NZ787410A - Sampling array devices and system for spectral analysis - Google Patents

Abstract

system for use in spectral analysis procedures can include a slide and a holder for carrying the slide. The slide includes a substrate forming a plurality of wells that are recessed relative to a surface of the substrate. Each of the wells forms a sample region that is recessed by a sample depth from the surface and a trough region that is recessed by a trough depth from the surface, the trough depth being greater than the sample depth. The holder includes a body defining a cavity between a first side and a second side of the body, a port for receiving the slide into the cavity, one or more first fenestrations on the first side, and one or more second fenestrations on the second side.

Description

SAMPLING ARRAY DEVICES AND SYSTEM FOR SPECTRAL ANALYSIS Background 10002] n aggregation phenomena are prevalent throughout the industrial bioprocess.

Proteins are expensive to express, isolate, and purify due to their complex physical-chemical characteristics. Aggregation is considered a primary mode of protein degradation, often leading to immunogenicity in patients, anti-drug antibody (ADA) response, and a loss of efficacy. The detection and determination of protein aggregates is a major objective in the biopharmaceutical industry and other areas of scientific research. The ion of protein aggregates is important in industrial ations e they can significantly affect the production of protein therapeutics (i.e, biologics or biosimilars), effectively lowering the production yields and sing risk of withdrawal. This is at the core of analytical technologies to understand characterization, comparability/similarity, release and stability testing of protein eutics.

The proposed technology also lends itself to high throughput multivariate analysis.

Summarv [0003[ The subject technology is illustrated, for example, according to various aspects described as numbered clauses (I, 2, 3, etc.) for convenience. Various examples of s of the subject logy are described below. These are provided as examples and do not limit the subject technology It is noted that any of the dependent clauses may be ed in any combination, and placed into a respective independent clause The other clauses can be presented in a r manner.

Clause t. Aslide comprising: a substrate forming a plurality of wells that are recessed relative to a surface of the substrate, wherein each of the wells forms a sample region that is recessed by a sample depth from the surface and a trough region that is recessed by a trough depth from the e, the trough depth being greater than the sample depth.

Clause 2. The slide of clause I, wherein the substrate transmits electromagnetic radiation.

Clause 3. The slide of clause I, wherein the substrate is a salt.

Clause 4. The slide of clause I, wherein the substrate comprises AgBr, AgCl, A12O3, AMTIR, BAF2, CaF2, CdTe, Csl, diamond, Ge, KBr, KCI, KRS-5, LiF, MgF2, NaCl, Si, $Si02, ZnS, ZnSe, and/or Zr02 Clause 5 The slide of clause l, wherein a periphery of the slide forms a bilaterally asymmetric shape Clause 6. The slide of clause I, wherein the trough region s entirely about the sample region.

Clause 7. The slide of clause I, n the sample region is concentrically within the trou gh .

Clause 8 The slide of clause l, wherein the plurality of wells are provided in a plurality of rows, wherein each row ses at least two of the plurality of wells.

Clause 9. A system comprising a slide comprising: a substrate forming a plurality of wells that are recessed relative to a surface of the substrate; a holder, comprising: a body defining a cavity between a first side and a second side of the body; a port for receiving the slide into the cavity; one or more first fenestrations on the first side, and one or more second fenestrations on the second side Clause 10. The system of clause 9, further comprising a block configured to secure the slide within the cavity when the block is placed within the port Clause I1. The system of clause 9, further comprising a cover configured to e each of the wells when placed upon the surface of the slide.

Clause 12. The system of clause l, wherein the cover is configured to transmit electrom agnetic ra diation.

Clause 13 The system of clause ll, wherein the cover and the slide have a substantially equal thickness in a direction orthogonal to the e of the slide when the cover is placed upon the surface of the slide.

Clause 14. The system of clause 9, wherein the body of the holder absorbs substantially all electromagnetic radiation incident to the holder.

Clause 15. The system of clause 9, wherein the plurality of wells are provided in a plurality of rows, wherein each row comprises at least two of the plurality of wells.

Clause 16. The system of clause 15, wherein the one or more first fenestrations comprises a number of first windows equal to a number of the plurality of rows, and the one or more second fenestrations comprises a number of second windows equal to the number of the plura li ty of ro ws.

Clause 17. The system of clause 16, wherein one of the first windows and one of the second windows are on opposite sides of one of the ity of rows when the slide is within the holder.

Clause 18 The system of clause 9, n the port is disposed on a third side of the holder.

Clause 19. The system of clause 9, n the first side is opposite the second side Clause 20. The system of clause 9, wherein at least one of the plurality of wells, the one or more first fenestrations, and the one or more second fenestrations are aligned along an axis when the slide is within the cavity.

Clause 21 . The system of clause 9, wherein the slide, the one or more first fenestrations, and the one or more second fenestrations are configured to transmit electro magnetic ra diation.

Clause 22. The system of clause 9, further sing a coating on an inner surface of the body.

Clause 23. The system of clause 22, wherein the coating comprises silicone.

Clause 24. The system of clause 9, further comprising a cap in thermal contact with an outer surfa ce of the holder, the cap comprising one or more third fe nestra tions.

Clause 25. The system of clause 9, r comprising a plate attached to an imaging device.

Clause 26. A method comprising. providing samples to each of a plurality of wells formed in a slide, each of the wells being recessed relative to a surface of the slide; enclosing the wells by applying a cover to the surface of the slide; inserting the slide and the cover into a cavity of a holder; and emitting electro magnetic ra diation thro ugh one or more fir st fe nestra tions of the holder, one or more second rations of the holder, and the sample.

Clause 27. The method of clause 26, further comprising inserting the holder into a receptacl e of a plate attached to an imaging .

Clause 28. The method of clause 27, n inserting the holder comprises positioning the sample at a focal length of the imaging device.

Clause 29. The method of clause 26, further comprising heating the samples to a target temperature by conducting heat through the holder.

Clause 30. The method of clause 26, further comprising placing a cap in th ermal contact with an outer surface of the bolder.

Clause 3I. The method of clause 30, wherein the emitting the electromagnetic radiation comprises emitting the electromagnetic radiation through one or more third fenestrations of the cap.

Clause 32. The method of clause 26, wherein the ing the slide and the cover comprises inserting the slide and the cover through a port of the holder.

Clause 33. The method of clause 32, further comprising, after ing the slide in the cover, obstructing the port with a block.

