1. MicroParticle 준비 <ol><li> 비오틴 NHS 화학을 사용하여 레이블 항체 </li><li> Biotinylation의 정도 결정 </li><li> Biotinylated 항체와 공액 Streptavidin 비즈 <ol><li> 1~2분에 대한 vortexing하여 원래의 약병에 비즈를 Resuspend </li><li> microcentrifuge 관으로 나누어지는 구슬 원하는 볼륨을 </li><li> 비즈에게 PBST로 3 회 씻어 : <ol><li> 이분에 대한 자석에 구슬이 들어있는 튜브를 배치 </li><li> 자석에 튜브로 흡인하여 표면에 뜨는를 제거합니다. 구슬을 방해하지 않도록주의 </li><li> 자석에서 튜브를 제거하고 1 ML의 PBST를 추가 </li><li> 구슬을 resuspend 20 초 소용돌이 </li><li> 뚜껑에서 비즈를 제거하는 5 초 동안 원심 분리기 </li><li> 두 번 더 2.3.1-2.3.5를 반복 </li></ol></li><li> 자석에 세 번째 세척 장소 관 후 PBST를 기음 </li><li> 비즈로 biotinylated 항체를 추가하고 resuspend </li><li> 필요한 경우하는 것이 PBS 300 μL에 biotinylated 항체의 볼륨을 가져 오십시오. 항체 볼륨이 이미있다면 큰 300 μL는 PBS를 추가하지 마십시오 </li><li> 와동 흔드는 또는 튜브 회전에서 RT 30 분 비드 / 항체 서스펜션을 품어 </li><li> 3 단계에서와 같이 PBST로 3 번 씻으십시오. </li><li> biotinylated BSA와 구슬 블럭 <ol><li> 세번째 세척이 10 μg / MG biotinylated BSA를 추가 후, PBS 필요한 경우 300 μL로 볼륨을 조정하고, 30 분 동안 교반과 부화 </li></ol></li><li> 3 단계에서와 같이 PBST로 3 회 씻어 </li><li> PBS/0.05 %의 BSA 100 μL에 비즈를 Resuspend. 나트륨 azide는 장기 보관을 위해 추가할 수 있습니다 </li></ol></li><li> 표준 NHS 플루오레신 프로토콜을 사용하여 탐지 항체를 fluoresceinate </li><li> Fluorescination에 노출된 정도를 확인하는 </li></ol> 2. HCP - 개발 <ol><li> 작업 솔루션을 준비 : <ol><li> 플루오레신 표시된 항체 (층 - AB) : 대부분의 CHO - HCP assays 400 NG / ML의 최종 농도에 층 - AB를 사용합니다. 층 - AB가 최종 분석 볼륨의 ¼로 추가됩니다 따라서 작업 솔루션의 농도는 1.6 μg / ML입니다. BioScale 비드 / FlAb 희석제와 함께 FlAb 주식을 diluting하여이 솔루션을 준비합니다. </li><li> 비즈 : 대부분의 CHO - HCP assays은 2x10 5 구슬 / ML의 최종 농도에 구슬을 사용합니다. 비드 시약은 최종 분석 볼륨의 ¼을 구성하므로 작업 솔루션의 농도 5 구슬 / ML 8x10입니다. BioScale 비드 / FlAb 희석제와 비드 주식을 diluting하여이 솔루션을 준비합니다. 와동 확실히 모든 비즈 있도록 잘 구슬 주식 사전 나누어지는를 제거하기 위해 중지되어 있습니다. </li><li> 버퍼를 실행 : 실행중인 버퍼의 구성은 그러나 몇 가지 CHO - HCP assays가 10 MM의 HEPES 버퍼, 1 % 트윈, 산도 7.5를 사용하여 개발되었습니다, 분석을 개발하는 데 사용되는 항체에 최적해야 할 수 있습니다. BioScale 사용하기 전에 1 % 농도로 버퍼에 추가됩니다 실행중인 버퍼 (개발 키트와 함께 제공된)에 첨가제를 개발하였습니다. </li></ol></li><li> BioScale 샘플 희석제와 calibrators 및 샘플을 희석. </li></ol> 3. 분위기 시스템 설정 <ol><li> 의 분위기 워크 스테이션 턴 </li><li> 시스템 공급 및 폐기물을 연결 </li><li> 프라임 시스템 </li><li> 분위기 카트리지를 삽입 </li><li> 루트 튜브에게 </li><li> &quot;샘플 설정&quot;이라는 시트를 작성하여 분석 템플릿을 준비합니다. BioScale 각 바이브 워크 스테이션 분석 한 분석 템플릿을 가지고 기능을 제공합니다. 