The research was supported by the University of Sydney. pVITRO1-Trastuzumab-IgG1/&#954; was a gift from Andrew Beavil (Addgene plasmid # 61883). We thank Tihomir S. Dodev for useful discussions regarding pVITRO1-Trastuzumab-IgG1/&#954;.

<ol>
	<li>Nelson, A. L., Reichert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+trends+for+therapeutic+antibody+fragments.">Development trends for therapeutic antibody fragments.</a> Nat Biotechnol. 27 (4), 331-337 (2009).</li><li>Drake, P. M., Rabuka, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emerging+playbook+for+antibody-drug+conjugates:+lessons+from+the+laboratory+and+clinic+suggest+a+strategy+for+improving+efficacy+and+safety.">An emerging playbook for antibody-drug conjugates: lessons from the laboratory and clinic suggest a strategy for improving efficacy and safety.</a> Curr Opin Chem Biol. 28, 174-180 (2015).</li><li>Kontermann, R. E., Brinkmann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bispecific+antibodies.">Bispecific antibodies.</a> Drug Discov Today. 20 (7), 838-847 (2015).</li><li>Beck, A., Wurch, T., Bailly, C., Corvaia, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+and+challenges+for+the+next+generation+of+therapeutic+antibodies.">Strategies and challenges for the next generation of therapeutic antibodies.</a> Nat Rev Immunol. 10 (5), 345-352 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Food and Drug Administration. 22, (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> European Medicines Agency. 13, (2014).</li><li>Lopez-Morales, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+and+biological+characterization+of+a+biosimilar+trastuzumab.">Physicochemical and biological characterization of a biosimilar trastuzumab.</a> Biomed Res Int. 2015, 427235 (2015).</li><li>Jung, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+characterization+of+Remsima.">Physicochemical characterization of Remsima.</a> MAbs. 6 (5), 1163-1177 (2014).</li><li>Visser, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+and+functional+comparability+between+the+proposed+biosimilar+rituximab+GP2013+and+originator+rituximab.">Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab.</a> BioDrugs. 27 (5), 495-507 (2013).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+different+vector+designs+for+the+mammalian+expression+of+recombinant+IgG+antibodies.">A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies.</a> J Immunol Methods. 318 (1-2), 113-124 (2007).</li><li>Fitzgerald, K. D., Semler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridging+IRES+elements+in+mRNAs+to+the+eukaryotic+translation+apparatus.">Bridging IRES elements in mRNAs to the eukaryotic translation apparatus.</a> Biochim Biophys Acta. 1789 (9-10), 518-528 (2009).</li><li>Ho, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRES-mediated+Tricistronic+vectors+for+enhancing+generation+of+high+monoclonal+antibody+expressing+CHO+cell+lines.">IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.</a> J Biotechnol. 157 (1), 130-139 (2012).</li><li>Dodev, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tool+kit+for+rapid+cloning+and+expression+of+recombinant+antibodies.">A tool kit for rapid cloning and expression of recombinant antibodies.</a> Sci Rep. 4, 5885 (2014).</li><li>Walsh, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+of+protein+biopharmaceuticals.">Post-translational modifications of protein biopharmaceuticals.</a> Drug Discov Today. 15 (17-18), 773-780 (2010).</li><li>Backliwal, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+vector+design+and+multi-pathway+modulation+of+HEK+293E+cells+yield+recombinant+antibody+titers+exceeding+1+g/l+by+transient+transfection+under+serum-free+conditions.">Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions.</a> Nucleic Acids Res. 36 (15), 96 (2008).</li><li>Bebbington, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+expression+of+a+recombinant+antibody+from+myeloma+cells+using+a+glutamine+synthetase+gene+as+an+amplifiable+selectable+marker.">High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker.</a> Biotechnology (N Y). 10 (2), 169-175 (1992).</li><li>Kaufman, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coamplification+and+coexpression+of+human+tissue-type+plasminogen+activator+and+murine+dihydrofolate+reductase+sequences+in+Chinese+hamster+ovary+cells.">Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells.</a> Mol Cell Biol. 5 (7), 1750-1759 (1985).</li><li>Dalton, A. C., Barton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over-expression+of+secreted+proteins+from+mammalian+cell+lines.">Over-expression of secreted proteins from mammalian cell lines.</a> Protein Sci. 23 (5), 517-525 (2014).</li><li>Pham, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+gene+expression+in+HEK293+cells:+peptone+addition+posttransfection+improves+recombinant+protein+synthesis.">Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis.</a> Biotechnol Bioeng. 90 (3), 332-344 (2005).</li><li>Pham, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+transient+transfection+of+serum-free+suspension-growing+HEK293+EBNA1+cells:+peptone+additives+improve+cell+growth+and+transfection+efficiency.">Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency.</a> Biotechnol Bioeng. 84 (3), 332-342 (2003).</li><li>Yang, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+valproic+acid+to+CHO+cell+fed-batch+cultures+improves+monoclonal+antibody+titers.">Addition of valproic acid to CHO cell fed-batch cultures improves monoclonal antibody titers.</a> Molecular Biotechnology. 56 (5), 421-428 (2014).</li><li>Backliwal, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid:+a+viable+alternative+to+sodium+butyrate+for+enhancing+protein+expression+in+mammalian+cell+cultures.">Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures.</a> Biotechnol Bioeng. 101 (1), 182-189 (2008).</li><li>Jiang, Z., Sharfstein, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+butyrate+stimulates+monoclonal+antibody+over-expression+in+CHO+cells+by+improving+gene+accessibility.">Sodium butyrate stimulates monoclonal antibody over-expression in CHO cells by improving gene accessibility.</a> Biotechnol Bioeng. 100 (1), 189-194 (2008).</li><li>Jordan, M., Wurm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+adherent+and+suspended+cells+by+calcium+phosphate.">Transfection of adherent and suspended cells by calcium phosphate.</a> Methods. 33 (2), 136-143 (2004).</li></ol>

