What are the key challenges - and advances - in fermentation and cell culture technology that industry faces over the next five years? To gain some insight, BioPharm International turned to six industry professionals for their forecasts: David A. Dodd is president, chief executive officer, and director of Serologicals Corporation; Sean Eicher is a product manager at New Brunswick Scientific Company, Inc.; William R. Tolbert, Ph.D, a BioPharm International editorial advisory board member, is president of WR Tolbert & Associates, LLC; Tim Ward is director of cell culture for The Automation Partnership; Larry West is director of sales and marketing for Broadley-James Corporation; and Frank Wicks is president of fine chemicals for Sigma-Aldrich Corporation. 	

BPI: What are the most difficult challenges facing the fermentation and cell culture sector during 2004 and within the next five years? How do you think the challenges will be met?

 In the early to middle 1960s, the earliest large-scale (50 liters to 1,000 liters) suspension culture of mammalian cells was accomplished. Then, during the 1970s, large-scale systems were further developed in Europe for biopharmaceutical production. Microcarrier technology for large-scale production of anchorage dependent cells was introduced in 1967 and later used for vaccine production in the Netherlands.	

FDA, however, prevented any use of "continuous cell lines" (that is, non-diploid, long-life cells that were suitable for scale-up) as substrates for human-use biologicals, until the middle to late 1980s. The first FDA-approved biological product - using the continuous Chinese hamster ovary (CHO) cell line as substarate - was Genentech's Activase (recombinant tissue plasminogen activator). This product has been produced both in the United States and Europe using bioreactors greater than 10,000 liters. During the 1990s and early 2000s, many more biopharmaceuticals have been approved using large-scale mammalian cell culture as the manufacturing technology.	

Currently, the manufacturing technology for commercial scale production of therapeutic recombinant proteins - including monoclonal antibodies - has matured to the extent that this technology (that is, large, stirred-tank bioreactors in many thousands of liters using batch, fed- batch, or perfusion methodology) is no longer a major hurdle in the development of these biopharmaceutical products. Development of well growing cell substrates; high-yield recombinant expression systems; animal product-free media; and efficient bioreactors has led to the economically feasible manufacture of hundreds of kilograms annually.	

With the large number of potential, new biopharmaceutical products, the availability of suitable mammalian cell manufacturing plants has become a problem for the next few years. This is a problem, however, of capital investment timing and plant construction planning, rather than of production technology. Future technological advances for these production systems likely will be incremental and focused on streamlining and increased efficiency, rather than on fundamental new approaches.	

The transfer of regulation for recombinant proteins and monoclonal antibodies from FDA's Center for Biologics Evaluation and Research (CBER) to the Center for Drug Evaluation and Research (CDER) may cause some disruption in the short term as companies adjust to the different regulatory polices and philosophies of CDER, but this should not be a long-term problem and may become an advantage.	

 The three greatest challenges to be faced by the cell culture and fermentation sector in the next five years will be navigating the governmental regulations concerning the development of products used for human consumption; dealing with the capacity shortage for larger-scale systems; and gathering and maintaining process data.		

The equipment used to produce compounds from microbial or cell culture batches must adhere to numerous manufacturing and processing regulations. To meet these regulations, the required documentation can range from basic material certification with test reports to full boroscopic examinations of all welds within the sterile envelope.	

Since the level of system validation is often unknown at the project onset, manufacturers need to accommodate the defined needs as well as anticipate additional requirements yet to be determined. One method to accomplish this is with a modular line of fermentation equipment that can be modified to incorporate components - that can be validated - into the system after delivery. This design would allow end-users to compensate for system features not defined in the original system specifications and further optimize their process with additional controls that could result in higher yields from the same equipment. 	

Many industry insiders are predicting a capacity shortfall in the next few years. With this in mind, scientists will need to venture from traditional protocols and investigate novel approaches to increase yields and efficiencies. Doing so could allow the scientist to use their current equipment to meet the increasing demand for certain compounds. Approaches such as fed and continuous batches in fermentation or perfusion with packed beds of non-woven polyester fiber disks in cell culture have shown promise in many applications. In some instances, employing a non-traditional approach has increased the production capability of a reactor tenfold compared to batch runs.	

In addition to these unique methods to increase yields, manufacturers will be pushed to deliver units with shorter lead times. The market is demanding that the current lead times of six to 12 months be cut by one half or one quarter to meet their needs. This will result in fewer custom-designed systems being sold and lead to more pre-packaged systems - which have already been tested and designed - emerging in the marketplace.	

With the increase in regulatory constraints concerning the generation and storage of electronic records and signatures, the integrity and availability of process data is becoming paramount in its importance. As a result, the data from the supplied systems will be required to be simply integrated into the end users' existing supervisory control and data acquisition (SCADA) or laboratory information mangement (LIM) system, without the need for custom drivers. This feature will allow scientists to use a software package they are already familiar with for data analysis and process optimization. In order to accomplish this, it is believed that proprietary control packages, as well as system components, will become obsolete, being replaced with industry standard components and controls. This will not only provide scientists with the open architecture they require but give them the reliability their process deserves.	

 The most difficult challenges all have the same origin: technology. In 2004 the availability of technology allowing sensors to monitor their own performance and predict failure will redefine the expectations on research, process development, and metrology as it relates to productivity. In addition, the availability of technology that allows on-line instruments to communicate with off-line instruments will put more information in the hands of operators. And the effective use of this information, its interpretation and documentation, could increase the initial workload of supervisors and validation.		

But technology - such as that already mentioned - will also alleviate its associated burden. The use of wireless communications, tablet PCs, remote interfaces, and the like will enable unprecedented multitasking by operators and supervisors alike. For example, a second shift operator in the United States could use his company's Intranet to contact a supervisor just getting into work in the UK to discuss a process alarm or unique cell activity.	

My recommendation to face these challenges is to embrace the technology that is becoming available, while ensuring optimum implementation.	

 The challenges facing the fermentation and cell culture industry include meeting the capacity for production of new, approved antibody-based therapeutic products; consistent assurance of viral clearance; and adequate quality systems throughout the entire development and manufacturing chain. Due to the continued industry-wide shortage in manufacturing capacity for approved antibody and recombinant protein therapeutic products, an increased number of contract manufacturing organizations (CMOs) can be expected to expand capacity.		