이것은 사용자가 실험의 완전한 기록을 유지 다른 assays에 대한 템플릿의 &quot;주의 사항&quot;시트에서 특정 노트를 편집하고 저장할 수 있습니다. </li><li> 초기 분석 템플릿은 설정 : &quot;예제 설정&quot;시트의 분석에 기준의 농도 범위의 테이블 (왼쪽 상단 모서리)를 작성하여 시작합니다. </li><li> 다음 긴 샘플 부화 필요한 방법 바이브 워크 스테이션을 지시하기 위해 표준의 오른쪽 (단순 칼럼 읽기 설정, 하나 시약 추가하고 두 시약 추가 설정)을 테이블에 추가 정보를 입력, 얼마나 시약의 부피가 전송하는 것입니다 , 샘플 축적되어야 얼마나 오래, 그리고 센서 컨디셔닝 및 프로브 및 카트리지의 오염 제거가 필요한지 여부 등 </li><li> 접시 레이아웃을 작성하십시오. </li><li> 접시에 샘플 / 시약을로드합니다. 느낌이 시약 판에 필요한 볼륨을 제공합니다. 샘플 볼륨은 다양하지만, 120 μL의 총 분석 볼륨의 절반에 좋은 시작 장소입니다 수 있습니다. </li><li> 플레이트 커버를 적용합니다. 워크 스테이션 데크에 번호판을로드합니다. </li><li> 마법사를 시작 분석을 실행 마법사를 통해 이동하여 분석을 시작합니다. </li></ol> 4. 대표 결과 <img src="/files/ftp_upload/2493/2493fig1.jpg" alt="그림 1" /> 그림 1 : AMMP 샌드위치 면역 분석법 </p&gt; 분위기 워크 스테이션에서 AMMP 분석은 항원 농도를 측정합니다. 시그널은 비드 - 항원 - 항체 hapten이 hapten / 안티 - hapten 상호 작용을 통해 센서에 바인딩 할 때 측정됩니다. 중생은 면역 단지를 제거하여 처음 상태로 다시 센서를 제공합니다. <img src="/files/ftp_upload/2493/2493fig2.jpg" alt="그림 2" /> 그림 2 : CHO - HCP AMMP 분석 교정 곡선 그림 위에 2 CHO HCP AMMP 분석 교정 곡선의 예입니다. 분석은 높은 정밀도와 재현성있는 반응기를 포함하여 정화 스트림 전역에서 표본으로 수행할 수 있습니다. <img src="/files/ftp_upload/2493/2493fig3.jpg" alt="그림 3" /> 그림 3 : 단백질 AMMP 분석 교정 곡선 그림 3은 느낌 잔여 단백질 어세이 키트와 함께 수행 잔여 단백질 AMMP 어세를위한 보정 곡선입니다. 동일한 키트는 30 분 잠복기와 빠른 분석, 또는 일반적으로 동급 엘리사의 사용자가보다 나은 둘 중 정밀도 또는 정확성의 손실없이 긴 잠복기와 더 민감한 결과와 넓은 다이나믹 레인지에 사용할 수 있습니다 &#39; 손 <img src="/files/ftp_upload/2493/2493fig4.jpg" alt="그림 4" /> 그림 4 : 제품 Titer AMMP 어세이 분위기 워크 스테이션 또한 제품 Titer 분석, 그림 4에 표시된 결과를 사용할 수 있습니다. 다른 AMMP의 Assays 다르게 수행,이 분석은 단순 희석, 또는 구슬보다 샘플 치료가 다른가 필요하지 않으며, quantitation는 viabilities 광범위한 이상 만 10 개 이상의 세포를 포함하는 &quot;아니오 스핀&quot;샘플로 확인되었습니다. <img src="/files/ftp_upload/2493/2493fig5.jpg" alt="그림 5" /> 그림 5 : GST - Gadd34 biomarker 분석을위한 AMMP 분석 표준 곡선 분위기 BioAnalyzer (패널) 또는 재조합 GST - Gadd34를 사용하여 AlphaScreen 기술로 얻은 신호의 표준 곡선은 그림 5에 표시됩니다. 바이브 플랫폼 결과가 동일한 항체를 사용하여보다 민감한 약 로그되었습니다. AMMP 아직 적은 큰 입자 솔루션 위상 캡처 및 감지 복잡한 형성에서 여전히 혜택의 측정을 활용합니다. <img src="/files/ftp_upload/2493/2493fig6.jpg" alt="그림 6" /> 그림 6 : 종양 샘플에서 GST - Gadd34에 대한 AMMP 분석 표준 곡선 그림 6에서 CWR22 xenografts에서 Gadd34의 유도는 느낌 BioAnalyzer (패널 A) 또는 AlphaScreen (패널 B)에 의해 분석으로 boronate proteasome 억제제의 존재 또는 부재에서 테스트되었습니다. 바이브 플랫폼 AlphaScreen 신호 반면, 생체내 종양 xenografts에있는이 단백질의 증가 μg로 침묵했다에 민감한, 재현성 결과를 얻을 수있었습니다. 이 예제는 매우 복잡한 샘플의 AMMP 기술의 파워를 보여줍니다. 칩 근처 자석의 민감한 감지 칩과 높은 효율성으로 인해, 자기 microparticles의 낮은 숫자는 효율적이고 reproducibly lysed 세포 (용해 시약 포함) 및 혈액 파편의 복잡 가운데 낮은 풍부한 대상에 대한 청소하실 수 있습니다.

