“… although enzymatic lysis was CGK, it was another matter as to how the Skilled Team would approach a suggestion to use it. That depends on the context.”
“A method for obtaining a polyunsaturated fatty acid-containing lipid, comprising the steps: a. providing a biomass which comprises microorganisms of the genus Schizochytrium, said biomass containing a polyunsaturated-containing fatty acid; b. contacting said biomass with an enzyme; and c. recovering said lipid, wherein said step of contacting said biomass with an enzyme comprises treating said biomass with a protease.”
“The extraction of a microbial or single cell oil, for example comprising one or more polyunsaturated fatty acids (PUFAs), directly from microbial cells is disclosed which avoids the need for solvents. After fermentation, the microbial cells are pasteurised, washed and the cell walls lysed or disrupted by a mechanical (e.g. homogenisation), physical (boiling or drying), chemical (solvents) or enzymatic (cell wall degrading enzymes) technique. The oil (containing the PUFA) is then separated from the resulting cell wall debris. This is achieved by centrifugation, which results in an oily phase (top layer) that contains the oil which that can be separated from an aqueous phase (containing the cell wall debris). The oil can then be extracted and if necessary the PUFA can be purified or isolated from the oil.”
“The present invention relates to the extraction (and then isolation) of a microbial (or single cell) oil, preferably comprising one or more polyunsaturated fatty acids (PUFAs), from single cell (or micro-) organisms. The process of the invention involves the disruption or lysis of microbial cell walls, followed by separating the oil from the resulting cell debris. The invention additionally relates to a microbial oil recovered by this process, preferably having a PUFA.”
“[0003] In most microbial PUFA production processes a microorganism is first cultured in a fermenter in a suitable medium. The microbial biomass is then harvested and treated to enable subsequent extraction of a lipid from the biomass with a suitable solvent. The lipid is usually subjected to several refining steps. Care must be taken during the process because degradation can occur if the lipids are subjected to lipolysis or oxidising conditions, for example heating (in the presence of oxygen) and/or due to lipases or lipoxygenases. The art teaches that to avoid oxidation (such as resulting from breaking open the cells and so exposing the contents to oxygen) PUFAs can be extracted from whole intact cells using a solvent …. The use of solvents is a common way of removing lipids from microbial biomass … [0004] Although these extraction processes [i.e. using a solvent] have been used for several years, the solvent needs to be removed and this results in extra cost. In addition, if the lipid is to be used in a foodstuff, it is important that certain solvents, such as hexane, are removed completely, or only remain in very small quantities. If the hexane is removed by evaporation then this may involve heating and that not only adds to costs but can cause lipid degradation. Furthermore, with increasing environmental considerations, the use of solvents for the extraction of lipids is becoming increasingly expensive and unpopular. [0005] The present invention therefore seeks to solve or at least mitigate these problems.
“…a process for obtaining an oil (or fat or lipid, the terms are used interchangeably) from microbial cells, the process comprising (a) disrupting (or lysing) the cell walls of the microbial cells to release the oil from the cells. The (microbial or single cell) oil can then be (b) separated from at least part of the resulting cell wall debris. One can then (c) recover, purify or isolate the microbial oil (or one or more PUFAs). A good yield of the oil can be achieved using this process without the need for a solvent. Preferably the oil will comprise one or more PUFAs.”
“Recent PUFA preparation processes advocate keeping the microbial cells intact … Throughout this process the cells are kept intact to prevent oxygen in the atmosphere contacting the PUFAs and causing undesirable oxidation. However, it has now been found that a good quality PUFA oil can be achieved if the cells are in fact lysed: any potential oxidation by the atmosphere is more than compensated by the advantage of avoiding the need for solvents.”
