PK thanks the Department of Chemical Engineering, Imperial College London, for his scholarship. RD thanks the U.K. Biotechnology and Biological Sciences Research Council for his studentship. MM is funded by the U.K. Biotechnology and Biological Sciences Research Council (Grant reference: BB/S006206/1). IAG thanks the Irish Research Council (Scholarship No. GOIPG/2017/1049) and CONACyT (Scholarship No. 438330).

1. DsiRNA design and reconstitution
<ol>
	<li>Use Safari, Firefox, or Microsoft Edge to access the IDT website (https://eu.idtdna.com). From the home page, select the&#160;Products and Services tab followed by RNA interference. To generate custom dicer-substrates (DsiRNA) constructs targeting the Fut8 gene, select Design tool followed by the Generate Custom DsiRNA tab.
	<ol>
		<li>Enter the Fut8 Accession Number or manually enter the gene sequence to begin. In this study, the proposed Fut8 sequences from both &#34;CHO-K1&#34; and &#34;Chinese Hamster&#34; entries were obtained from&#160;https://chogenome.org and used to generate DsiRNA. Most genes have several transcript variants, and it is important to verify that DsiRNA would be active on all reported variants in the current assembly.</li>
		<li>Select the option to perform a &#34;Manual BLAST&#34; search as the CHO cell genome is currently not available as a &#34;reference genome&#34; on the IDT website. This step searches for matches between custom DsiRNA sequences and the genome of interest.</li>
		<li>Click Search to generate DsiRNA sequences.</li>
		<li>In turn, assess the specificity of each DsiRNA using the &#34;Manual BLAST&#34; function with the tax id:10029 (CHO cell lines and Chinese hamster). Query coverage results indicate DsiRNA constructs match Fut8 (including Fut8 transcript variants) at 100%, whilst complementarity against unintended targets is &#8804;76%35.</li>
		<li>Check that the DsiRNA specifically targets Fut8 to ensure the GC content is between 30%-50%. A low GC content is associated with weak and unspecific binding, whilst a high GC content inhibits siRNA unwinding and loading into the RISC complex36.</li>
		<li>Select three DsiRNA for use in transfection studies (Table 1), each targeting a different location of the Fut8 gene. Purchase a predesigned non-targeting or scrambled DsiRNA from IDT for control experiments.</li>
	</ol>
	</li>
	<li>Upon arrival, centrifuge the DsiRNA at 13,300 x g for 1 min to pellet before opening the tube. Reconstitute each lyophilized DsiRNA in nuclease-free duplex buffer (provided by IDT) to a 100 &#956;M stock solution. For example, add 20 &#956;L of buffer to 2 nmol of DsiRNA to obtain a 100 &#956;M stock.
	<ol>
		<li>To ensure sufficient mixing, incubate the stock solutions at room temperature for 30 min whilst shaking gently on an orbital shaker (50 rpm, 16 mm orbit).</li>
		<li>Create a master mix by combining 20 &#956;L of each DsiRNA construct that targets Fut8 (100 &#956;M of each DsiRNA). Prepare aliquots and store at &#8722;20 &#176;C.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DsiRNA target</td>
			<td colspan="3">Sequence</td>
			<td>GC (%)</td>
		</tr>
		<tr>
			<td rowspan="2">Structure A</td>
			<td colspan="3">5&#39; GAGAAGAUAGAAACAGUCAAAUACC 3&#39;</td>
			<td rowspan="2">36%</td>
		</tr>
		<tr>
			<td colspan="3">5&#8217; GGUAUUUGACUGUUUCUAUCUUCUCUC 3&#39;</td>
		</tr>
		<tr>
			<td rowspan="2">Structure B</td>
			<td colspan="3">5&#39; AGAAUGAGAAUGGAUGUUUUUCCTT 3&#39;</td>
			<td rowspan="2">32%</td>
		</tr>
		<tr>
			<td colspan="3">5&#39; AAGGAAAAACAUCCAUUCUCAUUCUGA 3&#39;</td>
		</tr>
		<tr>
			<td rowspan="2">Structure C</td>
			<td colspan="3">5&#39; AGAGAAGAUAGAAACAGUCAAAUAC 3&#8217;</td>
			<td rowspan="2">32%</td>
		</tr>
		<tr>
			<td colspan="3">5&#39; GUAUUUGACUGUUUCUAUCUUCUCUCG 3&#39;</td>
		</tr>
	</tbody>
</table>
Table 1. DsiRNA sequences used for Fut8 knockdown. Sequences generated by IDT that target Fut8 in Chinese hamster and CHO K1 cell genomes. The sense and antisense sequences for each construct are shown (respectively), and the GC content of each structure is displayed. Reprinted from Kotidis&#160;et al.52.
2. DsiRNA transfection
<ol>
	<li>Revive CHO cells expressing an IgG monoclonal antibody37 using culture conditions suitable for the cell line of interest. 
	NOTE: The cells used in this study were generated using the glutamine synthetase (GS) system, where endogenous GS is inhibited by L-methionine sulfoximine (MSX) to enhance the selection of rare high-producing clones. Therefore, only use MSX if required by the cell line of choice.
	<ol>
		<li>Defrost a vial of cells for 2-3 min in a water bath set to 37 &#176;C. Clean the vial exterior with 70% (v/v) ethanol and continue all work in a class II biosafety cabinet.</li>
		<li>Transfer the cell suspension to a 15 mL centrifuge tube containing 9 mL of prewarmed medium appropriate for the cell line of choice. Pellet the cells by centrifugation at 100 x g for 5 min.</li>
		<li>Carefully remove and discard the medium without disturbing the cell pellet. Then, resuspend the cell pellet in 10 mL of prewarmed medium and take an aliquot for counting.</li>
		<li>Stain the cells with Trypan blue if counting with a hemocytometer, or an appropriate stain to distinguish live/dead cells when using an automated cell counter.</li>
		<li>Following either method of enumeration, transfer an appropriate volume of cell suspension to a 125 mL Erlenmeyer shake flask at a viable cell density of 3 x 105 cells&#183;mL-1 in 30 mL of medium (with optional supplementation of 50 &#956;M MSX).</li>
		<li>Transfer cells to an incubator set to 36.5 &#176;C, 5% CO2 and place on a shaking platform at 150 rpm (16 mm orbit).</li>
		<li>Passage cells every 3-4 days at a seeding density of 2 x 105 cells&#183;mL-1 and a working volume of 50 mL in a 250 mL Erlenmeyer shake flask. If using MSX, discontinue supplementation after the first passage.</li>
		<li>Passage cells 2x in addition to thawing.</li>
	</ol>
	</li>
	<li>Transfection
	<ol>
		<li>Assess the cell density and ensure the cell viability is greater than 90%.</li>
		<li>Carefully clean the biosafety cabinet and all equipment with 70% (v/v) ethanol and an RNase inhibitor solution to avoid contamination.</li>
		<li>Pellet cells at 100 x g for 5 min and resuspend in prewarmed medium to a viable cell density of 5 x 106 cells&#183;mL-1.</li>
		<li>Transfer 8 &#956;L (equivalent to 1 &#956;M) of the DsiRNA master mix or control to a sterile (0.4 cm) electroporation cuvette. Then, transfer 800 &#956;L of the cell suspension (equivalent to 4 x 106 cells) to the same cuvette and ensure both components are mixed.</li>
		<li>Deliver the following pulse conditions: 1200 V, 0.1 ms, square waveform.</li>
		<li>Transfer the cell suspension from the cuvette to one well of a 6-well plate, taking care to avoid foam-like material. Recover the cells in the incubator (36.5 &#176;C, 5% CO2) without shaking for 10 min.</li>
		<li>Add 800 &#956;L of prewarmed media to make a final volume of 1.6 mL per well and return the transfected cells to the incubator for growth (36.5 &#176;C, 5% CO2) while shaking at 150 rpm (16 mm orbit).</li>
		<li>Harvest the supernatants and cells at 48 h post-transfection. 
		Stopping point: The cell culture supernatant can be kept at -20 &#176;C; however, it is advisable that cell lysis should be performed immediately after cell pellet collection to avoid protein degradation. Supernatants are used in Step 3, Step 4, and Step 5, while cell pellets are used in Step 6.</li>
