Creating Products in the Absence of Markets: A Robust Design Approach

Keywords: New Product Development, uncertainty, high-technology venture

Purpose -The purpose of this study is to examine how firms deal with a situation of true uncertainty about their potential markets and technologies. Specifically, we ask how firms can create products when the corresponding market does not exist.

Design/methodology/approach -This paper is based on a longitudinal study of a high-tech firm, combined with analysis of existing theory in Product Design and Entrepreneurship.

Findings -Markets and products are usually a defining choice made early on by firms in their strategic process. Such a choice guides their development by providing a 'stable concept' to which decisions can be related to. When markets do not exist yet, however, this approach is not effective: Early choice of products and markets limits firms' flexibility by constraining their ability and willingness to adapt, while fundamental new technical and market information is likely to emerge during the project that will prove the initial assumptions wrong.

We show an alternative approach where products and markets actually result from a generic process of products and markets exploration driven by the firm. We suggest that this approach forms a robust design in that it allows the firm to deal with the uncertainty by simultaneously developing its products and exploring markets, while preserving the flexibility to adapt to the changing environment.

Practical implications -

The practical implication of this paper is to suggest an alternative approach to deliberate planning in high-tech ventures. With this approach, rather than markets and products, strategy defines a market and technology exploration process.

Originality/value -The paper is original in three ways: 1) It links the product design and market exploration processes in high-tech firm development; 2) It is based on an in-depth longitudinal study; and 3) It results from an academic-practitioner collaborative work.

INTRODUCTION

The purpose of this study is to examine how firms deal with a situation of true, "Knightian" [START_REF] Knight | Risk, Uncertainty and Profit[END_REF] uncertainty that characterizes emerging or disruptive environments. Using an in-depth case study approach, we seek to understand how the firm evolves from an initial situation of uncertainty about its potential markets and products to a situation where an opportunity has been defined and the corresponding products are created. The choice of products and markets are both essential components of a firm's strategy [START_REF] Grant | Contemporary strategy analysis[END_REF]. It is thought that these provide stability that will guide the firm's development, and possibly define its identity. The early and appropriate definition of these strategic components, even before the firm's creation, is assumed to be an important success factor, as exemplified by the institutional importance given to business planning [START_REF] Honig | Entrepreneurship Education: Toward a Model of Contingency-Based Business Planning[END_REF]. Underpinning this assumption is the predominant use of causal rationality in management. Causal rationality assumes pre-determined and well-structured ends, and formulates the decision problem as one of discovering the best possible means to achieve those ends. Causal reasoning proceeds inward by breaking given goals into sub-goals and sub-sub-goals to specific individual tasks [START_REF] Sarasvathy | Effectuation in the management of Knightian uncertainty: Evidence from the Realnetworks case[END_REF]. Causal rationality, with its emphasis on maximizing expected return or minimizing expected costs and avoiding surprises through accurate prediction and comprehensive competitive analysis, has long served as the normative foundation for both research and pedagogy in business management. It implies the definition of a clear goal, and works with a logic of optimization; i.e., given a particular goal, what is the optimal way (in terms of resources) to reach it. High-tech firms, however, seek opportunities in disruptive contexts where neither their products nor the corresponding market generally exist at the time of the disruption. In this context, goals cannot be defined easily, markets cannot guide product development, and causal rationality does not work anymore. How can firms create products when the corresponding market does not exist? We organize the paper as follows. We first highlight and discuss the core concepts of entrepreneurship and product design relevant to our issue. Following this discussion, we describe our methodology and then report on our findings. We then discuss those findings and draw their managerial implications for high-tech venture management.

THEORETICAL BACKGROUND

To characterize the disruptive contexts in which high-tech firms operate, [START_REF] Knight | Risk, Uncertainty and Profit[END_REF] distinguished between risk and uncertainty using the concepts of probability. Risk consists of a future with a known distribution, or a distribution that can be estimated by studying events over time. For instance, in the case of a box with six red balls and six blue ones the probability of a particular color being drawn (an event) is perfectly known. On the contrary, "Knightian" uncertainty consists of a future whose distribution is not only unknown, but objectively unknowable. The key difference between Knightian uncertainty and risk is that Knightian uncertainty involves dealing with a future that has no discernible distribution whatsoever, not even in theory. In this case, therefore, neither the calculus of a priori probability nor techniques of statistical estimation can work [START_REF] Sarasvathy | Effectuation in the management of Knightian uncertainty: Evidence from the Realnetworks case[END_REF], because the involved objects do not exist yet. To reuse the box example, we do not know whether there is a box, how many balls there are, and the concepts of box and balls might even not be defined. For instance, key elements of the Internet were invented in the late sixties, and commercial application only became significant in the early nineties (Slater, 2002). Such applications were totally unanticipated by the original inventors of the technology: they designed the Internet for military use, ie to provide a secure communication network in case of nuclear war; it is now used for rather different applications such as book buying, television watching, music distribution and game playing. While firms in established industries (e.g. banks, insurance companies) face risk, those in emerging industries face Knightian uncertainty. They trade in a context where prediction is impossible [START_REF] Knight | Risk, Uncertainty and Profit[END_REF], goals are not predetermined [START_REF] March | The technology of foolishness[END_REF], and the environment does not independently select the outcomes [START_REF] Weick | The Social Psychology of Organizing[END_REF]. Early choice of products and markets, in such a context, limits firms' flexibility by constraining their ability and willingness to adapt [START_REF] Bhide | How Entrepreneurs Craft Strategies that Work[END_REF][START_REF] Fredrickson | Inertia and creeping rationality in strategic decision processes[END_REF]; as a result, firms are particularly sensitive to disruptions and turbulence and every discontinuity generates an erratic trajectory. [START_REF] Sarasvathy | Causation and effectuation: toward a theoretical shift from economic inevitability to entrepreneurial contingency[END_REF] studied how entrepreneurs deal with a situation of Knightian uncertainty. She showed that they inverted the principles of causal reasoning and that the inversions together constituted a comprehensive new logic that she calls "Effectuation". Effectuation is a sequence of non-predictive strategies in dynamic problem solving that is primarily means-driven, where goals emerge as a consequence of stakeholder commitments rather than vice versa. An alternative to causal (predictive) rationality, effectuation suggests that "Knightian" actors succeed by taking a progressive approach to the definition of their products and markets [START_REF] Wiltbank | What to do Next? The Case for Non-Predictive Strategy[END_REF]. What matters, therefore, is not which products and markets they choose ex-ante, but how, in the absence of current markets for future products, such products get created by the firm [START_REF] Shane | The promise of entrepreneurship as a field of research[END_REF] and how this process allows the firm to resolve the Knightian uncertainty. Traditional models for product development projects have emerged from observations in relatively mature, industrial environments, such as the automobile industry (e.g. [START_REF] Wheelwright | Revolutionizing Product Development[END_REF]. Their focus is on developing a structured process with clearly defined and sequential phases for the development project through which the future product is defined, transferred to manufacturing, and rolled out to the market [START_REF] Iansiti | Shooting the Rapids: Managing Product Development in Turbulent Environments[END_REF]. Those models imply a clear separation between concept development, specification definition, and implementation. Underlying them is the assumption that products are developed in order to serve markets that already exist and have been selected by the firm. As such they adopt a causal logic and tend to pose the problems in terms of optimization. They work well in environments in which technology, product features and competitive requirements are predictable, and no new information is expected that would significantly change the specifications. In Knightian uncertainty, however, markets do not exist yet, and fundamental new technical and market information is likely to emerge during the project. Uncertainty is such that even the product concept itself can be modified, or even changed completely. For instance, RealNetworks, the current leader in media streaming technology over the Internet, started out as a project to create a TV channel [START_REF] Sarasvathy | Effectuation in the management of Knightian uncertainty: Evidence from the Realnetworks case[END_REF]. In this context, the success factor shifts from the capability for optimized project execution to the capability to react to new information during the course of the project by changing the product specifications [START_REF] Brown | Product Development: Past Research, Present Findings, and Future Directions[END_REF][START_REF] Iansiti | Shooting the Rapids: Managing Product Development in Turbulent Environments[END_REF], possibly to a large extent. To model the exploratory nature of the design process in such environments, and the blurring of the distinction between the specification and implementation phases, [START_REF] Hatchuel | C-K Theory: Notions and applications of a unified design theory[END_REF] introduced the Concept-Knowledge (CK) design theory. CK is based on the articulation of knowledge with the exploration of new products concepts. A unit of knowledge is a proposition with a logical status (true or false). For instance, in designing a car with a platform strategy, a unit might be "The car must use Platform X". A concept is a notion or proposition without a logical status. It is an innovative proposition used as a basis for initiating a design project. A "flying boat", for instance, is neither true, nor false, nor uncertain. The design process starts when unknowns are created by proposing a new concept (going from Knowledge 'K' to Concept 'C'). This initial concept is then expanded using properties that have a logical status in K. Concepts are expanded through design spaces, which need existing knowledge, and also generate discoveries and investigation processes that in turn expand the initial concept [START_REF] Hatchuel | Building innovation capabilities: the development of designoriented organizations[END_REF]. The design process stops when this dual expansion of concepts and knowledge leads to a concept that can be proved either true or false with the resulting knowledge (flying boat has become a "hydropter"). CK theory is directly applicable in design situations where the work is not based on detailed specifications or a well-identified definition of the object to be designed. It helps the researcher describe the design work not as a deterministic process of optimization, but as a creative exploration, thus meeting one of the design requirements in Knightian uncertainty [START_REF] Hatchuel | C-K Theory: Notions and applications of a unified design theory[END_REF]. With these core concepts as a starting point, we report empirical findings that suggest how a firm may overcome Knightian uncertainty. Taken together, the findings show that WSoft1 succeeded in resolving Knightian uncertainty by defining its stable concept as a generic process of products and markets exploration.

