Smaller recombinant antibody fragments as single-domain antibodies (sdAbs) are emerging as credible alternatives because of their target specificity, high affinity, and cost-effective recombinant production. sdAbs have been forged into multivalent and multispecific therapeutics, or targeting moieties, that are able to shuttle their linked therapeutic cargo (i.e., drugs, nanoparticles, toxins, enzymes, and radionuclides) to the receptor of interest. Their ability to permeate across the blood brain barrier is receiving industrial interest for neurological and neuro-oncological indications.


Biologic therapies such as monoclonal antibodies (mAbs) are enabling the targeted, personalized treatment of cancer, inflammatory diseases, infectious diseases, and diseases of the central nervous system. With 25 mAb products on the market and more than 100 undergoing clinical trials (1-3), it is evident that engineered antibodies have come of age as biopharmaceuticals (4).

Intact antibodies (immunoglobulins IgG, IgM, IgA, and IgE) are highly specific targeting reagents that form part of our key defence against pathogens and toxins. IgG, the main serum antibody, the intact form of which is almost 
exclusively used in therapeutic antibodies, is a Y-shaped, multidomain protein with antigen-binding sites located on the two Fab (antigen-binding fragment) tips and effector functions mediated by the stem Fc (crystallizable fragment) domain (see 

). Antibodies are bivalent, (i.e., able to bind to two antigens with high affinity and for long retention times [avidity] on many cell-surface receptors and polyvalent antigents). There is, however, a range of applications for which the Fc-mediated effects are neither required nor are desirable. Inappropriate activation of the Fc receptor-expressing cells by antibodies can lead to cytokine release and toxicity. On the other hand, their long circulation half-life results in poor contrast in imaging applications. 

In comparison to the integral antibody, the Fab shows an increased ability to penetrate dense tissues as solid tumors and the Fv (variable domain) appears to be even more effective (5). Hence, the reduction of the Fabs to a monomeric domain unit, as V

, should be, theoretically, more effective in the recognition of the associated antigen. Thus, dissecting IgGs initially through enzymatic proteolysis (e.g., with papain or pepsin) and later genetically engineering them into their constituent monovalent domains or bivalent fragments (Fab2, diabodies, and minibodies) (see 

) would improve their clinical value. Smaller recombinant antibody fragments such as 
  monovalent antibody fragments (Fab, scFv) and engineered variants (diabodies, triabodies, minibodies, sdAbs) along with antibody-drug or antibody-nanoparticle conjugates are emerging as credible alternatives. Many of these products are under clinical investigations (4) as Big Pharma and small and medium enterprises (SMEs) continue to make significant investments in bringing these new therapeutics to the market. The main drivers include the more economical production and unique and superior properties for a range of diagnostic and therapeutic applications. 


  In the late 1980s, a sdAb was firstly isolated by Greg Winter’s group at the MRC Laboratory of Molecular Biology in Cambridge, UK, comprising only a V

 domain of an antibody (6). It was four times smaller than a Fab and half of the size of Fv. Conventional antibodies are large (150 kDa), while small recombinant antibodies have a mass range between 25-50 kDa. A sdAb is defined as the smallest antigen-binding fragment of antibodies, ranging from 11 to 15 kDa, and consists of a variable domain of an antibody V

 chain containing three or six naturally occurring complementary determining regions (CDRs) that permit specific recognition of the antigen (7). 

sdAbs have several benefits compared to other antigen-binding units (8). Firstly, sdAbs are antibody-derived fragments that can be expressed in bacteria as active, soluble, and robust proteins. Additionally, single variable domains are known to be able to potentially target cryptic epitopes that are poorly accessible to intact antibodies but enable pathogen to bind to their target receptors (6, 9). Nevertheless, their use was originally limited because single variable domains rarely retained the affinity of the parent antibody and were also poorly soluble and prone to aggregation. 

The discovery that camelids (camels and llamas) and cartilaginous fish (wobbegong and nurse sharks) evolved into high-affinity, single, V-like domains (called VhH in camelids and V-NAR in sharks), mounted on an Fc-equivalent constant domain (8, 10-13), has resulted in translation of these camel, llama, and shark sdAbs into novel therapeutics. Camelid VhH and shark V-NAR domains display long surface loops, often larger than human antibodies and are able to penetrate cavities in target antigens (14). They are, in general, soluble and can be produced as stable I targeting reagents for sensitive diagnostic platforms and nano-sensors (14-17). However, for in-vivo administration, immunogenicity concerns require their humanization even though llama VhH domain have been claimed to be only minimally immunogenic (18). Thus, human single domains would be preferable for 

 applications provided problems of poor stability and solubility can be overcome. 

Crystal and solution structures of several sdAbs adopt a β-sheet structure similar to a V

 immunoglobulin fold in a conventional antibody. sdAbs have similar affinity to antigens as whole antibodies, but are more heat-resistant and stable towards detergents and high urea concentrations. Those derived from camelid and fish antibodies are less lipophilic and better soluble in water, owing to their complementary determining regions (CDR3) that form an extended loop covering the lipophilic site that normally binds to a light chain (19, 20). Examples include the camelid-origin Nanobodies that consist of a single variable domain and two constant domains C

3 (Ablynx). In contrast to common antibodies, two out of six sdAb survived a temperature of 90 C without losing their ability to bind antigens (21), a property that is attributed to a reversible unfolding behavior (7). Stability towards gastric acid and proteases depends on the amino-acid sequence. Some species have been shown to be active in the intestine after oral application, but their low absorption from the gut impedes the development of systemically active sAbs (22-26). The comparatively low molecular weight leads to a better tissue permeability, but also to short plasma half-lives and renal excretion (22). 