	</ol>
	</li>
</ol>
3. IgG quantification and purification
<ol>
	<li>Measure IgG concentration using biolayer interferometry or another method of choice.
	<ol>
		<li>Hydrate protein A biosensor tips in sample diluent for 10-30 min. In the meantime, transfer cell suspensions into 15 mL centrifuge tubes and pellet cells at 100 x g for 5 min or remove cells by filtration through a 0.45 &#181;m nitrocellulose filter.</li>
		<li>Without disturbing the pellet, carefully transfer the isolated supernatant containing IgG into a clean centrifuge tube.</li>
		<li>Use the following settings for biolayer interferometry: shaker speed, 2200 rpm; run time, 60 s. These parameters will be used to measure all samples and controls.</li>
		<li>Once the biosensor tips are hydrated, create a standard curve using an IgG standard (4 &#956;L of each concentration).</li>
		<li>Quantitate sample concentrations by loading 4 &#956;L of the cell supernatant. Clean the apparatus with lint-free wipes between each sample.</li>
		<li>Link the standard curve to the unknown samples to interpolate the binding rate of the unknown samples. Save the data and export as a csv or PDF file.</li>
	</ol>
	</li>
	<li>IgG purification
	<ol>
		<li>Prepare elution buffer containing 0.2 M glycine (pH 2.5) and sterilize by passing through a 0.22 &#956;m filter.</li>
		<li>Filter 1 mL of the isolated supernatant at room temperature using 0.22 &#956;m microcentrifuge filter tubes until the entire supernatant flows through.</li>
		<li>Pellet 150-200 &#956;L of protein A agarose beads in 1.5 mL centrifuge tubes at 100 x g for 3 min and discard the supernatant. Then, wash the protein A beads with 150-200 &#956;L of cell culture medium and repeat centrifugation. Discard the supernatant.</li>
		<li>Resuspend the equilibrated protein A beads with 1 mL of the prepared supernatants from Step 3.2.3. and load into a 1 mL polypropylene tube. Affix the polypropylene tube to a rotary mixer (15 mm orbit) and spin at 30 rpm for 60-90 min at room temperature or overnight at 4 &#176;C.</li>
		<li>After incubation is complete, collect the flow-through and wash the beads with 1 mL of 1x PBS to remove any unbound proteins. Collect the wash fraction as well.</li>
		<li>Elute IgG from the protein A beads by adding 3 mL of elution buffer to the polypropylene column. Collect sequential fractions that drain from the column (500 &#956;L each) into labeled tubes.</li>
		<li>Optional: If the purified antibody is to be stored, neutralize the elution buffer by adding 25 &#956;L of 1 M Tris pH 9.5. Skip this step if the antibody will be processed immediately via buffer exchange, etc. 
		NOTE: All parts of the purification (flow-through, washes, and all elution fractions) should be kept ensuring that the target protein is not lost. These samples can also help during troubleshooting if the purification is not successful.</li>
		<li>Use the first elution (elution A) for downstream processing but keep all other fractions should they be required in the future.</li>
	</ol>
	</li>
</ol>
4. Buffer exchange and sample concentration
<ol>
	<li>Exchange IgG elution buffer with 1x PBS.
	<ol>
		<li>Load elution A onto a 3 kDa molecular weight cutoff centrifugal concentrator. Refer to the manufacturer&#39;s guide when selecting an appropriate MWCO column. In this experiment, a smaller pore size ensures maximal retention of secreted protein but also increases the centrifugation time.</li>
		<li>Centrifuge at 13,300 x g for 40-50 min at 4 &#176;C. Centrifugation is complete once the residual volume is equal to or less than 50 &#956;L.</li>
		<li>Discard the flow-through and add 500 &#956;L of prechilled (4 &#176;C) 1x PBS to dilute the residual supernatant. Centrifuge the sample again using the same conditions (13,300 x g for 40-50 min at 4 &#176;C) until 50 &#956;L of residual supernatant remains.</li>
		<li>Add 500 &#956;L of prechilled (4 &#176;C) 1x PBS to dilute the residual supernatant and repeat the centrifugation process again to obtain a 100x dilution of the supernatant.</li>
		<li>Concentrate the supernatant to a final concentration of ~2.5 g&#183;L-1 in 40 &#956;L or less to ensure compatibility with the glycan analysis method. 
		NOTE: 100 &#181;g needs to be loaded for glycan analysis. 
		Stopping point: The concentrated IgG (in 1x PBS) can be stored at &#8722;20 &#176;C and thawed prior to glycan analysis.</li>
	</ol>
	</li>
</ol>
5. Glycan analysis
<ol>
	<li>Perform glycan analysis using capillary gel electrophoresis according to the manufacturer&#39;s instructions.
	<ol>
		<li>Transfer 200 &#956;L of the magnetic bead solution to a 0.2 mL PCR tube and place onto the magnetic stand to separate the beads from the supernatant.</li>
		<li>Carefully remove the supernatant and remove the tubes from the magnetic stand. Add the purified protein sample and vortex to ensure complete mixing.</li>
		<li>Add denaturation buffer (supplied) to the sample tube and incubate for 8 min at 60 &#176;C. Keep the sample tubes open for optimal reaction performance.</li>
		<li>Add PNGase F (500 units per sample) and incubate for 20 min at 60 &#176;C to cleave glycans from the purified antibodies.</li>
		<li>Following the release of N-glycans, close the sample tube and vortex. Add acetonitrile, vortex, and incubate at room temperature for 1 min.</li>
		<li>Place the sample tubes in the magnetic stand to separate the beads from solution. Use a pipette to carefully remove the supernatant without touching the beads.</li>
		<li>In a fume hood, add the glycan labeling solution containing a fluorophore to the sample. Vortex to ensure sufficient mixing and incubate at 60 &#176;C for 20 min (open lids).</li>
		<li>Wash the sample 3x in acetonitrile to remove excess dye. Then, elute the labeled glycans in DDI water (supplied).</li>
		<li>Place the sample tube in the magnetic stand to separate the beads from the supernatant containing purified and labeled glycans.</li>
		<li>Prepare and load the glucose ladder standard, bracketing standards, and samples into the designated tray positions. Run the glycan analysis protocol.</li>
		<li>Use appropriate software to analyze and identify the glycans present in the sample. 
		NOTE: Ensure the temperature in the heat block is accurate for efficient incubation.</li>
	</ol>
	</li>
</ol>
6. Western blot
<ol>
	<li>Quantify knockdown efficiency using western blot analysis with an anti-&#945;-1,6-fucosyltransferase antibody. Polyacrylamide gels and buffer recipes are available from commercial suppliers38,39.
	<ol>
		<li>At 48 h post transfection, count the cells using a hemocytometer or an automated cell counter and determine the volume of cell suspension equivalent to 5 x 106 cells.</li>
		<li>Transfer the appropriate volume of cell suspension from each experimental condition to a sterile 1.5 mL centrifuge tube and pellet at 13,200 x g for 10 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Lyse the cells with 200 &#956;L of lysis buffer (suitable for the extraction of proteins from mammalian cells) containing 1% v/v protease inhibitor cocktail at room temperature for 10 min. Shake the mixture gently during incubation (50 rpm, 16 mm orbit).</li>
		<li>To remove the cellular debris, centrifuge the lysate at 13,200 x g for 10 min and then transfer the cleared lysate to a sterile 1.5 mL tube.</li>
		<li>Measure the protein concentration of each lysate using a spectrophotometer at 280 nm. Subsequently, prepare aliquots of each sample adjusted to have the same protein concentration.</li>