The authors declare that they have no competing financial interests.

פרוטוקול זה מפרט את transfection, ביטוי וטיהור יציב של נוגדן רפואי בתאי HEK-293. ביטוי יציב של גני נוגדן הוא הצעד הראשון ביצירת קו תא מייצר נוגדנים לפיתוח והייצור של נוגדנים טיפוליים. בעוד שחלה של אוגר סיני (CHO) תאים נשארים פלטפורמת הביטוי מועדף על חלבונים תרופתיים, שורת תאי HEK-29...

ההצלחה של נוגדנים טיפוליים ממשיכה לנסוע השקעה ניכרת לתוך פיתוח נוגדן כגל של הרפוי דור הבאים מתחיל. שוק הנוגדנים צפוי לעצב מחדש מרסיסי נוגדן 1, conjugates 2, נוגדני bispecific סמי נוגדן 3 ונוגדנים מהונדסים עם תכונות חיוביות 4. מחלקה נוספת זוכה להתעניינות מחודשת התרופות הם biosimilars. נוגדני Biosimilar הם &quot;מאוד דומים&quot; לשכפל מוצרים של נוגדן טיפולי קבל אישור רגולטורים כבר. Biosimilar מוצעת חייבת להיות זהה לזו הנוגדן היוזם לגבי המבנה שלה, פונקציה, רעילויות בבעלי חיים, בטיחות ויעילות קליניות, פרמקוקינטיקה אדם (PK), פרמקודינמיקה (PD) ו חיסוני 5, 6. האישור rאטס של נוגדני biosimilar היה איטי בשל האילוצים הקפדנים על האיכות הסופית של המוצר. תהליכי הייצור המדויקים כגון ספציפי קווי תאים ותנאי culturing ועד השלבים לעיבוד הסופיים יכולים להישאר קניינית. יתרה מזאת, בייצור של נוגדני מטבעו כרוך מידה של השתנות אשר יכולים להוסיף האתגר בייצור מוצר דומה מאוד. אפיון physiochemical ו biophysical מקיף והשוואה קשה למדי, עדיין מספר מחקרים הוכחת המאפיינים של נוגדנים biosimilar הם מתעוררים בספרות 7, 8, 9. יצירת נוגדנים טיפוליים מתחילה עם transfection של תאים לארח יונקים עם וקטור הנושא את הגנים של הנוגדן בהתאמה. תנאי עיצוב וקטור, שורת התאים וההתרבות הם שיקולים מרכזיים להקמה לשעברמערכת pression. רצפי DNA של נוגדנים ניתן מקורות מבנק והתרופות (www.drugbank.ca), IMGT (www.igmt.org) או פרסומי מחקר לרבות פטנטים. לדוגמה, את הרצף של trastuzumab זמין באמצעות בנק והתרופות (ID DB: DB00072). רצף החומצות האמינו של האזורים המשתנים יכול לעבור עיצוב גן ואופטימיזציה לסינתזה של פונדקאי הרצוי. חשוב נוגדן biosimilar ששום שינוי הוא עשה את רצף החומצות האמיניות. לאחר מסונתז, גני נוגדן ניתן