Production of stable, high-affinity mAb fragments in high yield for preclinical and clinical trials can be a serious bottleneck in the product pipeline. Identification and design of mutations that minimize the hydrophobic interface and direct selection from phage libraries have contributed greatly towards overcoming solubility and stability problems (18-21). Bacteria are favored for expression of V-like domains, while human sdAbs can be expressed at high yields (> 50 mg L

 in Pichia pastoris shaker flask cultures) (4). Nevertheless, several strategies are developed to improve recombinant expression such as the use of terminal polypeptides (e.g., c-Myc, histidine, and the “flag” epitope, DYKDDDK) to enhance affinity purification after expression into the periplasm of 

Immunogenicity concerns in relation to these strategies are still under investigation by drug regulatory agencies. Favorable properties, such as good expression, thermal stability, and solubility, are co-selected with binding activity using phage libraries, such as in the case of human VH domain antibodies (21), a method that enables also cost-effective production (see 

Table I: Selected novel antibody fragments in development.

Nanobodies   (heavy-chain camelid antibodies)

Von   Willebrand Factor (vWF), Tumor necrosis factor alpha (TNFα), others

Thrombosis,   rheumatoid arthritis, inflammatory bowel disease, oncology, immune disorders,   Alzheimer’s disease

Domain   antibodies (heavy- or light-chain human antibodies)

TNFα/TNF1R   (tumor necrosis factor 1 receptor) CD40, IL13/IL4, other unspecified

Rheumatoid   arthritis, Crohn’s disease, COPD, autoimmune disease, asthma, oncology

Infectious   diseases, oncology, inflammation

Oncology,   autoimmune/inflammatory conditions

Ig =   immunoglobin, NAR = new antigen receptor, IL = interleukin. 

Given their high affinity and specificity, the small size of sdAbs might make them particularly suitable for targeting antigens in obstructed locations, such as tumors where penetration into poorly vascularized tissue is crucial to the success of the drug (7). The delivery of toxins or radioisotopes to diseased tissues would be another ideal function for a sdAb, which could provide specific delivery of the toxin to the tumor while minimizing the length of time that the toxin could cause damage to healthy cells in the blood (7). The short half-life of sdAbs is well suited to applications in which rapid clearance is essential. 


  For some applications such as treatment of brain disorders, rheumatoid arthritis, or cancers, the target antigens need to be available for binding in the bloodstream for a prolonged time requiring a prolonged serum half-life to increase time that the antibody has to act on its target and to reduce the frequency of administration (7). PEGylation, conjugation, or fusion to serum albumin, and fusion to either the Fc or complete antibody constant regions (7) have been employed; however, the latter method can lead to complement activation.

PEGylation increases the apparent hydrodynamic size of the antibody fragment above the glomerular clearance limit, thereby improving its circulating half-life, while also improving solubility, enhancing bioavailability by reducing losses at subcutaneous injection sites, and reducing immunogenicity and toxicity of therapeutic proteins (7). For example, conjugation of small human V domains (11-15 kDa) with a single PEG molecule extends the half-life from the normally rapid half-life of 20 minutes to 39 hours in mice (4). The site of modification needs to be carefully selected to ensure it does not interfere with the antigen-binding site. Conjugation/linkage of the sdAb fragment to a protein that has a 
  naturally extended serum half-life protein like serum albumin is another method to prolong serum half-life of sdAbs. Site-specific conjugation of a Fab fragment to serum albumin by maleimide crosslinking or generation of a bispecific Fab with specificity for serum albumin in one arm increased half life in rats (27). Fusion of peptides with high affinity for serum albumin to an anti-tissue factor Fab lead to 37-fold and 26-fold extension in rabbit and mouse serum half-life, respectively (7). Albumin is also known to accumulate in tumors (28) and in arthritic joints, potentially enabling a further level of targeting in disease-specific situations (29). A range of human sdAbs isolated by phage display that bind to mouse, rat, and/or human serum albumin were produced that have half-lives that match the half-life of serum albumin (~19 days) (AlbudAbs) compared to a terminal half-life of non-serum albumin binding sdAbs of less than 45 minutes (29). The fusion of an Albu/Ab to interleukin-1 receptor antagonist (AlbudAb/IL-1ra) resulted in improved in-vivo efficacy due to its extended serum half-life (29). AlbuAbs could be used to generate a range of long half-life versions of many drugs (such as methotrexate, interferon alpha, peptide antagonists of TNFa) (13) to reduce frequency of administration and improve clinical profiles (Domantis-GSK, Phase I clinical trials, PN0621).


  Due to prolonged aging, the prevalence of neurological disorders is growing, placing neurological disorders as a public health priority and an important cause of mortality constituting 12% of total deaths globally (30). The capillaries of the brain have evolved to constrain the movement of molecules and cells between the blood and brain, providing a natural defence against circulating toxic or infectious agents but also an effective barrier to drug delivery excluding 98% of therapeutic drugs. The relative impermeability of the blood brain barrier (BBB) results from tight junctions between capillary endothelial cells that are formed by cell-adhesion molecules, high expression of active efflux transport proteins, and large densities of mitochondria within endothelial cells (31). Thus, nearly all large molecules (molecular weight >1 kDa) such as recombinant proteins or gene-based medicines are unable to cross the BBB. 

Conventional antibodies are unable to cross the BBB. Antibodies (and other peptides, proteins, and nucleic acids), however, have been shown to be able to be transported across by binding to receptors selectively expressed on brain capillary endothelial cells such as transferrin or the insulin-like growth factor (IGF-1) by receptor-mediated endocytosis (32). An alternative strategy for enhancing their transport relied on adsorptive-mediated endocytosis of cationized antibodies prepared by conversion of the surface carboxyl groups of the antibody to amino groups (33-35). However, even if an antibody is able to permeate the BBB, it will likely be the target of brain endothelial cell metabolism. Thus, the stability advantages of sdAbs (e.g., high expression levels, high affinity, high solubility, small size, and good intracellular stability) make them ideal for central nervous system therapeutic or imaging applications (see B). 