MATERIAL AND METHODS

Approach

The paper is based on a longitudinal case study of WSoft, a small European firm developing mobile phone software. We seek to explain how the firm was able to resolve the Knightian uncertainty about its products and markets. Case study research, whether using single or multiple cases, has not only been used effectively for descriptive and exploratory purposes, but also for explanatory purposes including frame-breaking causal inferences about complex and important phenomena [START_REF] Sarasvathy | Effectuation in the management of Knightian uncertainty: Evidence from the Realnetworks case[END_REF]. As opposed to statistical generalization, case study research allows analytical generalizations [START_REF] Yin | Case Study Research: Design and Methods[END_REF]. This study contributes to the integration of concepts and theories by using the extended case method, which aims to integrate and synthesize existing bodies of work. In contrast to the Grounded Theory approach [START_REF] Glaser | The Discovery of Grounded Theory: Strategies for Qualitative Research[END_REF], the primary focus of the extended case study is not to build new theory. Rather, its method is to integrate and extend existing theories through an iterative process of traveling back and forth between the data, pertinent literature and emerging theory [START_REF] Danneels | The Dynamics of Product Innovation and Firm Competences[END_REF]. Specifically, we seek to integrate the entrepreneurial theory of opportunity and the product development theory in the context of emerging markets. As with any idiographic research, we recognize that the entrepreneurial processes that WSoft employed can be unique and that such processes may be difficult to identify and measure. In such cases, it is the generative mechanisms or underlying processes that are generalizable and not the unique phenomena described [START_REF] Tsoukas | The Validity of Idiographic Research Explanations[END_REF]. The challenge here, therefore, is in identifying these processes, and the drivers of such processes, in the creation and early market development of WSoft.

Choice of firm

We chose this firm as a case study because, as a player in the market for mobile phone software, it exemplifies the phenomenon of interest. The mobile phone industry is characterized by rapid and unpredictable evolution: pioneers have exited the market (e.g. Alcatel, Siemens, Philips), merged (e.g. Ericsson), or are currently in difficulties (e.g. Motorola). Asian competitors, once poised to overtake them, have had various degrees of success: while Korea's Samsung has become the second largest manufacturer worldwide in just a few years, Taiwan's BenQ's attempt has failed and the company has since exited the market. The industry is also characterized by high profile industry-wide product failures, such as the WAP mobile Internet in 2001, and unexpected successes such as text messaging. Repeated disruptions threaten to undermine the position of actors who cannot react fast enough. For instance, Nokia's leading position was compromised when the company failed to anticipate the sudden popularity of clam-shell phones or of the camera feature in 2003. While the origin of the mobile phone industry can be traced back to the 1970s, it is still undergoing significant disruptions, and as such is typical of a situation of Knightian uncertainty. It is in this context that WSoft attempts to develop its products and markets, and establish itself as leader. While this leadership position is not yet reached, and may not be reached, the firm has successfully dealt with a situation of Knightian uncertainty: it is growing profitably and is recognized as a key player, having won designs with important clients in its field.

Data collection

Data was collected through a two-year full-time presence in the company. Our primary source of data was the cofounder and CEO of the firm, who is part of the research team as a reflective practitioner [START_REF] Schön | The Reflective Practitioner[END_REF]) and a co-author of this article. Other sources of data were: 1) interviews with other founders of the firm; 2) accounts of actions provided by the firm in the form of press releases; 3) analysis of internal reports as well as emails exchanged during the studied period; and 4) analysts' reports (both on firm and industry) whenever available.

To gather industry related background information, we relied on multiple sources such as news.com and telecoms.com, online industry trade magazines, as well as electronic databases of press articles such as Factiva. As typical in inductive research [START_REF] Eisenhardt | Building Theories from Case Study Research[END_REF], the data collected from these multiple sources served as the basis for our own detailed case study on the mobile software industry and WSoft's role in it. To establish the validity of the reconstruction process, we compared our case history with studies in close fields of the software industry, such as [START_REF] Sarasvathy | Effectuation in the management of Knightian uncertainty: Evidence from the Realnetworks case[END_REF] study of RealNetworks, Cusumano and Yoffie's (1998) study of Netscape, as well as [START_REF] Battelle | The Search -How Google and its Rivals Rewrote the Rules of Business and Transformed our Culture, Portfolio[END_REF] study of Google. We used the case histories (our own and others) to identify the product and market related actions taken by the firm, and to create tables to organize this data chronologically. These different sources enabled us to examine the data from multiple vantage points [START_REF] Glaser | The Discovery of Grounded Theory: Strategies for Qualitative Research[END_REF] and triangulate facts and inferences. For example, press releases provide detailed information on entrepreneurial actions, often including top managers' vision of the market and expected consequences of these actions. However, analysis of press releases was limited: they tend to emphasize the positive aspects of the various actions [START_REF] Rindova | Continuous "Morphing": Competing Through Dynamic Capabilities, Form and Function[END_REF], and it was not possible to supplement the analysis with media reports examining the same actions owing to the limited visibility of the firm at that time.

