Contract Lab Using Flow Imaging Microscopy to Characterize Protein Aggregates
檢測(cè)機(jī)構(gòu)使用流動(dòng)成像顯微鏡來表征蛋白質(zhì)聚集體

Posted by Joyce Brown
Joyce Brown發(fā)表

Dan Berdovich knew there were protein aggregates in his customer’s parenteral sample but despite an assortment of high-tech instrumentation at his disposal, he couldn’t be completely confident he was seeing them all or getting a precise count.

Dan Berdovich了解他客戶的腸外樣品中有蛋白質(zhì)聚集體，但盡管有各種各樣的高科技儀器可供他使用，但他仍然無法完全確信他能看到它們或獲得精確計(jì)數(shù)。

For an authority on particle analysis who’d earned an award for a peer-reviewed paper on visual inspection standards of injectable drug products, it was disconcerting. Using imaging particle analysis, however, a technology available to industrial markets for less than a decade, Berdovich’s efforts to solve his customers’ problems led to his own business success.

對(duì)于顆粒分析的權(quán)威機(jī)構(gòu)，他們獲得了關(guān)于注射藥物產(chǎn)品目視檢查標(biāo)準(zhǔn)的同行評(píng)審論文獎(jiǎng)，這令人不安。 然而，使用顆粒成像分析，雖然在工業(yè)市場(chǎng)的技術(shù)應(yīng)用不到十年，但是Berdovich努力解決客戶的問題以至他自己的業(yè)務(wù)獲得成功。

His company, Wheeling, Ill.-based contract laboratory Micro Measurement Laboratories, Inc., does particulate matter testing, particle identification, method validation and visual inspection standards development for pharmaceutical and biotechnology companies world wide, among other services.

他的公司位于伊利諾伊州惠靈的實(shí)驗(yàn)室Micro Measurement Laboratories，Inc。為世界各地的制藥和生物技術(shù)公司以及其他公司進(jìn)行顆粒物測(cè)試，顆粒識(shí)別，方法驗(yàn)證和視覺檢測(cè)標(biāo)準(zhǔn)開發(fā)。

The company offers particular expertise in standards and recommendations set by the U.S. Pharmacopeial Convention (USP), including USP <787>, which covers the testing of sub-visible particulate matter in therapeutic protein injections; USP <788>, which covers testing for and reporting of sub-visible particulate matter in injections and parenteral infusions; and the forthcoming USP <790>, which covers the inspection of visible particulates in injectable drug products; along with the revisions USP <1787>, <1788> and <1790>.

該公司在美國藥典委員會(huì)（USP）制定的標(biāo)準(zhǔn)和建議方面提供了特殊的專業(yè)知識(shí)，包括USP <787>，其中包括治療性蛋白質(zhì)注射中亞可見顆粒物的測(cè)試; USP <788>，其中包括注射和腸胃外輸注中亞可見顆粒物的測(cè)試和報(bào)告; 以及即將出版的USP <790>，其中包括檢查注射藥品中的可見微粒; 以及USP <1787>，<1788>和<1790>的修訂版。

“I was getting very frustrated that when it came to sub-visible or semi-transparent particles like proteins, we were essentially in the dark,” says Berdovich, “and USP <788> is our bread and butter.”

“我非常沮喪的是，當(dāng)遇到像蛋白質(zhì)這樣的亞可見或半透明顆粒時(shí)，我們基本上處于黑暗中，”Berdovich說，“USP <788>是我們的面包和黃油。”

Why it hurt
為什么會(huì)沮喪

Berdovich’s team used light obscuration instruments to detecting protein aggregates and other particles, as prescribed in USP <788>. The problem, he realized, was that light typically travels through the particles without being scattered. Proteins and protein aggregates, along with other translucent and transparent particles, often defy detection by even the finest light obscuration devices, especially when in the sub-visible range of less than 20 um. Additionally, much larger particles are often severely undersized when measured, if detected at all.