 One challenge the biopharmaceutical industry faces is the replacement of animal-derived components. Industry increasingly will choose to outsource to medium development companies for medium optimization studies, spent medium analysis, and microarray analysis - to name a few - and also increase outsourcing to manufacturers of novel medium components. Another challenge is increasing productivity of fed-batch culture systems. This can be achieved by the use of bioreactor feeds or novel feed strategies in the production process and through protein expression in novel engineered cell types. In general, the biopharmaceutical industry is looking to increase the cost effectiveness of its processes at phase 2 and this helps drive outsourcing choices where cost-favorable.	

Small and medium industry players also seek  manufacturing capacity for preclinical recombinant proteins for purposes like toxicology studies. They can do this by outsourcing to nontraditional, non-CMO sources such as medium development companies with bioreactor capacity and this production capability.	

Other notable trends include the use of disposables to enable multiproduct manufacturing strategies, implementation of design controls, and antibody engineering to reduce the therapeutic dose required.	

 The challenge facing the fermentation and cell culture sector during 2004 and within the next five years is delivering large quantities of cells consistently and on time.	

BPI: What are the top technological advances industry will see in the fermentation and cell culture sector during 2004 and over the next five years?

 Cell culture will become a precise operation. The demands upon in-house cell culture teams will be to produce cells-to-order and just-in-time. Alternatively, companies will be investing in cell storage systems from which cells prepared in advance may be drawn. The significance of just-in-time delivery should not be underestimated, as it will place demands upon the suppliers of automation to provide machines that are capable of unattended operation 24 hours a day, seven days a week.		

In the next five years technological advances include more built-in intelligence, for example automatic cell life cycle management, and built-in QC functionality.	

 In 2004 and continuing throughout the course of the next five years, industry will see three significant technological advances related to on-line and off-line measurement and control in fermentation and cell culture applications. The first is the ability of sensors to monitor their own health and, in doing so, predict their own failure. The second is the ability of stand-alone instrumentation to communicate information to a single controlling device in the suite which will tie together control and data collection. Finally, the third is the ability of process monitoring and control to be portable through the use of wireless communications. All these will have the effect of reducing costs and improving reliability and repeatability. 	

 The industry will see advances in productivity from specialty supplements targeted toward specific cell lines; an increase in regulatory focus on the validated manufacturing processes relative to viral clearance objectives; and increased regulatory oversight of cell culture manufacturing to conform to tighter standards relative to viral clearance and quality systems.	

 Innovations for 2004 will include increased speed in development of animal component–free and defined media through the use of analytical methods, design of experiment techniques, and the use of microarrays. We'll see this because new tools are available to speed the process. Novel raw materials have been created through chemical synthesis and, in some cases, with transgenic proteins to replace animal-derived components. 		

Within the next five years, the creation of new, engineered production cell lines to compete with the CHO cell line will be achieved, leading to increases in productivity while maintaining normal mammalian post-translation modifications.	

 New and novel cell-based medical advances are likely to come in the area of gene and cell therapy modalities. Recombinant viral vectors that are directly injected into patients (in vivo gene therapy) may be produced in the same - or similar - bioreactors used for therapeutic protein production. The scale-up requirement for these products is directly proportional to the vector expression level and the required patient dose. These parameters vary widely for different products and vector and cell substrate systems. Annual production requirements for a commercial operation may range from a few hundred liters to many thousands of liters. Future development of new cell substrates, new viral vectors, and new media all will be important to the economic feasibility of these products. The technology for viral vector products is at a stage similar to where therapeutic proteins were in the early 1980s.	

 gene therapy, where cells are removed from a patient, genetically modified in culture, and then returned to the same patient, shares problems with patient-specific cell therapies (such as adult stem cells and activated dendritic cells). Here, aseptic manufacturing controls must be in place for every process step, as it is not possible to sterilize or final filter the live cell containing product, and the production process is very labor intensive with one product lot per patient instead of thousands of patient doses per product lot.	

However, academic research (at the preclinical and phase 1 and 2 clinical trial stages) has increased dramatically over the last few years, and there is the potential for substantial therapeutic advances, which may not be achievable by conventional pharmaceutical or biopharmaceutical drugs. It will be a major challenge to make any of these advances economically or commercially feasible for the hundreds of thousands of patients who might benefit from their use. In this field, innovative production technologies and novel manufacturing processes will be required to address the "one-patient-at-a-time" constraint and to effectively bring any of these therapies to the market. In order to properly address the regulatory issues associated with these new medical technologies, FDA raised the former Division of Cellular and Gene Therapies to office level within CBER.  

Articles

Product Development

What are the key challenges - and advances - in fermentation and cell culture technology that industry faces over the next five years?

Fermentation and Cell Culture Technology: Problems and Predictions

The distinct business specialties that we know collectively as the pharmaceutical industry — large pharmaceutical manufacturers ("big pharma"), biotechnology companies, specialty pharmaceutical manufacturers, generic manufacturers, contract research organizations (CROs), and distributors — are slowly converging. There has been a growing convergence of goals and activities between these sectors as firms refine their strategic focus to emphasize proprietary drugs and delivery systems.

 The big pharmaceutical manufacturers are becoming more focused on pure, research-based pharmaceutical and combination products with potential for significant sales and therapeutic impact. In order to focus on proprietary products, products that do not fit well with a pharmaceutical company's portfolio or have an overall sales potential below corporate targets will often be divested to specialty pharma companies or even to a competitor who sees a better fit with existing or emerging offerings. Pharmaceutical companies also have sold business lines to companies that have traditionally been service providers, in some cases to satisfy regulatory requirements of merger transactions.

 Historically, specialty pharmaceutical manufacturers identified opportunities to develop niche products and used those endeavors to grow and mature.  Now these companies are seeking to increase their investment in other competencies that have been the exclusive domain of big pharma.  The future will see more specialty pharmaceutical firms involved in manufacturing, sales, and marketing activities.

Collaborative efforts will emerge among specialty pharmaceutical firms to leverage technical capabilities upstream and downstream. The specialty sector will compete directly with the big pharma and biotech sectors for future product opportunities. As big pharma experiences future product shortages in the primary care market, direct competition between both specialty pharma and big pharma for the same biotech applications could emerge as a result of the search for the next blockbuster drug.

 Biotech companies, once cash-hungry R&D entities, have grown into significant research-based pharmaceutical discoverers, manufacturers, and marketers. As a result, their strategic needs are now quite similar to the rest of industry. This development has fueled overall industry growth, as over half of big pharma's current sales stems from relationships and alliances with biotech and specialty pharmaceutical companies.

