e47d38cf11b39c322c7749de571a9aee AppSci_a2021v11n14p6258.pdf 006883d40f530587eb049b4a76a8c3ffbd9dd610 AppSci_a2021v11n14p6258.pdf b80b108dee8ccc9e44c41611355e2be92fab612dbb6fe1477622e15f86d54d3e AppSci_a2021v11n14p6258.pdf Title: The Effect of the Expression of the Antiapoptotic BHRF1 Gene on the Metabolic Behavior of a Hybridoma Cell Line Subject: One of the most important limitations of mammalian cells-based bioprocesses, and particularly hybridoma cell lines, is the accelerated metabolism related to glucose and glutamine consumption. The high uptake rates of glucose and glutamine (i.e., the main sources of carbon, nitrogen and energy) lead to the production and accumulation of large amounts of lactate and ammonia in culture broth. Lactate and/or ammonia accumulation, together with the depletion of the main nutrients, are the major causes of apoptosis in hybridoma cell cultures. The KB26.5 hybridoma cell line, producing an IgG3, was engineered with BHRF1 (KB26.5-BHRF1), an Epstein–Barr virus-encoded early protein homologous to the antiapoptotic protein Bcl-2, with the aim of protecting the hybridoma cell line from apoptosis. Surprisingly, besides achieving effective protection from apoptosis, the expression of BHRF1 modified the metabolism of the hybridoma cell line. Cell physiology and metabolism analyses of the original KB26.5 and KB26.5-BHRF1 revealed an increase of cell growth rate, a reduction of glucose and glutamine consumption, as well as a decrease in lactate secretion in KB26.5-BHRF1 cells. A flux balance analysis allowed us to quantify the intracellular fluxes of both cell lines. The main metabolic differences were identified in glucose consumption and, consequently, the production of lactate. The lactate production flux was reduced by 60%, since the need for NADH regeneration in the cytoplasm decreased due to a more than 50% reduction in glucose uptake. In general terms, the BHRF1 engineered cell line showed a more efficient metabolism, with an increase in biomass volumetric productivity under identical culture conditions. Keywords: hybridoma; genome-scale metabolic model; antiapoptotic gene; mitochondrial transport; BHRF1 Author: Iván Martínez-Monge, Pere Comas, David Catalán-Tatjer, Jordi Prat, Antoni Casablancas, Carlos Paredes, Martí Lecina and Jordi Joan Cairó Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Jul 7 06:02:24 2021 CEST ModDate: Wed Jul 7 06:06:43 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2752011 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ECICOX+VnURWPalladioL Type 1 Custom yes yes no 10 0 AXSDKV+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 EZSBOU+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 SHBEWM+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 PRJKGQ+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 CRVMKC+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 36 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 58 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 62 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 66 0 MJSSOL+CMSY10 Type 1 Builtin yes yes no 81 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 85 0 WCFTGI+CMR10 Type 1 Builtin yes yes no 92 0 NTILRM+MSAM10 Type 1 Builtin yes yes no 103 0 AGPKIH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 112 0 AGPKJI+PalatinoLinotype CID TrueType Identity-H yes yes yes 115 0 AGPKJJ+PalatinoLinotype TrueType WinAnsi yes yes no 121 0 AGPKJL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 124 0 AGPMKA+TimesNewRoman CID TrueType Identity-H yes yes yes 127 0 AGPMMA+Arial CID TrueType Identity-H yes yes yes 133 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 144 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 148 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 152 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 156 0 TimesNewRomanPSMT CID TrueType Identity-H yes no yes 163 0 ArialMT TrueType WinAnsi no no no 173 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 