Clause 34. The method of clause 26, wherein the electromagnetic radiation is infrared li ght.

Clause 35. The method of clause 26, further comprising, after the ng the electromagnetic radiation, detecting a teristic of the electromagnetic radiation not absorbed by the sample.

Clause 36. The method of clause 26, further comprising: after the emitting the electromagnetic radiation, changing the temperature of the sample; emitting additional electromagnetic ion through the one or more first fenestrations and the one or more second rations of the holder and through the sample. 10004 onal features and advantages of the subject technology will be set forth in the description below, and in part will be apparent from the description, or may be learned by practice of the subject technology. The advantages of the t technology will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject logy as claimed.

Brief Description of the Drawings 10006] The accompanying gs, which are ed to provide further understanding of the subject technology and are incorporated in and tute a part of this description, illustrate aspects of the subject technology and, together with the specification , serve to explain principles of the subject technology. 10007] FIG. IA shows a perspective view of a slide in accordance with some embodiments of the subject technology. 10008] shows a top view of the slide of in accordance with some embodiments of the subject logy. 10009 shows a side view of the slide of FG 1A In accordance with some embodiments of the t technology. 10010] FIG. lD shows a bott om view of the slide of FIG. IA in accordance with some embodiments of the subject technology. [0011 FIG IE shows a sectional view of a portion of the slide of FIG 1A, ing a well, in accordance with some embodiments of the subject technology. 10012] FIG 2A shows a perspective view of a cover and the slide of FIG. A in ance with some embodiments of the subject technology. 10013] FIG 2B shows a perspective view of the cover of and the slide of in accordance with some embodiments of the subject technology. j0014 FIG 3A shows a perspective view of a cap in accordance with some embodiments of the subject technology FIG 3B shows a top vew of the cap of in accordance with some ments of the subject technology. shows a side view of the cap of m accordance with some embodiments of the subject technology. 10017] shows a front side view of the cap of in accordance with some embodiments of the subject technology. shows a perspective view of a holder in accordance with some embodiments of the t technology [0019 shows another perspective view of the holder of in accordance with some ments of the subject technology. 10020] shows a top view of the holder of in accordance with some embodiments of the subject technology. 10021] shows a front view of the holder of in accordance with some embodiments of the subject technology. 10022] shows a side view of the holder of in accordance with some embodiments of the subject technology. ]0023] shows a ctive view of a block in accordance with some embodiments of the t technology. shows a side view of the block of n accordance with some embodiments of the subject logy. 10025] shows a front view of the block of in accordance with some embodiments of the t technology.

FIG SD shows a top view of the block of FIG 5A in accordance with some embodiments of the subject technology. 10027] FIG 6A shows a perspective view of the slide of FIG 1A, the cover of FIG 2A, the cap of FIG 3A, and the holder of FG. 4A in accordance with some embodiments of the subject technology. shows a perspective view of the holder of FIG 4A and the block of in accordance with some embodiments of the subject technology 10029] shows a sectional view of portions of the slide of FIG 1A, the cover of , the cap of , and the holder of in accordance with some embodiments of the subject technology WO 84886 shows a perspective view of a slide in accordance with some ments of the subject technology. shows a perspective view of a cap in ance with some embodiments of the subject technology. 10031] FIG 9 shows a perspective view of a holder in accordance with some embodiments of the subject technology.

FIG 10A shows a top view of a plate in accordance with some embodiments of the subject technology 10033] FIG 1OB shows a top view of the holder of and the plate of in accordance with some embodiments of the subject technology. shows a tic view of the holder of and the plate of in accordance with some embodiments of the t technology.

Detailed Description In the following detailed description, specific details are set forth to provide an understanding of the subject technology. It will be apparent, however, to one ordinarily skilled in the art that the subject technology may be practiced t some of these specific s. In other ces, well-known structures and techniques have not been shown in detail so as not to obscure the subject technology 10036j The biologics and biosimilar industry is involved in the ch, development, and manufacturing of complex drugs known as protein therapeutics. The research and development efficiency can be undesirably low, which increases costs of drug development due to the high attrition rate of protein therapeutics. The costs of protein therapeutic development is significantly impacted by early and late stage failure. One way to lower research and development costs is to perform a series of evaluations of the protein therapeutic candidate early in the research and development phase. By performing the characterization of the therapeutic protein under varying ation conditions and stressors early in the research and development phase, a predictive e of the therapeutic candidate is generated to assess the risk of protein aggregation. This approach has been defined as a developability assessment This assessment can e important iformation for decision making, such as selecting protein therapeutic candidates for further development. When protein ation occurs the protein eutic typically has decreased efficacy and can elicit an immune se. In severe cases, such an immune response can be fatal. 10037 The problem of protein aggregation is complex and frequently involves several different chemical processes, which are difficult to discern. Aggregation may be stress induced and involve al or chemical changes such as agitation or stirring, oxidation, deamination and temperature changes. Even a slight change in pH, salt ions, protein concentration or formulation conditions can also induce protein aggregation. Again, aggregation leads to lower yields in production, loss of efficacy of the protein therapeutic, and safety concerns in relation to genicity risks. Currently, available techniques to assess aggregation do not address all of the factors that are involved in the process, such as the size, ty, ism and extent of aggregation, and stability of the protein therapeutic in solution. Several techniques have been developed to address the size of the ate or particulate, yet they do not determine the identity. Other techniques can determine the size and the identity of aggregates, but cannot determine the extent of ation or identify the aggregation prone s. The amino acid side chains present in a protein are important contributors to the stability of proteins. Yet to date, with the available routine high throughput bench instrumentati on the relationship between the weak chemical intera ctions involving side chains and the stab ili ty of the secondary stru cture of the pro tein candidate has not been ascertained.

The stability of the protein therapeutic is also critical for drug development, and cannot be fully characterized by simply identifying the thermal transition temperature of the protein. A greater level of understanding is needed to fully address the stability of n therapeutics. For example it would be beneficial to understand I) the relative ity of the domains within the protein of interest, 2) how the amino acid side chains contribute to the stability the domains, 3) whether the amino acid side chains are involved in the aggregation ism, and 4) if an excipient can stabilize weak interactions (e.g, in amino acid side chains) within the critical regions in ic domains of the protein therapeutic. There is a gap in understanding the factors that are important for determining the mechanism of protein aggregation.