Big pharma has an acute need to strengthen its fading proprietary product revenue streams. As significant patents expire over the next five years, the potential for alliances and convergence between biotech and other pharmaceutical players will increase. Biotechnology companies hold a strong position in terms of intellectual property, but their access to funding has been limited. Pharmaceutical companies have been reluctant to invest without proven results, and they have been negotiating for greater percentages of future royalties. This dynamic will likely rebalance as approval risks decrease in later development stages and prospective rewards grow.

 Generics manufacturers have benefited from a growing volume of overall prescriptions and the popularity of cheaper generics when patents expire. Strong growth and consolidation have resulted in large, well-financed organizations. Like their big pharma counterparts, these companies have invested in drug discovery, development, and alliances. They have explored developing proprietary drugs that complement their strong generic positions. While these companies are not abandoning their generic roots, they are adding new capabilities, establishing sales forces, and hiring contract sales organizations. They will increasingly go head-to-head with branded pharmaceutical and biotech firms, particularly if generic biologics become reality.

 Traditionally viewed as service providers, CROs and distributors have also joined the race to collect revenues from proprietary drugs and drug delivery systems. Often, these enterprises pursue opportunities in high-margin branded products using a risk-sharing collaboration model.

Success is breeding competition. Strategically, CROs have the R&D competencies to become viable players. By leveraging those strengths in collaboration with biotechs, CROs can gain access to intellectual capital and biotechs can obtain an additional source of funding in a tight investment cycle. As CROs will be challenged to grow at a rate similar to that of the 1990s, leveraging their strong clinical base and expanding into high-margin activities will be key to future success.

Similarly, distributors have become fully integrated healthcare companies, expanding into pharmaceutical packaging, services, and drug delivery niches. Additional high-margin strategic plays are likely as opportunities to broaden service offerings become available.

 As the various sectors increasingly overlap, the industry will change shape. Certainly, greater participation in various areas will increase competition between players; there will be more bidding and higher prices for deals ranging from licensing a drug to purchasing a subsidiary. However, there will also be tremendous opportunity for new forms of collaboration between the converging companies, a truly exciting prospect that will bring additional product offerings to the global health care markets.  

Columns and Departments

As the various industry sectors expand drug discovery and development activities, there will be more direct competition as well as opportunities for collaboration.

Components of the Pharmaceutical Industry Are Converging

This past year was filled with tremendous activity at FDA and other government agencies involved with healthcare, biomedical research, and national security. Scientists completed sequencing the human genome, increasing optimism for a period of unprecedented advances in biomedical discovery. Plus, the need to protect the nation from biological warfare promises to stimulate the development of new vaccines and medical treatments (see "Combating Bioterrorism, " below).

Scientific achievement was not alone in spurring industry and government action this year. Rising public pressure for more affordable medicines also shaped a wide range of policy initiatives in 2003. Congress tackled the daunting task of crafting a prescription drug benefit for the nation's more than 40 million Medicare beneficiaries. Meanwhile, consumers skirted existing law by purchasing less expensive drugs and biologics from foreign sources.

FDA responded to these developments with regulatory initiatives to lower drug costs. The agency continues its mission to improve the drug manufacturing process while accelerating the testing and approval of new drugs and biologics and low-cost generics. And, in an effort to reduce adverse drug events, it is promoting electronic prescribing and improving product labels.

In his first year in office, FDA Commissioner Mark McClellan unveiled a steady stream of initiatives designed to streamline FDA internal regulatory procedures and clarify testing requirements for new therapies. Such actions aim to reverse the recent slow-down in applications for innovative new drugs and biologics. Also, McClellan is emphasizing the application of risk management strategies to FDA policies and programs, a concept he explained in FDA's Strategic Action Plan, issued in August 2003. Risk management uses FDA's limited resources to best protect public health at the least cost by focusing more oversight on higher risk products and processes.

FDA also is crafting policies that encourage voluntary compliance with agency regulations. According to McClellan, initiatives that reduce the time, cost, and uncertainty of developing new biotech therapies can improve public health; cutting medical product development costs facilitates consumer access to effective new treatments. 

A number of FDA initiatives aim to further reduce healthcare costs by speeding new therapies to market and by reducing inappropriate drug use.

  Last January  the commissioner unveiled a plan to increase innovation in medical technology by clarifying regulatory standards and test data requirements. The goal is to process more applications in only one review cycle (Inside Washington, March 2003).

 The agency promised more guidance documents to better define the development requirements of new treatments for diabetes, cancer, and obesity — high priorities for government health agencies. 

 FDA continued its two-year campaign to overhaul FDA regulations governing GMPs. Launched in August 2002, the GMP update revises policies governing electronic records and signatures, manufacturing plant inspections, and postapproval manufacturing changes. Further initiatives and final rules are expected early next year.

 FDA held a three-day workshop in April to examine how risk management approaches can promote the safe use of medical products. Agency officials, manufacturers, and health professionals commented on FDA proposals for more extensive preapproval testing of new products to better identify and study safety concerns. Participants also discussed how manufacturers could develop pharmacovigilance plans to detect and analyze safety issues that arise after a product reaches the market. FDA wants sponsors to do more to identify risk "signals" and to develop postmarketing risk management programs for drugs with increased safety concerns.

FDA also is calling on manufacturers to print bar codes on drug packages, vials, and even individual dosage forms to ensure that the right patient receives the right dose at the right time. In March, FDA proposed a requirement that manufacturers print bar codes with national drug codes on packages. The agency is finalizing rules on these issues, and implementation will be a major challenge in coming months. 

 To facilitate public access to more affordable medicines, FDA is streamlining its generic drug oversight process and emphasizing the high quality and interchangeability of generics. Agency officials also have signaled interest in exploring avenues for authorizing generic versions of biotech therapies. In its October response to several citizen petitions, FDA asserted its authority to approve new therapies based on earlier published studies of the innovator drug. The agency is expected to seek more input from manufacturers, healthcare providers, and consumer groups on how to balance the desire for less expensive equivalent treatments while maintaining incentives for manufacturers to develop innovative biotech products.