		<li>Denature the protein samples by incubation at 100 &#176;C for 10-15 min in DTT-SDS sample loading dye; the final dye concentration is 1x.</li>
		<li>Load 15 &#956;L of the denatured samples and 5 &#956;L of prestained protein ladder into an SDS-PAGE gel with 12.5% resolving gel for efficient separation. Run the samples at 25 mA per gel for 90 min or until the dye front reaches the end of the gel.</li>
		<li>Carefully remove the gel from the cassette and incubate in 1x transfer buffer containing methanol.</li>
		<li>Prepare the wet transfer system and activate a PVDF membrane with methanol. Assemble the gel and PVDF membrane for wet transfer, place an ice block in the transfer tank, and submerge the entire tank in ice to ensure the transfer conditions remain cold. Run at 350 mA/100 V for 60 min.</li>
		<li>Block the membrane by incubating in a blocking solution for 30 min at room temperature while shaking gently on an orbital shaker at 50 rpm (16 mm orbit).</li>
		<li>Rinse the membrane with sterile water for 5 min and repeat. Then, use a clean scalpel to carefully cut the membrane horizontally at ~50 kDa, using the visible protein ladder as a guide.</li>
		<li>Incubate the membrane harboring proteins greater than 50 kDa with anti-&#945;-1,6-fucosyltransferase antibody at 1:1000 in an antibody diluent buffer. Incubate the membrane with immobilized proteins less than 50 kDa with anti-GAPDH at 1:10,000 in diluent buffer. Membranes may be incubated for 1 h at room temperature or overnight at 4 &#176;C.</li>
		<li>Wash the membranes for 5 min (x3) and then incubate with an appropriate secondary antibody for at least 30 min at room temperature.</li>
		<li>Perform 3x washes with a wash buffer for 5 min, followed by 3x washes with water. Then, develop the membrane with a chromogenic substrate (or appropriate detection reagent) until bands appear (1-60 min).</li>
		<li>Rinse the membrane 2x with water, and then allow it to dry. Capture an image of the membrane and perform densitometric analysis40.</li>
		<li>To calculate the relative protein expression in each sample, first calculate the ratio of the GAPDH signal in samples. This is the normalization factor for each sample that corrects for discrepancies in sample loading. 
		<img alt="Equation 1" src="/files/ftp_upload/63872/63872eq01.jpg" style="margin: auto;" /></li>
		<li>Divide the FUT8 signal intensity (for each lane) by the normalization factor of the corresponding lane to obtain the relative FUT8 protein expression. 
		<img alt="Equation 2" src="/files/ftp_upload/63872/63872eq02.jpg" style="margin: auto;" /></li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Glycosylation pathways involve a complex metabolic network of enzymes and accessory proteins. Dissecting the function of pathway constituents is daunting if reliant on conventional knockout or knockin genetic engineering strategies alone. An alternative approach is to preliminarily screen members of a pathway using a transient loss-of-function assay. To this end, two rapid protocols were combined, RNAi and CGE-LIF detection, to create a more efficient way to characterize glycosylation genes. The method described requires 5-7 days for completion compared to conventional methods that potentially take several weeks for completion. Further, research environments with automation capabilities could exploit this method to screen more gene candidates than feasible with manual handling.
The success of a transient glycoengineering campaign is largely dependent on the siRNA design. Custom DsiRNA designs must follow the rules previously outlined, or for ease, users may opt for commercially available predesigned sequences. Like other gene modification strategies, RNAi has the potential for off-target effects. Therefore, users are encouraged to assess unintentional gene targeting by computational methods41. Experimental design choices can also help limit off-target effects. Kittler et al. showed that the multiplexed delivery of siRNA led to a reduction in off-target effects42. Although this seems counterintuitive, it is suggested that a master mix reduces the concentration of each siRNA construct, thus limiting the opportunity for off-target gene silencing. A further benefit is that the simultaneous transfection of siRNA structures that target the same gene increases the likelihood of successful RNAi. The use of a master mix also ensures consistency between samples and replicates and speeds up the transfection process. Following an initial screen of mixed siRNA constructs, another experiment may be conducted using individual constructs to ascertain the RNAi efficiency of each sequence. In this and other knockdown studies, up to three siRNA have been pooled and delivered to cells43,44,45. However, it may be desirable to screen more than three siRNA simultaneously to efficiently target a single gene or to target several genes. Indeed, one study demonstrated multiplexed siRNA-mediated silencing of up to six genes at levels comparable to the silencing of individual genes46. However, further studies are needed to determine the maximum number of siRNA constructs that can be used in a pool without compromising the silencing efficiency. The multiplex strategy was proposed by Martin et al. to enhance the pace of RNAi library screening experiments46, and a similar concept may prove useful to screen glycosylation genes.
The protocol described herein serves as a proof-of-concept with the expectation that subsequent experiments will be performed to validate other glycosylation genes. New genes of interest may be uncharacterized or less popular than Fut8, and primary antibodies to detect gene silencing may be poor or unavailable. In this scenario, alternative methods such as RT-PCR may be used to quantify gene silencing47,&#160;but it should be noted that RT-PCR detects mRNA rather than protein. When antibodies for western blotting are available, a common issue is poor detection or the presence of non-specific bands. Troubleshooting guides to help users solve common issues are available, and these tend to include a range of solutions such as primary antibody titration, alternative blocking, and detection conditions48,49. In this study, FUT8 unexpectedly appeared as a double band at ~65 and ~70 kDa. It is possible that the ~70 kDa band represents glycosylated FUT8. Literature evidence from human cell lines describes O-linked glycosylation at Thr 56450,51, a site that is conserved in Chinese hamster, and CHO K1 FUT8 sequences.
As previously mentioned, glycosylation pathways often involve a complex array of enzymes. The current protocol has been developed, optimized, and demonstrated using a monogenic glycosylation reaction controlled by Fut8. Therefore, further studies are required to confirm the robustness of this method when the target gene encodes an enzyme with alternate kinetics and expression levels or a pathway regulated by isoenzymes with redundant functions.
Taken together, the ability to rapidly silence genes and detect modified IgG glycoprofiles is a useful tool in the effort toward custom glycoengineered antibodies. Insights from similar short-term studies can be applied to generate stable glycoengineered cells for use in long-term assays like fed-batch culture. Outside of the pharmaceutical context, this method contributes toward the study of glycan biology and highlights the important function of glycans in development, health, and disease.