Analysis

Our unit of analysis consisted of the decision-events that occurred in the creation of the firm pertaining to technology, products and markets choices. We performed a process trace of the decision-events as they occurred, using the event listing method prescribed by [START_REF] Miles | Qualitative Data Analysis[END_REF]. We began by listing the decision-events in their chronological order and then examining the relations between product-related, technology-related and market-related decision-events. We then performed a trace of the domain-knowledge explored by the firm as such decision-events occurred. Such tracing allowed us to examine the role of learning in the development of the firm in relation to these decision-events. As typical in qualitative research, the validity of our insights was checked with senior executives of the firm as well as with other academic members of the research team.

RESULTS

Products and markets do not define the stable concept

Most companies start with an initial target market or product, and WSoft is no exception. Starting in 1998, WSoft's initial business idea was to provide IT services in the field of Web sites development. One year later, trying to get into the product business, the firm initiated a project called SH aimed at creating an inter-personal communication platform over the Internet. The firm tried, but failed, to raise money for SH, and the project was discontinued. In early 2000, the firm got a contract to develop mobile phones services (using the WAP standard) for a large mobile operator, entering the mobile market for the first time. Based on this experience, and switching back to the product business, it developed a WAP simulator that was sold, with moderate success, to a few wireless operators. In 2002, this simulator was extended and transformed into a browser, and ported to personal digital assistants (PDA). The idea was to use it to develop mobile access to corporate information systems. WSoft would work with IT services providers who would sell the product to their corporate clients. However, the market didn't take off in large part due to the lack of maturity of mobile communication at that time, and the firm exited the market. The browser was then adapted to fit onto a mobile phone, to target mobile phone manufacturers. Despite a certain interest, the product was sold only to a few clients. In 2003, however, upon a client's suggestion, it was further transformed into a user interface engine. Introduced in 2004, the new product has since been successful, and the firm is now growing profitably. During the period, the firm's products and markets changed radically several times, yet the firm remained the same in the sense that its key constituents didn't change. Its business model, defined as the development and licensing of software, didn't change. Its management and shareholding didn't change. Its employee turnover was nil, and the firm didn't experience any traumatic change or crisis. Neither markets nor products, therefore, can adequately define the "stable concept" of the firm, which must therefore be defined differently.

The evolution of products and markets was not random

Was there, indeed, a stable concept besides the structure itself? The firm's trajectory might have been purely chaotic, with markets and products being randomly tested out. Upon close examination, however, the changes in markets and products clearly did not appear as random; the evolution of the firm followed an iterative, path-dependent process contingent upon (1) relationships forged by the firm, (2) the firm's product development thrust, and (3) its environment. For instance, the WAP contract was obtained for reasons falling into these categories. Firstly, the management happened to know the client's project manager. Secondly, the firm could claim it did know the technology, which it had learned with the SH project which played the role of complementary asset [START_REF] Tripsas | Unraveling the process of creative destruction: complementary assets and incumbent survival in the typesetter industry[END_REF] (i.e. an asset whose development had not been considered a priority but whose importance suddenly rose upon a change in the firm's context) and such competences were scarce at the time, and larger, established service providers were too slow to react. Thirdly, the client was in a hurry. The importance of the firm's product development thrust is exemplified by how the market for the user interface engine came about. The firm had been trying to sell its mobile phone browser with limited success for some time when a client suggested that the technology could be adapted to create a user interface (UI) engine. User interface had become a major headache for manufacturers as phones were becoming more and more sophisticated, and such engine could well provide a solution to this growing issue. Within six months, WSoft was able to transform the browser to that effect and begin addressing this opportunity with the resulting new product. More generally, our analysis of the firm's chronology of decision-events shows that each product/service and market iteration was possible because it was in some way connected to the existing trajectory, thus allowing a trajectory of cumulative learning. A simplified, high-level version of the chronology of action-events is shown in Figure 1. The firm started out as a provider of development services. It first focused on Web applications, then moved into the related field of mobile Web applications. It then used its newly acquired knowledge to create a mobile Web browser using the Java technology. This product was adapted for several platforms ending on a mobile phone. Another iteration transformed it into a UI engine, and a final iteration consisted into rewriting it from the Java programming language into C.

Figure 1. Chronology of Decision-events

The fact that (1) neither products nor markets can adequately define the 'stable concept' of the firm; and (2) the evolution of the firm's products and markets was not random, but driven by an iterative path-dependent learning process, this suggests that the firm defined its stable concept as a generic process of product and market exploration. This begs the following question to be asked: how does product design theory relate to this proposition?

Market exploration and product development based on lineage management

Complex software such as that for mobile phones takes months, sometimes years to develop. How was the firm able to drive this development while not knowing in advance which markets it would target? We observed that the last product the firm developed from scratch was the WAP simulator introduced in 2000, seven years ago. All of the firm's subsequent products derived from it, as illustrated in Figure 2.

Figure 2. Succession of products

While today's user interface engine for mobile phones (Product KR) and the initial simulator (WPW) have nothing in common, we created a CK [START_REF] Hatchuel | C-K Theory: Notions and applications of a unified design theory[END_REF] reconstitution of the design history which shows that the evolution followed an iterative, path-dependent process, from one product concept to another, always with a strong common technological base between the two. An extract of this reconstitution, describing the transition from WPM to KR (step 3 to 4), is shown in Figure 3. As Figure 3 shows, there is a constant exchange from C to K and K to C back and forth. Different types of knowledge are used (return from experience, market related and technical/design expertise) to every single logical component of the initial product, thus allowing a continuous progress from one product concept to another. We start from the concept: "A UI engine for mobile phones" (other approaches are possible to address the UI need), try a solution along one branch, backtrack, explore another branch, etc. until we have completely defined what "A UI engine for mobile phones" means in terms of knowledge: a functional layer using parameters and developed in the C language. In between, a series of intermediary products have been tried out, including the product WPM. KR, the end-product, is the result of this exploration. This sequence of products constitutes a lineage [START_REF] Hatchuel | Design-oriented Organizations: Towards a unified theory of design activities[END_REF], i.e. an evolving asset that provides both continuity and variation to the design process. Lineage management helped the firm drive the exploration in technology and customer needs on one side, and organize value-creating reuse of acquired knowledge on the other side. The trajectory of domain-knowledge explored by the firm, from its inception to the existing product-market, is depicted in Figure 4. Four main domains are distinguished: platform (for which WSoft creates products), customer, Field (the actual business problem solved by WSoft's product or service), and Technology (that needs to be mastered to address the business problem). Mapping the evolution of products with these domains shows the continuity of the process and the gradual stabilization of the domains, which stop changing. The first to be set is the platform, which is defined when WSoft focuses on embedded systems. Then comes the type of customer (mobile phones manufacturers), then the field (User Interface) and finally the technology, when the product is rewritten from the Java to the C language. When an opportunity was identified, instead of creating a product from scratch, which would have taken too long and de facto exclude the firm from this opportunity, the current product went through another iteration, leveraging hitherto accumulated knowledge. The technological solution embodied the learning and allowed the business concept to function.