FDA also is leading the campaign against importing more low-cost, but unapproved, drugs and biologics from Canada and other nations. McClellan and other federal officials insist that reimportation can bring unsafe counterfeit products into the United States, particularly via Internet pharmacies, many of which operate overseas. Despite these arguments, the House approved a bill in July permitting reimported drugs with no requirement that FDA first certify these drugs will not harm public health (Inside Washington, September 2003). The debate heated up in September when FDA asked the Department of Justice to file suit against RxDepot — a leading pharmacy website — for arranging drug imports from Canada. 

FDA's warnings continue to fall on deaf ears as several states and local governments devise plans to buy medicines from Canada to cut spending on drugs for Medicaid and local pharmacy benefit programs. The public generally believes that drugs and biologics from Canada are just as safe and effective as those made in the United States. A recent Harris Poll shows consumers oppose efforts by government and industry to prevent access to less expensive foreign medicines, and a growing number are purchasing or would like to shop abroad for prescription drugs.

According to FDA and manufacturers, the broader problem is that illegal reimportation opens the door for counterfeit and unsafe products to enter the US market. Counterfeiters have targeted several high-cost biologics, raising serious health concerns. FDA's Counterfeit Drug Task Force issued an interim report in October describing how counterfeit products slip into the US drug distribution system. The agency proposes an attack on counterfeiting through a mix of stricter licensing standards for wholesalers and distributors; technological innovations that manufacturers can use to identify bogus products; and more public education to help health professionals and consumers easily identify counterfeits.

One way to deter illegal importing and counterfeiting, says McClellan, is by reducing the price differences between US and foreign pharmaceuticals. At the National Press Club in October, McClellan spelled out FDA's concerns and pointed to international pricing disparities in drugs and biologics for escalating the reimportation problem. McClellan noted that Americans pay about half of all prescription drug costs worldwide (and support most pharmaceutical R&D). Consequently, Americans without insurance coverage are seeking cheaper, foreign medicines. Deeming this a global public health issue, he called for a "better, global solution to the problem of affordable drug pricing," and said that "if all countries pay their fair share," needed treatments will be "more affordable for everyone." 

Another high-profile challenge for FDA is to identify research paradigms for countermeasures to bioterrorism and facilitate emergency access to needed treatments. CBER sponsored a workshop in October to encourage industry development of counterterrorism vaccines, diagnostics, and therapies. FDA also is working with industry to identify antiterrorist uses of existing medical products, activities that may receive resources through Project BioShield legislation. 

Manufacturers are concerned, however, that the legislation fails to provide sufficient protections from liability for such high-risk products, which may limit private sector investment. A key challenge for the Bush administration is to clarify government commitments to providing a market and support for new biomedical counterterrorism measures.

FDA initiatives aim to spur development of more affordable treatments and ensure safe product use.

Inside Washington: Risk Management and Pricing Concerns Shaped FDA Policy in 2003

Bioconjugation chemistry is the joining of biomolecules to other biomolecules, small molecules, and polymers by chemical or biological means. This includes the conjugation of antibodies and their fragments, nucleic acids and their analogs, and liposomal components (or other biologically active molecules) with each other or with any molecular group that adds useful properties. These molecule groups include radionuclides, drugs, toxins, enzymes, metal chelates, fluorophores, haptens, and others.

The conjugation of monoclonal antibodies for therapeutic purposes is currently undergoing a flurry of research and development activity.

 These developments may not only change the way drugs are delivered, but could lead to safer protocols by dramatically reducing the dose to achieve efficacy without causing harmful side effects. Monoclonal antibodies can be utilized for drug or isotope deliveries, but as the biological functions of monoclonal antibodies are often sensitive to subtle variations in structure, attention must be paid to the chemical aspects of preparing, purifying, and characterizing these conjugates. The most commonly employed method for covalently crosslinking monoclonal antibodies to other molecules is the use of special reagents.	

This article will primarily focus on the experimental design, protocols, and procedures for the preparation of 

 therapeutic drug conjugates, such as the conjugation of monoclonal antibodies to other molecules using ligand and metal chelates, toxins, cancer drugs, and other proteins under cGMP or general large-scale manufacturing protocols. It focuses only on the chemical modification of the antibodies. Preparation of antibody fragments such as Fab' and F(ab')

 via enzymatic processes will not be discussed. For more information on the preparation of antibody fragmentation, see Hermanson (pages 478-482).

A simple illustration of conjugation of a biomolecule is depicted in Figure 1. It essentially involves the activation of a reagent and a biomolecule with Z-A and Z-B crosslinkers, respectively. The reactive moiety Z must react efficiently, yet must not crosslink with the reagent and biomolecule. The linkers A and B must be reactive enough to couple with high efficiency, yet stable enough to be stored in solution or circulated in the body for long periods. It is also important that no nonspecific (noncovalent) binding or sticking of molecules occurs, which would lead to potential reduction in the effectiveness of the drug conjugate and high background interference in any assay.	

This article will address the following specific topics: reactive groups of proteins (specifically, monoclonal antibodies) that are available for modification, including their naturally occurring amino acids and reactive groups introduced by chemical modification; reagents that can be used to couple molecules; the reaction environment; the current status of experimental procedures used in laboratory preparation; purification and isolation of the conjugates;  storage; and manufacturing.	

Monoclonal antibodies (and other proteins) are amino acid polymers containing a number of reactive side chains. In addition to, or as an alternative to, these intrinsic reactive groups, specific reactive moieties can be introduced into the polymer chain by chemical modification. These groups, whether they are naturally a part of the protein or are artificially introduced, may serve as "handles" for attaching a wide variety of drug molecules (center of Figure 2). The intrinsic reactive groups of antibodies are described below. 

Figure 1. A generic scheme for bioconjugation reaction				

 One of the most common reactive groups of proteins is the aliphatic ε-amine of the amino acid lysine. In general, nearly all antibodies contain abundant lysine. Lysine amines are reasonably good nucleophiles above pH 8.0 (pK

 and therefore react easily and cleanly with a variety of reagents to form stable bonds (Equation 1).	

Other reactive amines that are found in proteins are the α-amino groups of the 

-terminal amino acids. The α-amino groups are less basic than lysines and are reactive at pH~7.0. Some of them can be selectively modified in the presence of lysines. Since either 

-terminal amines or lysines are virtually always present in antibodies, and since they are easily reacted, these aliphatic amines provide the most commonly employed method of antibody modification.	