N-linked&#160;glycosylation is an enzymatic process by which oligosaccharide moieties are covalently linked to Asn residues. Unlike de novo protein synthesis, glycan synthesis is a non-templated reaction that results in heterogeneous glycosylation of proteins. The structure, composition, and distribution of glycans can affect protein conformation and function. Indeed, N-glycosylation in the crystallizable fragment (Fc) region of immunoglobulin G (IgG) regulates the therapeutic efficacy, immunogenicity, and half-life of the antibody1. As such, the quality by design (QbD) paradigm for the development of recombinant biotherapeutic protein products naturally identifies glycosylation as a critical quality attribute (CQA)2,3. Mammalian cells are often the preferred expression systems as they inherently produce human-like glycosylation patterns more closely than bacteria, yeast, insect, or plant cells. Moreover, Chinese hamster ovary (CHO) cells are selected over other mammalian cell lines because they are resistant to human virus infection, secrete products at high titers, and can be grown in suspension culture to high viable cell densities4. With respect to glycan formation, non-CHO murine production cells generate immunogenic glycans (&#945;(1-3)-linked galactose [&#945;(1-3)-Gal] and N-glycolylneuraminic acid [NeuGc]) that impinge on the safe use of monoclonal antibodies (mAbs)5. These benefits make CHO cells the foremost expression system, responsible for the production of over 80% of new biotherapeutics between 2014 and 20186. However, template-independent glycosylation is a conserved mechanism that leads to CHO-derived biotherapeutics with an array of glycoforms.
Biotherapeutic development strategies aim to control heterogeneity in CHO cells by genetic engineering. Some literature examples include the knockdown of sialidases (Neu1, Neu3)7, GDP-mannose 4,6-dehydratase (GMD) knockout8, and overexpression of glycosyltransferases (GnTIII)9. Advances in glycoengineering are possible due to a combination of publicly available resources, like the CHO genome10, and the ongoing development of genetic engineering tools, such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9)11,12,13,14. These tools are typically delivered to CHO cells as plasmid DNA or as purified ribonucleoprotein (RNP) complexes. Conversely, RNA interference (RNAi) is a genetic engineering technology that, in its simplest form, only requires the delivery of purified short interfering RNA (siRNA) oligonucleotides. Endogenous proteins process double-stranded siRNA into single strands, and the nuclease, RNA-induced silencing complex (RISC), forms a complex with siRNA to cleave target mRNA sequences15,16,17. Gene silencing via this method is transient due to RNA instability, but the investigation herein leverages this feature to assist rapid screening.
The model enzyme selected for the current study, &#945;1,6-fucosyltransferase (FUT8), produces N-glycans with &#945;-1,6 core-linked L-fucose (Fuc). This modification is a primary determinant of antibody-dependent cell cytotoxicity (ADCC) activity, as evidenced by studies of commercial antibodies. In the absence of core fucosylation, Rituximab (anti-CD20 IgG1) increases ADCC 50-fold and increases ADCC in Trastuzumab (anti-Her2 IgG1) by improving FcgRIIIa binding18,19. Core fucosylation is, thus, considered an undesirable feature of mAbs that warrants efforts to reverse this phenotype. There are examples of successful Fut8 gene targeting using siRNA with concomitant increases in ADCC20,21,22, albeit these examples deliver Fut8 siRNA encoded on plasmid DNA. Such experiments generate stable gene silencing as plasmid DNA serves as a template for siRNA synthesis. This allows cells to replenish siRNA molecules that are degraded by intracellular RNases and phosphatases. Conversely, the delivery of exogenous synthetic siRNA only permits transient gene silencing as siRNA cannot be replenished due to the lack of an intracellular template. Thus, users should consider if experimental designs are compatible with plasmid-derived or synthetic siRNA. For example, studies focused on peak mAb production, typically day six of the culture23,24, may opt for synthetic siRNA that can be delivered to cells a few days before peak expression. The benefits of a transient approach using synthetic siRNA include the ability to outsource production and the fact that multiple siRNA constructs can be generated in a fraction of the time taken to generate constructs in plasmids. Furthermore, synthetic siRNA is efficacious, as evidenced by literature examples of Fut8 gene silencing that are sufficient to reduce FUT8 protein expression25 and yield afucosylated IgGs with increased FCgRIIIa binding and ADCC26.
The success of this glycoengineering protocol was determined by the degree of Fc glycosylation. Mass spectrometry is normally the method of choice for glycomic analyses; however, capillary gel electrophoresis and laser-induced fluorescence detection (CGE-LIF) is perfectly amenable to resolving the glycoprofile of purified IgGs and has the advantage of greater rapidity and simplicity. Mass spectrometry protocols must combine the appropriate chromatographic and derivatization methods, ionization sources, and mass analyzers27,28,29. In addition to requiring a trained specialist, mass spectrometry protocols are lengthy, and the diversity of methods makes data difficult to compare between laboratories with different setups. In the context of biopharmaceuticals, CGE-LIF is a sensitive method that can provide sufficient details of an antibody glycoprofile and is easily scalable for high-throughput methods. For low abundance, highly complex mixtures with poorly characterized glycoproteins, the advantages of mass spectrometry might remain. However, the high-resolution and high-sensitivity mAb analytics afforded by CGE-LIF-based N-glycan analysis serve as a rationale to trial this method. Furthermore, sample preparation and analysis are complete in just a few hours30. Recent studies have shown that CGE-LIF can be used to monitor glycans derived from human plasma31, mouse32, and CHO IgGs33. These studies highlight the use of CGE-LIF for high-throughput sample analysis and small sample volumes.
The CGE-LIF method has limitations that should be taken into consideration. Cost is a significant barrier to the use of this and other devices for glycan analysis. However, these costs are typical within the field, and CGE-LIF is thought to be a cost-effective option34. Labs with smaller budgets may find it more practical to lease machinery or outsource samples analysis. Another consideration of any analytical method is repeatability. Evaluation of CGE-LIF was conducted using 48 replicates of the same sample that were assayed on different days. The relative standard deviation per capillary was determined for intrabatch and interbatch repeatability. The intrabatch comparison of replicates was found to have a relative standard deviation of 6.2%, indicating that capillary performance is not uniform. Further, a comparison of interbatch data showed a relative standard deviation of 15.8%31, indicating that the capillary performance changes over time. The operational shortcomings identified may not apply in the current study, which uses different machinery and proprietary reagents. If users intend to develop an in-house protocol, it would be worth considering the study by Ruhaak et al.31, which carefully evaluated the reagents for CGE-LIF. As such, the reagents for sample injection (Hi-Di Formamide and DMSO), glycan labeling (NaBH3CN or 2-picoline borane)31, and others have been optimized.
This study presents a time-efficient glycoengineering protocol that combines the rapidity of direct RNAi with downstream glycomic analysis. The methodology is illustrated using the Fut8 gene as a target for the reasons outlined above.