To date when commercially available techniques are used orthogonally , differences in the sensitivity of the available techniques is a concern. In general, such techniques focus on determining the size, purity and stability of the protein therapeutic, and evaluate the presence or absence of protein ates or particulates in a formulation, to achieve lot-to-lot consistency. 10040] There is a need for technology that can be used to better assess the developability of protein eutics, and for the comparability ments needed to maintain and ensure product integrity, efficacy and safety. Such a process would need to be ized as sufficient to ensure product integrity, efficacy and safety by the Food and Drug Administration (FDA) Center fo r Drug Evaluation (CDER) division and other relevant regulatory bodies. [0041[ s of the subject technology provide a fast, accurate, and reproducible technique to determine the size, identity, mechanism, and extent of aggregation and the stability of a protein therapeutic, or other chemicals, in a single experiment. Aspects of the subject technology address comparability assessment of different n therapeutic candidates and developability assessment of protein therapeutic candidates. 10042] Aspects of the subject technology provide a system that includes reusable components, requires small sample sizes, reduces or eliminates sample evaporation, provides excellent signal/noise ratio with no ed fringes, a fixed path length relative to an imaging device allowing for quantitative analysis, excellent comparability ment assays, developability assessment using Design of Experiment (DOE) appro ach, and assessment of aggregation reversibility during a l dependence study. 10043] Aspects of the subject technology provide a sample carrying device with enhanced thermal conductivity, ease of loading and ing, enhanced securement of samples, m temperature bution, optimal focusing with an imaging device, and high throughput data acquisition 10044] According to some embodiments, for example as shown in FIGS. IA-IE, a slide 100 can include a substrate 110 that forms a plurality of wells 150. Each of the wells 150 can be recessed relative to a surface 130 of the substrate 1IO . The surface 130 can be substantially flat or planar except where a well 150 is present. As used herein, a substantially flat or planar surface is one that varies from y flat or planar within a tolerance that is typical for an able manufacturing process. Each of the wells 150 can form a sample region 160 for receiving a sample to be analyzed and a trough region 170 for receiving excess portions of the sample 10045 The wells 150 can be provided in separate rows 140. For example, a first row 140 can include a fist number of wells 150, and a second row 140 can include a second number of wells 150. The rows 140 can include the same number of wells 150 or ent numbers of wells 150 The slide 100 can e any number of rows 140 and any number of wells 150 within each row 140. For example, the slide 100 can include one, two, three, four, five, six, seven, eight, nine, or more than nine rows 140. Each row 140 can include one, two, three,four, five, six, seven, eight, nine, or more than nine wells 150. The number of rows 140 and wells 150 can be selected based on a desired number of separate samples to be aneously analyzed on a single slide 100. [0046[ According to some embodiments, the slide 100 can be used to analyze a large number of samples along with a reference in the same slide 10O0. One of the wells 150 can be provided with the reference for is under the same conditions (e.g., temperature, humidity, etc) as those of the samples on the slide 100. According to some embodiments, features of the slide 100 can provide an indication regarding the identity of particular wells 150. For example, as shown in FIGS. 1A-1D, a shape of the slide 100 can include a number of corners. Some of the corners can be regular corners 120 that have common features (e.g., right angle at ecting surfaces).

One or more of the corn ers can be an irr egular corn er 122 that has at least one fe atur e that is different from the common feature of the regular corners 120. The resulting shape (e.g., perimeter) of the slide 10O can be asymmetric. For example, the shape of the slide 10O can be bilaterally asymmetric across one or more axes. As shown in FIG. ID, at least a portion of the irregular corner 122 can form an angle 124 that is different (e.g, 45 s) from the angle formed at the regular corners 120. The location of the irregular corner 122 can be used for identifying and distinguishing the separate wells 150. For e, a reference can be provided within the well 150 that is closest to the irregular corner 122. By further example, samples can be provided to the wells 150 other than the well 150 that is closest to the irregular corner 122. At various stages , during, and after analysis, the well 150 containing the reference can be identified based on the on of the irregular corner 122.

WO 84886 [0047 According to some embodiments, the slide 100 can include a substrate 110 Preferably, the substrate can transmit electromagnetic radiation, such as visible and/or infrared light, and is suitable for use in spectral analysis. The substrate 110 can be entirely of a single ate material or a composite of multiple materials. The material of the substrate 10 can be selected for facilitating spectral analysis of a reference or sample received within wells 150 of the slide 100. For example, the material of the substrate 1HO can transmit e and/or ed ight and be substantially non-reactive with the reference and/or the . The ate 110 can include a salt. The substrate 110 can include AgBr, AgCl, A1203, amorphous material transmitting infrared radiation (AMTIR), BaF2, CaF2, CdTe, water clear zinc sulfide (e.g , Cleartran19), CsI, d, Ge, KBr, KCl, red thallium bromoiodide ("KRS-5), LiF, MgF2, NaCl, Si, Si02, ZnS, ZnSe, Zr02, and/or combinations thereof. The substrate 110 can be coated or uncoated. Slides 100 of the type described herein can be reused multiple times in separate procedures with different references and samples. 10048] According to some embodiments, for example as shown in FIGS. 1B and IE, each of the wells 150 can form a sample region 160 that is recessed by a sample depth 162 from the surface 130. Each of the wells 150 can also form a trough region 170 that is recessed by a trough depth 172 from the surfa ce 13 0. The trou gh region 170 can extend entirely about the sample region 160. For example, the sample region 160 can be concentricall y within the trou gh region 170 with re spect to an axis extending orthogonall y to the surface 130. The sample region 160 and the tro ugh region 170 can be fo rmed by removing portions on the surfa ce 13 0 of the substrate 110. For example, a milling process or other process for removing material can be applied to the surface 130 to form the sample region 160 and the trough region 170 The sample region 160 of the trough region 170 can be formed in the same or separate processes.

The sample region 160 can span an outer sample cross-sectional dimension 164 in at least one direction. A volume of a reference or sample that can be ed within the sample region 160 can be defined, at least in part, by the sample depth 162 and the outer sample ectional dimension 164. At least a portion of the sample region 160 can be oned within a field of view of an imaging device 800 or other equipment. 10050] The trough region 170 can span a trough cross-sectional dimension 174 in at least one direction. The trough cross-sectional dimension 174 can be defined by the difference between an outer trough cross-sectional dimension 154 and the outer sample cross-sectional dimension 164.

