d42ac499a016c933397a2d97a648b2fc pharmaceutics_14_02138_v2.pdf 7afef3def40776ea33ba4701b2a5485c25b2812e pharmaceutics_14_02138_v2.pdf 7cf52454ac4670104a0c6b2b3dc46dd47bbb4f24ae3f4a64bf57739b8a96b57a pharmaceutics_14_02138_v2.pdf Title: Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation Subject: The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a representative example, we performed a preparative purification of this enzyme. The purified protein is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput functional and structural studies. Keywords: ECM proteins; sodium heparin; heparin binding; enhanced protein expression; carboxypeptidases; mammalian cells; HEK 293F cells; carboxypeptidase A6; carboxypeptidase Z; thrombin-activable fibrinolysis inhibitor Author: Javier Garcia-Pardo, Sergi Montané, Francesc Xavier Avilés, Sebastian Tanco and Julia Lorenzo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Oct 25 05:52:59 2022 CEST ModDate: Tue Oct 25 06:00:24 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2096352 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- DSWABJ+URWPalladioL-Bold Type 1 Custom yes yes no 10 0 EKFZSD+URWPalladioL-Roma Type 1 Custom yes yes no 15 0 WRAEKA+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 BTFGED+VnURWPalladioL-Bold Type 1 Custom yes yes no 23 0 NBMIUS+VnURWPalladioL Type 1 Custom yes yes no 28 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 47 0 UWJUYN+CMSY10 Type 1 Builtin yes yes no 75 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 79 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 100 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 104 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 108 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 112 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 128 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 132 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 136 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 140 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 156 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 160 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 164 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 168 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 184 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 188 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 192 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 196 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 212 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 216 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 220 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 224 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-08-01 02:36:16 CEST RepresentationInformation: pharmaceutics_14_02138_v2.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-07-31 12:06:36 CEST Size: 2096352 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 439 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Plasmids and Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Step-by-Step Protein Expression Procedure Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Optimized Protein Purification Protocol Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Children: Item: Title: Purification Step-1 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Purification Step-2 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Purification Step-3 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Cytotoxicity Experiments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Enzyme Activity Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Statistical Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Children: Item: Title: Heparin as an Additive to Enhance Recombinant Protein Expression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Enhanced Expression of Biomedically Relevant ECM/Heparin-Binding Proteins by Heparin Supplementation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Our Approach Is Compatible with a Straightforward Purification of ECM/Heparin-Binding Proteins Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Considerations