이 문서의 저자는이 문서에서 사용되는 악기와 시약의 일부를 생산 Bioscale, 병원에서 근무하고 있습니다.

상기 예제는 복잡한 샘플에 AMMP의 고감도 정량 결과를 보여줍니다. 시스템에 시약 서식의 용이성 및 제어 기능을 통해 전달이 성능, 다양한 어플 리케이션을 위해 사용됩니다. 이러한 응용 프로그램은 종양의 Gadd34에서와 같이, 대상에 대한 구체적으로, 또는 정화 스트림을 교차 샘플에 숙주 세포 단백질 감지 및 quantitation에서 표적의 스펙트럼에 대한 일반적인 수 있습니다. 매우 유연한 AMMP와 결합된 낮은 오버헤드 바이브 플랫폼은 중요한 assays를 전달하기위한 강력한 도구를 제공합니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NHS-Chromogenic-biotin or NHS-Chromogenic Biotin Labeling kit</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>If NHS-Chromogenic-biotin is purchased as a reagent the following additional materials will be required</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS) or other amine free buffer (pH 7 &#x2013; 9)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting columns or dialysis units for buffer exchange</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Spectrophotometer able to measure 354 and 280 nm &#x2013; for measuring protein and biotin concentrations</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated antibody (2-5 biotins/Ab)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin magnetic beads</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>(10 mg/mL) ~108 beads/mg</td>
</tr>
<tr>
<td>Magnet for microcentrifuge tubes</td>
<td>&nbsp;</td>
<td>New England BioLabs</td>
<td>cat # S1510S</td>
<td>&nbsp;</td>
<tr>
<td>Biotinylated BSA for blocking</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS 0.1% Tween 20</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vortex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microcentrifuge holder for Vortex or tube rotator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Antibody or protein to be labeled</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NHS-Fluorescein or NHS-Fluorescein Labeling kit</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>If NHS-Fluorescein is purchased as a reagent the following additional materials will be required</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water-miscible organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 mM borate, pH 8.5 or other amine free buffer (pH 7 &#x2013; 9)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting columns or dialysis units for buffer exchange</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Measurement of protein and fluorescein concentrations requires a spectrophotometer able to measure 493 and 280 nm.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Diluent</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>to dilute beads and FlAb</td>
</tr>
<tr>
<td>Sample Diluent</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>to dilute samples and calibrators</td>
</tr>
<tr>
<td>Running Buffer Additive</td>
<td>&nbsp;</td>
<td><a href="http://www.bioscale.com">BioScale</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

developing custom chinese hamster ovary-host cell protein assays using acoustic membrane microparticle technology