DISCUSSION

The purpose of this study was to understand how high-tech firms deal with a situation of true, Knightian uncertainty about their potential products and markets. We studied a wireless software firm that was successful in its attempt. The study highlighted three factors that we believe contributed to this success. Firstly, the firm did not define its stable concept in terms of products and markets. Secondly, this did not mean that the evolution of products and markets was random, but rather path-dependent based on cumulative learning, the result of an organized exploration. Thirdly, the exploration of markets and the products development were based on lineage management, reflecting the strategic importance of product design in the firm's strategy.

Lineage-based strategy for startup development

Product innovation has long been shown to drive learning about customers and technologies [START_REF] Maidique | The New Product Learning Cycle[END_REF]. Underpinning the firm's concept is a product design strategy relying on a generic technical solution evolving as a lineage that provides both continuity and variation to the design process. The continuity reflects the cumulative learning of previous iterations, while the variation allows local explorations. It was not, however, just a case of technology-push development process as is so common in technology ventures. The CK reconstitution of design and market trajectory shows a simultaneous process of products and markets design, in effect a combination of both technology push and market pull [START_REF] Berthon | To Serve or to Create: Strategic Orientations Towards Customers and Innovation[END_REF]. The design process allowed the creation of products that supported market exploration, but market exploration fed the design process in return, until both converged to an opportunity. This ability to adapt to the evolution of the target market differs from similar concepts such as mass customization, a concept introduced by [START_REF] Davis | Future Perfect[END_REF]. Mass customization is an effort to resolve the paradox of combining the ability to adapt a product to each customer while maintaining economies of scale. Mass customization differs at least in two ways with lineage management. Firstly, mass customization is effective in a clearly defined market where the customization accounts for minor differences among customers within an existing segment, such differences being predictable. Put another way, the variation occurs within a finite space defined by a small number of parameters, and all possible variations are perfectly known in advance. Secondly, mass customization is static: instances of the product are customized, but the product itself doesn't change: this is a condition to reap economies of scale. Lineage, on the contrary, involves profound changes in the product itself to allow addressing new and possibly unexpected market needs. Lineage management also differs from the platform approach. [START_REF] Gawer | Platform Leadership[END_REF] define a platform as "an evolving system made of independent pieces that can each be innovated upon" [START_REF] Gawer | Platform Leadership[END_REF]. By organizing the ex-ante sharing of given architecture and/or common elements of product or process, the platform embodies the firm's existing knowledge; what is expected from a derivative project is not new knowledge, but economies of scope. A platform is static by definition: while its constituting elements might evolve. The platform approach is therefore not a strategy for exploration learning, but an efficient one for exploitation learning. By defining the space of concepts within which the market exploration can take place, the iterative, cumulative, and path-dependent lineage-based product design process actually limits the opportunities that the firm can pursue: what can be achieved in terms of new product from an existing technology base with one iteration is necessary limited. While this might seem to be an inconvenience, [START_REF] Moore | Crossing the chasm[END_REF] showed the benefit of focus in uncertain environments. It limits unnecessary variation and avoids a random walk of products and markets. It supports the effectual approach of development by providing a product basis upon which the firm can base its 'next' effectual iteration.

Robust design through second order competencies

As noted by [START_REF] Teece | Dynamic capabilities and strategic management[END_REF], deciding, under significant uncertainty about the future states of the world, which paths to commit to and when to change paths is the central strategic problem confronting the firm. While technological and customer competences can be thought of as first-order competences, the ability to identify, evaluate, and incorporate new technological and/or customer competences into the firm (i.e. a competence at explorative learning and design by exploring and designing new markets or new technology can be thought of as a second order competence [START_REF] Collis | Research Note: How valuable are organizational capabilities?[END_REF]). Second order competences enable a company to renew itself through building new first-order competences related to technologies and markets [START_REF] Danneels | The Dynamics of Product Innovation and Firm Competences[END_REF]. They provide flexibility and allow cumulative learning. The definition of the stable business concept in terms of second order competences allows to simultaneously explore markets to create value with the generic technical solution, and learn from the exploration in terms of markets and products. It forms the basis of a "robust design" [START_REF] Hargadon | When innovations meet institutions: Edison and the design of the electric light[END_REF] of the firm, defined as the ability to simultaneously make advances while preserving the flexibility to respond to new situations.

Surviving Knightian uncertainty

This 'robust' approach allows the firm to solve the problem of Knightian uncertainty because it combines exploration and exploitation [START_REF] March | Exploration and Exploitation in Organizational Learning[END_REF]. The general model of strategy process is that of an initial period of exploration, often before the firm's creation, which ends up when an opportunity is identified, after which the firm turns to exploitation based on previously capitalized knowledge on products and markets. Firms such as Netscape [START_REF] Cusumano | Competing on Internet Time: Lessons from Netscape and its Battle with Microsoft[END_REF] or Google [START_REF] Battelle | The Search -How Google and its Rivals Rewrote the Rules of Business and Transformed our Culture, Portfolio[END_REF] are typical of such approach. In our case, however, explorative learning is not limited to the initial phase, but constant in the development of the firm. This learning process is structured in terms of reuse and leveraging of existing knowledge and competencies into revenue generating products on the one hand, and prudential exploration of new markets opportunities and technology development on the other. Knightian contexts are characterized by complete uncertainty about products and markets. In those contexts, the role of exploration is crucial. Developing customer and technology competences, and then exploiting them, is not enough. Envisioned markets might not come about. New products might fail. New opportunities might emerge unexpectedly that could alter the productmarket pair of the firm. Exploration results in greater ability to adapt to changes, and thus supports future viability [START_REF] Danneels | The Dynamics of Product Innovation and Firm Competences[END_REF]. While exploitation provides vital short-term resources, exploration enhances the adaptation of the organization to a changing environment because it increases the variance of organizational activities [START_REF] Mcgrath | Exploratory learning, innovative capacity, and management oversight[END_REF]. This suggests that the firm should maintain equilibrium in terms of exploration and exploitation, despite likely institutional pressures from shareholders that call for a switch to exploitation. More generally, the study suggests that Knightian uncertainty is resolved by the definition of the stable business concept of the firm in terms of product and market exploration. The concept allows an iterative process of technology push and market pull, driven by a deliberate strategy of learning.

Managerial implications

The practical implication of this paper is to suggest that high-technology firm management might go beyond two traditional oppositions. Firstly, between deliberate and emergent strategies [START_REF] Mintzberg | Of strategies, deliberate and emergent[END_REF], and secondly, between exploitation and exploration [START_REF] March | Exploration and Exploitation in Organizational Learning[END_REF]. A deliberate strategy of learning and lineage management is combined with an emergent strategy of market development: markets cannot be anticipated, but they can be created by actively driving a learning and technology development strategy. The articulation between the deliberate and emergent paths is provided by the product design strategy based on lineages. Exploration does not stop at an early stage but is pursued and combined with the exploitation of current products and markets. Rather than markets and products, the overall firm's strategy consists in defining a market and technology exploration process to resolve Knightian uncertainty.