180 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 184 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 188 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 192 0 TimesNewRomanPSMT CID TrueType Identity-H yes no yes 199 0 TimesNewRomanPSMT TrueType WinAnsi no no no 207 0 ArialMT TrueType WinAnsi no no no 212 0 AGPKIH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 223 0 AGPKJI+PalatinoLinotype CID TrueType Identity-H yes yes yes 226 0 AGPKJJ+PalatinoLinotype TrueType WinAnsi yes yes no 232 0 AGPKJL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 235 0 AGPMKA+TimesNewRoman CID TrueType Identity-H yes yes yes 238 0 AGPMMA+Arial CID TrueType Identity-H yes yes yes 244 0 AGPKIG+PalatinoLinotype,Bold CID TrueType Identity-H yes yes yes 257 0 AGPKIH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 263 0 AGPKJI+PalatinoLinotype CID TrueType Identity-H yes yes yes 266 0 AGPKJJ+PalatinoLinotype TrueType WinAnsi yes yes no 272 0 AGPKJL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 275 0 AGPKIH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 285 0 AGPKJJ+PalatinoLinotype TrueType WinAnsi yes yes no 288 0 AGPKJL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 291 0 AGPKIH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 301 0 AGPKJJ+PalatinoLinotype TrueType WinAnsi yes yes no 304 0 AGPKJL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 307 0 AGPKIH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 317 0 AGPKJJ+PalatinoLinotype TrueType WinAnsi yes yes no 320 0 AGPKJL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 323 0 AGPMKA+TimesNewRoman CID TrueType Identity-H yes yes yes 326 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 347 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2023-12-05 02:35:23 CET RepresentationInformation: AppSci_a2021v11n14p6258.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-12-04 19:13:59 CET Size: 2752011 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 530 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Cell Lines and Cell Maintenance Destination: subsection.2.1 Item: Title: Culture Medium Destination: subsection.2.2 Item: Title: Shake-Flasks Culturing Platform Destination: subsection.2.3 Item: Title: Bioreactor and Operational Conditions Destination: subsection.2.4 Item: Title: Analytical Methods Destination: subsection.2.5 Children: Item: Title: Cell Number Destination: subsubsection.2.5.1 Item: Title: Metabolite Concentrations Destination: subsubsection.2.5.2 Item: Title: Product Concentration Destination: subsubsection.2.5.3 Item: Title: Oxygen Uptake Rate (OUR) Destination: subsection.2.6 Item: Title: Estimation of Specific Consumption and Production Rates Destination: subsection.2.7 Item: Title: Reduced Genome-Scale Metabolic Model Destination: subsection.2.8 Item: Title: Flux Balance Analysis of the Reduced Model Destination: subsection.2.9 Item: Title: Results Destination: section.3 Children: Item: Title: Effect of BHRF1 on Cell Physiology Destination: subsection.3.1 Children: Item: Title: Noncontrolled Culture Conditions: Shake Flask Cultures Destination: subsubsection.3.1.1 Item: Title: Controlled Bioreactor Cultures Destination: subsubsection.3.1.2 Item: Title: Effect of BHRF1 on Cell Metabolism: Flux Balance Analysis Destination: subsection.3.2 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: The Effect of the Expression of the Antiapoptotic BHRF1 Gene on the Metabolic Behavior of a Hybridoma Cell Line Author: Iván Martínez-Monge, Pere Comas, David Catalán-Tatjer, Jordi Prat, Antoni Casablancas, Carlos Paredes, Martí Lecina and Jordi Joan Cairó Subject: One of the most important limitations of mammalian cells-based bioprocesses, and particularly hybridoma cell lines, is the accelerated metabolism related to glucose and glutamine consumption. The high uptake rates of glucose and glutamine (i.e., the main sources of carbon, nitrogen and energy) lead to the production and accumulation