Ablynx (Ghent, Belgium) is focused on the discovery and development of sdAbs, the Nanobodies, for a range of human diseases including inflammation, thrombosis, oncology, and neurology 
  with seven sdAb products in the clinic (three in Phase II and four in Phase III) (36, 37). BI 1034020 Nanobody is being developed in collaboration with Boehringer Ingelheim for Alzheimer’s disease and, currently, is undergoing Phase I studies. Bi-paratopic humanized half-life extended anti-aβ VhH were shown to be able to bind to two different epitopes on the free aβ peptide (N-terminus and central) with an extremely high affinity (≤ 1 pM), reducing aβ levels in plasma in a preclinical model for Alzheimer’s disease (amyloid precursor protein [APP] transgenic mice) (38), which is associated with prevention of amyloid plaque formation in the brain and clearance of existing plaques (37). Ablynx is collaborating with Merck and Merck Serono to co-develop other sdAbs for neurological or neuro-oncological indications. 

Transport across the BBB in human brain endothelial cells has been reported for llama sdAbs (39, 40). Using a llama sdAb phage display library (41), a new antigen-ligand system was identified for transvascular brain delivery (42).

 studies have been reported focused on the development of two sdAbs, FC5 and FC44 (39, 43, 44), which selectively recognized human cerebrovascular endothelial cells and transmigrated across the BBB. The transport of FC5 and FC44 across the human brain endothelial cells is polarized, charge-independent, and temperature-dependent, suggesting a receptor-mediated process. 

Abulrob et al. demonstrated that the transcytosis of FC5 is dependent on clathrin-coated vesicles and on the recognition of specific oligosaccharide antigenic epitopes on the surface of the human endothelial cells (39, 40). Following the internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes, and remained intact after the transcytosis. FC5 failed to recognize brain-endothelial cells-derived lipids suggesting that it binds luminal α(2,3)-sialoglycoprotein receptor, which triggers clathrin-mediated endocytosis (39). Moreover, Abulrod et al. also described the modification of the FC5 sdAb with a horseradish peroxidase (HRP)-tagged IgG to improve its potential as a BBB carrier vector (40). Briefly, a free cysteine was engineered at the C-terminal of FC5, 17 amino-acid residues distant from the antigen-binding site. The cysFC5 maintained the transmigration ability across an 

 BBB model. The cysteine group was used further to attach the HRP-IgG (~190 kDa). It was shown that only HRP-IgG conjugated to the FC5 transmigrated across the in-vitro BBB model, suggesting that cysFC5 is able to transport larger molecules into the target tissue. The receptor has been identified, and is related to a novel isoform of the transmembrane domain protein 30A (TMEM 30A) (31). TMEM30A is also known as CDC50A, which is responsible for the cell surface expression of ATP8B1 (hypothesized to be a flippase for phospatidylserine). FC5 is now being developed to deliver therapeutic amounts of doxorubicin to the brain after pentamerization and association to PEGylated liposomes (31). Functionalization of nanoparticles with sdAb such as FC5 increases their valency, as multiple number of antibodies can be conjugated to the nanocarriers by spontaneous binding of the histidine tag of the antibody to the nitrilotriacetic acid (NTA) of the lipid. After intravenous administration (8.9 mg kg-1), brain levels of FC5-targeted, doxorubicin-loaded, PEGylated liposomes were increased (~0.6 µcg g-1 brain, double those achieved by doxorubicin-loaded, PEGylated liposomes). The pentameric form of FC5 (P5) displays better binding to brain endothelial cells and is more efficiently transcytosed across brain endothelial cells than the monomeric form and is co-localized with neurons in brain tissue (see 

Table II: Selected single-domain antibodies (sdAbs) for brain targeting and neurological disorders.

Significant transcellular migration in an 

P5-doxorubicin loaded PEGylated liposomes

Double brain levels obtained with   doxorubicin loaded PEGylated liposomes (~0.15% of intravenously injected   dose)

ALX- 0081 prevented MCA thrombosis and   induced reperfusion when given immediately after and 15 minutes after   complete occlusion and reduced brain damage without inducing haemorrhage

ALX-0081, a nanobody against the A1 domain of von Willebrand factor (vWF) that blocks vWF binding to main platelet receptor glycoprotein (GPIb), was compared with thrombolytic agent recombinant tissue plasminogen activator (rtPA) and tirofiban (glycoprotein IIb/IIIa inhibitor, antiplatelet agent) in a middle cerebral artery (MCA) guinea pig thrombosis model. ALX-0081 prevented MCA thrombosis and induced reperfusion when given immediately and 15 minutes after complete occlusion and reduced brain damage without inducing hemorrhage, whereas tirofiban prevented thrombosis but did not induce reperfusion and induced striking brain hemorrhage. rtPA also induced reperfusion when given 60 minutes after occlusion but provoked brain hemorrhage (see Table II). Skin bleeding time was not modified or was moderately prolonged by ALX-0081, whereas tirofiban and rtPA prolonged it. Thus, the inhibition of the GPIb-vWF axis by nanobodies in guinea pigs prevented cerebral artery thrombosis and induced early reperfusion without provoking intracerebral bleeding, hence, reducing brain infarct area; ALX-0081 administration, early after onset of an acute stroke, may induce revascularisation and reduce brain damage with less haemorrhagic risk than rtPA.