 Another common reactive group in antibodies is thiol residue from the sulfur-containing amino acid cystine and its reduction product cysteine (or half cystine). Cysteine contains a free thiol group, which is more nucleophilic than amines and is generally the most reactive functional group in a protein. Thiols, unlike most amines, are generally reactive at neutral pH, and therefore can be coupled to other molecules selectively in the presence of amines (Equation 2). This selectivity makes the thiol group the linker of choice for coupling proteins. Methods that only couple amines (for example, amine reaction with gluteraldehyde) can result in formation of homodimers, oligomers, and other unwanted products (see Hermanson, pages 470-472).

Since free sulfhydryl groups are relatively reactive, proteins with these groups often exist in their oxidized form as disulfide groups. Immunoglobulin M is an example of a disulfide-linked pentamer, while the subunits of Immunoglobulin G are bonded by internal disulfide bridges. In such proteins, reduction of the disulfide bonds with a reagent such as dithiothreitol (DTT) is required to generate the reactive free thiol. However, this method also splits the chain linkage in these antibodies and reassembly of the chains to allow proper folding may not be possible. In addition to cystine and cysteine, some proteins also have the amino acid methionine containing sulfur in a thioether linkage. Selective modification of methionine is generally difficult to achieve and is seldom used as a method of attaching drugs and other molecules to antibodies. The literature describes the use of several thiolating crosslinking reagents such as Traut's reagent (2-iminothiolane), succinimidyl (acetylthio)acetate (SATA), and sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (Sulfo-LC-SPDP) to provide efficient ways of introducing multiple sulfhydryl groups via reactive amino groups. 

Carboxylic acids (aspartic acid, glutamic acid).

 Proteins contain carboxylic acid groups at the 

-terminal position and within the side chains of aspartic acid and glutamic acid. The relatively low reactivity of carboxylic acids in water usually makes it difficult to use these groups to selectively modify proteins and other biomolecules. When this is done, the carboxylic acid group is usually converted to a reactive ester by the use of a water-soluble carbodiimide and reacted with a nucleophilic reagent such as an amine, hydrazide, or hydrazine.

 The amine-containing reagent should be weakly basic in order to react selectively with the activated carboxylic acid in the presence of other amines on the protein. Protein crosslinking can occur when the pH is raised above 8.0.	

Figure 2. Three side groups provide the major antibody modification pathways. Starting from the unmodified antibody in the center, reactions with the amino group follow the red path, the alcohol group follows the blue path, and the carboxylic acid group follows the green path.	

 Sodium periodate can be used to oxidize the alcohol part of the sugar within the carbohydrate moiety to an aldehyde (see Hermanson, pages 340-341).

 Each group can be reacted with an amine, hydrazide, or hydrazine as described for carboxylic acids. Since the carbohydrate moiety is predominantly found on the crystallizable fragment (Fc) region of the antibody, conjugation can be achieved through site-directed modification of the carbohydrate away from the antigen-binding site. 	

This section will briefly describe many of the reagents used in protein modification. In order to understand how to use these reagents, it is necessary to know what reactive group(s) on the protein can be modified; which type of chemical reactions these reactive groups will participate in; and the nature of the chemical bonds that will result (Figure 2).				

 These reagents react primarily with lysines and the α-amino groups of proteins. Some amine-reactive reagents are more reactive, and therefore less sensitive, than others. It is necessary to consider reactivity when choosing the best reagent for modification of a specific protein. Table 1 lists commercially available amine-reactive reagents.	

Reactive esters (formation of an amide bond).

 Reactive esters, particularly N-hydroxy-succinimide (NHS) esters, are among the most commonly employed reagents for modification of amine groups. They are moderately reactive toward amines, with high selectivity toward aliphatic amines. Their reaction rate with aromatic amines, alcohols, phenols, and histidine is relatively low. The optimum pH for reaction in an aqueous environment is 8.0 to 9.0, and they form very stable aliphatic amine products. The NHS esters are slowly hydrolyzed by water, but are stable if stored well desiccated. Virtually any molecule that contains a carboxylic acid, or that can be chemically modified to contain a carboxylic acid, can be converted into its NHS ester. That explains why these reagents are among the most powerful protein-modification reagents available. A new class of NHS esters are now commercially available with sulfonate groups that have improved water solubility.	

Table 1. Some commonly used homo- and heterobifunctional crosslinking and chelating reagents	

Isothiocyanates (formation of a thiourea bond).

 Isothiocyanates behave like NHS esters. They are amine-modification reagents of intermediate reactivity and form thiourea bonds with proteins. They are somewhat more stable in water than NHS esters and react with protein amines in aqueous solution (optimally at pH 9.0 to 9.5). Since this is a higher pH than optimal for NHS esters (which undergo competing hydrolysis at pH 9.0 to 9.5), isothiocyanates may not be as suitable as NHS esters when modifying proteins that are sensitive to alkaline conditions.	

Aldehydes (formation of imine, Schiff's base, reduction to secondary amine bond).

 Aldehyde groups react under mild aqueous conditions with aliphatic and aromatic amines, hydrazines, and hydrazides to form an imine intermediate (Schiff's base). A Schiff's base can be selectively reduced with mild or strong reducing agents (such as sodium borohydride or sodium cyanoborohydride) to derive a stable alkyl amine bond. This method of amine modification can successfully be employed in situations in which the antibody is modified away from the antigen-binding site via the oxidation (typically with sodium periodate) of the alcohols on the carbohydrate moiety of the Fc region.	

Miscellaneous amine-reactive reagents (anhydrides).

 Other reagents that have been used to modify amines are acid anhydrides. For example, diethylenetriaminepentaacetic anhydride (DTPA) is a bifunctional chelating agent that contains two amine-reactive anhydride groups. It can react with 

-terminal and ε-amine groups of the proteins to form amide linkages. The anhydride rings open to create multivalent, metal-chelating arms able to bind tightly to metals in a coordination complex. This type of reaction is particularly useful in the preparation of radiolabeled immunoconjugates.

 Thiol-reactive reagents are those that will couple to thiol groups on proteins, forming thioether-coupled products. These reagents react rapidly at slight acidic to neutral pH and therefore can be reacted selectively in the presence of amine groups.	

Haloacetyl derivatives (formation of a thioether bond).

 These reagents (usually iodoacetamides) are common reagents for thiol modification. In antibodies, the reaction takes place at cysteine groups that are either intrinsically present or that result from the reduction of cystine's disulfides at various positions of the antibody. The thioether linkages formed from any reaction of haloacetamides are very stable. However, iodoacetamide modification reagents are unstable in light, especially in solution. They must be protected from light during reaction and in storage. The level of control to achieve reproducibility required for large scale manufacturing conditions may be difficult to achieve.		