<table><tbody><tr><td>32 Karat software</td><td>SCIEX</td><td>contact manufacturer</td><td>Software for glycan data acquisition and analysis using the Fast Glycan analysis protocol and separation method.</td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Sigma Aldrich</td><td>34851</td><td>Solvent.</td></tr><tr><td>Anti-FUT8 antibody</td><td>AbCam</td><td>ab198741</td><td>Rabbit polycloncal to Fut8. Use this antibody to quantify Fut8 protein expression; replace this antibody if using siRNA targeting a different gene.</td></tr><tr><td>Anti-GAPDH antibody</td><td>AbCam</td><td>ab181602</td><td>Rabbit monoclonal to GAPDH. Alternative housekeeping genes exist and might be preferred by the user.</td></tr><tr><td>BioDrop Spectrophotometer</td><td>Biochrom</td><td>80 3006 55</td><td>Instrument used to quantify protein concentration.</td></tr><tr><td>BLItz</td><td>ForteBio</td><td>45 5000</td><td>Instrument. Label-Free Protein Analysis System.</td></tr><tr><td>BRAND Haemocytometer</td><td>Sigma Alrich</td><td>BR717810</td><td>Counting chamber device</td></tr><tr><td>Capillary cartridge</td><td>SCIEX</td><td>A55625</td><td>Pre-assembled capillary cartridge with window (30 cm total length, 375 &amp;micro;m outer diameter (o.d), x 50 &amp;micro;m inner diameter (i.d).</td></tr><tr><td>C100HT Glycan analysis&amp;mdash;capillary electrophoresis</td><td>SCIEX</td><td>contact manufacturer</td><td>Capillary gel electrophoresis instrument, the CESI 8000 Plus instrument is now used.</td></tr><tr><td>CD CHO Medium</td><td>Thermo Fisher Scientific</td><td>10743029</td><td>Replace this with a culture medium appropriate for the cell line of choice.</td></tr><tr><td>Centrifuge tubes, 15 mL</td><td>Greiner Bio</td><td>188261</td><td>Sterile polypropylene tube.</td></tr><tr><td>Centrifuge tubes, 50 mL</td><td>Greiner Bio</td><td>227270</td><td>Sterile polypropylene tube.</td></tr><tr><td>CHO IgG</td><td>MedImmune</td><td>Gift</td><td>Chinese Hamster Ovary cells expressing an IgG monoclonal antibody (CHO T127). Created using the GS system.</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>14190144</td><td>1x PBS, without calcium or magnesium.</td></tr><tr><td>Erlenmeyer Flasks with Vent Cap, 125 mL</td><td>Corning</td><td>431143</td><td>Replace this with a culture vessel suitable for growing the cell line of choice.</td></tr><tr><td>Erlenmeyer Flasks with Vent Cap, 250 mL</td><td>Corning</td><td>431144</td><td>Replace this with a culture vessel suitable for growing the cell line of choice.</td></tr><tr><td>Fast Glycan Labelling and Analysis kit</td><td>SCIEX</td><td>B94499PTO</td><td>Labels N-glycans with APTS and then uses a magnetic-bead based clean up system to remove excess APTS.</td></tr><tr><td>Fut8 DsiRNA</td><td>IDT</td><td>Custom</td><td>Custom designed DsiRNA targetting Fut8.</td></tr><tr><td>Gene Pulser cuvettes, 0.4 cm</td><td>Bio-Rad</td><td>1652088</td><td>Electroporation cuvette.</td></tr><tr><td>Gene Pulser Xcell Eukaryotic System</td><td>Bio-Rad</td><td>165 2661</td><td>Insturment. Xcell main unit with Capacitance Extender (CE) Mocdule and ShockPod.</td></tr><tr><td>Immobilon-FL PVDF&amp;nbsp; membrane</td><td>Merck-Millipore</td><td>IPFL00010</td><td>Immunoblot transfer membrane, low background.</td></tr><tr><td>L-Methionine sulfoximine (MSX)</td><td>Sigma Aldrich</td><td>M5379</td><td>Only necessary for CHO cell lines using the glutamine synthetase (GS) selection system.</td></tr><tr><td>Kimwipes</td><td>Thermo Fisher Scientific</td><td>10623111</td><td>Low-lint, high absorbency and chemically inert wipes.</td></tr><tr><td>M-PER Mammalian Protein Extraction Reagent</td><td>Thermo Fisher Scientific</td><td>78505</td><td>Alternative lysis buffers such as RIPA are also appropriate.</td></tr><tr><td>Methanol, HPLC grade</td><td>Fisher Scientific</td><td>10365710</td><td>Solvent.</td></tr><tr><td>Microcentrifuge tubes, 1.5 mL</td><td>Eppendorf</td><td>616201</td><td>Autoclavable tubes.</td></tr><tr><td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell system</td><td>Bio-Rad</td><td>1658035FC</td><td>Instrument. 4-gel capacity, for 1.0 mm thick handcast gels, with Mini Trans-Blot Module and PowerPac HC Power Supply.</td></tr><tr><td>NC-Slide A8</td><td>ChemoMetec</td><td>942 0003</td><td>8-chamber slide for use with NucleoCounter NC 250.</td></tr><tr><td>Negative Control DsiRNA, 5 nmol</td><td>IDT</td><td>51 01 14 04</td><td>Non-targeting DsiRNA.</td></tr><tr><td>Nuclease-free duplex buffer</td><td>IDT</td><td>11-01-03-01</td><td>Reconstitution buffer for DsiRNA.</td></tr><tr><td>NucleoCounter NC-250</td><td>Chemometec</td><td>contact manufacturer</td><td>Instrument. Automated Cell Analyzer</td></tr><tr><td>Page-Ruler ladder, 10 to 180 kDa</td><td>Thermo Fisher Scientific</td><td>26616</td><td>Mixed blue, orange and green protein standards for SDS PAGE and western blotting.</td></tr><tr><td>PCR tubes</td><td>Greiner Bio</td><td>608281</td><td>Autoclavable tubes for DsiRNA aliqouts and glycan preparation.</td></tr><tr><td>Pipette filter tips sterilised&amp;nbsp; (10, 200, 1000 &amp;micro;L)</td><td>Gibson</td><td>F171203, F171503, F171703</td><td>Sterile filter tips to avoid RNA contamination.</td></tr><tr><td>PNGase F enzyme</td><td>New England Biolabs</td><td>P0704S</td><td>Enzymatic cleavage of glycans from glycoproteins.</td></tr><tr><td>Polypropylene columns, 1 mL</td><td>Qiagen</td><td>34924</td><td>Columns for gravity-flow chromatography.</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma Aldrich</td><td>P8340</td><td>Inhibition of serine, cysteine, aspartic proteases and aminopeptidases</td></tr><tr><td>Protein-A Agarose Beads</td><td>Merck-Millipore</td><td>16 125</td><td>For purification of human, mouse and rabbit immunoglobulins.</td></tr><tr><td>Protein-A biosensor</td><td>ForteBio</td><td>18 5010</td><td>Tips functionalised with Protein A for rapid antibody quantification.</td></tr><tr><td>RNaseZap</td><td>Invitrogen</td><td>AM9780</td><td>Removes RNAse contamination.</td></tr><tr><td>Sample dilutent</td><td>ForteBio</td><td>18 1104</td><td>&lt;strong&gt;Activate Protein A tips.&lt;/strong&gt;</td></tr><tr><td>Serological pipets (5, 10, 25 mL)</td><td>Corning</td><td>4487, 4488, 4489</td><td>Used for sterile cell culture tecniques.</td></tr><tr><td>Sodium cyanoborohydride solution 1 M in THF</td><td>Sigma Aldrich</td><td>296813</td><td>Reducing agent.</td></tr><tr><td>Solution 18</td><td>ChemoMetec</td><td>910-3018</td><td>Staining reagent containing acridine orange (AO) and 4&#039;,6-diamidino-2-phenylindole (DAPI)</td></tr><tr><td>Spin-X Centrifuge Tube Filters</td><td>Corning</td><td>8161</td><td>&amp;nbsp;0.22 &amp;micro;m pore, Cellulose Acetate membrane.</td></tr><tr><td>Suspension plate with lid, 6-well</td><td>Greiner Bio</td><td>657 185</td><td>Hydrophobic culture plate for growth of suspension cultures.</td></tr><tr><td>Syringe filters,&amp;nbsp; 0.22 &amp;mu;m</td><td>Sartorius</td><td>514 7011</td><td>Surfactant-free cellulose acetate (SFCA)</td></tr><tr><td>Syringes with Luer lock tip, 20 mL</td><td>Fisher Scientific</td><td>10569215</td><td>For secure connection with syringe filter.</td></tr><tr><td>Trypan Blue solution</td><td>Gibco</td><td>15250061</td><td>Stains dead and dying cells.</td></tr><tr><td>Vivaspin 500, 3,000 MWCO</td><td>Sartorius</td><td>VS0191</td><td>Polyethersulfone</td></tr><tr><td>WesternBreeze Chromogenic Kit, anti-rabbit</td><td>Thermo Fisher Scientific</td><td>WB7105</td><td>Western blot detection kit, alternative blocking buffers and antibody diluents can be made by the user using recipes available online.</td></tr></tbody></table>