of large amounts of lactate and ammonia in culture broth. Lactate and/or ammonia accumulation, together with the depletion of the main nutrients, are the major causes of apoptosis in hybridoma cell cultures. The KB26.5 hybridoma cell line, producing an IgG3, was engineered with BHRF1 (KB26.5-BHRF1), an Epstein Barr virus-encoded early protein homologous to the antiapoptotic protein Bcl-2, with the aim of protecting the hybridoma cell line from apoptosis. Surprisingly, besides achieving effective protection from apoptosis, the expression of BHRF1 modified the metabolism of the hybridoma cell line. Cell physiology and metabolism analyses of the original KB26.5 and KB26.5-BHRF1 revealed an increase of cell growth rate, a reduction of glucose and glutamine consumption, as well as a decrease in lactate secretion in KB26.5-BHRF1 cells. A flux balance analysis allowed us to quantify the intracellular fluxes of both cell lines. The main metabolic differences were identified in glucose consumption and, consequently, the production of lactate. The lactate production flux was reduced by 60%, since the need for NADH regeneration in the cytoplasm decreased due to a more than 50% reduction in glucose uptake. In general terms, the BHRF1 engineered cell line showed a more efficient metabolism, with an increase in biomass volumetric productivity under identical culture conditions. Keywords: hybridoma; genome-scale metabolic model; antiapoptotic gene; mitochondrial transport; BHRF1 Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Jul 07 06:02:24 CEST 2021 ModDate: Wed Jul 07 06:06:43 CEST 2021 ID: 0xfea2b050f989276c09dbead6bd45ee7a, 0xfea2b050f989276c09dbead6bd45ee7a Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: MJSSOL+CMSY10 FontSubset: true FirstChar: 0 LastChar: 15 FontDescriptor: FontName: MJSSOL+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: EZSBOU+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: EZSBOU+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: NTILRM+MSAM10 FontSubset: true FirstChar: 6 LastChar: 78 FontDescriptor: FontName: NTILRM+MSAM10 Flags: Symbolic FontBBox: 8, -463, 1331, 1003 FontFile: true Font: BaseFont: SHBEWM+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: SHBEWM+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ECICOX+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: ECICOX+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: PRJKGQ+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: PRJKGQ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: WCFTGI+CMR10 FontSubset: true FirstChar: 61 LastChar: 93 FontDescriptor: FontName: WCFTGI+CMR10 Flags: Symbolic FontBBox: -40, -250, 1009, 750 FontFile: true Font: BaseFont: AXSDKV+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 121 FontDescriptor: FontName: AXSDKV+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 175, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 155, 659, 166, 673 Annotation: Subtype: Link Rect: 113, 358, 154, 367 Annotation: Subtype: Link Rect: 35, 346, 130, 355 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 286, 170, 293, 178 ActionDest: 344 Annotation: Subtype: Link Rect: 293, 170, 300, 178 ActionDest: 344 Annotation: Subtype: Link Rect: 334, 82, 341, 91 ActionDest: 344 Annotation: Subtype: Link Rect: 342, 82, 349, 91 ActionDest: 344 Annotation: Subtype: Link Rect: 121, 17, 259, 28 Annotation: Subtype: Link Rect: 409, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 458, 87, 477 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 199, 732, 206, 741 ActionDest: 344 Annotation: Subtype: Link Rect: 209, 732, 216, 741 ActionDest: 344 Annotation: Subtype: Link Rect: 218, 682, 225, 691 ActionDest: 344 Annotation: Subtype: Link Rect: 443, 644, 450, 653 ActionDest: 344 Annotation: Subtype: Link Rect: 549, 619, 556, 628 ActionDest: 344 Annotation: Subtype: Link Rect: 482, 594, 494, 603 ActionDest: 344 Annotation: Subtype: Link Rect: 494, 594, 506, 603 ActionDest: 344 Annotation: Subtype: Link Rect: 317, 556, 324, 565 ActionDest: 344 Annotation: Subtype: Link Rect: 368, 531, 380, 540 ActionDest: 344 Annotation: Subtype: Link Rect: 380, 531, 392, 540 ActionDest: 344 Annotation: Subtype: Link Rect: 385, 506, 397, 515 ActionDest: 344 Annotation: Subtype: Link Rect: 400, 506, 412, 515 ActionDest: 344 Annotation: Subtype: Link Rect: 201, 481, 213, 490 ActionDest: 344 Annotation: Subtype: Link Rect: 321, 456, 333, 465 ActionDest: 344 Annotation: Subtype: Link Rect: 362, 431, 373, 440 ActionDest: 344 Annotation: Subtype: Link Rect: 544, 431, 556, 440 ActionDest: 344 Annotation: Subtype: Link Rect: 348, 406, 360, 415 ActionDest: 344 Annotation: Subtype: Link Rect: 345, 381, 357, 390 ActionDest: 344 Annotation: Subtype: Link Rect: 357, 380, 369, 390 ActionDest: 344 Annotation: Subtype: Link Rect: 528, 343, 540, 352 ActionDest: 351 Annotation: Subtype: Link Rect: 378, 293, 390, 302 ActionDest: 351 Annotation: Subtype: Link Rect: 265, 280, 272, 289 ActionDest: 344 Annotation: Subtype: Link Rect: 273, 280, 285, 289 ActionDest: 351 Annotation: Subtype: Link Rect: 278, 268, 290, 277 ActionDest: 351 Annotation: Subtype: Link Rect: 505, 267, 517, 277 ActionDest: 351 Annotation: Subtype: Link Rect: 467, 104, 479, 113 ActionDest: 344 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 532, 678, 544, 687 ActionDest: 344 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 251, 443, 263, 452 ActionDest: 351 Annotation: Subtype: Link Rect: 264, 443, 275, 452 ActionDest: 351 Annotation: Subtype: Link Rect: 212, 392, 224, 402 ActionDest: 351 Annotation: Subtype: Link Rect: 225, 392, 237, 402 ActionDest: 351 Annotation: Subtype: Link Rect: 534, 342, 546, 351 ActionDest: 351 Annotation: Subtype: Link Rect: 198, 138, 210, 147 ActionDest: 351 Annotation: Subtype: Link Rect: 474, 126, 486, 135 ActionDest: 351 Annotation: Subtype: Link Rect: 422, 100, 433, 110 ActionDest: 351 Annotation: Subtype: Link Rect: 434, 100, 446, 110 ActionDest: 351 Annotation: Subtype: Link Rect: 496, 75, 508, 84 ActionDest: 351 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 500, 707, 512, 716 ActionDest: 351 Annotation: Subtype: Link Rect: 211, 619, 223, 628 ActionDest: 351 Annotation: Subtype: Link Rect: 386, 594, 398, 603 ActionDest: 351 Annotation: Subtype: Link Rect: 301, 568, 313, 578 ActionDest: 351 Annotation: Subtype: Link Rect: 446, 481, 458, 490 ActionDest: 351 Annotation: Subtype: Link Rect: 544, 468, 556, 477 ActionDest: 351 Annotation: Subtype: Link Rect: 387, 313, 393, 325 ActionDest: 99 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 452, 268, 459, 280 ActionDest: 215 Annotation: Subtype: Link Rect: 247, 205, 254, 217 ActionDest: 251 Annotation: Subtype: Link Rect: 274, 130, 281, 142 ActionDest: 279 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 308, 338, 320, 347 ActionDest: 351 Annotation: Subtype: Link Rect: 308, 275, 320, 284 ActionDest: 351 Annotation: Subtype: Link Rect: 479, 250, 491, 259 ActionDest: 351 Annotation: Subtype: Link Rect: 360, 210, 367, 222 ActionDest: 279 Annotation: Subtype: Link Rect: 386, 210, 393, 222 ActionDest: 295 Page: Sequence: 8 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 549, 106, 556, 118 ActionDest: 279 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 368, 654, 375, 666 ActionDest: 311 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 544, 556, 556, 565 ActionDest: 344 Annotation: Subtype: Link Rect: 261, 478, 268, 490 ActionDest: 335 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 551, 692, 558, 704 ActionDest: 338 Annotation: Subtype: Link Rect: 333, 313, 345, 322 ActionDest: 351 Annotation: Subtype: Link Rect: 437, 288, 449, 297 ActionDest: 351 Annotation: Subtype: Link Rect: 396, 235, 403, 247 ActionDest: 279 Annotation: Subtype: Link Rect: 406, 235, 412, 247 ActionDest: 338 Annotation: Subtype: Link Rect: 196, 200, 208, 209 ActionDest: 351 Annotation: Subtype: Link Rect: 348, 150, 360, 159 ActionDest: 351 Annotation: Subtype: Link Rect: 451, 150, 