Tips for Preparing Therapeutic Monoclonal Antibody Conjugates	

Maleimides (formation of a thioether bond).

 The reaction of maleimides with thiol-reactive reagents is essentially the same as with iodoacetamides. Maleimides react rapidly at slight acidic to neutral pH. Above pH 8.0, maleimides can undergo hydrolysis to form nonreactive maleamic acids.	

Aldehyde and carboxylic acid-reactive reagents.

Amines, hydrazides, and hydrazines (formation of amide, hydrazone, or alkyl amine bonds). 

Amines, hydrazides, and hydrazines can be coupled to carboxylic acids of proteins after the activation of the carboxyl group by a water-soluble carbodiimide. As mentioned previously, the amine-containing reagent must be weakly basic so that it reacts selectively with the carbodiimide-activated protein in the presence of the more highly basic ε-amines of lysine to form a stable amide bond. 	

Amines, hydrazides, and hydrazines also can react with aldehyde groups, which can be generated on antibodies by periodate oxidation of the carbohydrate residues on the antibody. In this scenario, a Schiff's base intermediate is formed, which can be reduced to an alkyl amine through the reduction of the intermediate with sodium cyanoborohydride (mild and selective) or sodium borohydride (strong) water-soluble reducing agents.	

 Bifunctional crosslinking reagents are specialized reagents that will form a bond between different groups, either on the same molecule or two different molecules. These reagents can be divided into two kinds, homobifunctional reagents (those with the same reactive group at each end of the molecule) and heterobifunctional reagents (those with different reactive groups at each end of the molecule). Recent trends appear to strongly favor the use of heterobifunctional cross-linkers where the bifunctional reagent has two reactive sites, each with selectivity toward different functional groups (for example, an amine reactive and a thiol reactive). Many bifunctional reagents are commercially available with variable chain lengths and water solubility.

Table 2. Status of immunoconjugate drug products	

 Photoactive reagents capable of being activated by light can produce a reactive intermediate for coupling to various functional groups on biomolecules. Generation of highly reactive intermediates such as nitrene, carbene, and diradical species employed in photoaffinity labeling is of little value since such species are highly nonspecific and often capable of reaction even with alkyl side-chains.

 These reagents have not been widely utilized in the modification and coupling of monoclonal antibodies.	

It is imperative that the scientist understands the practical aspects of reacting with large, complex, conformationally sensitive, water-soluble antibodies and other biomolecules or small organic molecules. The buffers, the cosolvents, and the reaction conditions all must be considered in order to obtain a scalable process that can be validated and used under aseptic, GMP manufacturing conditions. 				

 In general, conjugation should be carried out in a well-buffered system at an optimal pH for the reaction. The ionic strength of the buffer in most cases should be around 10 to 100 mM. Phosphate buffers should be well suited for the modification of thiol groups and α-amino groups, because this occurs selectively at physiological pH (7.0 to 7.5). For lysine amines, which require a more alkaline pH (8.0 to 9.2), carbonate-bicarbonate or borate buffers work well. In some cases, the choice of buffer will be dictated by the compatibility of the protein. For example, since many of the conjugations of antibodies involve the utilization of amine groups, it is necessary to avoid amine-containing buffers like tris(hydroxy-methyl)aminomethane (TRIS). 	

 Many of the available crosslinking reagents are sulfonated and water-soluble at millimolar to micromolar concentrations. These reagents need no cosolvent for solubilization. If the reagents are not soluble in aqueous medium, water-miscible cosolvent - such as dimethylformamide (DMF), dimethyl-sulfoxide (DMSO), and some alcohols - must be used to dissolve the reagent without causing decomposition. Furthermore, the cosolvent must not cause irreversible denaturation or precipitation of the antibody. DMSO and DMF are the most commonly used cosolvents, usually up to 10% (w/v) concentrations. 	

 In general, conjugation reactions involving antibodies (and most other proteins) should be done between room temperature and near-freezing temperature (~25Â°C to 2Â°C). Lower temperature tends to slow down the reaction rate and increase the selectivity and control of the reaction. This could result in fewer side reactions and more consistent and reproducible results. 	

 Typically, a 1 to 10 mg/mL protein concentration is recommended for protein conjugation with an optimum range of 5 to 10 mg/mL. Concentrations of less than 1 mg/mL have been successfully reported, but these require a longer incubation time to achieve adequate conjugation (based on unpublished data).	

 During the modification of primary amines, only the unprotonated form is reactive and, therefore, it is necessary to maintain a pH at which a significant proportion of amines are unprotonated. When working with lysine, it was found that at an  average pK

 above 9, the reaction runs at a faster rate. However, some crosslinking reagents may undergo hydrolysis and some antibodies (and other proteins) tend to be unstable at higher pHs. The α-amino groups of the 

-terminal amino acids are less basic than lysines and are reactive at pH ~7.0. These amino groups are sometimes preferentially modified when the reaction is run at neutral pH. Thus if the protein and crosslinking reagents are stable, the reaction should be performed close to pH 9.0. For less stable reagents, the reaction should be performed near pH 7.0.	

 In general, one to two hours should suffice for conjugation reactions to go to completion. Longer reaction times  are acceptable, since the degree of labeling is generally limited by the molar ratio of the reagent to antibody, not the reaction time. Many published procedures indicate overnight reaction times and incubations performed at 2Â°C to 8Â°C.

 The more reactive the reagent is, the shorter the reaction time.	

 Antibodies are relatively sensitive to substitution, since there are usually reactive amino acid side chains (amines, thiols) in or near the antigen binding sites. For this reason, a low to moderate degree of labeling is preferable in order to preserve binding specificity. Over-labeling can result in decreased solubility which may reduce the overall activity of the conjugates. In some cases it causes precipitation of the antibody conjugate.	

The methods below are widely employed for conjugating amine-reactive and thiol-reactive and other reagents to antibodies. The wide variety of experimental conditions required for activating and coupling antibodies with bifunctional and other reagents make it difficult to generate a simple general procedure. The reader is advised to consult the literature for specific procedures. We recommend three books for initial consultation: Hermanson,

 Although the procedures described below are for preparing antibody conjugates, they can also be utilized to prepare many other proteins and biological molecules. 				

