The Importance of Immunogenicity Testing in Biotherapeutic Development

Immunogenicity, defined as the ability of a substance to produce an immune response, is key to a successful and safe biopharmaceutical drug development program. Therapeutic Antibodies, enzyme therapies, peptides and combination products can elicit an immune response that may impact their safety and efficacy.

Example of an ELISA assay plate

How Biologics Create an Immune Response

Many types of substances administered to the human body may pose a chance of eliciting an immune response. However, biotherapeutics can be especially immunogenic because the large size of these molecules can trick the body into thinking that they are foreign invaders, triggering action by the immune system. These large molecules can also denature, which changes their profile, or aggregate, creating even larger particles. Ultimately, these characteristics can change or increase a biotherapeutic’s immunogenicity profile.

New technologies have been created - for example, PEGylation –to reduce the immunologic effect of biotherapeutics. However, the efficacy of these attempts to reduce immunogenicity varies. It is for this reason that testing for immunogenicity of a product in development is so important. Furthermore, immunogenicity testing is still necessary in a biosimilar development program. This is because the small changes in the manufacturing of biotherapeutics can effect large changes in their immunogenicity profile. Thus biosimilars, which almost invariably will be manufactured under different conditions than their parent biologics, still need to be tested for immunogenicity.

What is Immunogenicity Testing

Immunogenicity assays provide a way to measure the potential immune responses of biologics and biosimilars. Often a single biologic will require a panel of assays to produce a thorough picture of potential immunogenicity. The FDA stipulates that assays should be designed in such a way that they provide sufficient sensitivity, are free from confounding interference, can detect physiological consequences, and account for potential risks based on the profile of the therapeutic and the target patient population.

By designing assays with these factors in mind, it is possible to gather predictive data about the strength and type of immune response that a drug may produce in humans.

Types of Immunogenicity Analysis

For some biologics, a total antibody assay (to measure the antibodies that are part of the immune response) that includes screening, confirmatory and titer components will be sufficient to develop an immunogenicity profile.  In other cases a neutralizing antibody assay or cell based bioassay may be necessary and informative. ELISA (enzyme-linked immunosorbent assay) is also a useful method to detect antibody-antigen complexes.

In addition, aggregation potential of a biotherapeutic can be measured using size-exclusion chromatography and HPLC combined with laser-light scattering. This can help to provide a fuller picture of the overall immunogenic profile of a compound.

The goal of all these tests is, ultimately, to predict the clinical effect of patient immune responses to biotherapeutics. The role immunogenicity plays in drug development is an important one. As such, a well thought out and well executed program is a key to producing safe and efficacious biologic therapies.

To learn more about Pacific BioLabs Immunogenicity Testing Services, visit or contact us at 510-964-9000.

You can also view a PDF download of PBL's Immunogenicity Testing Services.


Genetic Engineering and Biotechnology news: Proposed Standards for Immunogenicity Testing -

ANP Tech: Immunogenicity Testing and Immunogenicity Assays -

FDA: Immunogenicity of Protein-Based Therapeutics -

Wikipedia: Immunogenicity -

A Note on Chemical Characterization

glass molecule.jpg

According to the ISO standard 10993, a medical device should be biocompatible i.e. not induce any adverse effects when it comes in contact with the patient. The standard provides a specific roadmap for planning a thorough biocompatibility study program for a device using both in vivo and in vitro techniques. But before evaluating the final or prototype device for biocompatibility, information should be gathered with regard to the “fitness of purpose” of the material(s) selected for the manufacture of the device. To begin, questions like the following can be asked. What are the chemical constituents of the material(s)? Will the chemical, physical, biological and other characteristics of the material(s) have any effects on the toxicological profile of the device? In other words- is the device in its entirety, safe? 

Extractables and Leachables

Chemical characterization essentially consists of two main tests. The first test is for extractables, which are substances, both organic and inorganic that can be “extracted” from the test material in the presence of a solvent under controlled conditions, and may be indicators of potential leachables. The second test is for leachables, which can leach out from the material during normal use via contact with liquids (including water or aqueous solutions). These chemicals can pose a potential risk to patients and hence should be detected before the final production and marketing of the device.

Analytical Instrumentation

GC (gas chromatography) and GC/MS (gas chromatography/mass spectrometry) are used for analyzing these chemicals and such work is routinely conducted by Pacific BioLabs’ analytical lab, where our team of chemists designs the most appropriate extraction conditions for a device after considering the device material, use of the device, and the solvent. Moreover, our analytical team can also characterize the chemical’s identity and concentration using HPLC, and can also conduct routine tests including moisture determination by Karl Fischer, pH, conductivity, viscosity and osmolarity.