463, 159 ActionDest: 344 Annotation: Subtype: Link Rect: 346, 74, 358, 83 ActionDest: 351 Annotation: Subtype: Link Rect: 359, 74, 371, 83 ActionDest: 351 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 378, 732, 390, 741 ActionDest: 351 Annotation: Subtype: Link Rect: 390, 732, 402, 741 ActionDest: 351 Annotation: Subtype: Link Rect: 201, 707, 213, 716 ActionDest: 351 Annotation: Subtype: Link Rect: 523, 594, 535, 603 ActionDest: 351 Annotation: Subtype: Link Rect: 323, 519, 335, 528 ActionDest: 351 Annotation: Subtype: Link Rect: 544, 292, 556, 301 ActionDest: 344 Annotation: Subtype: Link Rect: 424, 216, 560, 227 Annotation: Subtype: Link Rect: 166, 204, 269, 215 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 393, 640, 430, 651 Annotation: Subtype: Link Rect: 59, 618, 97, 628 Annotation: Subtype: Link Rect: 59, 561, 97, 571 Annotation: Subtype: Link Rect: 131, 503, 169, 513 Annotation: Subtype: Link Rect: 109, 469, 146, 479 Annotation: Subtype: Link Rect: 215, 434, 253, 444 Annotation: Subtype: Link Rect: 145, 377, 182, 387 Annotation: Subtype: Link Rect: 188, 377, 224, 387 Annotation: Subtype: Link Rect: 213, 353, 251, 364 Annotation: Subtype: Link Rect: 274, 318, 311, 329 Annotation: Subtype: Link Rect: 317, 318, 353, 329 Annotation: Subtype: Link Rect: 421, 295, 459, 306 Annotation: Subtype: Link Rect: 191, 272, 228, 283 Annotation: Subtype: Link Rect: 451, 249, 489, 260 Annotation: Subtype: Link Rect: 519, 226, 557, 237 Annotation: Subtype: Link Rect: 59, 216, 95, 226 Annotation: Subtype: Link Rect: 182, 192, 219, 203 Annotation: Subtype: Link Rect: 367, 169, 404, 180 Annotation: Subtype: Link Rect: 357, 146, 395, 157 Annotation: Subtype: Link Rect: 59, 112, 97, 122 Annotation: Subtype: Link Rect: 103, 112, 139, 122 Annotation: Subtype: Link Rect: 490, 88, 528, 99 Annotation: Subtype: Link Rect: 464, 65, 501, 76 Annotation: Subtype: Link Rect: 507, 65, 543, 76 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 450, 731, 488, 742 Annotation: Subtype: Link Rect: 494, 731, 530, 742 Annotation: Subtype: Link Rect: 277, 708, 314, 719 Annotation: Subtype: Link Rect: 320, 708, 356, 719 Annotation: Subtype: Link Rect: 277, 685, 315, 696 Annotation: Subtype: Link Rect: 108, 662, 146, 673 Annotation: Subtype: Link Rect: 152, 662, 188, 673 Annotation: Subtype: Link Rect: 374, 639, 412, 650 Annotation: Subtype: Link Rect: 204, 616, 241, 627 Annotation: Subtype: Link Rect: 59, 593, 97, 604 Annotation: Subtype: Link Rect: 95, 559, 133, 569 Annotation: Subtype: Link Rect: 187, 524, 225, 535 Annotation: Subtype: Link Rect: 479, 501, 517, 512 Annotation: Subtype: Link Rect: 454, 478, 492, 489 Annotation: Subtype: Link Rect: 260, 420, 298, 431 Annotation: Subtype: Link Rect: 438, 397, 475, 408 Annotation: Subtype: Link Rect: 481, 397, 517, 408 Annotation: Subtype: Link Rect: 204, 362, 241, 373 Annotation: Subtype: Link Rect: 248, 362, 284, 373 Annotation: Subtype: Link Rect: 305, 339, 342, 350 Annotation: Subtype: Link Rect: 349, 339, 385, 350 Annotation: Subtype: Link Rect: 108, 317, 146, 327 Annotation: Subtype: Link Rect: 152, 317, 188, 327 Annotation: Subtype: Link Rect: 179, 270, 217, 281 Annotation: Subtype: Link Rect: 131, 248, 169, 258 Annotation: Subtype: Link Rect: 232, 224, 270, 235 Annotation: Subtype: Link Rect: 292, 201, 329, 212 Annotation: Subtype: Link Rect: 335, 201, 371, 212 Annotation: Subtype: Link Rect: 217, 178, 254, 189 Annotation: Subtype: Link Rect: 261, 178, 297, 189 Annotation: Subtype: Link Rect: 271, 155, 308, 166 Annotation: Subtype: Link Rect: 315, 155, 351, 166 Annotation: Subtype: Link Rect: 59, 133, 97, 143 Annotation: Subtype: Link Rect: 103, 133, 139, 143 Annotation: Subtype: Link Rect: 511, 109, 548, 120 Annotation: Subtype: Link Rect: 205, 86, 243, 97 Checksum: ec6f4e84 Type: CRC32 Checksum: e47d38cf11b39c322c7749de571a9aee Type: MD5 Checksum: 006883d40f530587eb049b4a76a8c3ffbd9dd610 Type: SHA-1 Checksum: b80b108dee8ccc9e44c41611355e2be92fab612dbb6fe1477622e15f86d54d3e Type: SHA-256