A volume of a reference or sample that can be received within the trough region 170 can be defined, at least in part, by the trough depth 172 and the trough cross-sectional dimension 174 The trough depth 172 can be greater than the sample depth 162. For e, the trough depth 172 can be more than double, more than five times, or more than ten times the sample depth 162 The outer sample cross-sectional dimension 164 can be about 2.0 mm. The sample depth 162 can be about 0.04-0.08 mm ±00l mm ideal for DO containing samples or 0.004­ 0.012 mm ± 0.002 mm HO containing samples. For example, the sample depth 162 can be about 0.04 mm or about 0.07 mm. A greater sample depth 162 can facilitate a greater concentration range of proteins for is. The trough cross-sectional dimension 174 can be about 1.0 mm. The outer trough cross-sectional dimension 154 can be about 6.0 mm. The trough depth 172 can be about 0.60 mm. Spacing between centers of adjacent wells 150 can be greater than the outer trough cross-sectional dimension 154. These exemplary dimensions can be modified as needed to suit a given purpose. 10052] According to some embodiments, the trough region 170 can receive an amount of a reference or sample that exceeds the volume of the sample region 160. For example, as a sample or refe rences appli ed to the sample region 160, and the amount that does not fi t within the sample depth 162 and the outer sample cros s-sectional dimension 164 can move to the tro ugh region 170. Such action may occur dur ing appli cation of the refere nce or sample or dur ing appli cation of a cover 200 t0 the sli de 100. The tro ugh re gion 170 can be of adequate volume to accommodate all ive amounts of the nce or sample. The trough region 170 can be wide and deep enough to prevent portions of the reference or sample from owing out of both the sample region 160 and the trough region 170 (ie., onto the surface 30). As such, an entire amount of the reference or sample can be contained entirely within the well 150 and prevent cross contamination with the contents of any other well 150 According to some embodiments, for example as shown in FIGS. 2A-2B, a cover 200 can be d to the slide 100 An ng surface 230 of the cover 200 can be substantially flat or . Some features of the cover 200 can be complementary or identical to features of the slide 100. For example, a shape of the cover 200 can include a number of corners. Some of the corners of the cover 200 can be regular corners 220 that are identical or similar to the regular s 120 of the slide 100. One or more of the corners of the cover 200 can be an irregular corner 222 that are identical or similar to the irregular corner 122 of the slide 100. The resulting shape (e.g., perimeter) of the cover 200 can be identical or similar to the shape (e.g, perimeter) of the slide 100. 10054] According to some embodiments, the cover 200 can include a substrate 210. ably, the substrate can transmit electromagnetic radiation, such as visible or infrared light, and is suitable for use in al analysis. The substrate 210 can be of the same material as or a different material from that or the substrate 1O of the slide 100. The al of the substrate 210 can be selected for facilitating spectral is of a reference or sample ed within wells 150 of the slide 100. For example, the al of the substrate 210 can transmit e and/or infrared hight and be substantially non-reactive with the referenc e and/or the sample Covers 200 of the type described herein can be reused multiple times in separate procedures with different references and samples. 10055[ The opposing surface 230 of the cover 200 can be brought into contact with the surface 130 of the slide 100. In such a configuration, the opposing surface 230 can be parallel to or coplanar with the e 130. When the cover 200 is brought into contact with the slide 100, the opposing sur fa ce 230 can extend over one or more of the wells 150 of the slide 100. The opposing surface 230 can enclose each of the wells 150, such that each well 150 is ed from an external environment and from every other well 150. The cover 200 can prevent evaporation of the reference or sample from the well 150. The boundary of each of the sample region 160 and the trough region 170 can be defined, in part, by the opposing surface 230. Application of the cover 200 to the slide 100 can cause at least a portion of a reference or sample within the sample region 160 to move into the trough region 170 10056] The cover 200 can define a cover thickness 212 in a dimension that is orthogonal to the opposing surface 230. Additionally, the slide 100 can define a slide thickness 112 and a dimension that is orthogonal to the surface 130. The cover thickness 212 can be equal to or substantially equal to the slide thickness 112. As used herein, a substantially equal thickness is one that varies from exactly equal within a tolerance that is typical for an applicable manufacturing process. [0057 ing to some embodiments, for example as shown in FIGS. 3A-3D, a cap 300 can be ed for conduction of heat across portions of the slide 100 and/or the cover 200.

The cap 300 can include a cap surface 330 that is substantially flat or planar. One or more t rails 360 can de from the cap surface 330. One or more cap fenestrations 350 can extend through a portion of the cap 300, including the cap surface 330. The one or more cap fenestrations 350 can form windows that are each configured to be aligned with a corresponding one of the rows 140 of the slide 100. The number of cap fenestrations 350 can be equal to the number of rows 140 of the slide 100. Each of the cap fenestrations 350 can span a length that encompasses all of the wells 150 of the corresponding row 140. The one or more cap fenestrations 350 provide transmission of electromagnetic radiation to and/or from the wells 150 of the slide 100. Heat can be transmitted between the cap 300 and adjacent components. At least a portion of the cap 300 can be of a material with high thermal conductivity. Exemplary materials for the body 410 include aluminum, polytetrafluoroethylene (PTFE), brass, bronze, copper, silver, gold, and other metal alloys or ceramic. 10058} According to some embodiments, for example as shown in FIGS. 4A-4E, a holder 400 can n and support the sli de 100 and/or the cover 200. A body 410 of the holder 400 can define a cavity 420 between a first side 412 and a second side 414 of the body 410. A port 470 es access to the cavity 420 for receiving the slide 100 and/or the cover 200 into the cavity 420. 10059 According to some embodiments, one or more first fenestrations 450 can extend through a portion of the first side 412 of the body 410. One or more second fenestrations 460 can extend through a portion of the second side 414of the body 410. The first fenestrations 450 and/or the second fenestrations 460 can form windows that are each configured to be aligned with a corresponding one of the rows 140 of the slide 100 when the slide 100 is placed in the holder 40 The number of first fenestrations 450 and/or the number of second fenestrations 460 can be equal to the number of rows 140 of the slide I0O Each of the first fenestrations 450 and/or the second rations 460 can span a length that encompasses all of the wells 150 of the corresponding row 140. The first fenestrations 450 and/or the second fenestrations 460 provide transmission of electromagnetic radiation to and/or from the wells 150 of the slide 100.