 Use the following general procedure for the first trial. It is adaptable to amine-reactive molecules such as tags and probes, biotin, homobifunctional and heterobifunctional crosslinkers, haptens, carbodiimides, bifunctional chelating agents (for radioimmunoconjugates), and toxins. The procedure may be modified after the degree of substitution has been determined in analysis of the final product.	

For the basic lysine amines, prepare the antibody at 1 to 10 mg/mL in 50 to 100 mM sodium carbonate-bicarbonate (or borate) buffer pH 8.2 to 9.2 at room temperature. For less-basic α-amino groups, which react selectively at physiological pH, prepare the antibody in 10 to 50 mM sodium phosphate buffer, with 150 mM sodium chloride, pH 7.2 to 7.5. Note that amine-containing buffers such as TRIS are not recommended, since these buffers will compete with the antibody in reacting with the modifying reagent. 	

Bioconjugation chemistry is the joining of biomolecules to other biomolecules, small molecules, and polymers by chemical or biological means.

Monoclonal Antibody Conjugation via Chemical Modification

The annual meeting of the American Association of Pharmaceutical Scientists (AAPS) is one of the best windows on the state of the contract services industry. This year's meeting in Salt Lake City, UT was no exception.	

The mood of most vendors was strongly positive. CRO and CMO staffers at AAPS confirmed that product development activity and spending are picking up. Most said that they are seeing the upturn in RFP activity that the public CROs are reporting in their quarterly earnings conference calls. One CMO that finishes active pharmaceutical ingredients (APIs) reported that it had worked on almost 90 new chemical entities this year, most in preclinical development.	

Most small, privately-held providers of laboratory and clinical trials materials (CTM) services reported good results for 2003; according to the representatives with whom I spoke, revenues were up 15% or more at most companies. This is encouraging news, as some of the large public CROs have reported disappointing results in their lab services business this year. The smaller labs have a fairly diversified client base, so they have been less exposed to big pharma mergers and the financing problems of biopharmaceutical companies (contract labs that focus on biopharmaceutical companies reported having an off-year). In addition, smaller labs are usually willing to do more in order to meet a client's needs than larger CROs, and that flexibility has helped them retain business in this slow market.	

Table 1. September 30, 2003 Quarterly Results; Source: PharmSource	

One focus for service providers is the expansion of CTM formulation manufacturing capacity, especially for sterile products.  Both DSM Pharmaceuticals and Quintiles Transnational (at its facility in Kansas City, MO) will bring on new clinical-scale sterile manufacturing operations in 2004. Just before the meeting, Baxter announced a strategic alliance between its Baxter Pharmaceutical Solutions unit and Althea Technologies; Althea will provide phase 1 and 2 CTM services to clients, and Baxter will handle phase 3 and commercial manufacturing. Patheon is expanding its offerings of development services at its Cincinnati, OH facility, which it acquired from Aventis last year.	

Drug delivery technology was the hot topic at AAPS once again, with many of the presentations and poster sessions dedicated to related scientific and technical issues. Some 75 exhibitors were highlighting their proprietary drug delivery technologies and expertise. While dosing convenience and product differentiation are important, formulation challenges may be the biggest incentive of all - by some estimates, as many as 50% of all new drug candidates involve poorly soluble APIs.	

Publicly traded contract services providers reported good results for the July through September quarter, but the big news was the strength of their new business activity. After a slow first half, proposal and contract activity has really picked up, and contractors expect 2004 to be an especially strong year.				

Overall, revenues for the third quarter were up an average of 15% over the same quarter in 2002, with profits growing at a slightly higher rate. The strongest results were in phase 1 clinical research, where most CROs reported very high utilization rates, and preclinical toxicology, especially in Europe. Phase 2 and 3 research was slowed by weak business development activity in the first half of the year and by patient recruitment problems in the startup of new studies.	

CRO executives were especially optimistic about 2004. All experienced a substantial increase in RFPs in the third quarter, and several experienced record quarters for new business signings. There was a general sense that big pharma is finally getting its pipeline moving, while biopharmaceutical companies are somewhat more willing to spend now that the financial markets are more conducive to fundraising. The strong performance in preclinical and phase 1 have increased optimism about future phase 2 and 3 activity, which account for 60% of total R&D spending. Executives at Charles River Laboratories see R&D funding continuing to favor development over research (target and lead identification and optimization), which has hurt their discovery services business.	

Cardinal Health once again got the industry's attention with an acquisition, this time of UK-based Intercare. Intercare is a manufacturer and wholesaler of generic pharmaceuticals in the UK, as well as a leading contract sterile products manufacturer. Its sterile manufacturing operations include Martindale, located in the UK, and Federa, with sites in Brussels, Belgium and Limoges, France. It also owns L.C.O. Sante, a contract manufacturer of hormonal products. According to George Fotiades, president of Cardinal Health's Pharmaceutical Technologies and Services business unit, Intercare's sterile manufacturing operations are a "strategic platform" for expansion of Cardinal Health's business in Europe.				

The acquisition heats up the rivalry in sterile manufacturing on several fronts. Cardinal Health will now compete more directly with Patheon for FDA-compliant sterile manufacturing in Europe. Federa is building a new manufacturing facility in Brussels that is intended to be FDA-compliant, while Patheon has two FDA-compliant sites in Italy. In addition, it will intensify competition between Cardinal Health and Baxter Pharmaceutical Solutions by increasing Cardinal Health's capabilities in pre-filled syringes. Cardinal Health is already installing a 60-million-unit syringe manufacturing operation in Puerto Rico, and Federa's new facility will have complementary capabilities.	

Cardinal Health is paying $530 million in cash and assumption of debt for publicly-traded Intercare, whose annual sales are about $550 million. The sale is pending approval by Intercare stockholders. Fotiades says that Cardinal Health has a strategic interest in Intercare's generic products business, which is focused on niche products used in hospitals, but has yet to decide what it will do with the distribution business, which accounts for just 25% of Intercare's operating profits. 	

Publicly traded CROs and CMOs reported solid financial results in their most recent quarter, but the big news at October's AAPS meeting was the substantial rise in new RFPs and contracts.

Outsourcing Outlook

With the threat of a bioterrorist attack still weighing heavily on Americans, an antidote for anthrax has yet to be introduced. 	

True, antibiotics can stem the effects of anthrax if discovered early on, but once the anthrax toxin enters the bloodstream, there's not much doctors can do. The toxin destroys the human antibodies that would otherwise track down and kill anthrax bacteria.	