subcloned לתוך הווקטור המתאים בחירה. נוגדני IgG אדם מורכבים משתי שרשרות כבדות זהות ושתי רשתות אור זהות. בחוזקה ביטוי מוסדר הן שרשראות חיוני לייצור אופטימלי של חלבון IgG Heterologous בתאי יונקים 10. התוך וכן אג&quot;ח דיסולפיד הבין-שרשרת צריך להיווצר ומספר שלאחר translational שינויים צריך להיותtroduced במהלך ביוסינתזה חלבון. מספר הווקטורים זמינים אשר עוצב במיוחד כדי לבטא גני נוגדן (עיינו בטבלה של חומרים). וקטורי נוגדנים ספציפיים אלה בדרך כלל להביע האזורים המתמידים הוא שרשרות כבדות ואור כך שרק האזורים המשתנים של כל שרשרת דורשים שיבוט. Transfection של תאים עם שני מבנים עצמאיים (-transfection שיתוף) הוא הגישה הנפוצה ביותר להעברת גני קידוד שרשרת כבדה וקלים. כלומר, כל גן הוא מונע על ידי אמרגן משלה עבד כמו שרשרות נוגדן נפרדות בטרם התכנס reticulum endoplasmic. מצד השני, וקטורים רבים cistronic יש אתר כניסה הריבוזום (IRES) אלמנטים משולבים המאפשרים ביטוי של גנים מרובים כמו תעתיק mRNA יחיד עם תרגום מותר מאזורים פנימיים של mRNA 11. במקרה זה, הגנים המקודדים-שרשרת כבדה ואור הם מצמידים בבית arrangement להשיג ביטוי שיתוף של שני שרשראות נוגדן 10, 12. בעוד תאי transfected זמני להניב מספיק חלבון כדי לבצע מספר מוגבל של ניסויים, שורות תאי transfected ביציבות שעברו בחירה לשילוב הגנום יכולות לספק תשואות גבוהות יותר. כמויות חלבון גבוהות לאפשר התפתחות assay הנוגעת אפיון במבחנה יכולות לספק אינדיקציה של איכות נוגדן בתמורת יישומים במורד כגון שורת תאים משובטת ובחירת מועמד מובילה. מטרת מאמר זה היא לתאר את הביטוי וטיהור היציבים של נוגדן רפואי היוצר מערכת ביטוי יונקת. אכן, שיטה זו יכולה להיות מיושמת על הביטוי של נוגדן biosimilar. השיטה יכולה לשמש עבור האפיון הראשוני של נוגדנים לפני שתמשיך הלאה אל קריטי, אם כי זמן רב STEps של זיהוי שיבוט רצוי לייצור בקנה מידה גדול יותר. יתר על כן, בשיטה זו ניתן להשתמש כדי לבטא חלבונים אחרים ולא רק נוגדנים. הפרוטוקול המפורט הבא מתאר את הביטוי של trastuzumab נוגדנים הטיפולי. זה מורכב של הכנת DNA וקטור ואחריו transfection יציבה שורת תאים HEK-293 וטיהור של חלבון נוגדנים על ידי שיטה chromatographic אוטומטיות.