for Selecting ECM/Heparin-Binding Proteins Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Info: Title: Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation Author: Javier Garcia-Pardo, Sergi Montané, Francesc Xavier Avilés, Sebastian Tanco and Julia Lorenzo Subject: The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a representative example, we performed a preparative purification of this enzyme. The purified protein is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput functional and structural studies. Keywords: ECM proteins; sodium heparin; heparin binding; enhanced protein expression; carboxypeptidases; mammalian cells; HEK 293F cells; carboxypeptidase A6; carboxypeptidase Z; thrombin-activable fibrinolysis inhibitor Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Oct 25 05:52:59 CEST 2022 ModDate: Tue Oct 25 06:00:24 CEST 2022 ID: 0x630b022844c3a53411e6e34b32e98f9f, 0x630b022844c3a53411e6e34b32e98f9f Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: WRAEKA+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: WRAEKA+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: BTFGED+VnURWPalladioL-Bold FontSubset: true FirstChar: 178 LastChar: 178 FontDescriptor: FontName: BTFGED+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: DSWABJ+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: DSWABJ+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: UWJUYN+CMSY10 FontSubset: true FirstChar: 0 LastChar: 21 FontDescriptor: FontName: UWJUYN+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: NBMIUS+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 243 FontDescriptor: FontName: NBMIUS+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: EKFZSD+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: EKFZSD+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZXDKIP+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ZXDKIP+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 64, 323, 152, 332 Annotation: Subtype: Link Rect: 34, 311, 109, 320 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 168, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 130, 642, 141, 655 Annotation: Subtype: Link Rect: 214, 642, 225, 655 Annotation: Subtype: Link Rect: 332, 642, 343, 655 Annotation: Subtype: Link Rect: 422, 642, 433, 655 Annotation: Subtype: Link Rect: 521, 642, 532, 655 Annotation: Subtype: Link Rect: 484, 256, 491, 265 ActionDest: 236 Annotation: Subtype: Link Rect: 494, 256, 501, 265 ActionDest: 236 Annotation: Subtype: Link Rect: 272, 218, 279, 227 ActionDest: 236 Annotation: Subtype: Link Rect: 280, 218, 287, 227 ActionDest: 236 Annotation: Subtype: Link Rect: 344, 193, 351, 202 ActionDest: 236 Annotation: Subtype: Link Rect: 351, 193, 358, 202 ActionDest: 236 Annotation: Subtype: Link Rect: 359, 193, 366, 202 ActionDest: 236 Annotation: Subtype: Link Rect: 465, 168, 472, 177 ActionDest: 236 Annotation: Subtype: Link Rect: 472, 168, 479, 177 ActionDest: 236 Annotation: Subtype: Link Rect: 399, 143, 406, 152 ActionDest: 236 Annotation: Subtype: Link Rect: 406, 143, 413, 152 ActionDest: 236 Annotation: Subtype: Link Rect: 386, 117, 392, 126 ActionDest: 236 Annotation: Subtype: Link Rect: 298, 92, 305, 101 ActionDest: 236 Annotation: Subtype: Link Rect: 306, 92, 313, 101 ActionDest: 236 Annotation: Subtype: Link Rect: 305, 80, 317, 89 ActionDest: 236 Annotation: Subtype: Link Rect: 318, 80, 330, 89 ActionDest: 236 Annotation: Subtype: Link Rect: 135, 17, 311, 28 Annotation: Subtype: Link Rect: 385, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 420, 87, 439 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 389, 698, 401, 707 ActionDest: 236 Annotation: Subtype: Link Rect: 401, 698, 413, 707 ActionDest: 236 Annotation: Subtype: Link Rect: 343, 673, 355, 682 ActionDest: 239 Annotation: Subtype: Link Rect: 182, 648, 194, 657 ActionDest: 236 Annotation: Subtype: Link Rect: 195, 648, 207, 657 ActionDest: 239 Annotation: Subtype: Link Rect: 487, 635, 499, 644 ActionDest: 239 Annotation: Subtype: Link Rect: 499, 635, 511, 644 ActionDest: 239 Annotation: Subtype: Link Rect: 507, 623, 519, 631 ActionDest: 239 Annotation: Subtype: Link Rect: 320, 610, 332, 619 ActionDest: 239 Annotation: Subtype: Link Rect: 422, 585, 434, 594 ActionDest: 239 Annotation: Subtype: Link Rect: 435, 585, 447, 594 ActionDest: 239 Annotation: Subtype: Link Rect: 225, 560, 237, 569 ActionDest: 239 Annotation: Subtype: Link Rect: 529, 245, 541, 254 ActionDest: 239 Annotation: Subtype: Link Rect: 544, 245, 556, 254 ActionDest: 239 Annotation: Subtype: Link Rect: 520, 207, 532, 216 ActionDest: 239 Annotation: Subtype: Link Rect: 427, 157, 439, 166 ActionDest: 239 Page: Sequence: 3 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 485, 144, 497, 154 ActionDest: 239 Annotation: Subtype: Link Rect: 498, 145, 509, 154 ActionDest: 239 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 361, 622, 373, 631 ActionDest: 239 Annotation: Subtype: Link Rect: 252, 371, 264, 380 ActionDest: 239 Annotation: Subtype: Link Rect: 295, 269, 307, 278 ActionDest: 239 Annotation: Subtype: Link Rect: 208, 219, 220, 228 ActionDest: 239 Annotation: Subtype: Link Rect: 221, 219, 232, 228 ActionDest: 239 Annotation: Subtype: Link Rect: 423, 191, 430, 203 ActionDest: 92 Annotation: Subtype: Link Rect: 288, 141, 295, 153 ActionDest: 92 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 230, 735, 242, 744 ActionDest: 239 Annotation: Subtype: Link Rect: 493, 720, 500, 732 ActionDest: 120 Annotation: Subtype: Link Rect: 515, 723, 527, 732 ActionDest: 239 Annotation: Subtype: Link Rect: 527, 723, 539, 732 ActionDest: 239 Annotation: Subtype: Link Rect: 238, 708, 245, 720 ActionDest: 120 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 549, 227, 556, 239 ActionDest: 148 Annotation: Subtype: Link Rect: 408, 202, 416, 214 ActionDest: 148 Annotation: Subtype: Link Rect: 443, 205, 455, 214 ActionDest: 239 Annotation: Subtype: Link Rect: 458, 205, 470, 214 ActionDest: 239 Annotation: Subtype: Link Rect: 191, 105, 203, 113 ActionDest: 239 Annotation: Subtype: Link Rect: 304, 105, 316, 113 ActionDest: 239 Annotation: Subtype: Link Rect: 472, 104, 484, 113 ActionDest: 239 Annotation: Subtype: Link Rect: 522, 102, 529, 114 ActionDest: 148 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 348, 180, 359, 189 ActionDest: 239 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 492, 644, 500, 656 ActionDest: 176 Annotation: Subtype: Link Rect: 546, 644, 554, 656 ActionDest: 176 Annotation: Subtype: Link Rect: 512, 621, 524, 631 ActionDest: 239 Annotation: Subtype: Link Rect: 501, 531, 508, 543 ActionDest: 176 Annotation: Subtype: Link Rect: 375, 518, 382, 530 ActionDest: 176 Annotation: Subtype: Link Rect: 425, 506, 432, 518 ActionDest: 176 Annotation: Subtype: Link Rect: 331, 483, 343, 493 ActionDest: 239 Annotation: Subtype: Link Rect: 343, 483, 355, 493 ActionDest: 239 Annotation: Subtype: Link Rect: 456, 123, 467, 131 ActionDest: 239 Annotation: Subtype: Link Rect: 451, 85, 462, 93 ActionDest: 239 Annotation: Subtype: Link Rect: 463, 85, 474, 93 ActionDest: 242 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 544, 720, 556, 729 ActionDest: 242 Annotation: Subtype: Link Rect: 236, 692, 243, 704 ActionDest: 120 Annotation: Subtype: Link Rect: 544, 657, 556, 666 ActionDest: 239 Annotation: Subtype: Link Rect: 381, 582, 393, 591 ActionDest: 242 Annotation: Subtype: Link Rect: 425, 554, 432, 566 ActionDest: 204 Annotation: Subtype: Link Rect: 447, 557, 459, 566 ActionDest: 239 Annotation: Subtype: Link Rect: 459, 557, 471, 566 ActionDest: 242 Annotation: Subtype: Link Rect: 471, 557, 483, 566 ActionDest: 242 Annotation: Subtype: Link Rect: 244, 529, 251, 541 ActionDest: 204 Annotation: Subtype: Link Rect: 545, 141, 556, 149 ActionDest: 239 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 196, 708, 202, 720 ActionDest: 148 Annotation: Subtype: Link Rect: 252, 683, 259, 695 ActionDest: 204 Annotation: Subtype: Link Rect: 505, 497, 517, 506 ActionDest: 239 Annotation: Subtype: Link Rect: 228, 446, 240, 455 ActionDest: 239 Annotation: Subtype: Link Rect: 460, 434, 472, 443 ActionDest: 242 Annotation: Subtype: Link Rect: 255, 409, 267, 418 ActionDest: 242 Annotation: Subtype: Link Rect: 465, 396, 477, 405 ActionDest: 242 Annotation: Subtype: Link Rect: 