Berdovich的團(tuán)隊(duì)使用光阻法儀檢測(cè)蛋白質(zhì)聚集體和其他顆粒，如USP <788>所述。 他意識(shí)到，問題在于光線通常穿過顆粒而不會(huì)散射。 蛋白質(zhì)和蛋白質(zhì)聚集體以及其他半透明和透明的顆粒，即使是最好的光阻法裝置也經(jīng)常無法檢測(cè)，特別是在小于20微米的亞可見光范圍內(nèi)。 另外，如果檢測(cè)到的話，更大的顆粒通常在測(cè)量時(shí)測(cè)量尺寸會(huì)小于實(shí)際尺寸。

Characterizing protein aggregates is even more challenging using manual visual inspection-looking at individual vials under controlled lighting conditions and determining whether visible particles are present using the naked eye. This method has limitations similar to those using light obscuration, and because it is inherently subjective, it can yield unreliable results, especially in some biologics and lyophilized drugs.

使用手動(dòng)目視檢查表征蛋白質(zhì)聚集體更具挑戰(zhàn)性 - 在受控光照條件下觀察單個(gè)樣品瓶，并使用肉眼確定是否存在可見顆粒。 該方法具有類似于使用光阻法的局限性，并且因?yàn)槠浔举|(zhì)上是主觀的，它可以產(chǎn)生不可靠的結(jié)果，尤其是在一些生物制劑和凍干藥物中。

At present, 100% visual inspection is required for visible particulates and two methods, light obscuration and membrane microscopy, are accepted by USP to test for the presence of sub-visible particles in parenteral formulations.

目前，可見顆粒需要100％目視檢查，USP接受兩種方法，即光阻法和膜顯微鏡法來測(cè)試腸胃外制劑中亞可見顆粒的存在。

Since most manufacturers aim primarily for compliance, there is little incentive to learn more about the formation or introduction of particles present in compliant formulations. For example, under USP <788>, sub-visible particle testing is typically done in a lab away from the line where visual inspections are conducted for visible particles. Vials may be rejected from either test, but are not often evaluated together using orthogonal methods to determine what particles are triggering the rejections. Concern is focused on effectively rejecting off-spec vials rather than on identifying the type of particle detected and understanding why it is present.

由于大多數(shù)制造商主要致力于合規(guī)性，因此幾乎沒有動(dòng)力去了解合規(guī)配方中存在的顆粒的形成或引入。 例如，在USP <788>下，亞可見顆粒測(cè)試通常在遠(yuǎn)離對(duì)可見顆粒進(jìn)行目視檢查線的實(shí)驗(yàn)室中進(jìn)行。 可以從任一測(cè)試中拒絕小瓶，但是通常不使用正交方法一起評(píng)估小瓶以確定哪些顆粒觸發(fā)不可接受條件。 關(guān)注的重點(diǎn)是有效地拒絕不合格的樣品瓶，而不是確定檢測(cè)到的顆粒類型并理解其存在的原因。

The situation faced
面臨的情況

According to Berdovich, this mind set is changing. “Most of my customers want to know more about their products than compliance requires, such as particle shape - it’s invaluable in case problems arise later in formulation - but you can’t find out much more if you’re using laser scatter or light obscuration devices,” he says.

根據(jù)Berdovich的說法，客戶的這種心態(tài)正在發(fā)生變化。 “我的大多數(shù)客戶都希望了解他們的產(chǎn)品，而不僅是合規(guī)要求，例如顆粒形狀 - 如果在配方中出現(xiàn)問題，這是非常寶貴的 - 但如果您使用激光散射法或光阻法，您就無法發(fā)現(xiàn)更多“他說。

The problem came to a head during another test that required measuring particles in a liquid or gel formed on a membrane. Since the particle shape deformed in minutes, the analysis need ed to be done quickly. Manual micros copy proved far too slow. Moreover, the particles deformed when filtered and squeezed between the glass slides. “We were bumping up against a wall and thought there was no way to do this,” says Berdovich.

在另一個(gè)需要測(cè)量膜上形成的液體或凝膠中的顆粒的測(cè)試中，又一個(gè)問題出現(xiàn)了。 由于顆粒形狀在幾分鐘內(nèi)變形，因此需要快速完成分析。 手動(dòng)微拷貝證明太慢了。 此外，顆粒在過濾并在載玻片之間擠壓時(shí)變形。 “我們碰到了一堵墻，并認(rèn)為沒有辦法做到這一點(diǎn)，” Berdovich說。

Then he read in a trade magazine about new instrumentation called the FlowCAM^®, an imaging particle analysis system that automatically detects the presence of particles and microorganisms in a sample, including transparent, semi transparent and sub-visible particles. 