CONCLUSION

Firms in disruptive environment have to deal with a situation of true uncertainty about their potential markets and technologies. We asked how firms can create products when the corresponding market does not exist. This case study suggests that instead of defining the firm in terms of markets or products, a definition as a generic process of learning and organizational development constitutes a robust approach that allows the firm to resolve the uncertainty by simultaneously developing its products and exploring markets, while preserving the flexibility to respond to new situations. This paper results from a research initiated two years ago on the development mode of high-tech startups. Insight on the relation between the design process and the market exploration has already been gained. While our findings are largely consistent with the recent literature on Product Development and Entrepreneurship, they must be confronted to a larger sample of firms in order to mitigate the issue of contingency. The idea of a second order stable concept was not deliberately defined by WSoft but rather emerged empirically from an initial lack of market and technology success. We think entrepreneurial firms would benefit from having a normative framework from the outset to increase their effectual performance. Future research will aim at creating such framework.

Figure 3 .

 3 Figure 3. Reconstitution of Product Design history, from product concept WPM to product concept KR

Figure 4 .

 4 Figure 4. Domain-knowledge explored by the firm

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Julien Camperi

Audrey Combès

Thierry Fournier

Valérie Pichon

Nathalie N Delaunay
email: nathalie.delaunay@espci.fr

Valerie Pichon

Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS

Keywords: high resolution mass spectrometry, human Chorionic Gonadotropin, hydrophilic interaction chromatography, intact protein, reversed-phase liquid chromatography, time-of-flight

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Introduction

The human Chorionic Gonadotropin (hCG) is an hormone essential for the preservation of the pregnancy and the development of the placenta and fetus [START_REF] Cole | hCG physiology[END_REF]. It is a heterodimeric glycoprotein, consisting of an α (hCGα) and a β (hCGβ) subunit. The α-subunit has identical amino acid sequence to the pituitary gonadotropin hormones such as the follicle-stimulating hormone, the thyroidstimulating hormone, and the luteinizing hormone, containing two N-glycosylation sites [START_REF] Kessler | Structure and location of the O-glycosidic carbohydrate units of human chorionic gonadotropin[END_REF][START_REF] Liu | Silencing of the gene for the alpha-subunit of human chorionic gonadotropin by the embryonic transcription factor Oct-3/4[END_REF], while the β-subunit represents the specific part of hCG and has 2 N-and 4 O-glycosylation sites [START_REF] Liu | Mass spectrometric characterization of the beta-subunit of human chorionic gonadotropin[END_REF].

Nowadays, it is well-known that changes in the glycosylation pattern of a protein affect its biological activity and half-life. Therefore, the detection and identification of the numerous hCG glycoforms is essential to be able to use one or several of them as biomarkers [START_REF] Guibourdenche | Hyperglycosylated hCG Is a Marker of Early Human Trophoblast Invasion[END_REF].

With 8 potential glycosylation sites, hCG is a highly heterogeneous protein and the characterization of all its glycoforms constitutes an important analytical challenge. The analysis of the protein at the intact level by liquid chromatography (LC) or capillary electrophoresis (CE) hyphenated with mass spectrometry (MS) is a fast and easy approach that allows the determination of the glycosylation profile [START_REF] Camperi | Separation Methods hyphenated to Mass Spectrometry for the Characterization of the Protein Glycosylation at the Intact Level[END_REF]. Recently, we optimized for the first time a CE method hyphenated with MS for the separation of hCG glycoforms [START_REF] Camperi | First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level[END_REF]. The MS analyzer was a triple quadrupole that was useful for comparing glycosylation profiles but prevented the glycoform identification, which requires a highresolution (HR) MS. Then, we developed for the first time a LC separation in reversed phase (RP) mode coupled with quadrupole/time-of flight (qTOF) MS for the analysis of hCG at the intact level [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF]. This method led to the separation of some α-subunit glycoforms, but did not allow the detection of the hCGβ glycoforms, perhaps due to a higher heterogeneity of hCGβ and/or a less efficient ionization with its potential 2 N-and 4 O-glycans. With regard to selectivity in RPLC, a correlation between the retention time of the hCGα glycoforms and the number of their sialic acids present at the terminal position on the N-glycans antennas was observed. Surprisingly, the retention time of hCGα glycoforms increases with the number of their sialic acids whereas this elution order would be more expected with hydrophilic interaction liquid chromatography (HILIC), which is complementary and orthogonal to RPLC [START_REF] Periat | Potential of hydrophilic interaction chromatography for the analytical characterization of protein biopharmaceuticals[END_REF][START_REF] Tetaz | Hydrophilic interaction chromatography of intact, soluble proteins[END_REF]. Indeed, the retention mechanism in HILIC is partially based on partitioning of the compounds between a water-enriched layer formed at the surface of the polar stationary phase and the hydro-organic mobile phase when containing a high content of organic solvent. Additional interactions can then occur between the compounds and the stationary phase including hydrogen bonding, ionic, and dipole-dipole interactions [START_REF] Buszewski | Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique[END_REF][START_REF] Gama | Hydrophilic interaction chromatography[END_REF]. The highly organic mobile phase is particularly well adapted to MS detection, leading to an improvements in sensitivity for a large variety of compounds [START_REF] Nguyen | The advantages of ESI-MS detection in conjunction with HILIC mode separations: Fundamentals and applications[END_REF]. Therefore, the HILIC mode is nowadays largely used for the analysis of small polar compounds, glycopeptides or N-and O-glycans released from glycoproteins [START_REF] Mechref | Recent advances in glycomics, glycoproteomics and allied topics[END_REF][START_REF] Bones | Ultra Performance Liquid Chromatographic Profiling of Serum N -Glycans for Fast and Efficient Identification of Cancer Associated Alterations in Glycosylation[END_REF][START_REF] Takegawa | Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography[END_REF] and could help to separate the glycoforms of a protein according to their glycan moieties.

For the analysis of glycoproteins at intact level in HILIC, only few studies reported its use [START_REF] Rinaldi | Application of a rapid HILIC-UV method for synthesis optimization and stability studies of immunogenic neo -glycoconjugates[END_REF][START_REF] Tengattini | Hydrophilic interaction liquid chromatography-mass spectrometry as a new tool for the characterization of intact semi-synthetic glycoproteins[END_REF][START_REF] Pedrali | Characterization of Intact Neo-Glycoproteins by Hydrophilic Interaction Liquid Chromatography[END_REF][START_REF] Domínguez-Vega | High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry[END_REF][START_REF] Zhang | Polyacrylamide brush layer for hydrophilic interaction liquid chromatography of intact glycoproteins[END_REF][22][START_REF] Gargano | Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry[END_REF][START_REF] Gargano | Capillary HILIC-MS: A New Tool for Sensitive Top-Down Proteomics[END_REF]. Its potential was demonstrated for the analysis of intact neo-glycoproteins obtained after a glycosylation procedure from ribonuclease A, and two antigenic proteins, namely TB10.4 and Ag85B [START_REF] Rinaldi | Application of a rapid HILIC-UV method for synthesis optimization and stability studies of immunogenic neo -glycoconjugates[END_REF][START_REF] Tengattini | Hydrophilic interaction liquid chromatography-mass spectrometry as a new tool for the characterization of intact semi-synthetic glycoproteins[END_REF][START_REF] Pedrali | Characterization of Intact Neo-Glycoproteins by Hydrophilic Interaction Liquid Chromatography[END_REF]. These studies allowed the monitoring of the glycosylation process and glycoprotein stability.

More recently, Domínguez-Vega et al. [START_REF] Domínguez-Vega | High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry[END_REF] developed , for the first time, a HILIC-HRMS method for assessing the glycosylation pattern of one highly glycosylated protein, erythropoietin having 3 N-and 1 O-glycosylation sites [START_REF] Domínguez-Vega | High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry[END_REF]. Although a partial separation was obtained, the qTOF analyzer allowed the identification of 51 glycoforms, in addition to glycoforms with other post-translational modifications such as succinimide, oxidation, and N-terminal methionine-loss products [START_REF] Domínguez-Vega | High-resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography-mass spectrometry[END_REF].

With regard to hCG, we optimized a HILIC-UV method to separate its glycoforms [START_REF] Camperi | First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms[END_REF]. Two hCGbased drugs were successfully analyzed using an amide-based column and a mobile phase containing ACN and water with 0.1% of trifluoroacetic acid (TFA). The resolution obtained made it possible to distinguish the two drugs thanks to their different glycoforms in number and nature. However, the hyphenation with HRMS is mandatory to significantly improve the level of information about the nature of these hCG glycoforms. Therefore, the potential of HILIC-(qTOF) MS for the hCG characterization is here evaluated and compared to the results obtained with RPLC-(qTOF) MS in terms of selectivity, sensitivity, orthogonality, and complementary.

Materials and methods

Reagents and analytes

HPLC-grade acetonitrile (ACN) and formic acid (FA) were supplied by Carlo Erba (Val de Reuil, France). TFA was purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The ultra-pure water was obtained with a Direct-Q3 UV system (Millipore, Molsheim, France). Ovitrelle®(Organon, Oss, The Netherlands) was presented as a solution containing 500 mg L -1 of hCG (recombinant mammalian cell, r-hCG). Pregnyl® (Serono Europe Ltd, London, UK) is a lyophilized powder containing 5,000 IU of hCG isolated from the urine of pregnant women (u-hCG) (1 IU is equivalent to a concentration of 0.092 µg L -1 [START_REF] Berger | The molecular relationship between antigenic domains and epitopes on hCG[END_REF][START_REF] Berger | The ISOBM TD-7 Workshop on hCG and related molecules. Towards user-oriented standardization of pregnancy and tumor diagnosis: assignment of epitopes to the three-dimensional structure of diagnostically and commercially relevant monoclonal antibodies directed against human chorionic gonadotropin and derivatives[END_REF]).

The Ovitrelle® solution was reconstituted in 0.5 mL of water to obtain a stock solution containing 500 µg mL -1 of r-hCG. For Pregnyl®, the lyophilized content of each ampoule was dissolved in 1 mL of water to obtain a concentration of 460 µg mL -1 . All the solutions were aliquoted and stored at -20°C.

Before injection into LC-MS, the hCG aliquots were washed and pre-concentrated to 1 mg mL -1 by centrifugation with ultra-centrifugal units (Merck, Darmstadt, Germany) having a molecular weight cut-off of 10 kDa.

Instrumentations and columns

HILIC-(qTOF) MS

The HILIC-(qTOF) MS experiments were performed on a bio-compatible 1260 Infinity™ system composed of a quaternary pump, auto-sampler, column oven, and a diode array UV detector (Agilent Technologies, Les Ulis, France) coupled to a MicrOTOF-Q II Mass Spectrometer (Bruker Daltonics, Wissembourg, France) with an electrospray ionization (ESI) source. An Accucore Amide column (150 x 2.1 mm, 2.6 µm, 150 Å) purchased from ThermoFisher (Le Pecq, France) was used. The mobile phase was composed of acetonitrile and water both containing 0.1% TFA. The mobile phase gradient was from 85 to 40% ACN in 30 min, a plateau for 5 min, and a return to the initial composition within 1 min and an equilibrium step for 15 min. The column temperature was maintained at 60°C. The injection volume was fixed at 2 µL, the hCG sample was at 1 mg mL -1 in water, and the flow-rate at 0.4 mL min -1 . The MS experiments were operated in the positive mode and the mass-detection range was set to m/z 2200-2500. The parameters of the ESI source were as follows: drying gas (N 2 ) flow rate, 9.0 L min -1 , drying gas temperature, 200°C, nebulizing gas pressure, 30 psi, capillary voltage, 4500 V. The system was controlled by the MicroTOF Control Software (Version 3.4, Bruker Daltonics) and the Hystar (Version 3.2, Bruker Daltonics) software was used to interface the HPLC and MS systems. To ensure optimal conditions for the detection, the calibration of the qTOF was performed once a week using cesium clusters (2 g L -1 in water-isopropanol (1:1, v/v)) allowing a mass accuracy of about 10 ppm.

RPLC-(qTOF) MS

The RPLC-(qTOF) MS method was previously optimized [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF]. It was performed on a 1100 LC system (Agilent Technologies) hyphenated to the same (MicroTOF-Q II) MS detector. The RPLC separation was achieved using an Aeris WIDEPORE XB-C18 column (150 x 2.1 mm, 200 Å) packed with 3.6 µm core-shell silica-based particles (Phenomenex, Le Pecq, France). The mobile phase was composed of 0.1% FA in water and ACN. The mobile phase gradient was an increase from 4.5 to 31.5% of ACN in 50 min, a plateau for 5 min, and a return to the initial composition within 1 min and an equilibrium step for 10 min. The column temperature was maintained at 65°C, the injection volume was 5 µL, the hCG sample was at 0.1 mg mL -1 in water, and the flow-rate was set at 0.4 mL min -1 . The MS detection was carried out with the same parameters as those used for the HILIC experiments, except for the mass detection range set to m/z 1000-2500. Table S1 lists MS parameters recommended by the manufacturer (Bruker Daltonics) for the analysis of macromolecules (with m/z > 1000) that were used.

Results and discussions

HILIC-UV vs HILIC-HRMS

The separation of hCG glycoforms in HILIC-UV was previously optimized, the reliability of the method in terms of retention time and peak area was demonstrated, and its application to two hCG-based drugs showed its potential for fingerprinting approach (see Figure S1 in the supplementary data) [START_REF] Camperi | First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms[END_REF]. The content and nature of the acidic additive (FA and TFA) and the addition of a volatile salt (ammonium formate) in the mobile phase on the retention and the resolution were studied. It was observed that TFA, an ion-pairing agent, was mandatory to obtain a high number of peaks and led to a nice separation. It is worthwhile to notice that only reagents compatible with MS were investigated since, for identification purposes, the hyphenation with HRMS detection is required and this is the objective of this work. The best HILIC separation was obtained with an amide-based stationary phase and a mobile phase composed of water/ACN containing 0.1% of TFA. However, no peaks were observed in MS using these conditions and a m/z range of 1000-2500, as we did it in RPLC-MS [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF].

This may be due to the presence of TFA in the mobile phase, which induced a dramatic decrease in the MS signal, but our previous study showed that TFA was essential to achieve a good HILIC separation of hCG glycoforms. In addition, TFA has a stabilizing effect on protein species in highly organic solutions and is considered as a good solubilizing agent at low concentrations (e.g. ∼0.1%) [START_REF] Bobály | Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach[END_REF]. Therefore, a compromise between LC resolution/protein solubilization and MS sensitivity was done.

To overcome this detection problem, two modifications were considered. First, the injected amount of hCG was increased by increasing the injection volume, because the hCG concentration was already high (1 mg mL -1 ). In our previous study with UV detection [START_REF] Camperi | First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms[END_REF], the hCG-based drug samples were dissolved in water and an injection volume of 1 µL was used. Even if a water-based sample is not favorable in HILIC, as a partial precipitation of hCG was observed with an ACN content higher than 30%, a fully aqueous sample was maintained and the injection volume was increased to 2 µL.