rapid antibody glycoengineering in chinese hamster ovary cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

Western blot analysis showed reduced FUT8 protein expression in cells transfected with a mixture of three Fut8 DsiRNA constructs. In control samples transfected with non-targeting DsiRNA, FUT8 appeared as a double band at ~65 and 70 kDa. Since the predicted molecular weight of FUT8 is 66 kDa, a reduction in the signal intensity of the lower molecular weight band is indicative of gene silencing. To confirm and quantify gene silencing, the level of FUT8 protein was normalized to the relative GAPDH protein level. Western blot analysis detected two bands for GAPDH at ~37 and 35 kDa. The higher molecular weight band corresponds to the predicted protein size and is, therefore, used in normalization calculations. When normalized against GAPDH protein levels, FUT8 protein expression was reduced by up to 60% (Figure 1).
In line with the observation of gene knockdown at 48 h post transfection, corresponding mAb samples were processed for analysis by CGE- LIF. Glycan structures from knockdown cells showed a decrease in fucosylation. This trend was most pronounced in agalactosylated structures (G0F) and observed to a lesser extent in galactosylated structures (G1F, G1F&#39; and G2F). From this dataset, the total IgG core fucosylation decreased to ~75%, down from ~95% core fucosylation observed for the negative control condition (Figure 2). A greater reduction in core fucosylation was anticipated given the ~60% decrease in FUT8 protein levels. Upon reflection, it is noteworthy that the glycoprofile represents glycosylated mAbs that accumulated over a period of 48 h since transfection, while gene silencing represents protein levels present at the time of harvesting only.
Further scrutiny of this knockdown method involved varying the DsiRNA concentration, harvest time, and electroporation conditions. Each factor was individually probed to determine its relevance. The impact of electroporation pulse conditions on core fucosylation and cell viability is captured in Experiments B, C, D, and E. These results demonstrate a two-fold reduction in core fucosylation from electroporation using two square wave pulses (Experiment C) compared to a single square wave pulse (Experiment B), without significant differences in cell viability (Table 2). Electroporation condition e3 (Experiment D) led to the lowest cell viability (~90%) and IgG yield at this timepoint. However, cells that survived the electroporation event were moderately transfected, as evidenced by the ~10% decrease in core fucosylation (Table 2). Interestingly, Experiment D used electroporation conditions that provided the greatest reduction in core fucosylation (14.7%) but were evidently detrimental to cell viability (91%-93% viability). This limited set of experiments illustrates the need to determine electroporation settings that enable sufficient permeabilization of the cell membrane without causing irrevocable damage. It is also interesting to note the role of siRNA concentration and harvest time on core fucosylation. Overall, increasing siRNA concentration has a greater influence on core fucosylation than increasing the harvest time (Experiments B, F, G versus Experiments A, B, H). In future experiments, it would be interesting to titrate the siRNA concentrations delivered by the electroporation method e2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63872/63872fig01.jpg" /> 
Figure 1. Experimental flow chart. Glycoengineering and sample analysis steps are depicted with the associated time required for each step. SiRNA design takes a few hours, depending on the number of gene targets or constructs per gene target. CHO cell transfection with siRNA is complete in a few hours, and transformed cells are left to grow for 48 h. Cell pellets and supernatants are harvested within a few hours. Cell pellets are lysed, and the intracellular proteins are separated on an SDS PAGE and subsequently blotted and probed with antibodies against the target gene. Glycans are cleaved from purified antibodies and analyzed by CGE-LIF. These assays may require 1 day each. <a href="https://www.jove.com/files/ftp_upload/63872/63872fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63872/63872fig02.jpg" /> 
Figure 2. Confirmation of RNA interference. Western blot detection of &#945;-1,6-fucosyltransferase (FUT8) protein levels in samples treated with Fut8 or non-targeting control DsiRNA. Bands corresponding to FUT8 are more intense in control than Fut8 knockdown samples. The GAPDH protein level was also assessed in order to normalize target gene expression. All samples were taken from Experiment G (see Table 2). Reprinted from Kotidis&#160;et al.52. <a href="https://www.jove.com/files/ftp_upload/63872/63872fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63872/63872fig3v2.jpg" /> 