<table><tbody><tr><td>pFUSE vector series</td><td>InvivoGen</td><td>N/A</td><td>Heavy and light antibody genes expressed in separate vectors that require co-transfection.</td></tr><tr><td>mAbXpress vector series</td><td>ACYTE Biotech Pty Ltd.</td><td></td><td>Heavy and light antibody genes expressed in separate vectors that require co-transfection. Refer to: Jones, M. L. et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. J Immunol Methods. 354 (1-2), 85-90, doi:10.1016/j.jim.2010.02.001, (2010).</td></tr><tr><td>pVITRO1 vector</td><td>N/A</td><td>N/A</td><td>Heavy and light antibody genes are each driven by a separate promoter in a single vector.&amp;nbsp; Refer to: Dodev, T. S. &lt;em&gt;et al.&lt;/em&gt; A tool kit for rapid cloning and expression of recombinant antibodies. Sci Rep. 4 5885, doi:10.1038/srep05885, (2014).</td></tr><tr><td>GS vector series</td><td>Lonza</td><td></td><td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript.</td></tr><tr><td>Multi-cistronic vector series 1</td><td>N/A</td><td>N/A</td><td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Li, J. &lt;em&gt;et al.&lt;/em&gt; A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J Immunol Methods. 318 (1-2), 113-124, doi:10.1016/j.jim.2006.10.010, (2007).</td></tr><tr><td>Multi-cistronic vector series 2</td><td>N/A</td><td>N/A</td><td>Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Ho, S. C. &lt;em&gt;et al.&lt;/em&gt; IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol. 157 (1), 130-139, doi:10.1016/j.jbiotec.2011.09.023, (2012).</td></tr><tr><td>pVITRO1-Trastuzumab-IgG1/&amp;kappa;</td><td>Addgene</td><td>61883</td><td>Mammalian expression vector containing trastuzumab antibody genes with hygromycin resistance gene; pVITRO1-Trastuzumab-IgG1/&amp;kappa; was a gift from Andrew Beavil.</td></tr><tr><td>Fast-Media Hygro Agar</td><td>Jomar Life Research</td><td>fas-hg-s</td><td>Used to prepare low salt LB agar containing 75 &amp;micro;g/ml hygromycin.</td></tr><tr><td>Fast-Media Hygro TB</td><td>Jomar Life Research</td><td>fas-hg-l</td><td>Used to prepare low salt TB broth containing 75 &amp;micro;g/ml hygromycin.&amp;nbsp;</td></tr><tr><td>Glycerol, BioXtra, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>G6279</td><td>Prepare to 80% with water and autoclave. Store at room temperature.</td></tr><tr><td>Jestar 2.0/LFU Plasmid Maxi Kit</td><td>Astral Scientific</td><td>G221020</td><td>Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5).</td></tr><tr><td>FreeStyle 293-F Cells</td><td>Life Technologies</td><td>R790-07</td><td>HEK-293 cell line adapted to suspension culture in serum-free media.</td></tr><tr><td>FreeStyle 293 Expression Medium</td><td>Life Technologies</td><td>12338-018</td><td>Serum-free media specially formulated for maintaing 293-F cell line and high protein expression.