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Antigen-antibody complexes are small and soluble when in antigen excess. Therefore, precipitation near the center of the circle is usually less dense than it is near the circle's outer edge, where antigen is less concentrated.

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Expansion of the circle reaches an end point and stops when free antigen is depleted and when antigen and antibody reach equivalence. For most antigens, the area and the square of the diameter of the circle at the circle's end point are directly proportional to the quantity of antigen and are inversely proportional to the concentration of antibody.

Circles that small quantities of antigen create reach their end points before circles that large quantities create. While circles are still expanding, a graph that compares the quantities or concentrations of the antigen on a logarithmic scale with the diameters or areas of the circles on a linear scale may be a straight line kinetic method. The quantity and concentration of insoluble antigen-antibody complexes at the outer edge of the circle increase with time.

Measurements of large circles are more accurate than are those of small circles. From Wikipedia, the free encyclopedia. Clinical Chemistry. Archived from the original PDF on Retrieved Rose, Noel; Friedman, Herman eds. As is clear from D4, communicated before the priority date of the patent in suit, CEA is a glycoprotein of molecular weight kD page , first paragraph.

The Appellant has obtained from Professor Mach, co-author of D1, cell residues originating from the clone which produced Mab 23 certified by Professor Mach's letter of 4 June This is confirmed by cross-inhibition experiments using Gold epitopes Gold 1 to Gold 5. The Respondent's argument that Mab 23 and Mab 3d are not corresponding because Mab 23 recognises the same determinant as does Mab P2 and Mab has a different epitopic specificity from Mab 3d P3 is not valid; only experiments in which both antibodies to be compared take part can provide a reliable conclusion. The Respondent's further argument that D1 is not an enabling disclosure because it has not been proved that Professor Mach would supply the cell line producing Mab 23 to anyone requesting it is also not valid; the authors of P2 under acknowledgements were also able to obtain the cell line from Professor Accolla.

As is shown in P1, page , chart 4A, kD CEA has a number of epitopes, of which only one, specific for Mab 3d, is not found on any of the lower molecular weight constituents. Moreover P2 chart 4 shows that Mab 23 recognises the same antigenic determinant as does Mab , whereas P3 Table 9 shows that Mab has a different epitopic specificity from Mab 3d. Mab 23 and Mab 3d therefore cannot be corresponding.

As to the Maurer report, the quality of the photographs is such that no conclusion can be drawn. The Gold epitope experiments were only explained at the oral proceedings and no protocol for these has been provided; these should be disregarded. Moreover D1 is not an enabling disclosure because it relies on Professor Mach making available the hybridoma which produces Mab There is no certainty that he would have done so, as is evidenced by the fact that the Appellant only acquired the cell residues after several attempts.

That the authors of P2 received samples is not surprising, since P2 is a publication originating from Professor Mach's laboratory. As compared with the granted patent, Claims 1 and 3 according to the main and first auxiliary requests now include a definition of the term "corresponding" derivable from page 7, lines 2 to 12 of the description and also from the original application documents.

No objection therefore arises under Articles 2 and 3 EPC. It is convenient first of all to dispose of the second auxiliary request, because the Appellant stated at the end of the oral proceedings that the request for revocation of the patent did not apply to this request. This is in accord with a corresponding statement made during the opposition procedure to the effect that the objections under Articles 54, 56 and 83 EPC were maintained only in connection with the expression "corresponding to" paragraph 11 of Facts and Submissions in the Opposition Division's decision.

Moreover the Board sees no reason to disagree with the Opposition Division's decision, to the extent that it relates to the part of the subject-matter of the granted claims covered by the second auxiliary request, which is therefore allowable. The main and first auxiliary requests have substantially the same scope and can be dealt with together. The Board has noted the conflicting evidence adduced by the parties as to whether the antibody Mab 23 corresponds to Mab 3d or not, but finds it unnecessary to take a position on this issue or for that matter on whether D1 is an enabling disclosure, because the requests are not allowable for another reason.