Enter ABthrax, a monoclonal antibody which acts as a proxy for the antibodies that are destroyed by the anthrax toxin. This promising new antidote has already proven successful in treating laboratory monkeys subjected to lethal doses of anthrax. In clinical tests, ABthrax saved 90% of the monkeys. Now, ABthrax is being tested on healthy humans as a way to beat the deadly bacterium at its own game by strangling it before it can act.	

Great news, right? Well, it could get even better. What if I told you that ABthrax could be one of the first in a long line of commercial success stories from the world of genomics?	

This industry, in recent years, has taken its fair share of lumps from critics who complained that genomics was all show and no dough. Not any more, advocates say. 	

Human Genome Sciences (HGS), which is using its genomics expertise to develop ABthrax as well as other protein and antibody drugs, is one of a growing list of companies that are looking to harness the power of genomics and bring revolutionary new drugs and medicines to the masses. Genomics and genomics-related firms like Agilent Technologies, Amersham BioSciences, Apogent Technologies, Isis Pharmaceuticals, Nanogen, Millennium Pharmaceuticals, and PerkinElmer are also looking to make their mark on the biopharm market and on Wall Street.	

Table 1. Of the 75 companies that currently make up the Nasdaq Biotechnology Index, genomics companies Human Genome Sciences and Millennium Pharmaceuticals are among the 20 largest, based on market value. 	

Given time and patience - two traits that Wall Street has in short supply - advocates say we can expect myriad drugs derived from work based on the human genome. The technology isn't exactly new - the term genome goes back to the 1930s when it was used to denote the totality of genes on all chromosomes in the nucleus of a cell. In 1944, DNA (which is now - thanks to OJ Simpson and his 1995 murder trial - a widely used, if not understood,  term) was recognized as the genetic fabric of all living organisms. For the past decade, human genome scientists have spent their days slogging through the estimated 30,000 human genes to pinpoint mutations that cause disease and find the genes that may help prevent diseases. Finally, indicators show that they may be on to something - and just in the nick of time.	

To be candid, I, like some other Wall Street observers, get antsy when the term genomics comes up. In recent years, publicly traded companies delving into the tricky science of human genes have come out bloodied and bruised, at least in terms of stock performance. 	

Just how bloodied and bruised? The 20 or so genomics companies that are publicly traded, as tracked by the financial newsletter BioCentury, fell off by a whopping 71% in 2002. And critics like Associated Press biotechnology writer Paul Elias note that the "genomics revolution has indeed led to a few medical breakthroughs - but it has cost investors billions of dollars with few returns." Things turned so dour in the industry that one company - Palo Alto, CA-based Incyte -dropped Genomics from its name after its stock fell from $140 to $4 per share.	

Evidently, solving the genomics puzzle is one thing, finding commercially viable uses for it is another. 				

Industry enthusiasts are high on ABthrax and HGS because the firm has provided a road map on how companies can turn genomic concepts and business models into viable products, transforming themselves from wooly-headed gene theorists into money-making drug development companies. 	

Biopharmaceutical companies have taken note. 	

According to Scope Marketing & Information, the overall US genomics market will rise at an average annual growth rate of 18% through 2007. Right now, the market is a small one - about $1 billion in 2003, according to Scope. The company also reports that the genomics industry is highly fragmented but is seeing increased consolidation in the form of alliances and acquisitions primarily concentrated in the United States and Europe. 	

In genomics, where cash is king, companies like HGS and Millennium Pharmaceuticals are sitting pretty with cash reserves of $1.5 billion and $1 billion, respectively. Perhaps it's that "breathing room" factor that nearly doubled HGS's stock price earlier this year and increased Millennium's share price by 98%. Analysts say that Millennium's strategy to acquire another operation with a strong existing product pipeline (it recently acquired COR Therapeutics) is a winner that other industry players may take to heart.	

Other genomics companies forging inroads into profitability include Genencor International, which has reported strong quarterly sales so far in 2003. Genencor is another "comfort level" company with deep pockets - it was founded in the early 1980s as a joint start-up between heavyweights Genentech and Corning.	

Admittedly, you've got to kick some tires and look under some rocks to really figure out what's going on in the genomics market. 				

One mutual fund that's doing a fairly good job of that lately is the GenomicsFund, which returned a hefty 60% year-to-date through October 2003. It wasn't always so rosy at the fund, which is managed by healthcare industry guru Robert Sullivan of Scituate, MA-based Satuit Capital Management. Sullivan's only been aboard since early 2003, so he doesn't have to explain the fund's atrocious performance in 2001 when it lost 43% of its value and in 2002 when the fund was labeled by Lipper Analytical Resources as one of the "Top 10 Worst Performing Funds" of the year. During that time, fund inflows fell from $50 million to a paltry $7 million. 	

To turn things around, Sullivan has taken a different tack than the fund's previous management, opting to reduce volatility whenever possible in order to attract fund inflows from more risk-averse investors. He also has widened the scope of the fund to include even more genomic industry stocks (although he has proven not to be wedded to too many or too few genomics-only firms). Now, as 2004 opens, the GenomicsFund is what the industry really needed on Wall Street - a benchmark to gauge its overall success.	

So, can the genomics industry - boosted by good news on the research and development and market performance fronts - do what it promises and find faster, less expensive, and better performing drugs?				

So far, the answer isn't entirely clear. Yes, genomics is changing how the biopharmaceutical industry thinks about drug discovery and its links to human genes. Just about every leading biopharm company has either forged alliances with genomics companies or created an in-house research department. If genomics companies take the HGS route and emphasize shorter-term drug development over long-range thinking - a blueprint that blew up in the sector's face in 2001 and 2002 - then the sky might just be the limit.	

I'm not usually given to "magic bullet" theories, but the genomics play book for 2004 seems worth considering.	

Call me an optimist, but I guess it's just hard-wired into my genes.  	

Genomics companies have taken a beating on Wall Street. Will newly developed therapies like ABthrax help the sector return?

BioPharm International-12-01-2003

Fermentation and Cell Culture Technology: Problems and Predictions

Components of the Pharmaceutical Industry Are Converging

Inside Washington: Risk Management and Pricing Concerns Shaped FDA Policy in 2003

Monoclonal Antibody Conjugation via Chemical Modification

Outsourcing Outlook

StreetTalk