Figure 3. Effect of Fut8 knockdown on cumulative IgG glycosylation at 48 h. A shift in glycan distribution is detected in knockdown samples. In particular, the relative abundance of the main core-fucosylated structures (G0F) is reduced while the afucosylated species are increased in the knockdown experiment. Measurements were performed from Experiment G samples (see Table 2). Biological triplicates performed for each experiment were mixed after harvesting to reduce the burden of downstream analysis. Reprinted from Kotidis et al.52. <a href="https://www.jove.com/files/ftp_upload/63872/63872fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Experiment name</td>
			<td colspan="2">Electroporation method</td>
			<td colspan="2">DsiRNA concentration (n&#924;)</td>
			<td>Harvest time (h)</td>
			<td>Viability (%)</td>
			<td colspan="2">Xv (106 cells&#183;mL-1)</td>
			<td>IgG titer (mg&#183;L-1)</td>
			<td colspan="2">Difference in core-fucosylation (%)</td>
		</tr>
		<tr>
			<td colspan="3">ExpA_Negative</td>
			<td colspan="2">e1</td>
			<td colspan="2">500</td>
			<td>24</td>
			<td>98.3</td>
			<td colspan="2">4.71</td>
			<td>122.5</td>
			<td colspan="2">-</td>
		</tr>
		<tr>
			<td colspan="3">ExpA_Knockdown</td>
			<td colspan="2">e1</td>
			<td colspan="2">500</td>
			<td>24</td>
			<td>98.3</td>
			<td colspan="2">4.9</td>
			<td>110.3</td>
			<td colspan="2">4.08</td>
		</tr>
		<tr>
			<td colspan="3">ExpB_Negative</td>
			<td colspan="2">e1</td>
			<td colspan="2">500</td>
			<td>48</td>
			<td>95.6</td>
			<td colspan="2">9.55</td>
			<td>453.3</td>
			<td colspan="2">-</td>
		</tr>
		<tr>
			<td colspan="3">ExpB_Knockdown</td>
			<td colspan="2">e1</td>
			<td colspan="2">500</td>
			<td>48</td>
			<td>96.7</td>
			<td colspan="2">9.61</td>
			<td>469</td>
			<td colspan="2">5.38</td>
		</tr>
		<tr>
			<td colspan="3">ExpC_Negative</td>
			<td colspan="2">e2</td>
			<td colspan="2">500</td>
			<td>48</td>
			<td>96.3</td>
			<td colspan="2">9.91</td>
			<td>449.3</td>
			<td colspan="2">-</td>
		</tr>
		<tr>
			<td colspan="3">ExpC_Knockdown</td>
			<td colspan="2">e2</td>
			<td colspan="2">500</td>
			<td>48</td>
			<td>96.7</td>
			<td colspan="2">11</td>
			<td>454.6</td>
			<td colspan="2">11.42</td>
		</tr>
		<tr>
			<td colspan="3">ExpD_Negative</td>
			<td colspan="2">e3</td>
			<td colspan="2">500</td>
			<td>48</td>
			<td>90.6</td>
			<td colspan="2">6.25</td>
			<td>318.5</td>
			<td colspan="2">-</td>
		</tr>
		<tr>
			<td colspan="3">ExpD_Knockdown</td>
			<td colspan="2">e3</td>
			<td colspan="2">500</td>
			<td>48</td>
			<td>89.1</td>
			<td colspan="2">6.09</td>
			<td>311.85</td>
			<td colspan="2">9.71</td>
		</tr>
		<tr>
			<td colspan="3">ExpE_Negative</td>
			<td colspan="2">e4</td>
			<td colspan="2">500</td>
			<td>48</td>
			<td>91.1</td>
			<td colspan="2">7.2</td>
			<td>380.3</td>
			<td colspan="2">-</td>
		</tr>
		<tr>
			<td colspan="3">ExpE_Knockdown</td>
			<td colspan="2">e4</td>
			<td colspan="2">500</td>
			<td>48</td>
			<td>93.3</td>
			<td colspan="2">7.79</td>
			<td>422.8</td>
			<td colspan="2">14.7</td>
		</tr>
		<tr>
			<td colspan="3">ExpF_Negative</td>
			<td colspan="2">e1</td>
			<td colspan="2">750</td>
			<td>48</td>
			<td>96.2</td>
			<td colspan="2">9.7</td>
			<td>501</td>
			<td colspan="2">-</td>
		</tr>
		<tr>
			<td colspan="3">ExpF_Knockdown</td>
			<td colspan="2">e1</td>
			<td colspan="2">750</td>
			<td>48</td>
			<td>95.7</td>
			<td colspan="2">9.76</td>
			<td>504.6</td>
			<td colspan="2">9.9</td>
		</tr>
		<tr>
			<td colspan="3">ExpG_Negative</td>
			<td colspan="2">e1</td>
			<td colspan="2">1000</td>
			<td>48</td>
			<td>96.1</td>
			<td colspan="2">11.1</td>
			<td>422.6</td>
			<td colspan="2">-</td>
		</tr>
		<tr>
			<td colspan="3">ExpG_Knockdown</td>
			<td colspan="2">e1</td>
			<td colspan="2">1000</td>
			<td>48</td>
			<td>95.9</td>
			<td colspan="2">9.73</td>
			<td>499.3</td>
			<td colspan="2">17.26</td>
		</tr>
		<tr>
			<td colspan="3">ExpH_Negative</td>
			<td colspan="2">e1</td>
			<td colspan="2">500</td>
			<td>72</td>
			<td>94.4</td>
			<td colspan="2">14.3</td>
			<td>925.8</td>
			<td colspan="2">-</td>
		</tr>
		<tr>
			<td colspan="3">ExpH_Knockdown</td>
			<td colspan="2">e1</td>
			<td colspan="2">500</td>
			<td>72</td>
			<td>95</td>
			<td colspan="2">13.5</td>
			<td>1018.4</td>
			<td colspan="2">7.37</td>
		</tr>
	</tbody>
</table>
Table 2. Transfection optimization. Iterative modifications of the electroporation method, DsiRNA concentration, and harvest time led to changes in cell viability, viable cell density, IgG titer at the harvest time, and differences in core fucosylation. Each experiment compared the knockdown and the respective negative control to determine if the modification produces the desired effect (i.e., a decrease in fucosylation). Electroporation settings were as follows: e1: 1200 V, 0.1 ms, square waveform; e2: 1200 V, 2x 0.1 ms, 5 s between pulses, square waveform; e3: 150 V, 20 ms, square waveform; e4: 250 V, 500 &#956;F, exponential decay. Reprinted from Kotidis&#160;et al.52.