This is the case in the patent in suit, because the unique specificity of Mab 3d for kD CEA provides an improved tool for diagnosing colorectal carcinoma. The question arises whether the Patentee, having disclosed this monoclonal antibody, and also taught for the first time that there is an antigenic determinant on kD CEA, not shared by other components of CEA, for which Mab 3d is specific, is entitled to claim any monoclonal antibody having this same property that is, a corresponding antibody.

In convectively bound systems, much higher fluid velocities and pressure drops may be used. For perfusion systems, increases in bed velocity at the outset increase productivity in a manner similar to diffusively bound systems. However, above a threshold bed velocity, when the Peclet number in the throughpores becomes greater than 1, or convective flow velocity exceeds diffusive flow velocity within the pores, the perfusive realm is entered.

Further increases in velocity serve to increase convection within the pores and increase mass transport. At some high flow rate, the perfusive system becomes diffusively bound because the time it takes for a solute molecule to diffuse to and from a throughpore to an interactive surface region becomes much greater than the time it takes a solute molecule to move by convection past the region. However, the distance over which diffusion must act as the transport mechanism is much smaller than in conventional diffusion bound systems.

Thus, optimal perfusive performance continues at least through the bed velocity where the subpore diffusion time is ten times as great as the throughpore convective time. To evaluate the implications of perfusive kinetics on chromatography bed sorption, existing models were modified and used to simulate the sorption process. Column sorption behavior often is shown in the form of solute "breakthrough" curves which comprise a plot of effluent concentration vs.

For a given column, if the flow rate of the feed to the sorptive surface is sufficiently slow to permit the contact time between the solute and the sorbent to be long enough to overcome finite mass transfer rates, equilibrium sorption is achieved. In this case, the initial amount of solute loaded onto the column is sorbed and no solute appears in the column effluent.

When sufficient solute is loaded onto the column to saturate the sorbent phase, no more solute can be sorbed and the solute concentration in the effluent matches that of the feed. In practice, in diffusively bound systems, sorption deviates from the equilibrium limit due to slow mass transport rates.


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As illustrated in FIG. As linear bed velocity increases, mass transfer rates begin to dominate and premature solute breakthrough occurs. At very high bed velocities, e. In contrast, for a similar column having the same simulation condition wherein the matrix is perfusive, the predictive solute breakthrough curve is much sharper and is similar to the equilibrium sorption limit.

This predicted behavior was verified by experiment, as is discussed below. The amount of feed processed until that point defines the column capacity. This capacity term is an important determinant to overall productivity in the system, and typically decreases as the bed velocity increases in a diffusive particle column. In contrast, as shown in FIG. From the foregoing description many of the basic engineering goals to be pursued in the fabrication of matrix materials suitable for the practice of perfusion chromatography will be apparent to those skilled in the art.

Thus, what is needed to practice perfusion chromatography is a matrix which will not crush under pressure having a bimodal or preferably multimodal pore structure and as large a surface area per unit volume as possible. The first and second pore sets which give the material its bimodal flow properties must have mean diameters relative to each other so as to permit convective flow through both sets of pores at high V beds.

The provision of subpores in the matrix is not required to conduct perfusion chromatography but is preferred because of the inherent increase in surface area per unit volume of matrix material such a construction provides. The matrix can take the form of a porous, one-piece solid of various aspect ratios height to cross-sectional area.

Cross-sectional areas may be varied from a few millimeters to several decimeters; matrix depth can vary similarly, although for high fluid flow rates, a depth of at least 5 mm is recommended to prevent premature breakthrough and what is known as the "split peak" phenomenon. The structure of the matrix may comprise a rigid, inert material which subsequently is derivatized to provide the interactive surface regions using chemistries known to those skilled in the art.

Alternatively, the structure may be made of an organic or inorganic material which itself has a suitable solute interactive surface. Methods of fabricating suitable matrices include the construction of particles which are simply packed into a column. These optionally may be treated in ways known in the art to provide a bond between adjacent particles in contact.