然后他在一本行業(yè)雜志上閱讀了一種名為FlowCAM^®的新儀器，這是一種顆粒成像分析系統(tǒng)，可自動(dòng)檢測(cè)樣品中顆粒和微生物的存在，包括透明，半透明和亞可見顆粒。

The instrument’s software package does typical spreadsheet analysis operations on particle data while presenting the results visually as images- as opposed to tabular representation. It incorporates statistical pattern recognition for particle characterization, a big improvement over simple value filtering.

該儀器的軟件不僅對(duì)顆粒數(shù)據(jù)進(jìn)行典型的電子表格分析，同時(shí)又以圖像形式呈現(xiàn)結(jié)果 - 而不僅僅是表格。 它結(jié)合了用于粒子顆粒表征的統(tǒng)計(jì)模式識(shí)別，這是對(duì)簡單值過濾的重大改進(jìn)。

Not relying on scatter
不依賴于分散

Rather than rely on light scatter, the FlowCAM’s imaging technology captures a high-resolution, digital image of each individual particle passing in front of its flow cell. Imaging thousands of particles in seconds, it measures each one based on its actual image to yield data based on particle shape, size and transparency.

FlowCAM的成像技術(shù)不是依賴光散射，而是捕獲通過流動(dòng)池前方的每個(gè)粒子的高分辨率數(shù)字圖像。 在幾秒鐘內(nèi)成像數(shù)千個(gè)顆粒，它根據(jù)其實(shí)際圖像測(cè)量每個(gè)顆粒，根據(jù)粒子形狀，大小和透明度產(chǎn)生數(shù)據(jù)。

Clicking an image or touching the image on screen reveals the measurement data, which may be graphed several different ways depending on how the data needs to be visualized and the types of particles targeted.

單擊圖像或觸摸屏幕上的圖像會(huì)顯示測(cè)量數(shù)據(jù)，這些數(shù)據(jù)可能會(huì)以幾種不同的方式表達(dá)，具體取決于數(shù)據(jù)需要如何顯示以及所針對(duì)的顆粒類型。

More than 30 different properties, including length, width, diameter, volume and aspect ratio, as well as advanced morphological types such as circle fit, elongation, perimeter and roughness are measured. Each image is saved with its corresponding measurement data for review, analysis and sharing online or by email.

通過測(cè)量了30多種不同的特性，包括長度，寬度，直徑，體積和縱橫比，以及先進(jìn)的形態(tài)類型，如圓形擬合，伸長率，周長和粗糙度。 每個(gè)圖像都保存有相應(yīng)的測(cè)量數(shù)據(jù)，以便在線或通過電子郵件進(jìn)行查看，分析和共享。

“You can get more information from the FlowCAM, says Berdovich.

Berdovich說，您可以從FlowCAM獲得更多信息。

From the advent
自從出現(xiàn)

Now that he and his analytical staff have more than three years of experience running analyses on the instrument, he says, “Going to the FlowCAM with a particle problem is just the best feeling in the world because it turns data into useful information a customer can use to solve a problem.”

現(xiàn)在，他和他的分析人員在儀器上運(yùn)行分析已有三年多的經(jīng)驗(yàn)，他說，“帶有顆粒問題地去使用FlowCAM只是世界上最好的感覺，因?yàn)樗�可以將�?shù)據(jù)變成有用的信息，可以幫助客戶解決問題。“

For one application, FlowCAM measures the coverage of coatings on medical devices. If the coating process is insufficient, particles may be released that could affect the patient. “These types of particles simply can’t be effectively characterized by anything but a FlowCAM, says Berdovich.

對(duì)于一種應(yīng)用，F(xiàn)lowCAM可測(cè)量醫(yī)療設(shè)備上涂層的覆蓋范圍。 如果涂覆過程不充分，可能會(huì)釋放可能影響患者的顆粒。 Berdovich說：“除了FlowCAM之外，這些類型的顆粒根本無法有效地表征。”

Similarly, the FlowCAM may be used to optimize microencapsulation processes by looking at the shell layer around individual particles.