After a decade of intensive engineering followed by preclinical and clinical testing, antibody fragments joined mAbs as powerful therapeutic agents particularly for targeting cancer, inflammatory, autoimmune, and viral diseases. With recent advances in scaffold design, construction, and selection methodologies, there is now a rapid process for recombinant synthesis of specific, high-affinity antibody fragments against virtually any target. sdAbs are able to penetrate antigen clefts (e.g., enzyme active sites, cell surface receptors) targeting both antigens and immunosilent epitopes. sdAbs have shown impressive tumor-to-blood ratios and are increasingly applied towards the discovery of new cancer or inflammatory biomarkers and nanosensors while they are currently explored as highly refined biopharmaceutical drugs in challenging therapeutic areas such as neurology and oncology. 

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*To whom all correspondence should be addressed.

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Articles

Cover Story

Single-domain antibodies are emerging as credible alternatives due to their target specificity, high affinity, and cost-effective recombinant production.

Single-Domain Antibodies for Brain Targeting

Global healthcare systems are currently undergoing major transformations spurred by increasing regulation, record public debt, and shrinking budgets. The United States has the dubious distinction of leading the way in this regard. The mandates of the Affordable Care Act, along with shrinking reimbursement to providers, are fundamentally changing the operation of the US healthcare system. At the same time, patients are being challenged to assume greater accountability for their own health, and the industry is moving to a model that better aligns stakeholder incentives to deliver on cost and quality.  Healthcare providers and payers that have historically been siloed and fragmented are now exploring ways to collaborate and coordinate efforts to reduce costs while improving patient safety and healthcare quality.

Amidst lower reimbursement from payers and declining productivity in R&D, manufacturers are looking to develop high-value, cost effective and targeted drugs. It has long been maintained that personalized medicine offers the prospect of improved health, better outcomes, and less harm. It should not be surprising that the concept of personalized medicine has become a buzzword throughout the industry as manufacturers increasingly see it as the solution to many of their business needs. In fact, the number of personalized medicine products has more than quadrupled in recent years, from only 13 on the market in 2006 to more than 70 in 2012, and a recent report projects that the US market for predictive personalized drugs will double, increasing from $9.2 billion in 2013 to $18.2 billion in 2019 (1). 

FDA defines personalized medicine as “the tailoring of medical treatment to the individual characteristics, needs, and preferences of a patient during all stages of care, including prevention, diagnosis, treatment, and follow-up” (2). Other definitions include specific language around using biological information and biomarkers on the level of molecular disease pathways, genetics, proteomics as well as metabolomics to stratify patients. Some examples of personalized therapies, devices, or technologies that illustrate the broad nature of the term include an implanted 3D-printed tracheal splint, an artificial pancreas device system, drugs aimed at molecular targets specific to a patient’s disease state (e.g., Kadydeco for cystic fibrosis and Zykadia for melanoma), immunotherapies that combat tumors using the body’s own immune system, and disease risk assessment through genetic testing.  

Regardless of how the term is defined, one thing is clear--data and analytics have emerged at the center of this patient-centered and personalized ideal.  Technologies that can extract large amounts of data from samples or biopsies are allowing for previously unknown factors involved in disease to be identified and used as drug targets or disease biomarkers. “Big data can help us personalize medicine, and also glean new insights from big numbers of patients in the real world like never before,” according to Geno Germano, head of Pfizer’s Global Innovative Pharma division (3). Many stakeholders including manufacturers, however, are still struggling with how to effectively leverage massive data sets to improve efficiencies, reduce costs, and advance patient-centric treatments. This article explores data analytic strategies that pharmaceutical companies are considering to capitalize on the opportunities of personalized medicine.


Pharma has always been a data-driven sector; however, the volume of data available today is unprecedented. According to Eric Schmidt, chairman of Google, we generate as much information in a couple of days as all of mankind has since the dawn of recorded history up to 2003 (4). This notion certainly applies to the life-sciences industry, which has seen an explosion of documented patient data over the past decade.  This has been spurred by dramatic changes that include advancements in genome sequencing technologies; the adoption of electronic health records (EHRs) across health systems; the sharing of clinical-trial data; and the rise in patient registries, social media networks, and medical and nonmedical device data systems (e.g., smartphones and fitness monitors). These changes have created a virtual explosion of genomic, clinical trials, claims, EHR, and research study data. When paired with the adoption of advanced analytic tools to generate insights from these data, manufacturers are positioned to create more targeted therapies and to transform the way that biopharmaceutical drugs are discovered, developed, and marketed.  

Personalized medicine is rooted in the hypothesis that diseases are heterogeneous in their causes, rates of progression, and their response to drugs. Simply put, each person’s disease is potentially unique, and therefore, that person needs to be treated as an individual. Thus, the shift toward personalized medicine has required that manufacturers develop rigorous strategies to understand the cause or origin of a disease as opposed to simply treating patients’ symptoms. Ted Driscoll, digital health director at Claremont Creek Ventures, believes that mining data on genomics, proteomics, and metabolomics alongside clinical-trial data and real-world clinical data will enable manufacturers to more thoroughly understand the particular structure of a disease. This understanding will lead to faster and more accurate identification and validation of targets and biomarkers for designing personalized therapies (5). Some forward-thinking manufacturers are already participating in public-private research initiatives to harness the power of “big data” and genome sequencing to improve the success rate for discovering new personalized medicines (

). Such strategies will enable manufacturers to implement patient-centered approaches across all stages of translational and clinical research in therapeutic areas where targeted therapies are lacking.  

Traditional clinical trials enroll a tightly selected group of patients to show, under highly controlled circumstances, that a drug’s effects are statistically significant. As manufacturers invest greater resources in drug discovery focused on the etiology and mechanism of specific diseases, diagnostic biomarkers will help target a patient population that will optimally respond to therapy. Clinical trials designed to test these personalized medicines will likely become more structured as well. Using personal medical and population genomics data, manufacturers now have tools to design more targeted clinical trials by matching cell types and individual metabolic response to the drug candidate, recruiting subjects who will be more likely to respond and excluding those likely to have treatment-limiting side effects.  