199, 356, 206, 368 ActionDest: 120 Annotation: Subtype: Link Rect: 226, 356, 232, 368 ActionDest: 148 Annotation: Subtype: Link Rect: 454, 346, 466, 355 ActionDest: 239 Annotation: Subtype: Link Rect: 384, 293, 391, 305 ActionDest: 204 Annotation: Subtype: Link Rect: 189, 246, 196, 255 ActionDest: 236 Annotation: Subtype: Link Rect: 471, 208, 478, 217 ActionDest: 236 Annotation: Subtype: Link Rect: 342, 158, 354, 167 ActionDest: 239 Annotation: Subtype: Link Rect: 355, 158, 366, 167 ActionDest: 242 Annotation: Subtype: Link Rect: 367, 158, 379, 167 ActionDest: 242 Annotation: Subtype: Link Rect: 207, 133, 219, 142 ActionDest: 239 Annotation: Subtype: Link Rect: 220, 133, 231, 142 ActionDest: 239 Annotation: Subtype: Link Rect: 232, 133, 244, 142 ActionDest: 239 Annotation: Subtype: Link Rect: 249, 107, 261, 117 ActionDest: 242 Annotation: Subtype: Link Rect: 262, 107, 274, 117 ActionDest: 242 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 277, 733, 284, 745 ActionDest: 176 Annotation: Subtype: Link Rect: 301, 647, 313, 657 ActionDest: 239 Annotation: Subtype: Link Rect: 314, 647, 326, 657 ActionDest: 239 Annotation: Subtype: Link Rect: 326, 648, 338, 657 ActionDest: 242 Annotation: Subtype: Link Rect: 536, 558, 560, 569 Annotation: Subtype: Link Rect: 165, 546, 424, 557 Annotation: Subtype: Link Rect: 266, 296, 303, 307 Annotation: Subtype: Link Rect: 479, 284, 516, 295 Annotation: Subtype: Link Rect: 348, 261, 385, 272 Annotation: Subtype: Link Rect: 59, 239, 96, 249 Annotation: Subtype: Link Rect: 398, 215, 436, 226 Annotation: Subtype: Link Rect: 59, 182, 96, 192 Annotation: Subtype: Link Rect: 392, 169, 430, 180 Annotation: Subtype: Link Rect: 417, 146, 455, 157 Annotation: Subtype: Link Rect: 179, 112, 217, 123 Annotation: Subtype: Link Rect: 389, 89, 427, 100 Annotation: Subtype: Link Rect: 478, 77, 515, 88 Annotation: Subtype: Link Rect: 521, 77, 557, 88 Annotation: Subtype: Link Rect: 349, 66, 387, 77 Annotation: Subtype: Link Rect: 393, 66, 429, 77 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 144, 722, 182, 733 Annotation: Subtype: Link Rect: 188, 722, 224, 733 Annotation: Subtype: Link Rect: 104, 688, 142, 698 Annotation: Subtype: Link Rect: 401, 676, 438, 687 Annotation: Subtype: Link Rect: 312, 652, 349, 664 Annotation: Subtype: Link Rect: 59, 630, 96, 640 Annotation: Subtype: Link Rect: 354, 606, 392, 617 Annotation: Subtype: Link Rect: 135, 573, 173, 583 Annotation: Subtype: Link Rect: 445, 560, 483, 571 Annotation: Subtype: Link Rect: 268, 537, 306, 548 Annotation: Subtype: Link Rect: 312, 537, 348, 548 Annotation: Subtype: Link Rect: 59, 504, 96, 514 Annotation: Subtype: Link Rect: 102, 504, 138, 514 Annotation: Subtype: Link Rect: 82, 469, 119, 479 Annotation: Subtype: Link Rect: 339, 435, 377, 445 Annotation: Subtype: Link Rect: 117, 400, 155, 410 Annotation: Subtype: Link Rect: 196, 365, 233, 376 Annotation: Subtype: Link Rect: 351, 342, 388, 353 Annotation: Subtype: Link Rect: 443, 308, 481, 318 Annotation: Subtype: Link Rect: 147, 274, 185, 284 Annotation: Subtype: Link Rect: 518, 261, 556, 272 Annotation: Subtype: Link Rect: 244, 238, 282, 249 Annotation: Subtype: Link Rect: 378, 204, 415, 215 Annotation: Subtype: Link Rect: 59, 170, 96, 180 Annotation: Subtype: Link Rect: 383, 146, 421, 157 Annotation: Subtype: Link Rect: 372, 135, 501, 146 Annotation: Subtype: Link Rect: 59, 101, 96, 111 Annotation: Subtype: Link Rect: 174, 78, 212, 88 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 171, 722, 208, 733 Annotation: Subtype: Link Rect: 490, 710, 527, 721 Annotation: Subtype: Link Rect: 131, 688, 168, 698 Annotation: Subtype: Link Rect: 319, 653, 357, 664 Annotation: Subtype: Link Rect: 59, 630, 96, 640 Annotation: Subtype: Link Rect: 102, 630, 138, 640 Annotation: Subtype: Link Rect: 59, 607, 96, 617 Annotation: Subtype: Link Rect: 250, 584, 288, 594 Annotation: Subtype: Link Rect: 294, 584, 330, 594 Annotation: Subtype: Link Rect: 113, 561, 150, 571 Annotation: Subtype: Link Rect: 153, 538, 190, 548 Checksum: 0c8983d6 Type: CRC32 Checksum: d42ac499a016c933397a2d97a648b2fc Type: MD5 Checksum: 7afef3def40776ea33ba4701b2a5485c25b2812e Type: SHA-1