ISO 10993-18:2005 Biological evaluation of medical devices – Part 18: Chemical characterization of materials

PK Studies at PBL

Pharmacokinetic (PK) and Toxicokinetic (TK) analysis is an important aspect of drug development.  These studies may be exploratory in nature or more extensive and formal.  Understanding the bioavailability, exposure, half-life, clearance and metabolism of your drug may be the difference between success or failure in the clinic. Pacific BioLabs has integrated toxicology and analytical services departments that can coordinate the in-life and bioanalytical aspects of a PK study

beaker and vials.png

Pacific BioLabs operates GLP compliant systems and validated instrumentation.  We have several LC/MS/MS systems as well as ELISA platforms to provide sensitive, high throughput evaluation of drug concentration in a variety of matrices.  Stability studies and metabolite identification can be conducted to understand the strengths and liabilities of your compound.

We can quickly develop and validate a sensitive, accurate and robust PK assay in a number of different matrices from different species.  We have experience working with plasma, serum, urine, CSF, and tissue.  Our chemists will optimize the extraction conditions to provide a reproducible and robust method.  Non-GLP exploratory PK assays can be implemented in a few days and use 4-6 reference standards, internal standard, and quality control samples.  Pivotal GLP compliant Toxicokinetic studies require method validation protocols, validation reports and analytical SOP.  We have the systems and Quality resources in place to guide your project, comply with the latest regulatory requirements and ensure high quality, timely data.

Learn more about PBL's PK studies or request a quote

AM2PED Grant to Accelerate California Bay Area Biomanufacturing

Source: NIST

Source: NIST

Last week I was privileged to be able to participate in a focus group for California East Bay biomanufacturing.

The purpose of the focus group was to generate ideas on how best to utilize the $2.2 million in Federal grant funds given to the East Bay Economic Development Alliance (among others) in the form of the AMP2ED Grant.

According to Manex Consulting, one of the partner organizations for the grant, "The Advanced Medical/Biosciences Manufacturing Pipeline for Economic Development (AM2PED) will build a regional innovation ecosystem for technology transfer, economic development, and workforce development in medical device and bio-science manufacturing along the I-80/880 Corridor of the Inner San Francisco East Bay Area."

For those who might not be aware, the San Francisco Bay Area is one of the strongest and most innovative clusters for biotech and medical device innovation in the world. And the East Bay region contains 469 bioscience companies, generating over $6 billion per year. As a CRO, PBL serve this cluster both in development testing, and also in lot release testing - thus we function as part of the supply chain for many local manufacturers.

It was in this capacity, as part of the bioscience manufacturing supply chain that PBL was able to provide perspective that will hopefully lead to the encouragement of more small companies and bioscience manufacturing in the East Bay region.

If you are part of a small company here in the East Bay, you would be well-served by learning more about some of the resources available to you here - including the Tech Futures Group (, a nonprofit that helps support small tech companies.

All in all, it's exciting to be part of the innovation taking place here, and to support so many of this region's bioscience companies.


UC Berkeley News Center:

Manex Consulting:

BBL Contributes to Successful Phase 3 Trial of BioMarin Orphan Drug

Keratan Sulfate

Keratan Sulfate

Bay Bioanalytical Laboratory (BBL), recently acquired by Pacific BioLabs (PBL), provided important biomarker data to the successful Phase 3 trial of GALNS for Morquio Syndrome ( “We are very proud of our contribution to the success of this innovative treatment for an orphan disease,” said Bob Cunico, Principal Scientist and founder of BBL.

“BBL’s work on the GALNS trial is a fine example of the sophisticated analytical expertise we can now offer to all of PBL’s clients,” said Tom Spalding, President of PBL. “We are excited to add these capabilities to our existing services in Toxicology and Microbiology.”

In 2007, BioMarin Pharmaceuticals approached BBL to develop and validate a novel LC/MS/MS method to measure plasma and urine concentrations of Keratan Sulfate. Keratan Sulfate is a glycosaminoglycan (GAG) that accumulates in the cells and tissues of patients afflicted with Morquio Syndrome.  The children and young adults with the disease have a defective enzyme (GALNS) and cannot properly process certain GAGs. The GAGs accumulate and cause damage to muscles, bone, and other organs.

BioMarin sought the bioanalytical expertise of BBL to measure Keratan Sulfate in blood and urine to support their preclinical and clinical studies of GALNS. The effort between BBL and BioMarin was extensive and resulted in peer reviewed publications from Bob Cunico and Erik Foehr, a successful IND submission, and now a Phase 3 trial that demonstrated the efficacy of the GALNS treatment. The urinary biomarker data demonstrated a dramatic and statistically significant decrease in urinary keratan sulfate with weekly treatment with GALNS.

The work involved many team members from BBL, including excellent analytical skills of Jayoung Ohh, timely and expert support by the QA group, Kate Lancaster and Samantha McCoy, as well as logistics and lab support by Steve Guthrie and Andrea Parungao.


Martell LA, Cunico RL, Ohh J, Fulkerson W, Furneaux R, Foehr ED. (2011.) Validation of an LC-MS/MS assay for detecting relevant disaccharides from keratan sulfate as a biomarker for Morquio A syndrome. Bioanalysis 3(16):1855-66.