Some global manufacturers have already launched personalized medicine clinical trial strategies that could change how drugs are studied and potentially cut the time and cost of bringing them to market (see 

). Multi-drug, multi-arm, biomarker-driven clinical trials are one strategy manufacturers are putting into practice. According to Maria Freire, PhD, president and executive director of the Foundation for the National Institutes of Health, this clinical trial design establishes a model of clinical testing that more efficiently meets the needs of both patients and drug developers. “This strategy will validate biomarkers and facilitate drug development in one infrastructure, to more rapidly provide safer and more effective treatments to patients” (6).   

Table II: Examples of more personalized clinical trial strategies. 

Finally, as greater volumes of patient reporting information are captured and tracked, manufacturers should be reviewing that data and correlating the real-world reactions of patients with their clinical data to ensure that the intended outcome of the drug or device is actually happening. This especially holds true for manufacturers of companion diagnostic tests as it is often more difficult for these developers to achieve broad coverage and value-based reimbursement for their product. To this point, some manufacturers have already created proprietary data networks through partnerships to gather, analyze, share, and respond to real-world outcomes and claims data. One example involves Genomic Health’s Oncotype DX for breast cancer recurrence, where the manufacturer analyzed the real-world outcomes and costs of 925 Humana patients managed with diagnostic and demonstrated significant cost savings (7).   


The volume of health-related data is exploding at an unprecedented rate. While having access to these data is important, access alone does not advance the science. The real challenge that manufacturers must address is asking the right questions and effectively discerning which data are of value in answering these questions. Given the sheer volume of information available, framing appropriate questions along with data and analytic capabilities will be key differentiators in personalized medicine. Some organizations may be able to do these analytics in-house, while others may need to consider investing in data through innovative partnerships with healthcare-focused and even non-healthcare focused entities. Regardless, manufacturers at the leading edge of personalized medicine will be those that invest resources to determine where true medical unmet needs exist and what data and analytics can answer key questions around improving patient outcomes.  

From a drug discovery and development standpoint, the ability to manage, integrate, and link data across functions (e.g., safety, clinical studies, regulatory, patient reported outcomes, etc.) will enable manufacturers to conduct comprehensive data mining that can facilitate lead generation as well as identify related applications or potential safety issues.  In order for this to be possible, however, silos that have historically separated internal functions will need to be removed to allow for end-to-end data integration. R&D heads can also use genomic data, clinical-trial data, and real-world data to prioritize investments in specific disease areas. Criteria such as potential clinical relevance of biomarkers and feasibility of discovering biomarkers should also be considered given the increasing importance of biomarkers and companion diagnostics in personalized medicine.  

As manufacturers continue to develop more targeted therapies, clinical trials must be more targeted as well. Essentially, clinical trials must be designed so that they can show, early on, if a drug is efficacious for the first cohort of patients. Manufacturers must identify appropriate patients using more data sources (e.g., patient registries and social media) and additional criteria (e.g., genetic composition). Targeting specific populations will allow for trials that are smaller, shorter, less expensive, and more powerful. When appropriate, manufacturers that develop or invest in companion diagnostic tests early on in their R&D processes will be better-positioned to conduct such studies.
With payers increasingly driving value-based reimbursement strategies for services, therapeutics, and devices, the availability of data around individual patients’ medical history, clinical interventions including prescription history, outcomes, and cost will help drug manufacturers develop better target therapies and stratify populations thereby optimizing value and patient outcomes.  

What You Need to Know to Create or Sustain a Personalized Medicine Strategy for Your Company

Paving the Way for Personalized Medicine: FDA’s Role in the New Era of Medical Product Developmen

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7.	K. Pothier and G. Gustavsen, 


Jill E. Sackman, DVM, PhD, is a senior consultant, and Michael Kuchenreuther, PhD, is a research analyst, both at Numerof     & Associates, Inc., St. Louis, MO, www.nai-consulting.com.   

Features

Data analytic strategies can help companies capitalize on personalized medicine.

Marrying Big Data with Personalized Medicine

Manufacturing biopharmaceuticals carries great responsibility. The drugs produced must be available when needed, effective for their intended purpose, and safe to use. At an operational level, it is vital that biopharmaceutical production is carefully controlled within defined parameters and there is no possibility of contamination. Suppliers share a commensurate responsibility, as the quality of raw materials has a direct impact on these goals. There is an acknowledged gap between current capabilities of many suppliers and what will be needed in the future to support the industry in an increasingly demanding regulatory and end-user focused market.

This gap is especially true when the meaning of supplier is defined widely as any player including manufacturers and distributers along the inbound supply chain going up through the layers of tier 1, tier 2, etc., to the original source material. The scope of material is similarly broad: excipients, actives, reagents, and product contact materials must all be included. While tier 1 suppliers are largely reliable and aligned to needs of the pharmaceutical industry, there is a whole array of connected players that need to understand and adopt pharmaceutical best practices.


  The starting point for helping transform the inbound supply chain is to define and articulate what these best practices are and to set out a road map for their implementation. With this goal in mind, a BioPhorum Operations Group (BPOG) team comprising 20 leading biopharmaceutical manufacturers has been working collaboratively together to create a 10-point checklist with the intent of sharing it with the industry. The mindset of the manufacturers is one where they grasp the need to work cooperatively with the supply base and that the journey to supply excellence will be a long one.