“[0014] The cell walls of the microbial cells can then be disrupted (or lysed). This can be achieved using one or more enzymatic, physical or mechanical methods or techniques, for example at high shear conditions. Physical techniques include heating and/or drying the cells to a sufficient temperature whereby the cell walls are ruptured. This may comprise boiling. [0015] Enzymatic methods include lysis by one or more enzymes, e.g. cell wall degrading enzymes. The cell wall degrading enzyme may be a lytic enzyme. Other enzymes include (e.g. alkaline) proteases, cellulases, hemicellulases, chitinases and/or pectinases. Other cell wall degrading substances may be used instead of or in combination with one or more enzymes, e.g. salts, alkali, and/or one or more surfactants or detergents. A combination of physical, mechanical and/or enzymatic methods is also contemplated.”
“[0021] The microbial oil is then separated from at least part of the cell wall debris formed. At this stage the PUFA may be in an oily or lipid layer. This may be a top or upper layer, which is (or has risen) above an aqueous layer containing cell wall debris. The oily layer comprising the PUFA can then be separated from the aqueous phase. One or more surfactants or detergents may be present or added to assist this process. [0022] The separation of the oil from at least some of the cell wall debris is preferably achieved or assisted by using a mechanical method, in particular by centrifugation. … Centrifugation may result in either a 2-phase system (a fatty or oily top layer and a lower aqueous layer) or a 3-phase system (a fatty or oily top layer, a middle aqueous layer and a bottom layer, usually containing the cell debris).”
“… result[ed] in an arachidonic acid-enriched oily top layer (that was recovered from the centrifuge) and a lower aqueous layer containing the cell debris. A crude PUFA oil was recovered: the yield of oil was 95% (based on the oil in the cell). The crude oil had the following approximate composition: 1 to 2% sterols and cell debris; 3 to 4% phospholipids; 4% monoglycerides; 6% diglycerides; and the remainder being triglycerides.”
“… This resulted in a DHA-enriched fatty top layer (crude oil) and a lower aqueous layer. A crude PUFA oil was recovered from the fatty top layer.”
“The cross-examination of the respondents’ expert followed with customary skill the familiar ‘step by step’ course. I do not find it persuasive. Once an invention has been made it is generally possible to postulate a combination of steps by which the inventor might have arrived at the invention that he claims in his specification if he started from something that was already known. But it is only because the invention has been made and has proved successful that it is possible to postulate from what starting point and by what particular combination of steps the inventor could have arrived at his invention. It may be that taken in isolation none of the steps which it is now possible to postulate, if taken in isolation, appears to call for any inventive ingenuity. It is improbable that this reconstruction a posteriori represents the mental process by which the inventor in fact arrived at his invention, but, even if it were, inventive ingenuity lay in perceiving that the final result which it was the object of the inventor to achieve was attainable from the particular starting point and in his selection of the particular combination of steps which would lead to that result.”
“A central question on Bijl is whether the Skilled Team would be motivated to apply the suggestion of enzymatic lysis, either at all, or to Schizochytrium microbial cells. Another key question is whether the Skilled Team appreciated that an emulsion from enzymatic lysis would be easier to break than an emulsion generated by mechanical lysis.”
“283. …. No-one said that the Skilled Team would spontaneously understand that the emulsion formed from enzymatic lysis would be easier to break than an emulsion from mechanical lysis. If the Skilled Team turned their mind to that specific question, they might well hypothesise that it would and of course, if the Skilled Team started experimenting with enzymatic lysis, they would be likely to discover that the emulsion so formed was easier to break. 284. The problem for Mara is that they could not rely on Dr Kyle’s evidence on obviousness of EP155 and the evidence from Mr Dueppen (and Dr Wynn) was firmly based on their experience in commercial production. I did not receive any (reliable) evidence as to what would have been the reaction to Bijl amongst those accustomed to working at lab scale.”