Watch this Scientific Journal Video about Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells at JoVE.com

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

rapid-antibody-glycoengineering-in-chinese-hamster-ovary-cells

Research

JoVE Journal

Bioengineering

3.1K Views.  Imperial College London. Dit protocol kan worden gebruikt om het effect van meerdere glycosylatie-enzymen op het glycoprofiel van een doeleiwit te screenen en bij te dragen aan een beter begrip van de functionaliteiten van het enzym. De aard van het protocol maakt de screening van glycosylatie-enzymen op een voorbijgaande, maar toch high-throughput manier mogelijk. Veranderde glycosylatieprofielen kunnen leiden tot verbeterde antilichaamactiviteiten en daardoor de werkzaamheid van het eindproduct verhogen.Biof...

Video: Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

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.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

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

Biochemistry

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)1-3. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP4,5, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP.
Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF6-8. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition.
OBJECTIVE: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay.
METHODS: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMR&alpha;) with high purity9-13. The recombinant sGMR&alpha; was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions14,15. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMR&alpha; than those produced by other insects or yeasts.
RESULTS: The cell-free assay system using sGMR&alpha; yielded the data with high plasticity and reliability. GM-CSF binding to sGMR&alpha; was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMR&alpha; is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells.
CONCLUSIONS: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.

We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

Immunology and Infection

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

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.

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

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Biology

A Technique to Assess the In Vivo Localization of Tumor-Specific Antibodies

Source: Shivange, G., et al. <a href="https://app.jove.com/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody.</a> J. Vis. Exp. (2020).
This video demonstrates a technique to study the in vivo localization of tumor-specific antibodies in a mouse tumor xenograft model. Upon subcutaneously injecting cancer cells into mice for tumor formation, fluorescently-labeled antibodies are injected that bind to tumor-specific receptors, enabling in vivo assessment of antibody localization via imaging.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

A Method to Produce Recombinant Antibodies Against Metallopeptidase

Grow the pET-28a(+) recombinant antibodies aminopeptidase in BL21-transformed bacteria in the presence of 0.4 millimolar beta-D-1-thiogalactopyranoside in an orbital shaker at 37 degrees Celsius for 10 hours. Seed 100 microliters 0.5 x 105 Chinese hamster ovary cells per well into a 96-well plate for incubation at 37 degrees Celsius in a 6% carbon dioxide atmosphere for 18 to 24 hours.
When the cells reach 80% to 90% confluence, dilute the pIRES2-ZsGreen1 recombinant antibodies aminopeptidases in plasmid with Opti-MEM to a final concentration of 0.1 micrograms per microliter. After five minutes, mix the 50 microliters of diluted pIRES2-ZsGreen1 recombinant antibodies aminopeptidases in plasmid with one microliter of Lipofectamine 2000 and 49 microliters of Opti-MEM.
After 20 minutes of incubation, add 100 microliters of the mixture to each well of a 96-well plate containing CHO cells. At four to six hours post-transfection, replace the medium with DMEM/F12 supplemented with 10% FBS.
After 48 hours of incubation, add 400 micrograms per microliter G418 to each well to select the stably transfected cells. After 10 days of selection using DMEM/F12 medium supplemented with 10% FBS and 400 micrograms per microliter G418, sort the cells with fluorescence-activated cell sorting. Serially dilute the harvested positive cells. Seed them at an average of 0.5 to two cells per well in a 96-well plate, and culture in 37 degrees Celsius 6% carbon dioxide incubator.

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

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