Suitable matrices also may be fabricated by producing fiber mats containing porous particles which provide the chromatography surface. These may be stacked or otherwise arranged as desired such that the intersticies among the fibers comprise the first pore set and the throughpores in the particles the second pore set. Matrices also may be fabricated using laser drilling techniques, solvent leaching, phase inversion, and the like to produce, for example, a multiplicity of anisotropic, fine pores and larger pores in, for example, sheet-like materials or particulates which are stacked or aggregated together to produce a chromatography bed.

Preferably, after fabrication of the particles, the interactive surface regions are created by treating the high surface area particles with chemistries to impart, for example, a hydrophilic surface having covalently attached reactive groups suitable for attachment of immunoglobulins for affinity chromatography, anionic groups such as sulfonates or carboxyl groups, cationic groups such as amines or imines, quaternary ammonium salts and the like, various hydrocarbons, and other moities known to be useful in conventional chromatography media.

Protides of the Biological Fluids, Volume 31 - 1st Edition

Methods are known for producing particles of a given size and given porosity from porons ranging in diameter from 10 nm to 1. The particles are fabricated from polymers such as, for example, styrene cross-linked with divinylbenzene, or various related copolymers including such materials as p-bromostyrene, p-styryldiphenylphosphine, p-amino sytrene, vinyl chlorides, and various acrylates and methacrylates, preferably designed to be heavily cross-linked and derivatizable, e.

Generally, many of the techniques developed for production of synthetic catalytic material may be adapted for use in making perfusion chromatography matrix particles. For procedures in the construction of particles having a selected mean diameter and a selected porosity see, for example, Pore Structure of Macroreticular Ion Exchange Resins, Kunin, Rohm and Haas Co. Polymer Sci.

Physiology

Part C, No. These, and other technologies known to those skilled in the art, disclose the conditions of emulsion and suspension polymerization, or the hybrid technique disclosed in a Ugelstad patent, which permit the production of substantially spherical porons by polymerization. These uniform particles, of a predetermined size on the order of a few to a few hundred angstroms in diameter, are interadhered to produce a composite larger particle of desired average dimension comprising a large number of anisotropic throughpores, blindpores, and various smaller throughpores well suited for the practice of perfusion chromatography.

The difference between the chromatography particles heretofore produced using these prior art techniques and particles useful in the practice of this invention lies in the size of the throughpores required for perfusion chromatography. PL sells a line of chromatography media comprising porons of polystyrene cross-linked with divinylbenzene which are agglomerated randomly during polymerization to form the particles. Actually, the mean pore diameter of the particles represents an average between throughpores and subpores and thus bears little significance to the perfusion properties of these materials.

These types of particle geometries can be made to perfuse under appropriate high flow rate conditions disclosed herein. Its interactive surfaces are hydrophobic polymer surfaces which interact with the hydrophobic patches on proteins.


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A second type of particle has interactive surface elements derivatized with polyethyleneimine and act as a cationic surface useful for anionic exchange. Both types of particles were produced in an ongoing effort initiated by F.

T 0349/91 () of 10.3.1993

Regnier to increase intraparticle diffusion of large solutes such as proteins by increasing pore size. These particles were used by the inventors named herein in the initial discoveries of the perfusive chromatography domain. While they are by no means optimal for perfusion chromatography, the pores defined by the intraparticle space in a packed bed of these materials and the throughpores in the particles have an appropriate ratio for achieving perfusion chromatography under practical flow conditions.

Referring to FIGS. As shown in FIG. The perfusive pores are anisotropic, branch at random, vary in diameter at any given point, and lead to a large number of blind pores in which mass transport is dominated by diffusion.

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As illustrated, the particles are approximately 10 micrometers in diameter. Circle B in FIG. Here, the microstructure of the bed on a scale of approximately 1 micrometer is illustrated. The particles comprise clusters of porons illustrated as blank circles The intersticies among the poron clusters define throughpores The individual porons making up clusters 24 here are illustrated by dots.