“In my view, the best way to evaluate this case is to apply the well-known Pozzoli approach. As for the first two stages, I have identified the Skilled Team and their CGK above. The third stage is to identify the differences between Bijl and claim 1 of EP155. The clearest difference is the use of Schizochytrium cells, so I refer to the choice of Schizochytrium as Step 1. A more subtle difference is the bringing to the fore of the use of enzymatic lysis with a suitable protease from (a) the general list in Bijl of CGK methods of lysis and (b) the general list of possible enzymes which could be used in enzymatic lysis, plus the later processing to obtain the desired DHA-rich oil. This would involve the following steps: i) Step 2: decide to investigate the use of enzymatic lysis. ii) Step 3A: make a choice as to the enzyme(s) to try. iii) Step 3B: do a literature search as to whether there was any information as to the cell wall composition of Schizochytrium cells to aid in the selection of a suitable enzyme and/or iv) Step 3C: perform some lab scale experiments to find out whether certain well-known enzymes would lyse Schizochytrium cells e.g. alkaline proteases, cellulases. v) Step 4: decide to use an alkaline protease to enzymatically lyse Schizochytrium cells. vi) Step 5: try the separation techniques taught in Bijl at [0028](f)-(h) and in the Examples to see whether the desired DHA could be obtained with a sufficiently good yield or use other separation techniques.”
“309. It should not be a surprise that the above series of steps can be formulated to lead from the prior art Bijl to claim 1 of EP155, but the key question is whether those are a series of Technograph steps or whether the combination of all those steps was obvious to the Skilled Team in 2002. 310. I have come to the conclusion that EP155 was not obvious over Bijl. Although it is clear that Mr Dueppen and Dr Wynn took somewhat of a negative view of Bijl in their written evidence, those views were ameliorated in the answers they gave in cross-examination. I also consider that Dr Wynn was overly resistant to suggestions that the Skilled Team would be able to decide on a suitable enzyme. If the Skilled Team had decided to investigate enzymatic lysis, they would have found a suitable enzyme or combination of them (e.g. a protease and a carbohydrase). However, overall the evidence extracted from Mr Dueppen and Dr Wynn in cross-examination did not, in my view, establish a case of obviousness for four main reasons. 311. First, when one stands back from the detail, the steps I discussed above show that in fact, Mara’s case uses Bijl as nothing more than a hook to pick up the suggestion of enzymatic lysis. Beyond that, Mara’s case appeared to me largely to discard the rest of the teaching of Bijl, so this case is not significantly different from a case of obviousness over the CGK concept of enzymatic lysis. 312. The related, second, point is that what was new in Bijl (for the Skilled Team) was not the mention of the CGK methods of lysis but the suggestion of a solventless separation step using centrifugation, with the optional addition of one or more salts. This separation step was demonstrated in two examples, each of which featured mechanical lysis. There was no example drawing attention to enzymatic lysis. 313. Third, enzymatic lysis was CGK and the notion had been around for some years. What was unexplained in Mara’s case was why this particular mention of enzymatic lysis in Bijl would trigger the Skilled Team to investigate it, whereas other mentions (e.g. in conferences recorded in the SCO Book) had not. Bijl was published in February 2002, only a few months before the EP155 Priority Date of3 May 2002 . However, in view of the way Mara’s case was developed the question: if it was obvious, why was it not done before? has greater significance, to which no answer was supplied. 314. Fourth, despite Mara’s best efforts, there was no evidence to support their suggestion that the Skilled Team, on reading Bijl, would understand the emulsion ‘problem’ was caused by the violence of mechanical lysis, that enzymatic lysis would produce a gentler emulsion and this thought process would point the Skilled Team towards taking up enzymatic lysis. 315. Overall, it is difficult to avoid the conclusion that there was an undue focus on enzymatic lysis, caused by hindsight. 316. The issue was reasonably finely balanced such that, with better expert evidence (i.e. which gave a reason why the Skilled Team would focus on enzymatic lysis without hindsight), I might well have been able to find that EP155 was obvious, but, on the basis of the evidence I received, EP155 was valid.”
“The Skilled Microbiologist would find these results very surprising, as it is well known that when lipids and water are mixed, particularly under high-turbulence conditions, such as those present in a homogeniser, it is likely an emulsion would form.”
“Q. Sure. The reason that you give is the fear of an emulsion which you say is a particular problem using the high turbulence condition of a homogenizer? A. Which emulsion is the fear that would particularly happen if you did it as described in the examples. Q. So an enzyme would be better on that score; yes? A. If you could get an enzyme system to work, it would potentially be better, it would be better than that; yes.”