</td></tr><tr><td>Kolliphor P188</td><td>Sigma-Aldrich</td><td>K4894</td><td>Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 &amp;mu;m filter. Store at 4 &amp;deg;C.</td></tr><tr><td>DMEM, high glucose&amp;nbsp;</td><td>Life Technologies</td><td>11995-065</td><td></td></tr><tr><td>Heat-Inactivated Foetal Bovine Serum</td><td>Life Technologies</td><td>10082-147</td><td></td></tr><tr><td>Polyethylenimine, Linear, MW 25,000&amp;nbsp;</td><td>Polysciences, Inc.</td><td>23966</td><td>Prepare to 1 mg/ml in water. Adjust to pH 7.0 with 1 M HCl (solution becomes clear) and filter-sterilize using 0.22 &amp;mu;m filter. Store at -80 &amp;deg;C until use.</td></tr><tr><td>OptiPro SFM</td><td>Life Technologies</td><td>12309-050</td><td>Transfection formulated serum-free media</td></tr><tr><td>Hygromycin B Solution</td><td>Jomar Life Research</td><td>ant-hg-1</td><td></td></tr><tr><td>Dimethylsulphoxide (DMSO)</td><td>Thermo Fisher Scientific</td><td>AJA2225</td><td></td></tr><tr><td>Tryptone (casein peptone)</td><td>Thermo Fisher Scientific</td><td>LP0042B</td><td>Prepare to 20% in PBS and filter-sterilize using 0.22 &amp;mu;m filter. Store at 4 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml</td><td>Astral Scientific</td><td>09-2051-100</td><td></td></tr><tr><td>HiTrap Protein A High Performance, 1 x 5 ml column</td><td>Sigma-Aldrich</td><td>GE17-0403-01</td><td></td></tr><tr><td>AKTApurifier 100</td><td>GE Healthcare</td><td>28406266</td><td>Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector.</td></tr><tr><td>Glycine-HCl</td><td>Sigma-Aldrich</td><td>G2879</td><td></td></tr><tr><td>Citric Acid, monohydrate</td><td>Astral Scientific</td><td>BIOC2123</td><td></td></tr><tr><td>Sodium Citrate, trisodium salt dihydrate&amp;nbsp;</td><td>Astral Scientific</td><td>BIOCB0035</td><td></td></tr><tr><td>1 M Tris-HCl solution pH 9.0</td><td>Astral Scientific</td><td>BIOSD8146</td><td></td></tr><tr><td>Amicon Ultra Centrifugal Filters (30 MWCO)</td><td>Merck Millipore</td><td>UFC803008/UFC903008</td><td>Used to buffer exchange and concentrate purified protein.</td></tr><tr><td>Pierce Bicinchoninic Acid (BCA) Assay Kit</td><td>Thermo Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>BLItz System</td><td>fort&amp;eacute;BIO</td><td>45-5000</td><td>Instrument used for bio-layer interferometry (BLI) measurements.</td></tr><tr><td>Protein A biosensors</td><td>fort&amp;eacute;BIO</td><td>18-5010</td><td></td></tr><tr><td>Acrylamide/Bisacrylamide (37.5:1), 40% solution</td><td>Astral Scientific</td><td>786-502</td><td></td></tr><tr><td>Ammonium Persulfate (APS)</td><td>Astral Scientific</td><td>AM0486</td><td></td></tr><tr><td>TEMED</td><td>Astral Scientific</td><td>AM0761</td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Astral Scientific</td><td>786-498</td><td></td></tr><tr><td>Precision Plus Dual-Color Protein Standard</td><td>Bio-Rad</td><td>1610374</td><td></td></tr></tbody></table>