독특한 단백질에 대한 사용자 정의 assays은 종종 시간이 많이 걸리는 수동 검색 및 엘리사 또는 대체 플랫폼 개발의 복잡으로 인해 서양 blots로 quantitation 기술로 제한됩니다. BioScale의 독점 음향 막 MicroParticle (AMMP) 기술은 더 나은 감도, 재현성 및 자동 작동을 제공 샌드위치 immunoassays 쉽게 바이브 플랫폼에서 사용하기 위해 개발된 수 있습니다. 예를로 제공,이 프로토콜은 사용자 정의 중국어 햄스터 난소 - 숙주 세포의 단백질 (CHO - HCP) 분석을 개발하기위한 절차를 설명합니다. 여기에 설명된 일반 원칙은 immunoassays의 다양한 발전을 위해 다음 수 있습니다. 계산하는 센서 표면에 microparticles의 바인딩에 의한 진동 주파수의 변화를 측정하여 AMMP 분석 측정 항원 농도. 그것은 네 개의 주요 컴포넌트로 구성되어 있습니다 : 기능화 팔 센서 칩이 포함되어 있습니다 (1) 카트리지 (2) 항체 라벨 자기 microparticles, 기능화 칩의 표면 (4)의 항원을 포함하는 샘플에 바인딩 (3) hapten 태그 항체 관심. BioScale의 바이오 센서는 별도의 유체 경로의 8 각 세포막을 포함하는 공진 장치입니다. 점막 대량의 표면에 축적이 주파수 변경이 추가 대량의 금액을 quantitate하는 데 사용됩니다에 대한 응답으로 진동 주파수를 변경합니다. 센서 방지 hapten 항체로 작용합니다 immunoassays의 다양한 사용을 촉진합니다. 검정 특정 항체 한 항체 및 다른 비오틴에 hapten 태그의 공유 결합 활용을 통해 수정할 수 있습니다. 비오틴 레이블은 hapten - 태그 항체와 함께, 샌드위치에서 analyte를 캡처하는 데 사용됩니다, 결합 자기 구슬을 streptavidin 수있는 항체를 바인딩하는 데 사용됩니다. 단지 anti-hapten/hapten 상호 작용을 통해 칩을에 바인딩합니다. 각 분석의 끝에 센서가 96 - 웰 플레이트에서 열 순서 분석을 가능하게 희석 염산과 청소됩니다 실행합니다. 여기, 우리는 bioprocess 개발을위한 사용자 정의 CHO - HCP AMMP 분석을 개발하는 방법을 제시한다. AMMP의 assays을 개발하거나 AMMP assays로 기존의 assays를 수정하는 것은 복잡한 샘플 및 감소 연산자는 시간에 더 나은 성능 (재현성, 감도)를 제공할 수 있습니다. 프로토콜 개발을위한 단계를 보여줍니다 및 토론 섹션 대표 결과를 검토합니다. 분석 최적화 및 사용자 정의 매개 변수의보다 깊이 설명 BioScale에 문의하십시오. 이 키트는 잔여 단백질, 제품 titer, 그리고 CHO - HCP와 같은 일반 bioprocess 개발 assays을 제공합니다.

Watch this Scientific Journal Video about Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology at JoVE.com

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

복잡한 매트릭스에서 더 민감한, 재현성, 자동 검색 결과에 대한 바이브 플랫폼에서 사용자 정의 assays의 개발을 설명합니다. 유니버설 카트리지는 assays 쉽게 사용자 정의 assays 사용하기 위해 적응 수 있습니다. 이 다재 다능한이 소설 assays 본 동영상에 exemplified 기존의 수동 assays의 자동 버전의 신속한 개발 및 검증이 가능합니다.