  Suppliers of raw materials need to understand what GMPs mean as they apply to the biopharmaceutical industry. Suppliers need to have a system for assuring the quality of their products in line with GMPs or an appropriate fit-for-purpose classification approach. Suppliers at all tier levels should similarly require their suppliers to understand GMP and have quality control systems that meet the needs of biopharmaceutical GMP. All suppliers need to appreciate the legal (regulatory) obligations that are placed on biopharmaceutical manufacturers including the control of raw materials.


  Suppliers at all points in the inbound supply chain must have a proper process for notifying their customers and hence, end users, of changes to raw material manufacturing. The timing of the notification must, if possible, allow end users to evaluate and plan for the effects of the change. ‘Changes’ are defined as anything that may impact end user product quality, manufacturability, or material availability. Change notification must include all planned (foreseen) changes and unexpected (unplanned) changes.

Elimination and control of sources of variation


  Suppliers need to understand how variability in the manufacturing process affects the quality attributes of their product. They need to have tools that are fit for purpose, including measurement procedures for monitoring and controlling variation (e.g., tools and concepts such as statistical process control, process capability, and critical process parameters). Suppliers need to understand how eliminating sources of variation is crucial for continuous improvement. They need to understand how variability in raw material quality can have a significant downstream impact on bioprocessing capability and biopharmaceutical product safety.

Traceability, visibility, and transparency


  Suppliers need to understand that the origin of all materials flowing through the inbound supply chain needs to be entirely visible to intermediaries and end users in pharmaceutical manufacturing. The source and time of supply for individual batches need to be traceable so out-of-specification (OOS) situations and adverse events are managed. All players in the raw material supply chain must have an attitude of openness so that problems can be identified and resolved. Suppliers must be ready, willing, and able to participate in supply-chain mapping exercises to enable transparency. End users need to be equally open and clear to ensure that suppliers can provide their guidance as to the effective use of the raw material.

Effective (root cause) investigation approaches and tools


  Suppliers need to commit to the notion that identified issues (e.g., contamination events, OOS situations, and manufacturing deviations) must be investigated to identify a root cause, possibly by going back several steps in the supply chain. They need to have capabilities and processes for participating in (multi-company) root-cause analysis and investigations. Suppliers need to understand and apply the concept of corrective action preventive action (CAPA), and they need to be able to produce information and reports to support the paperwork trail.


  Suppliers must have the capability to use risk-management approaches to assess and manage risks in their own business. They must be willing and able to participate in risk assessments with customers and end users on a technical and business level. Suppliers must understand their own process and its part in the supply chain, what risks there are and how to mitigate those risks as they impact end-user manufacturing and potentially cause harm to the patient.


  Suppliers must have robust systems and tools for collecting data in their production processes and the causes of variation. They must be open to sharing these data with customers and end users and to driving continuous improvement. Suppliers need to understand how data capture in the manufacturing process can be a source of value and competitive advantage in the pharmaceutical context.

Comprehensive (and electronic) information and knowledge flow


Suppliers need to understand that product and process data over and above that shown on a certificate of analysis are needed by end users. They must understand the importance of proactive trending of product and process data and its relevance to the control of quality. Suppliers must be able to provide data in a standard electronic format and transfer data where appropriate to customers and end users. 


Suppliers must be open to working with end users to align material specifications. They need to help end users maximize the use of standard specifications and to minimize customized specifications. Suppliers must understand why end users need to drive toward ‘zero particles’ in raw materials and cooperate in achieving this goal. Knowledge, understanding, and dialogue are foundations for success.


Suppliers need to appreciate the intended use of the medicinal and therapeutic products that their raw materials have been used to produce. They need to understand the role that their materials have played and be able to judge their responsibilities in this context. The employees of suppliers should be provided with this information. The management in suppliers must demonstrate appropriate leadership behaviors.

The meaning of each point requires definition and explanation. Capabilities will need to be developed and tools and techniques learned. The drivers for change are powerful and unrelenting, but biopharmaceutical companies are ready for the challenge.

At some point in the not too distant future there will be two types of suppliers: those that understand and have adopted biopharmaceutical raw material manufacturing best practice and those that no longer supply the industry.  


  Simon Chalkis director of the BioPhorum Operations Group, simon@biophorum.com  


Manufacturing Best Practices

Defining best biopharmaceutical practices is necessary to ensure the safety of the supply chain.

Applying GMPs to the BioPharma Supply Chain

Two years into the Generic Drug User Fee Act (GDUFA) program, enacted in July 2012, FDA officials are striving to meet goals and timelines for speeding new generic drugs to market. Agency reviewers have made some progress in eliminating the huge backlog in abbreviated new drug applications (ANDAs) pending in the Office of Generic Drugs (OGD), but not as quickly as manufacturers had anticipated. New guidance from FDA aims to help manufacturers meet standards and to streamline oversight. But a continual rise in submissions to the agency makes it hard for reviewers to get ahead, especially in the wake of a surge of 600 ANDAs filed in June to beat new stability testing requirements.

An efficient and comprehensive generic-drug review program is important for FDA to facilitate consumer access to high quality, low-cost generic drugs. Unfortunately, manufacturing problems with generic drugs continue to precipitate recalls and shortages. FDA has blocked the import and sales of numerous generic products from leading manufacturers such as Wockhardt and Dr. Reddy’s Laboratories. And dissolution problems with beta blocker metoprolol have raised new concerns about the safety and efficacy of extended-release generic drugs. 

Quality lapses make it difficult for the generic-drug industry to challenge policies linked to enhanced drug safety and prescribing. An FDA proposal to require label updates when new safety issues arise has generic-drug makers up in arms. Brand manufacturers support the FDA policy as a way to reduce their liability for quality and safety issues involving generic drugs. But generic-drug firms fear that differing labels on brands and generics will confuse and alarm patients and prescribers. A recent survey sponsored by the Generic Pharmaceutical Association (GPhA) indicates that physicians and pharmacists would be less willing to prescribe and dispense generic drugs if the rule is adopted, and many members of Congress have voiced concerns over the FDA proposal (1).