laboratory scale production and purification of a therapeutic antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

ייצור יציב של trastuzumab ידי תאי HEK-293 טרנספקציה אושר באמצעות אינטרפרומטריה השכבתי ביו (BLI) כמוצג באיור 1. עקומת תקן IgG נוצר על ידי מדידת קצב מחייב בין תקן נוגדן IgG ו חלבון biosensor (איור 1 א). מדגם supernatant הגולמי נמדד באופן דומה, אז ריכוזו אינטרפו...

Watch this Scientific Journal Video about Laboratory Scale Production and Purification of a Therapeutic Antibody at JoVE.com

Laboratory Scale Production and Purification of a Therapeutic Antibody

פרוטוקול זה מתאר את הייצור של נוגדן טיפולי מערכת ביטוי יונקת. השיטות שתוארו כוללות הכנת DNA וקטור, transfection היציבה והתאמת סרום ללא של קו תא 293 כליות עוברי אנושי, להקים תרבויות בקנה מידה גדולות טיהור באמצעות כרומטוגרפיה זיקה.

הפקה בקנה מידה מעבדתי וטיהור של נוגדן רפואי

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

laboratory-scale-production-and-purification-of-a-therapeutic-antibody

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

17.3K Views.  University of Sydney.  פרוטוקול זה מתאר את הייצור של נוגדן טיפולי מערכת ביטוי יונקת. השיטות שתוארו כוללות הכנת DNA וקטור, transfection היציבה והתאמת סרום ללא של קו תא 293 כליות עוברי אנושי, להקים תרבויות בקנה מידה גדולות טיהור באמצעות כרומטוגרפיה זיקה. 

Video: Laboratory Scale Production and Purification of a Therapeutic Antibody

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

פלטפורמה תפוקה גבוהה אוטומטיות לפיתוח קווי ייצור עבור Cell Therapeutics חלבון

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Medicine

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

פרוטוקול הניסוי לזיהוי<em&gt; כחוליות</em&gt; בדגימות נוזלים ומוצקים עם צ&#39;יפ Microarray נוגדן

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Environment

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

ניתוח גידול ורקמות התפלגות של נוגדן טיפולי ספציפי אנטיגן היעד

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

Cancer Research

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

השימוש תפוקה גבוהה אוטומטית Microbioreactor מערכת לייצור של מודל IgG1 בתאים צ'ו

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Bioengineering

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

טיהור וניתוח של נוגדן חד שבטיים מן הסינית שחלות אוגר תאים באמצעות מערכת Microbioreactor חקן אוטומטי

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

יצירת נוגדנים: ייצור נוגדנים חד שבטיים באמצעות היברידיות

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

Education

JoVE Science Education

Advanced Biology

Immunology

Production and Purification of Recombinant Antibodies from Mammalian Cells

Source: Shivange, Gururaj, et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen-Specific Therapeutic Antibody.</a> J. Vis. Exp.(2020).
The video explains the process of producing antibodies in mammalian host cells, from gene expression to secretion. It involves transcription, translation, signal recognition, correct folding, and eventual release into the extracellular medium via secretory vesicles.

ייצור וטיהור נוגדנים רקומביננטיים מתאי יונקים

1. Expression and purification of antibodies

	Maintenance of CHO cells
	
		Grow CHO cells in FreeStyle CHO Media supplemented with commercially available ...

JoVE Encyclopedia of Experiments

Antibody-Based Technologies

Production and Purification

A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Source: Velugula-Yellela, S. R., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58947">Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System.</a> J. Vis. Exp. (2019).
This video demonstrates an assay for purifying monoclonal antibodies from cell culture fluid harvested from Chinese Hamster Ovarian cells. It employs a porous glass resin with immobilized protein A, which binds to the Fc region of the antibodies in the sample, effectively separating them from contaminants. The use of a low-pH elution buffer weakens the interaction, facilitating the retrieval of purified antibodies.

טכניקה לטיהור נוגדנים חד שבטיים המיוצרים על ידי תאי שחלה של אוגר סיני

1. Purification of antibody
NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M ...

Antibody Production from a Batch Overgrowth Culture of Transfected Mammalian Cells

Source: Elgundi, Z. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a>&#160;J. Vis. Exp.&#160;(2017)
The video demonstrates a method for producing therapeutic antibodies using stably transfected mammalian cells. Tryptone supplementation stimulates cell division, resulting in overgrowth and supporting a higher antibody yield. The antibody-containing medium is harvested, filtered, and stored for further use.

ייצור נוגדנים מתרבית גידול יתר של תאי יונקים נגועים

NOTE: A suitable mammalian expression vector must be used for this protocol. Here, a single construct containing two expression cassettes is used ...

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

טכניקת כרומטוגרפיית זיקה לטיהור נוגדנים חד-שבטיים

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...