“Following the evidence, it is clear that the following were CGK methods for seeking to break an emulsion: i) Use of a polar organic solvent such as isopropanol (as in the FRIOLEX process). This worked by the water-miscible solvent making the aqueous phase even more polar, and so less favoured by the non-polar lipids such as TAGs …. ii) Addition of salt. The mechanism of this was to increase the density of the heavy (water-containing) phase and encourage better separation, particularly upon centrifugation …. iii) Heating, which worked by increasing the energy in the system and increasing the rate at which droplets of oil can meet and coalesce …. iv) Stirring (gentle agitation), which works on a similar principle to hearing, by increasing the rate at which droplets may come into contact and coalesce …. Vigorous agitation, however, can promote formation of emulsion …. v) Centrifugation, which when used alone could break weak emulsions, and for stronger emulsions would be used along with other approaches …. vi) Combinations of the above. For instance, heating and stirring would have been used with one of more of the other techniques. Also, centrifugation amplifies the effect of gravity, so works in combination with the increased density differential caused by addition of salt.”
“In my judgment, the evidence established the following: i) The various techniques covered in [656] above were CGK as possible methods for breaking an emulsion. Which ones would actually work effectively in practice would depend on the strength of the emulsion. If in doubt, the Skilled Team would conduct a simple lab test at the bench to see which techniques or combinations would work effectively. ii) As a matter of fact, no solventless method had been developed at a commercial scale. iii) But the absence of such a method at a commercial scale was adequately explained by the commercial barriers, namely Martek’s patent portfolio.”
“The present invention relates to processes for obtaining a lipid from a cell by lysing the cell, raising a pH of the cell and/or contacting the cell with a salt, and separating the lipid. The scope of protection is defined by the process as set out in the claims.”
“Therefore, there is a need for a process for obtaining lipids from a cell which does not use an organic solvent. Several processes have been proposed for separating a lipid from a cell without the use of an organic solvent. For example, U.S. Patent No. 6,750,048 discloses an aqueous washing process whereby an emulsion is washed with aqueous washing solutions until a substantially non-emulsified lipid is obtained. However, in some embodiments, this process requires multiple washing steps, which require substantial cost and time. U.S. Patent No. 7,431,952 discloses a process whereby lysed cells are centrifuged to remove cell wall debris and then oils are extracted and purified. However, this process provides a crude oil that requires extensive further purification. Thus, what is needed is a process that does not utilize a volatile solvent to extract a lipid from a cell, and which can be performed using readily available equipment and a minimum number of steps to provide a highly pure lipid.”
“[0044] In some embodiments, the lipid is a crude lipid. In some embodiments, the crude lipid optionally has less than 5% by weight or volume of an organic solvent. [0052] In some embodiments, the extracted microbial lipid is a crude lipid or a crude oil[. ]The crude lipid has less than 5% by weight or volume of an organic solvent.”
“There is further disclosed a process for obtaining a lipid from a cell composition, the process comprising raising the pH of the cell composition to 8 or above to lyse the cell composition and demulsify the cell composition, adding a salt to the cell composition, and separating a lipid from the demulsified cell composition, wherein the lipid optionally contains less than 5% by weight or volume of an organic solvent. …”
“In some embodiments, the process does not add an organic solvent to the lysed cell composition. …”
“Generally, the processes of the present invention do not utilize an organic solvent in order to extract or otherwise separate a lipid. Thus, in some embodiments, an organic solvent is not added to a cell broth comprising plant material or fermentation broth comprising a microbial cell, is not added to a cell composition, is not added to a lysed cell composition, or is not added to a lipid during a process of the present invention in an amount or concentration sufficient to extract a lipid. In some embodiments, an organic solvent can be added to a cell composition, a lysed cell composition, or a demulsified cell composition. In such embodiments, the organic solvent is added in a concentration less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.05% by volume. … An organic solvent as defined herein can be optionally added to a lysed cell composition, for example, as a component of a base and/or a salt for contacting with the lysed cell composition. However, in such embodiments the organic solvent is present in a concentration such that the lipid is not substantially extracted from the cell composition, lysed cell composition, or demulsified cell composition by the solvent (i.e., in a concentration of less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or less than 0.05% by volume or weight).”