Similar issues regarding the “sameness” of generic and brand-name drugs have ignited battles over the labeling and distribution of future biosimilars. Brands are fighting generic-drug manufacturers over whether follow-on biologics can use the same name of the reference product, while also seeking curbs on automatic dispensing of biosimilars by pharmacists. 


  FDA officials have long maintained that generic drugs that meet its rigorous standards for bioequivalence, stability, dissolution, and quality are just as safe and effective as the original brand. But ongoing problems with generic-drug quality have spurred new efforts by the Center for Drug Evaluation and Research (CDER) to ensure drug quality throughout the product lifecycle. CDER director Janet Woodcock’s strategy is to establish a new Office of Pharmaceutical Quality (OPQ) to implement a “one quality voice” approach. OPQ will feature review teams that assess the chemistry and controls aspects of a drug product from new drug application (NDA) through generic and over-the-counter versions. While seeking higher-level approval of the OPQ reorganization, CDER is piloting this approach, explained Woodcock at GPhA’s CMC workshop in June, noting that one team reviewing the reference drug and all later versions will avoid duplication. 

OGD also looks to a risk-based approach to its chemistry review program to focus efforts on products that raise more crucial issues. OGD is implementing risk-based quality assessment for solid oral-dosage forms, based on a “failure mode, effects and criticality analysis” algorithm that assigns a risk score to each product. This approach is being tested now, explained Susan Rosencrance, OGD acting deputy director for chemistry, with the intent of expanding it to immediate release solid orals and other dosage forms. 

OGD efforts to accelerate its review process have been hampered, though, by the steady rise in ANDAs and supplements. During GDUFA negotiations two years ago, industry and FDA estimated that approximately 700 new ANDAs would be filed each year, she noted. In fact, the volume exceeded 1000 last year and continues to climb, making it impossible for the agency to eliminate the backlog as fast as expected and creating concerns about how FDA will meet faster review goals that start to phase in next year. 

The solution, from FDA’s perspective, is for manufacturers to submit more complete and high quality applications in the first place. This step would reduce review cycles and the need for multiple amendments that extend the review time frame. OGD acting director Kathleen Uhl emphasized the importance of high quality submissions at the GPhA workshop. Just filing more data does not necessarily make for a quality application, Uhl noted. OGD wants submissions to clearly demonstrate an understanding of the product and process and to avoid “superfluous data” that tries to cover up problems. 

To this end, FDA issued draft guidance on ANDA content and format in June, identifying basic requirements for a complete, high-quality application (2). The guidance specifies the information to be provided in each section of an application, following the format of the Common Technical Document (CTD) developed under the International Conference on Harmonization (ICH). It also summarizes the statutory and regulatory requirements for ANDAs and provides references to other guidance documents. 

The content and format guidance builds on an advisory issued last year on what ANDAs should include to avoid rejection under the agency’s Refuse to Receive (RTR) policy. FDA’s draft RTR guidance indicates what can trigger an RTR, including faulty listing of active and inactive ingredients, poor packaging information, lack of dissolution studies, and inadequate stability data (3). Last year, FDA issued multiple RTR letters to companies that failed to pay GDUFA facility user fees on time, but recently revised its policy to limit RTRs to applications clearly in arrears on fees or with serious shortcomings.

FDA also is updating and clarifying stability testing and assessment requirements for generic drugs as key to minimizing queries, reducing stability failures, and enhancing generic-drug quality overall. A final stability guidance for ANDAs was published in June 2013 (4), and FDA sought to clarify policy specifics in a Question & Answer guidance issued in May (5). Rosencrance acknowledged that the new policy represents “a huge change for industry,” and manufacturers raised multiple questions at the GPhA workshop about how FDA will implement the many specific requirements. But FDA is hopeful that the revision will be beneficial over the long run. 

Despite these and other challenges, Uhl reported at the workshop that OGD has made progress in meeting key GDUFA goals. Some initial action has been taken on almost 50% of backlog applications, and she sees progress towards achieving parity in domestic and foreign plant inspections. OGD aims to finalize the RTR policy this year and to issue additional guidance on ANDA amendments and on manufacturing supplements. FDA is hiring more chemistry reviewers, as supported by GDUFA, and hopes to bring on board 140 new reviewers by fall. These efforts aim to help assure the high quality of generic medicines, which, said Uhl, is “imperative in assuring the American public that the products you make are of high quality.”  

 (2014),  accessed July 10, 2014.
  2.	FDA, 

Draft Guidance, Content and Format of Abbreviated New Drug Applications

Draft Guidance, ANDA Refuse-to-Receive Standards

Final Guidance, Stability Testing of Drug Substances and Products 

Final Guidance: Stability Testing of Drug Substances and Products, Questions and Answers

’s Washington editor, Chevy Chase, MD, 301.656.4634, jwechsler@advanstar.com. Read Jill’s blogs at

Regulatory Beat

FDA seeks high quality applications and products to facilitate approvals and reduce safety and supply problems.

Changes and Challenges for Generic Drugs

This article is the second in a series designed to offer industry leaders and managers at all levels a road map to excellence in human performance and human error prevention. The benefits of human error prevention are many. High reliability organizations enjoy reduced costs, increased efficiencies, and a sharp competitive edge. Achieving human performance excellence, the hallmark of a high-reliability organization, is not a fantasy; it is a proven, eminently achievable method of cultivating and sustaining exceptional levels of performance. This proven approach is a coordinated implementation and integration of three key elements: human factors training, the establishment of an open reporting system, and a thorough incident investigation process. Such a system is complementary to the biotechnology industry’s cGMP requirements for training and corrective action, as human errors pose risk and must be investigated for appropriate corrective action commensurate with that risk. Through human factors training and an open reporting system, the organization can be transformed to one where the workforce is empowered, engaged, and proactive in the pursuit to reduce human error and achieve human performance excellence. 