According to some embodiments, the cavity 420 of the holder 400 is sized such that an inner surface 422 of the holder contacts at least a portion of the slide 100 and/or the cover 200. The inner surface 422 can provide thermal contact with at least one of the slide 1O0 and/or the cover 200. For example, the inner surface 422 on the first side 412 can contact the cover 200 and the inner surface 422 on the second side 414 can contaet the slide 100. Heat can be transmitted between the holder 400 and its contents. At least a portion of the body 410 of the holder 400 can be of a material with high thermal tivity, sturdy construction, and low­ electromagnetic radiation (e.g, visible light, infrared light, quantum cascade laser) reflection.

Exemplary materials for the body 410 include anodized aluminum, PTFE, bronze, and copper.

The holder 400 can absorb a substantial amount of omagnetic radiation that is incident thereon. For example, the holder 400 can absorb at least 90%, at least 95%, or at least 99% of the electromagnetic radiation that is incident thereon. As the holder 400 absorbs more electromagnetic radiation than it reflects, the holder 400 reduces erence with accurate readings during a spectral analysis. At least a portion of the inner surface 422 can be provided with a coating to reduce or eliminate damage to the contents of the holder 400 when moving or ng therein. Exemplary materials for the coating include silicone. j0061} According to some embodiments, one or more extensions 430 can be provided at an outer periphery of the holder 400. The extensions 430 can provide guidance and slideable engagement with a portion of other equipment fo r aligning the refe rence and samples within a field view of an imaging device 800. 10062 According to some ments, the port 470 can be provided on any side of the holder 400. For e, the port 470 can be provided on a side of the holder 400 that has a length shorter than at least one other side of the holder 400. Alternatively or in combination, the port 470 can be provided on a side of the holder 400 that has a length longer than at least one other side of the holder 400. The port 470 can be adjacent to a receptacle 480 for ing a block 500. The acle 480 can include a , recess, channel, rail, or other guide for receiving the 502 partially or entirely obstructing the port 470. 10063] According to some embodiments, for example as shown in FIGS. 5A-5D, a block 500 can be used to secure items within the cavity 420 of the holder 400. The block 500 may include a r 520 configured to fit within the receptacle 480 of the holder 400. The barrier 520 can be further configured to partially or entirely obstruct the port 470, such that contents within the cavity 420 of the holder 400 remain within the cavity 420 until the block 500 is removed. For example, the slide 100 and/or the cover 200 can be secured within the cavity 420 while the block 500 is within the receptacle 480. The block 500 can also include a handle S30 for use during operation by a user. 10064 According to some embodiments, for example as shown in FIGS 6A-6B, an ly can be formed of the components described herein. One or more references and samples are provided to each of a plurality of wells 150 formed in the slide 100. The wells 150 are enclosed by applying the cover 200 to of the slide 10O. For example, the opposing surface 230 of the cover 200 can be applied to the surface 130 of the slide 100. The slide 100 and/or the cover 200 can be inserted through the port 470 and into the cavity 420 of the holder 400. ing to some embodiments, the cap 300 can be applied to the holder 400. For example, the Surface 330 can be applied to a side of the holder 400, such as the first side 412. In such a configuration, the cap fenestrations 350 can be aligned with the first fenestrations 450. At least one of the support rails 360 of the cap 300 can t and/or rest on one or more of the extensions 430 of the holder 400. The cap 300 can be in thermal conta ct with one or more sides of the holder 400 to fa cilitate heat distribution acro ss the holder 400. The cap 300 can pro vide thermal control through intimate contact with an etched foil that is equipped with resistance temperature detectors, thermocouples, and/or other sensors. Features {e.g., thermocouples and/or s) of the cap 300 can be connected to one or more control components, such as a PLC controller. By heating in a homogeneous fashion, the cap 300 allows a continuous and gradientfree heat transfer along a region of contact with the holder 400. The cap 300 can shield the holder 400 from a cool draft of a microscope purge. One or more of the features of the cap 300, as bed herein, can be provided exclusively on the cap 300 and not on the holder 400, such that the one or more features can be omitted when the holder 400 is used without the cap 300.

Additionally, es of the cap 300 connecting to other ents of the system (e.g., PLC controller, etc.) can remain connected to a cap 300 while various holders 400 can each be used in conjunction with the cap 300 without ing connection and disconnection of the es.

According to some embodiments, with the contents (not shown in ) within the cavity 420 of the holder 400, the block 500 can be inserted into the receptacl e 480 of the holder 400 to secure the contents within the cavity 420 The cap 300 can e an etched foil heater that controls the ature of samples in the slide. The etched foil heater spans a surface of the cap 300. A foil layer of the heater can be n one or both of a base laminate layer and a cover laminate layer. The laminate layers can include a dielectric material (e.g., polyimide) for electrically isolating the etched foil from the cap 300 and/or other structures. The etched foil can have a pair of terminals with leads on a side of the cap 300 that allow a user to connect a power supply (e.g, DC power) to the etched foil heater. Between the terminals, the etched foil heater can define a pathway that traverses the cap 300. For e, the pathway can extend about and between each and every one of the cap fenestrations 350. The etched foil heater can distribute heat evenly across the cap 300 and thereby transfer heat evenly to the slide, so that temperature gradients across the cap 300 and the slide are reduced or eliminated. The etched foil heater can have a ature threshold, above which the heater activity will cease automatically. According to some embodiments, a base (not shown) similar to the cap 300 can be provided for application to the cover 200. The base can be provided on a side of the cover opposite the cap 300. The base can be a mirror image of the cap 300 and have fe atur es that are identical to that of cap 300. For example, the base can inclu de fe nestra tions that ali gn with the second fenestration 460. By fur ther e, the base can include an etched foil heater.

According to some embodiments, for example as shown in , the components described herein can be stacked to align a reference or sample with an imaging axis 1000. Along the imaging axis 1000, the cap fenestration 350, the first fenestration 450, the cover 200, the slide 100 (including the sample region 160), and/or the second fenestration 460 can be aligned.