“[0167] In some embodiments, a broth comprising a microbial cell or a broth comprising plant material is concentrated to provide a lipid concentration of at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% by weight of the broth. In some embodiments, a broth comprising a microbial cell or a broth comprising plant material is concentrated to provide a lipid concentration of 4% to 40%, 4% to 30%, 4% to 20%, 4% to 15%, 5% to 40%, 5% to 30%, 5% to 20%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to 40%, 15% to 30%, 20% to 40%, 20% to 30%, 25% to 40%, or 30% to 40% by weight of the broth. [0168] In some embodiments, a cell composition or a lysed cell composition is concentrated to provide a lipid concentration of at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% by weight of the lysed cell composition. In some embodiments, a cell composition or a lysed cell composition is concentrated to provide a lipid concentration of 4% to 40%, 4% to 30%, 4% to 20%, 4% to 15%, 5% to 40%, 5% to 30%, 5% to 20%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to 40%, 15% to 30%, 20% to 40%, 20% to 30%, 25% to 40%, or 30% to 40% by weight of the lysed cell composition.”
“… In some embodiments, the crude [microbial] lipid has less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% by weight or volume of an organic solvent. …”
“… In some embodiments, the crude lipid has less than 5% by weight or volume of an organic solvent. …”
“A process for obtaining a lipid from a microbial cell, said process comprising: a) lysing a cell to form a lysed cell composition; b) adding a base to the lysed cell composition, raising the pH of the lysed cell composition to 8 or above to demulsify the cell composition; c) one or more of cl, c2, c3 and c4; c1) adding a salt to the lysed cell composition to demulsify the cell composition c2) heating the lysed cell composition to demulsify the cell composition c3) agitating the lysed cell composition to demulsify the cell composition c4) adding a second base to the lysed cell composition to demulsify the cell composition; and d) separating a lipid from the demulsified cell composition; wherein the lipid contains less than 5% by weight of an organic solvent and wherein the lysing comprises enzymatic treatment.” c1) adding a salt to the lysed cell composition to demulsify the cell composition c2) heating the lysed cell composition to demulsify the cell composition c3) agitating the lysed cell composition to demulsify the cell composition c4) adding a second base to the lysed cell composition to demulsify the cell composition; wherein the lipid contains less than 5% by weight of an organic solvent and wherein the lysing comprises enzymatic treatment.”
“(i) The first overarching principle is that contained in Art.69 of the European Patent Convention. (ii) Art.69 says that the extent of protection is determined by the claims. It goes on to say that the description and drawings shall be used to interpret the claims. In short the claims are to be construed in context. (iii) It follows that the claims are to be construed purposively—the inventor’s purpose being ascertained from the description and drawings. (iv) It further follows that the claims must not be construed as if they stood alone—the drawings and description only being used to resolve any ambiguity. Purpose is vital to the construction of claims. (v) When ascertaining the inventor’s purpose, it must be remembered that he may have several purposes depending on the level of generality of his invention. Typically, for instance, an inventor may have one, generally more than one, specific embodiment as well as a generalised concept. But there is no presumption that the patentee necessarily intended the widest possible meaning consistent with his purpose be given to the words that he used: purpose and meaning are different. (vi) Thus purpose is not the be-all and end-all. One is still at the end of the day concerned with the meaning of the language used. Hence the other extreme of the Protocol—a mere guideline—is also ruled out by Art.69 itself. It is the terms of the claims which delineate the patentee’s territory. (vii) It follows that if the patentee has included what is obviously a deliberate limitation in his claims, it must have a meaning. One cannot disregard obviously intentional elements. (viii) It also follows that where a patentee has used a word or phrase which, acontextually, might have a particular meaning (narrow or wide) it does not necessarily have that meaning in context. … (xi) Finally purposive construction leads one to eschew the kind of meticulous verbal analysis which lawyers are too often tempted by their training to indulge.”