Part one of this series discussed how an organization can begin making the journey to human performance excellence by learning from the experts and adopting a new perspective. The recognition that human performance requires a systemic approach to error reduction is a pivotal first step.  As the aviation and nuclear power industries have shown, deviations reflect weaknesses in an organization’s systems, not in its people. When human performance is embraced, knee-jerk, corrective responses to errors disappear and are replaced by meaningful, effective reforms, not the least of which is the implementation of an open reporting system that all can learn from (see 

The second article in this series explores the fundamental principles of high-reliability organizations and key knowledge from BioPhorum Operations Group (BPOG) members.


  The nuclear power and aviation industries have emerged as world-class leaders in the field of human performance. Their hard-won experiences underline the need for “Right First Time, Every Time” operational excellence. In such organizations, human performance is an integral part of the culture, embraced at all levels and demanded by regulators.  From these high-reliable organizations, the following practical principles for success can be drawn:
  
•	Human performance is not a project or a program. Human performance excellence doesn’t have a beginning, middle, and end. It is simply a part of the way things get done every day. It is ingrained in the organization’s DNA, and a proactive, open reporting culture is the norm (see 


  •	CEO sponsorship and top executive buy-in are essential for success. The quest for human performance excellence requires champions, effective advisory boards, investments, and the time and means to deliver the expected returns on investments.
  •	Behaviors and standards are clearly defined at every level, in all areas. Be clear about what human performance excellence requires. Requirements might include discussion of prior events during briefings prior to high-risk activities, expectations for shop-floor after action reviews following these activities, and organizing simple informal learning group activities to teach techniques and best practices.
  •	Performance is managed. Human performance objectives are visible and aligned with corporate and site performance goals, and managers ensure that everyone understands how their actions contribute to the achievement of the organization’s objectives.
  •	Management adopts a strong presence. Managers at every level need to observe their people regularly.  They know what the right behaviors look like and they coach their people effectively. Managers must regularly step out of the office and onto the work floor to ensure that human performance tools are being used in the right way and performance is meeting expectations.
  •	Technology is used wisely. Knowledge management programs have a large technology component that can be used to search for prior incidents, share lessons learned, and help staff learn from events in other industries. 
  •	Metrics are visible and consistent. Human error types are categorized and applied consistently. Examples of error types include lack of knowledge or understanding, cognitive process, problem detection/diagnosis, execution errors, and skill/performance errors. Reports are timely and show the rate of learning as well as error reductions, and are communicated effectively in ways that people can relate to and understand.
  •	Effective learning from failures and successes is supported. The open reporting process and follow-up actions must be executed under an environment of a “just culture.” In a “just culture,” there is an atmosphere of trust, where honest mistakes are treated fairly. Errors are learning opportunities and people are encouraged to speak up about their issues, which must then be acted upon. People must be confident that reported issues will be handled in a fair and just manner that not only protects the integrity of the company but also and of equal importance the individuals involved. In the same manner, those individuals who are found to have knowingly violated company policies are treated firmly but fairly.
•	Leaders lead. Leaders should demonstrate and reward the right behaviours.

Learning From BPOG Members’ Experience 


  BPOG members engage in internal and external benchmarking activities, regularly share lessons from  experience in online  meetings, co-create tools and other assets and meet face-to-face to synthesize ideas and recommendations. Key among their recommendations form experience are the following:
  •	Provide human performance awareness training for everyone. No exceptions. Begin with top management. Training typically can take at least two hours to half a day; don’t underestimate the effort that is required to enable people to understand what human performance is all about and what the organization is doing to embrace it. Plan on several months to put everyone through the training.
  •	Implement causal factors analysis (CFA). Establish investigation teams, including key managers, and train them to effectively use CFA.
  •	Provide superior human performance skills training. Training should be directed to three target audiences: workforce, managers, and engineers/other specialists. Begin with core skills training. After core skills, train cross-functionally as much as possible. Implement an effective process and resources system (e.g., learning management systems) that enables people to continually refresh, test, and develop their skills in using human performance tools. 
  •	Implement a leader observation and coaching program. At all levels from site lead to line, have managers conduct 6-12 active observations to support the use of human performance thinking and tools in practice.  This can be run as part of existing practices such as regular gemba walks and safety checks. Show and train your leaders to do it well. 
  •	Introduce a human performance practitioner role. Individuals who have the respect of their peers and colleagues should be selected to drive change and coach the leaders and teams in human performance. Be sure to provide them sufficient time to accomplish this properly.  
  •	Speak with data. Have meaningful human error categories and establish true root causes, making ‘effectiveness and performance,’ and not simply compliance, the incentive for corrective and preventive action.
The final installment of this series will review the eight crucial steps for success in human performance.  


Gerry McAuley is an experienced business consultant and facilitator to the Human Performance Group at the BioPhorum Operations Group.

Featured Article

Part II of this three-part series presents a roadmap to human error prevention.

Optimizing Human Performance, Part II: A Road Worth Traveling

Issue PDF

Click the title above to open the BioPharm International August 2014 issue in an interactive PDF format.

BioPharm International-08-01-2014

Single-Domain Antibodies for Brain Targeting

Marrying Big Data with Personalized Medicine

Applying GMPs to the BioPharma Supply Chain

Changes and Challenges for Generic Drugs

Optimizing Human Performance, Part II: A Road Worth Traveling

BioPharm International, August 2014 Issue (PDF)