Secondly, the detection mass range was reduced in order to increase the MS signal intensity, even if only a single charge state of the glycoforms was observed instead of three generally. Therefore, different small m/z ranges of 300 instead of 1500 were tested and it was observed that the m/z range between 2200 and 2500 gave the best signal intensities.

With these conditions, the resulting base peak chromatogram (BPC) is presented in Figure 1A analyzing the recombinant hCG-based drug sample (r-hCG). [START_REF] Camperi | First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms[END_REF]. In HILIC-MS, the first zone corresponds to the same first elution zone observed in HILIC-UV, but no peaks were detected in the last two elution zones. Two explanations can be proposed. First, the detection mass range used (m/z 2200-2500) may not be suitable for the detection of glycoforms present in the second and third elution zones. Therefore, different mass ranges were then tested (m/z 100-800, m/z 800-1500 and, m/z 1500-2200), but no peaks were detected. Secondly, these glycoforms are difficult to ionize, which may be due to a high content of glycans and/or the presence of TFA in the mobile phase. Regarding the detected peaks in MS, the average spectrum between 19.8 and 21.8 min on the BPC (see arrow Figure 1A) is presented in Figure 1B. As expected, the mass range used (m/z 2200-2500) enabled to detect m/z values corresponding to hCG glycoforms with a charge state (z) equal to 6 for all peaks.

However, the observation of a single charge state does not allow the application of a conventional deconvolution method for the determination of glycoform mass values requiring 3 charge states.

In conclusion, the applicability of the HILIC-MS method for the analysis of intact hCG is not trivial. Indeed, the presence of TFA, essential to separate the hCG glycoforms as previously demonstrated [START_REF] Camperi | First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms[END_REF], leads to a very low ionization of glycoforms in MS and thus to a dramatic loss of information on glycoforms present in the last two elution zones which are detected in UV. Nevertheless, the HILIC-HRMS method gave some m/z values corresponding to several hCG glycoforms in the first elution zone. In order to evaluate the potential of HILIC for hCG characterization, the number of detected glycoforms and the sensitivity of the analysis were then compared to the values obtained by RPLC-HRMS [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF].

Comparison of RPLC and HILIC

The RPLC-HRMS analysis of r-hCG led to about 12 chromatographic peaks (see Figure S2A in the supplementary data) [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF]. At least three charge states were observed (z = 9, z = 8, and z = 7) with m/z values between 1300 and 2200 on the average MS spectrum (Figure S2B) obtained between 28.5 and 32.5 min on the BPC (see arrow Figure S2A). After deconvolution, these peaks were assigned to glycoforms of the α-subunit and no glycoforms of the β-subunit were detected [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF].

The sensitivity between HILIC-and RPLC-HRMS was first compared and it is worthwhile to notice that the qTOF system, r-hCG provider, and LC columns dimensions were the same. In addition, based on the hypothesis that 1 nmole corresponds to 35 μg of hCG (MW≈35 kDa), a quantity of 56.8 pmole was injected in HILIC-HRMS (2 µL at 1 mg mL -1 ), instead of 14.2 pmole in RPLC-HRMS method (5 µL at 100 µg mL -1 ). With these conditions, the signal intensity observed on the BPC for the HILIC separation (Figure 1A, approx. 3.0E10 3 ) is 10 times less intense than for the RPLC mode (Figure S2A, approx.

3.0E10 4 ). Therefore, the RPLC-HRMS method appears to be 40 times more sensitive than the HILIC-HRMS method, despite a larger mass range of detection (m/z 1000-2500 in RPLC-HRMS instead of m/z 2200-2500 in HILIC-HRMS), which clearly demonstrates a dramatic decrease in the sensitivity of the HILIC-HRMS method compared to RPLC-HRMS. As mentioned above, this high sensitivity difference is mainly due to the presence of TFA in HILIC that was required to obtain a high resolution LC separation.

The complementarity of RPLC and HILIC modes was next studied. All the peaks observed on the spectrum obtained in HILIC-HRMS between m/z 2200 and 2500 (Figure 1B) were selected and their eXctracted Ion Chromatograms (XIC) were plotted (Figure 2A). The absolute mass values, calculated according to a charge state of z=6, are reported. The XIC chromatograms were also plotted for all the peaks observed on the spectrum obtained in RPLC between m/z 1700 and 1900 (z=8) (Figure S2B). They are presented in Figure 2B. First, it is worthwhile to notice the presence of several not resolved peaks associated with a given mass in RPLC (e.g. 14,316.0 Da, Figure 2B), while there is only one peak for a given mass in HILIC (Figures 2A).

Although it is difficult to conclude at this stage, two hypotheses will be discussed later. 2A and2B, it is interesting to note that 5 glycoforms with masses of 13,440.8, 13,733.7, 14,024.8, 14,316.0, and 14,972.0 Da were detected with both methods. These hCGα glycoforms were previously identified during the RPLC-HRMS study [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF] and their glycan structures are represented in Figures 2A and2B. These hCGα glycoforms contain sialylated N-glycans with two antennas, except for one form (14,972.0 Da) having an N-glycan with three antennas. Table 1 shows all the hCGα glycoforms detected during the analysis of r-hCG by HILIC-and RPLC-HRMS. In HILIC, even if one glycoform (13,566.4 Da) was not observed, 4 were detected in addition to RPLC (13,716.6, 14,006.4, 14,389.2, and, 14,682.6 Da) and 2 out of 4 were identified (a x means that no peak was detected corresponding to its isoform and unidentified that a peak was present on the spectrum but the N-glycans structures were not identified). They contain 2 N-glycans with 2 and 3 antennas. A mass difference corresponding to an N-acetylneuraminic acid was observed between some of them (291 Da between 13,716.6 and 14,006.4 and between 14,389.2 and 14,682.6). It can be noticed that a higher number of glycoforms composed of N-glycans with 3 antennas are detected in HILIC-than in RPLC-HRMS (3 in HILIC vs 1 in RPLC). These results demonstrate the complementarity of both separation modes.

When comparing Figures

Table 1: List of absolute mass values and N-glycan structures of the α-subunit glycoforms of r-hCG

analyzed by HILIC-or RPLC-HRMS. x: no peaks corresponding to its isoform. Unidentified: a peak is present on the spectrum but the N-glycans structures were not identified. Green circle: mannose; yellow circle: galactose; pink diamond: N-acetylneuraminic acid; blue square: N-acetylglucosamine.

To confirm this complementarity, a second hCG-based drug prepared from urines of pregnant women, u-hCG, was then analyzed by HILIC-HRMS, as it was already carried out by RPLC-HRMS [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF].

The resulting BPCs showed in both cases at least 10 peaks with retention times between 19.1 and 20.8 min in HILIC (Figure 3A) and between 28.5 and 32.5 min in RPLC (Figure 3B). As for r-hCG, a decrease in sensitivity was observed in HILIC-HRMS compared to RPLC-HRMS. For all the peaks Conditions: see experimental section.