類似地，F(xiàn)lowCAM可用于通過觀察單個(gè)顆粒周圍的殼層來優(yōu)化微囊化過程。

The ability to automatically differentiate one type particle from another, such as a globular protein aggregate from a curly fiber, round oil droplet or air bubble, also proves useful. Foreign matter, such as silicone oil droplets, for example, may come into contact with a sample from a rubber stopper. To a light obscuration device, these opaque oil droplets are counted just as if they were a protein or any other particle, since no distinction from one to the other may be made. This artificially increases the total particle count and may trigger destruction of an entire batch of quality product for failing to meet specifications.

自動(dòng)區(qū)分一種粒子與另一種粒子的能力，例如來自卷曲纖維，圓形油滴或氣泡的球狀蛋白質(zhì)聚集體，也證明是有用的。 例如，諸如硅油滴的異物可能與來自橡膠塞的樣品接觸。 對(duì)于光阻法儀器，這些不透明的油滴就像它們是蛋白質(zhì)或任何其他顆粒一樣被計(jì)數(shù)，因?yàn)榭梢圆粚?duì)它們進(jìn)行區(qū)分。 這會(huì)人為地增加總顆粒數(shù)，并且可能觸發(fā)破壞整批優(yōu)質(zhì)產(chǎn)品而不符合規(guī)范。

While microscopic membrane analysis can verify that these droplets are not discrete, it cannot sufficiently characterize their concentration, since they pass through the membrane. This can be troublesome because the concentration is typically needed to solve a problem.

雖然膜式顯微鏡分析可以證實(shí)這些液滴不是離散的，但由于它們穿過膜，因此不能充分表征它們的濃度。 這可能很麻煩，因?yàn)檎f明濃度來解決問題。

To the outcome
結(jié)果

In contrast, the FlowCAM images each individual oil droplet and the total count of droplets - or of any other type particle - may then be characterized as an isolated population, or even removed from the total particle count. Again, you just can’t do this with anything but a FlowCAM. The data tells a lot,” Berdovich adds.

相比之下，F(xiàn)lowCAM對(duì)每個(gè)單獨(dú)的油滴成像，并且液滴或任何其他類型顆粒的總數(shù)可以被表征為分離的群體，或甚至從總顆粒計(jì)數(shù)中去除。 再說一次，除了FlowCAM之外你不能做任何事情。 數(shù)據(jù)說明了很多，“Berdovich補(bǔ)充道。

The lab has provided several customers with FlowCAM data for sub mission to the FDA. “When you’re submitting data to the FDA, you want to understand the issues and you don’t want any doubt about the accuracy, Berdovich says. “I feel very confident in the FlowCAM because it helps us look under every rock when solving a problem, and it has also opened a lot of doors for us.”

該實(shí)驗(yàn)室為幾個(gè)客戶提供了FlowCAM數(shù)據(jù)，用于FDA的子任務(wù)。 Berdovich說：“當(dāng)您向FDA提交數(shù)據(jù)時(shí)，您希望了解這些問題，而且您不希望對(duì)準(zhǔn)確性產(chǎn)生任何疑問。” “我對(duì)FlowCAM非常有信心，因?yàn)樗鼛椭�我們�(cè)诮鉀Q問題時(shí)能夠看到每一個(gè)顆粒，它也為我們打開了很多大門。”

Since adding the FlowCAM, Berdovich has substantially increased his laboratory services customer base among medical device and pharmaceutical product manufacturers. Growth triggered a facility expansion, with an added dean room and staff. “With the FlowCAM and our knowledge base, we can take on applications that other labs won’t,” says Berdovich.

自配置FlowCAM以來，Berdovich已大幅增加其醫(yī)療設(shè)備和醫(yī)藥產(chǎn)品制造商的實(shí)驗(yàn)室服務(wù)客戶群。 增長引發(fā)了設(shè)施擴(kuò)建，增加了管理和工作人員。 “通過FlowCAM和我們的知識(shí)庫，我們可以接受其他實(shí)驗(yàn)室所不具備的應(yīng)用程序，”Berdovich說。