Electromagnetic radiation can be transmitted through each of the above components in either or both directions along the imaging axis 1000. [0067 ing to some embodiments, for example as shown in FIGS 7-9, any number of samples can be evaluated by providing an appropriate number of wells in a slide and corresponding ures to accommodate the wells. For example, as shown in the slide 100 can include a substrate that forms a plurality of wells 150 provided in te rows 140. As shown in three rows 140 can be provided. For example, a fist row 140 can include a first number of wells 150, a second row 140 can include a second number of wells 150, and a third row 140 can include a third number of wells 150. The rows 140 can include the same number of wells 150 or different numbers of wells 150. According to some embodiments, the cap 300 can include a number of cap fenestrations 350 that ponds to the number of rows 140 of the slide 100. For example, three cap fenestrations 350 can form windows that are configured to be aligned with the three rows 140 of the slide 100. According to some ments, the holder 400 can include a number of first fenestrations 450 and second fenestrations 460 that that correspond to the number of rows 140 of the slide 100. For example, three first fenestrations 450 and three second fenestrations 460 can form s that are configured to be aligned with the three rows 140 of the slide 100 10068] According to some embodiments, for example as shown in FIGS. 10A-11, a plate 700 can receive the holder 400, its contents, and/or the cap 300. The plate 700 can be a component of an imaging device 800 or be configured to attach to an imaging device 800 during operation thereof. The imaging device 800 can include a microscope, a camera, a mirror, a lens, m cascade lasers, or an infrared source with a beam splitter, a detector, a sensor, or combinations thereof. The imaging device 800 can be configured to capture information regarding electromagnetic radiation incident upon the reference or sample within the sample region 160.

The plate 700 can include a body 710 and a window 750 that transmits omagnetic radiation to or from the slide 100. At the location of the window 750, the plate 700 can provide a recessed region for receiving the holder 400. 10069] According to some embodiments, the plate 700 can facilitate alignment to bring the sample region 160 within a field of view and a focal length and/or focal plane of the imaging device 800. For example, the plate 700 can include one or more guide 740 for ing the holder 400. At least a portion of the holder 400, such as the extensions 430, can be received within the guides 740. The holder 400 can move along a positioning axis 790 The positioning axis 790 can be parallel to one or more of the rows 140 of the slide 100. As the holder 400 is moved along the positioning axis 790, a selected one or more of the sampling regions 160 can be aligned to be within a field of view of the imaging device 800. The holder 400 can move along the positioning axis 790 manually or by an automated or mmed ism 80, such as a servo-motor and/or stepper motor. The ism 810 can be controlled by a controller, such as the controller connected to the cap 300. For a variety of positions of the holder 400 within the plate 700, the sample region 160 that is within a field of view of the imaging device 800 will be a fixed and consistent distance (e.g., focal distance) from a reference point (e.g., electromagnetic radiation source) Accordingly, a fixed path length for an electromagnetic radiation beam can be determined and maintained throughout a procedure in which multiple references and/or s are analyzed.

According to some ments, the path length for an electromagnetic radiation beam can vary for multiples samples in multiple wells of a slide. The path length can vary to ensure that sample concentration is the same or similar for ent samples. The path length can be determined based on the Beer-Lambert law (or Beers law), which trates the linear relationship between absorbance and concentration of an ing species. The general Beer­ Lambert law is usually written as: where A is the measured absorbance, cf.) is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the e concentration. When working in concentration units of ty, the Beer-Lambert law is written as: A = e b e where z is the wavelength-dependent molar absorptivity coefficient with units of M' cm' Experimental measurements can be made in terms of transmittance (T), which is d as: T =I/ I% where I is the light intensity after it passes through the sample and Io is the initial light intensity.

The relation between A and T is: A=-log T=-log(/ lo) [0071 Modern absorption instruments can usually display the data as either transmittance, %-transmittance, or absorbance. An unknown concentration of an analyte can be determined by measuring the amount of light that a sample absorbs and applying Beers law. If the absorptivity coefficient is not known, the n concentration can be determined using a g curve of absorbance versus concentration derived from standards. [0072 Calibration of path lengths (i.e., well depths) for wells of a slide can be performed, for example, by analyzing absorbance results. Where the measurements of absorbance can be linearly correlated with the known path lengths and where deviations from this correlation are observed, corrections to the path lengths can be made to account for the lack of ity. The variable path lengths can then compensate for the deviations from the linear ation, so that further tests during a calibration stage provide s that are tent with the linear correlation. 10073] The plate 700, for example as shown in FIGS. 10B, can include features (e.g., recesses 760) to accommodate other components of the sampling system I, for example as shown in FIGS. 11, such as heating elements 830, cooling elements 840 (e.g., Peltier, embedded microfluidic/TEG system), and/or ature sensors 850 (e.g., thermocouples) to uniformly heat and monitor temperature conditions of the plate 700. The plate 700 can also be provided with humidity sensors 860 for determining a humidity level in a sampling compartment of the imaging device 800. Alternatively or in combination, the cap 300 and/or the holder 400 can include features (e.g., recesses 390) to include heating elements, cooling elements (e.g., Peltier), a ture sensors (e.g., couples), and/or tion port s connecting to one or more of the above to uniformly heat and r temperature conditions of the holder 400 and/or its contents. Output of one or more sensors of the system can provide data to a controller 820, such as a Peripheral Interface Controller (PI€") microcontroller. Temperature conditions can be regulated by the controller 820 based on data from sensors. The ller 820 can further control operation of any heat elements are cooling elements to provide uniform temperature conditions across the slide 100. Heat can be transmitted across the plate 700, the holder 400, and/or its ts. At least a portion of the body 710 of the plate 700 can be of a material with high thermal conductivity, sturdy construction, and low electromagnetic radiation tion.

Exemplary materials for the body 710 include anodized aluminum, PTFE, bronze, and copper. 10074] The controller 820 can raise or lower temperatures in multiple (e.g, 3 or more) positions according to a user defined instructions and/or inputs. Once a target temperature has been reached, the assembly (e.g, holder 400) can move to a first measuring position. Multiple (eg., 3 or more) measuring positions can be achieved to capture information with t to all wells in the assembly. At a given target temperature, a measuring cycle can include of the following steps: (I) reach correct temperature in all heaters; (2) send a "ready to measure" signal to the microscope; (3) wait until the microscope returns an "end of ement" signal; (4) move the assembly to the next position. Relative humidity and temperature for each well can be measured and ed. The system can control a servo or stepper motor to move the assembly through appropriate well alignments. Each position can be maintained for a time specified as part of the ment parameters. The limits of assembly movement can be ted by end limit switches. The controller 820 can sense and be ed with an indication when the target well is in position for measurement. The controller 820 can provide a user interface for a user to enter and modify one or more target atures and/or other ters for an experiment. A full cycle can include ascending temperatures and/or descending temperatures. The controller 820 can include a feature that provides an indication (e.g., alarm) to a user based on occurrence of particular conditions, such as failure to maintain temperature during measurements and/or failure to maintain relative humidity within a range 10075] According to some embodiments, multiple (e.g, four) different zones will define the sampli ng arra y of the sampling system 1 which will be fu ll y automated with the servo-motor to all ow fo r the advancement of the holder 400 throu gh the fi eld of view of the g device 800 The control of the servo-motor to predefined positions and the cycling thereof can be facilitated by a controller 820. Thus, data acquisition can be fully automated along with temperature control of the holder 400 and its ts.