Table 2 presents the glycoforms detected with both methods. Seven glycoforms of hCGα were detected in both modes. They are composed of sialylated N-glycans with 1 or/and 2 antennas except for 1 glycoform (13,010.4 Da) which was not identified. Furthermore, 3 additional hCGα glycoforms were detected in HILIC: 12,960.0, 13,659.0, and 13,666.8 Da. Regarding the glycoform with a mass value of 13,659.0 Da, the N-glycan structures can be proposed thanks to a mass difference of 291 Da with another already identified glycoform (13,949.9 Da). Unfortunately, the two other hCGα glycoforms were not identified. In RPLC, 2 glycoforms were detected in addition to HILIC: one oxidized form (13,310.4 Da) and one with 2 sialylated N-glycans with 2 antennas (Table 2). These results confirm again the complementary of both modes for the identification of hCGα glycoforms.

Table 2:

List of absolute mass values and N-glycan structures of the α-subunit glycoforms of u-hCG analyzed by HILIC-or RPLC-HRMS. Ox: Oxidized isoforms. x: no peaks corresponding to its isoform.

Unidentified: a peak is present on the spectrum but the N-glycans stcutures were not identified.

Green circle: mannose; yellow circle: galactose; pink diamond: N-acetylneuraminic acid; blue square: N-acetylglucosamine. In Tables 1 and2, it appears that a higher number of hCGα glycoforms was detected in HILIC compared to RPLC for both drugs: 10 and 10 vs 6 and 9 for r-and u-hCG, respectively. As it was already concluded after analyzing both drugs in RPLC [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF], despite the additional glycoforms detected in HILIC, none are common to both drugs. As an example, no hCGα glycoforms with 3 antennas were observed for u-hCG but some with a very large number of hybrid glycans. This confirms the strong difference in the glycosylation patterns of the 2 drugs previously mentioned [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF].

Finally, the orthogonality of the HILIC and RPLC separation modes was investigated. Figure 4A presents the retention time in HILIC as a function of the retention time in RPLC for the most intense peak of the 4 identified hCGα glycoforms of r-hCG having N-glycans each with 2 antennas. It appears that the number of terminal sialic acids present on this type of glycoform contributes significantly to an increase in its retention time in both modes, which could indicate a poor orthogonality of these methods for the r-hCG characterization. A similar representation was done with the glycoforms identified during the analysis of u-hCG with both methods (Figure 4B). According to Tables 1 and2, the structures of the corresponding N-glycans are different for u-hCG since at least one out of the four antennas is based on mannose. It appears here that the elution order in HILIC does not seem to be related to the number of sialic acids of the glycoforms. In this case, some glycoforms vary only in the number of mannose sugars they contain, such as 13,293.7, 13,455.7, and 13,618.4 Da having 6, 7, and 8 mannose sugars, respectively. They co-elute in RPLC but not in HILIC. Looking at glycoforms with similar N-glycan structures but where a mannose has been replaced by an antenna (one Nacetylglucosamine plus one galactose plus one N-acetylneuraminic acid) on one of the two N-glycans, such as the glycoforms of 13,455.7 and 13,949.9 Da, respectively, they co-elute in HILIC but not in RPLC. This clearly demonstrates that HILIC and RPLC are orthogonal separation modes for the separation of u-hCG glycoforms, which have a more heterogeneous composition than r-hCG glycoforms.

As seen previously, it is also important to notice the presence of several peaks associated with a given mass in RPLC for both drugs (see Figure 2B and 3D), while there is only one peak for a given mass in HILIC (Figures 2A and3C). This difference between both modes clearly appears also in Figures 4C and4D, where all peaks for one mass were plotted for r-and u-hCG, respectively. Two explanations can be proposed. First, the different peaks could correspond to different isomers varying by the localization of the sialic acids on the ends of glycans. However, this hypothesis cannot be retained because several peaks were also observed for non-sialylated glycoforms, having a mass value of 13440.8 Da, as shown in Figure 2B. Secondly, the main differences between RPLC and HILIC are the presence of 0.1% TFA in the HILIC mobile phase instead of formic acid in RPLC and the nature of the stationary phase. Indeed, the injection solvent (H 2 O), the flow rate (0.4 mL min -1 ), and the H 2 O/ACN-based mobile phase are the same. Even the column temperature was almost identical (65°C instead 60°C). It was already observed that TFA can induce the dissociation of the two subunits of hCG, since they are not covalently linked [START_REF] Liu | Mass spectrometric characterization of the beta-subunit of human chorionic gonadotropin[END_REF]. Therefore, it could be assumed that the TFA present in the HILIC mobile phase leads to the dissociation of the two subunits during their analysis, giving different retention times for the α-and β-subunits. This is in agreement with experimental results. Indeed, as observed above, the elution zone observed in HILIC-HRMS corresponds to glycoforms of the α-subunit and the two other elution zones observed in our previous experiments in HILIC-UV that are not detected in HILIC-HRMS could correspond to glycoforms of the β-subunit [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF]. Unfortunately, it was impossible to confirm this hypothesis with UV detection and the too low MS signal intensity, due to the presence of TFA and the difficulty to ionize highly glycosylated glycoforms of the β-subunit that have potentially 2 N-and 4 O-glycans and should be therefore also more heterogeneous. In RPLC-HRMS, the absence of TFA in the mobile phase and the observation of different chromatographic peaks for one single mass of hCGα could be explained by the fact that the dimeric form of hCG is preserved during the separation.

One single hCGα glycoform, which can be more easily ionized with only two N-glycosylation sites, could be associated with different hCGβ glycoforms, which are not well ionized and consequently not detected in MS [START_REF] Camperi | An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level[END_REF]. However, it is still not possible to conclude at this stage of the hCG study.

Conclusion and perspectives

For the first time, a HILIC-HRMS method for the characterization of the highly glycosylated hCG protein at the intact level was developed. Despite the presence of TFA in the mobile phase, which affects the performance of the MS detection while favoring the separation resolution, the HILIC-HRMS method allowed the identification of different hCGα glycoforms in 2 hCG-based drugs. HILIC allowed the detection of a slightly higher number of hCGα isoforms than RPLC. However, both are Table S1: MS parameters recommended by the manufacturer (Bruker Daltonics) for the analysis of macromolecules (with m/z > 1000) and used for the analysis in RPLC-(qTOF) MS.

Figure 1 :

 1 Figure 1: (A) BPC and (B) average MS spectrum between 19.8 and 21.8 min (see arrow) obtained by

Figure 2 :

 2 Figure 2: XICs of hCG glycoforms with N-glycan structures obtained with the analysis of r-hCG by (A)

  mass spectrum, the XICs were plotted for each absolute mass value and the glycan structures are presented in Figures3C and 3Dfor HILIC and RPLC, respectively.

Figure 3 :

 3 Figure 3: BPCs of u-hCG analyzed by (A) HILIC-HRMS or (B) RPLC-HRMS and XICs of the hCG

  Isoform mass (Da) u-hCGαPossible N-glycan structures (2 N-glycans by isoform)

Figure 4 :

 4 Figure 4: Retention times in HILIC as a function of retention times in RPLC for the common hCGα

complementary because some isoforms were only detected in RPLC or HILIC and, even more so, they are orthogonal. Several perspectives can be considered, such as improving sensitivity in HILIC-HRMS or developing two-dimensional approaches coupling HILIC in the first dimension and RPLC in the second.

This work has received the support of "Institut Pierre-Gilles de Gennes" (laboratoire d'excellence, "Investissements d'avenir" program ANR-10-IDEX-0001-02 PSL and ANR-10-LABX-31).

Supplementary data