“The present invention is directed to high-quality lipids, and in particular lipids with low anisidine values. Methods are provided for producing high-quality lipids that include the step of liberating lipids from biomass, such as algal biomass, using enzymatic treatment.”
“Problems with prior methods include poor product quality due to chemically aggressive conditions of high temperature and high pH, high costs due to the need to dry the biomass or for additional equipment such as homogenizers and pressure vessels.”
“[0015] The use of protease enzymes, or protease enzymes in combination with surfactants, provides an economical and simple way of releasing the lipid from the biomass under mild conditions conducive to making high-quality lipid. The lipid can then be isolated from the rest of the fermentation broth by centrifugation of the mixture. … [0016] The use of protease enzymes can help break down emulsion-stabilising proteins present, thereby aiding in the breaking of an emulsion. …”
“In some cases, after the lipids are liberated from the biomass, the lipids can be separated directly from the undesired materials (e.g., cellular debris), such as by centrifugation, or other appropriate methods. In other cases, an agent such as an alcohol or other polar organic solvent can be added to facilitate the separation of the liberated lipid from the other material. In still other cases, a solvent can be added that will dissolve the lipid and facilitate the separation of the liberated lipid from the other material, e.g., by solvent extraction. …”
“[864] In light of the CGK and [0027]-[0030] of Kobzeff, Mr Dueppen agreed that it would be obvious to use salt and/or heat, without any polar organic solvent (Dueppen XX [T2/296/8 - 297/20]). This satisfied step (c) by means of (c1) and/or (c2). [865] Alternatively, it would also be obvious to adopt Kobzeff’s suggestion of using an organic polar solvent such as isopropanol to break the emulsion, in a FRIOLEX approach, in combination with stirring. The solvent would then go in the aqueous phase upon separation of the oil, leaving the oil free of solvent (Dueppen 2 ¶¶122-126; Dueppen XX [T2/298/11-25]). As Mara submitted, this is also within the claims (see the construction of ‘less than 5% of organic solvent’ above).”
“877. However, these arguments only work on DSM’s construction of the < 5% integer, which I have rejected. Development of a solventless method is not required. 878. For all these reasons, I find that claims 1A, 6A and 7A of EP801 were obvious over Kobzeff. I accept Mara’s submissions as recorded in [855]-[868] above.”
“Q. It means the part of the document that addresses techniques to deal with an emulsion, specifically paragraph [0030] that we looked at earlier, is going to be of interest; yes? A. Yes. Q. As we discussed earlier, it would be routine for there to be agitation in the form of continual stirring, in any event; yes? A. Yes. Q. Paragraph [0030] of Kobzeff identifies several different techniques that can be used as treatment to release the lipid from the emulsion; yes? A. Yes, those where it had been used to break emulsions, other types of emulsions, yes. Q. One of which is the use of polar solvents? A. Yes, which is similar to the way they talked about in one of the examples in paragraph [0036]. Q. But there are others; yes? A. There are others. Q. Including, for example, adding salt? A. Yes. Q. That, as we have covered, would be understood as a technique for breaking an emulsion; yes? A. It could break an emulsion, correct Q. And we saw specifically that paragraph [0036] was expressly contemplating that in some cases, when no extraction solvent was used, there would also be no polar organic solvent added; yes? A. Yes. Q. It is the first scenario it mentions, direct separation such as by centrifugation; yes? A. Yes. Q. That is without using alcohol or other polar organic solvent; yes? A. That is what I understand, yes. Q. Of course, the addition of salt operates so that the centrifugation has an amplified difference in density, as we discussed this morning; yes? A. Yes. Q. Surely using salt is one of the obvious things to do? A. I do not disagree with that. Q. The skilled person would know that other techniques listed in paragraph [0030] could also be used with salt; yes? A. They could, yes. Q. Heating would be another obvious thing to do too; yes? A. It is possible; yes. Q. It would be equally obvious to add the salt and then heat it or to heat it up and then add the salt; yes? A. Again, you have to set up an experimental design to test all these potential scenarios out. It is not obvious which ones work, how they work in combination. These are things that have worked in the past in other emulsions, so yes, any combination could potentially do that but I do not know if it will work or not.”