According to some embodiments, multiple data acquisition stages can be performed in which a plurality of references and/or samples are evaluated under a first set of conditions (eg., temperature, humidity, etc ), followed by a change in the conditions and further evaluation of at least some of the same references and/or samples under the new conditions. [0077 According to some embodiments, additional data acquisition stages can be performed in which the ity of references and/or samples are returned to the first set of conditions(e.g., temperature, humidity, etc.), followed by further tion to assess reversibility under one or more condition dependencies (e.g., thermal dependency).

According to some embodiments, the sampling system I described herein can be used for determining the mechanism of aggregation and the amount of aggregation in protein, peptide or peptoid formulation, in solution or lyophilized state without the use of probes or additives by performing a Fourier transform infrared (FT-IR) and two-dimensional correlation analysis ("2DCOS") is, for e as described in US. Patent No. 8,268 ,628, hereby incorporated herein by reference. Such is can be performed by a computing system 890. FT-IR spectroscopy allows for a high degree of flexibility and speed in the determination of protein aggregates, with limited manipulation and t the use of exogenous probes. A sample and/or reference of the sampling system I is heated and left to equilibrate ed by spectral acquisition at the desired temperature and the determination of protein, peptide and peptoid, stabili ty, aggregation and viability can be performed. The method can also be applied to the study of lipids, membrane proteins, hydrophilic proteins, peptides and peptoids as a single component or in binary or ternary mixtures with other peptides, or lipid mixtures. 10079 When studying two protein components in a mixture or complex, one of the components can be ically labeled to allow for the aneous detection of each component. FT-IR spectroscopy can be combined with the 2DCOS which allows for determination of the presence of ates and ination of the mechanism of aggregation.

This information can then be used to alter the protein manufacturing process to generate a more viable protein for development. In on, the thermal transition of the protein can also be determined and a 2DCOS plot generated to compare with the established viable n, ng for quality control, ity, and viability of the desired protein product. 10080] According to some embodiments, the sampling system l is not limited to analyzing proteins, es or peptoids, but may be used to analyze any desired ition such as a liquid sample, lipid, or polymers during thermal or other perturbation . According to one aspect of the subject technology, the dual cell holder can be applied to the study of substances (organic or inorganic), materials or reagents, and liquids in general. According to some embodiments, the dual cell holder can he used in spectrophotometers where a shuttle or automated method of sampling is used. According to some embodiments, the use of the sampling system I is not limited to the infrared range, but can also be used in UV and visible range, as well as circular dichroism (CD), vibrational circular dichroism, and Raman spectroscopies, for example, for the analysis of desired materials and substances. 10081] According to some embodiments, the sampling system I can be used to determine n-protein interactions (PPIs") or n-macromolecules (protein-lipid interactions, protein DNA or n-RNA ctions or protein drug interactions). Also, the sampling system l can be used for the analysis of c solutions, polymers, gels, nanostructures or small liquid crystals, etc.

A phrase such as "an aspect" does not imply that such aspect is essential to the t technology or that such aspect s to all configurations of the subject technology. A disclosure relating to an aspect may apply to all configurations, or one or more configurations. An aspect may provide one or more examples of the sure. A phrase such as an aspect"may refer to one or more aspects and vice versa. A phrase such as an embodiment"does not imply that such embodiment is essential to the subject technology or that such embodiment applies to all configurations of the subject technology. A disclosure relating to an embodiment may apply to all embodiments, or one or more embodiments. An embodiment may provide one or more examples of the disclosure. A phrase such an embodiment' may refer to one or more embodiments and vice versa. A phrase such as a configuration? does not imply that such uration is essential to the subject technology or that such configuration applies to all confi gura tions of the subject technology. A discl osur e relating to a confi gura tion may apply to all configurations, or one or more configurations. A configuration may e one or more examples of the disclosure, A phrase such as "a configuration may refer to one or more configurations and vice versa. 10083] The foregoing description is provided to enable a person skilled in the art to practice the various configurations described herein. While the subject technology has been particularly described with reference to the various figures and configurations, it should be understood that these are for illustration purposes only and should not be taken as limiting the scope of the subject technology. 10084] There may be many other ways to implement the subject technology. Various functions and elements described herein may be partitioned differently from those shown without departing from the scope of the subject technology. Various modifications to these configurations will be y apparent to those skilled in the art, and generic principles defined herein may be applied to other configurations. Thus, many changes and modifications may be made to the subject technology, by one having ordinary skill in the art, t departing from the scope of the subject technology.

It is understood that the specific order or hierarchy of steps in the processes disclosed is an illustration of exemplary approaches. Based upon design preferences, it is understood that the specific order or hierarchy of steps in the processes may be nged. Some of the steps may be performed simultaneously. The accompanying method claims present elements of the various steps in a sample order, and are not meant to be limited to the specific order or hierarchy presented. 10086] As used herein, the phrase "at least one of ing a series of items, with the term and" or "or" to separate any of the items, es the list as a whole, rather than each member of the hist (ie., each item). The phrase at least one of does not require selection of at least one of each item listed; rather, the phrase allows a meaning that includes at least one of any one of the items, and/or at least one of any combination of the items, and/or at least one of each of the items. By way of example, the phrases "at least one of A, B, and C or at least one of A, B, or C each refer to only A, only B, or only C; any combination of A, B, and C; and/or at least one of each of A, B, and C. 10087} Terms such as "top," bottom, "front," rear and the like as used in this disclosure should be tood as referring to an arbitrary frame of reference, rather than to the ordinary gravitational frame of reference. Thus, a top surface, a bottom surface, a front surface, and a rear surface may extend upwardly, rdly, diagonally, or horizontally in a gravitational frame of reference 10088] Furthermore, to the extent that the term include," "have," or the like is used in the description or the claims, such term is intended to be inclusive in a manner similar to the term comprise as comprise is interpreted when ed as a transitional word in a claim. 10089} The word "exemplary"is used herein to mean ng as an example, instance, or illustration." Any embodiment bed herein as "exemplary is not necessarily to be construed as preferred or advantageous over other embodiments.