어쿠스틱 막 Microparticle 기술을 사용하여 개발 사용자 지정 중국어 햄스터 난소 - 숙주 세포의 단백질 Assays

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

developing-custom-chinese-hamster-ovary-host-cell-protein-assays

Research

JoVE Journal

Bioengineering

13.1K 조회수.  BioScale, Inc.. 복잡한 매트릭스에서 더 민감한, 재현성, 자동 검색 결과에 대한 바이브 플랫폼에서 사용자 정의 assays의 개발을 설명합니다. 유니버설 카트리지는 assays 쉽게 사용자 정의 assays 사용하기 위해 적응 수 있습니다. 이 다재 다능한이 소설 assays 본 동영상에 exemplified 기존의 수동 assays의 자동 버전의 신속한 개발 및 검증이 가능합니다.

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

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

어쿠스틱 Microstreaming를 사용하여 표준 실험실 역방향 Transcriptase 반응에서 mRNA의 단일 세포로부터 cDNA 수량 산출 증가

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

연구

생체공학

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

단백질 A 멤브레인 흡착제 및 간소화된 워크플로우를 사용하여 키메라 인간 Fc 발현 단백질의 격리 및 특성화

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

자동화 된 Microbioreactor 기술을 사용 하 여 분리 단백질 생산의 최적화를 위한 일반 프로토콜

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

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.

자동화된 미생물체 시스템을 사용하여 중국 햄스터 난소 세포의 단일 클론 항체의 정화 및 분석

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

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

중국 햄스터 난소 세포 재배에서 고처리량 자동화 마이크로 바이오리액터를 사용한 공정 최적화

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

세포가 없는 세균 추출물, 리포좀 및 에멀젼 전달을 사용하여 합성 세포를 생산하는 단백질 을 준비합니다.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

음향 분리기사용 CHO 수확세포 배양액의 1차 해명

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Formulation and Acoustic Modulation of Optically Vaporized Perfluorocarbon Nanodroplets

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These microbubbles can be condensed or formulated as perfluorocarbon nanodroplets (PFCnDs) that are small enough to extravasate and then be triggered acoustically at the target site. These nanoparticles can be further enhanced by including an optical absorber such as near infrared organic dye or nanoparticles (e.g., copper sulfide nanoparticles or gold nanoparticles/nanorods). Optically tagged PFCnDs can be vaporized through laser irradiation in a process known as optical droplet vaporization (ODV). This process of activation enables the use of high boiling point perfluorocarbon cores, which cannot be vaporized acoustically under the maximum mechanical index threshold for diagnostic imaging. Higher boiling point cores result in droplets that will recondense after vaporization, resulting in "blinking" PFCnDs that briefly produce contrast after vaporization before condensing back into nanodroplet form. This process can be repeated to produce contrast on demand, allowing for the background free imaging, multiplexing, super-resolution, and contrast enhancement through both optical and acoustic modulation. This article will demonstrate how to synthesize optically-triggerable, lipid shell PFCnDs utilizing probe sonication, create polyacrylamide phantoms to characterize the nanodroplets, and acoustically modulate the PFCnDs after ODV to improve contrast.

Optically activated perfluorocarbon nanodroplets show promise in imaging applications outside of the vascular system. This article will demonstrate how to synthesize these particles, crosslink polyacrylamide phantoms, and modulate the droplets acoustically to enhance their signal.

광학적으로 기화된 퍼플루오로카본 나노방울의 제형 및 음향 변조

Microbubbles are the most commonly used imaging contrast agent in ultrasound. However, due to their size, they are limited to vascular compartments. These ...

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.

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.

중국 햄스터 난소 세포의 신속한 항체 당공학

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

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Here we present a protocol to express and purify recombinant Drosophila caspases Dronc and Drice, and their use in in vitro cleavage assays.

체외 기질 스크리닝을 위해 정제된 재조합 초파리 카스파제를 사용한 절단 분석

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis ...