetric or chemiluminescent
reaction which can be measure by a plate reader. Direct ELISA is
used to detect and quantify the amount of antigen present in a
7. Indirect ELISA is similar to direct ELISA but requires a secondary
antibody that binds the primary antibody. The secondary antibody
typically binds the Fc region of the primary antibody and will is
conjugated to an enzyme that is able to catalyze a colormetric
or chemiluminescent reaction when exposed to the appropriate
substrate. Secondary antibodies are easy to find commercially and
therefore most studies are performed using indirect ELISA over direct
ELISA which would often require the user to conjugate the enzyme to
the primary antibody themselves.
When performing an indirect ELISA, the antigen is attached to the
bottom of the plate and the plate is blocked just like direct ELISA. The
primary antibody is then added and binds to the antigen. Excess
primary antibody is washed away and the secondary antibody is
added which binds to the primary antibody. Substrate is then added
and the enzyme linked to the secondary antibody catalyzes
a colormetric or chemiluminescent reaction which is detected using a
In sandwich ELISA, the primary antibody, which is bound to the
bottom of the plate, binds the antigen and then a secondary antibody
also binds the antigen forming an antibody-antigen-antibody
sandwich. Because the antibody that binds the antigen (primary
antibody) is often not commercially available with an enzyme
conjugated to it, a secondary antibody is added which is conjugated to
an enzyme that can catalyze the colormetric or chemiluminescent
reaction when exposed to substrate.
The name “Competitive ELISA” derives from the competitive binding
between the sample antigen and antigen that has been added in. The
procedure for competitive ELISA differs from the other types of
ELISA. For competitive ELISA, the primary antibody is added to the
sample which contains the antigen. The primary antibody will bind the
antigen forming an antibody-antigen complex. The sample is then
8. added to 96 well plates which has antigen bound to each well.
Primary antibodies have already been bound to the antigen in the
sample can not bind to the antigen on the plates and are therefore
washed away. The more antigen in the sample the more primary
antibody gets washed away. A secondary antibody is then added to
the wells which binds the primary antibody. The secondary antibody
is again bound to an enzyme which can catalyze a colormetric
or chemiluminescent reaction. For competitive ELISA, a low signal
from the enzyme means that there is high amount of antigen in the