A reference to an element in the singular is not intended to mean "one and only one unless specifically stated, but rather "one or more." Pronouns in the masculine (e.g., his) include the feminine and neuter gender (e.g., her and its) and vice versa. The term "some" refers to one or more. Underlined and/or italicized headings and subheadings are used for convenience only, do not limit the subject technology, and are not referred to in connection with the interpretation of the description of the subject technology. All structural and functional lents to the elements of the various configurations described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly orated herein by reference and intended to be encompassed by the subject technology. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of wheth er such disclosure is explicitly d in the above description. 10091] While certain aspects and embodiments of the subject technology have been described, these have been presented by way of example only, and are not ed to limit the scope of the subject technology. , the novel methods and systems described herein may be embodied in a variety of other forms without departing from the spirit thereof. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the subject technology.

Claims (28)

WHAT IS D IS:

1. A system sing a slide sing: a substrate forming a plurality of wells that are recessed relative to a surface of the substrate; a holder, comprising: a body defining a cavity between a first side and a second side of the body; a port for receiving the slide into the cavity; one or more first fenestrations on the first side; and one or more second fenestrations on the second side.

2. The system of claim 1, further comprising a block configured to secure the slide within the cavity when the block is placed within the port.

3. The system of claim 1, r comprising a cover configured to enclose each of the wells when placed upon the surface of the slide.

4. The system of claim 3, wherein the cover is configured to transmit electromagnetic radiation.

5. The system of claim 3, wherein the cover and the slide have a substantially equal thickness in a direction orthogonal to the surface of the slide when the cover is placed upon the surface of the slide.

6. The system of claim 1, wherein the body of the holder absorbs substantially all electromagnetic radiation incident to the holder.

7. The system of claim 1, wherein the plurality of wells are provided in a plurality of rows, wherein each row comprises at least two of the plurality of wells.

8. The system of claim 7, wherein the one or more first fenestrations comprises a number of first windows equal to a number of the plurality of rows, and the one or more second fenestrations comprises a number of second windows equal to the number of the plurality of rows.

9. The system of claim 8, wherein one of the first s and one of the second s are on opposite sides of one of the plurality of rows when the slide is within the holder.

10. The system of claim 1, wherein the port is disposed on a third side of the holder.

11. The system of claim 1, n the first side is opposite the second side.

12. The system of claim 1, wherein at least one of the plurality of wells, the one or more first fenestrations, and the one or more second fenestrations are aligned along an axis when the slide is within the .

13. The system of claim 1, wherein the slide, the one or more first rations, and the one or more second fenestrations are configured to transmit electromagnetic

14. The system of claim 1, further comprising a coating on an inner surface of the body.

15. The system of claim 14, wherein the coating comprises silicone.

16. The system of claim 1, further comprising a cap in l contact with an outer surface of the holder, the cap comprising one or more third fenestrations.

17. The system of claim 1, further comprising a plate attached to an imaging device.

18. A method comprising: providing s to each of a plurality of wells formed in a slide, each of the wells being recessed relative to a surface of the slide; ing the wells by applying a cover to the surface of the slide; inserting the slide and the cover into a cavity of a holder; and emitting electromagnetic radiation through one or more first fenestrations of the holder, one or more second fenestrations of the holder, and the sample.

19. The method of claim 18, further comprising inserting the holder into a receptacle of a plate attached to an imaging device.

20. The method of claim 19, wherein inserting the holder comprises oning the sample at a focal length of the imaging device.

21. The method of claim 18, further comprising heating the samples to a target temperature by conducting heat through the holder.

22. The method of claim 18, further comprising placing a cap in thermal contact with an outer surface of the holder.

23. The method of claim 22, wherein the emitting the electromagnetic radiation comprises ng the electromagnetic radiation h one or more third fenestrations of the cap.

24. The method of claim 18, n the inserting the slide and the cover comprises inserting the slide and the cover through a port of the holder.

25. The method of claim 24, r comprising, after inserting the slide in the cover, cting the port with a block.

26. The method of claim 18, wherein the electromagnetic radiation is infrared light.

27. The method of claim 18, further comprising, after the emitting the electromagnetic radiation, detecting a characteristic of the electromagnetic radiation not absorbed by the sample.

28. The method of claim 18, further comprising: after the emitting the electromagnetic radiation, changing the temperature of the sample; and emitting additional electromagnetic radiation through the one or more first fenestrations and the one or more second fenestrations of the holder and through the sample. WO 84886 122 150 150 100 150 150 150 110 130 120 150 150 150 120 150 150 150 120 ·, ·-, ·-, I -.... -.... l a - .... l a - .... ! « ! s f l ) I f l ) I f l ) I f l ) I f "l s) I f "l s) I f "l s f l f l \ \ --- \ \ --- \ \ \ \ \ \ ) I ) I ) I ' ......,,/ ' ......,,/ ---4 , ......,,/ ~ '..... .,,/--- \ \'..... .,,/--- \ \ --- \ \ 4 '..... - \ \'...... .,,/--.... -.... -.... -.... - .... -.... -.... -.... - .... %fl" ) I fl" ) I fl" " + fl fl" fl fl" fl" fl" \ \-' \ \ \ \ ) I \ \ ) I ) I \ \ \ \---) I ) I ) I ) I -1? z°? ,-- ' ✓--- '-.,,,/--- ' \ \ \ \ \ \ } ,.......,,.,--- ' ,-- ' ✓ } a --- 150 150 150 110 130 120 : 174 4 150 WO 84886 WO 84886 o c uo co co co co c■ co c■ uo LL LL o o 0o co 0o 0o o o co 0o c■ WO 84886 400-, 400-, WO 84886 400-, 400-, 412 410 400-, 412 410 WO 84886 uo co c uc uc c■ c■ ' LL r LL o o o o uo uo <( G uc o CV uc c■ uo c■ o r o o uo o WO 84886 o <( CV co