检测机构使用流动成像显微镜来表征蛋白质聚集体——中国生物器材网

Contract Lab Using Flow Imaging Microscopy to Characterize Protein Aggregates
检测机构使用流动成像显微镜来表征蛋白质聚集体
Posted by Joyce Brown
Joyce Brown发表
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了解他客户的肠外样品中有蛋白质聚集体，但尽管有各种各样的高科技仪器可供他使用，但他仍然无法完全确信他能看到它们或获得精确计数。
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.
对于颗粒分析的权威机构，他们获得了关于注射药物产品目视检查标准的同行评审论文奖，这令人不安。 然而，使用颗粒成像分析，虽然在工业市场的技术应用不到十年，但是Berdovich努力解决客户的问题以至他自己的业务获得成功。
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.
他的公司位于伊利诺伊州惠灵的实验室Micro Measurement Laboratories，Inc。为世界各地的制药和生物技术公司以及其他公司进行颗粒物测试，颗粒识别，方法验证和视觉检测标准开发。
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 .
该公司在美国药典委员会（USP）制定的标准和建议方面提供了特殊的专业知识，包括USP 787 ，其中包括治疗性蛋白质注射中亚可见颗粒物的测试; USP 788 ，其中包括注射和肠胃外输注中亚可见颗粒物的测试和报告; 以及即将出版的USP 790 ，其中包括检查注射药品中的可见微粒; 以及USP 1787 ， 1788 和 1790 的修订版。
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.
我非常沮丧的是，当遇到像蛋白质这样的亚可见或半透明颗粒时，我们基本上处于黑暗中， Berdovich说， USP 788 是我们的面包和黄油。
Why it hurt
为什么会沮丧
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的团队使用光阻法仪检测蛋白质聚集体和其他颗粒，如USP 788 所述。 他意识到，问题在于光线通常穿过颗粒而不会散射。 蛋白质和蛋白质聚集体以及其他半透明和透明的颗粒，即使是最好的光阻法装置也经常无法检测，特别是在小于20微米的亚可见光范围内。 另外，如果检测到的话，更大的颗粒通常在测量时测量尺寸会小于实际尺寸。
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.
使用手动目视检查表征蛋白质聚集体更具挑战性 - 在受控光照条件下观察单个样品瓶，并使用肉眼确定是否存在可见颗粒。 该方法具有类似于使用光阻法的局限性，并且因为其本质上是主观的，它可以产生不可靠的结果，尤其是在一些生物制剂和冻干药物中。
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，可见颗粒需要100％目视检查，USP接受两种方法，即光阻法和膜显微镜法来测试肠胃外制剂中亚可见颗粒的存在。
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.
由于大多数制造商主要致力于合规性，因此几乎没有动力去了解合规配方中存在的颗粒的形成或引入。 例如，在USP 788 下，亚可见颗粒测试通常在远离对可见颗粒进行目视检查线的实验室中进行。 可以从任一测试中拒绝小瓶，但是通常不使用正交方法一起评估小瓶以确定哪些颗粒触发不可接受条件。 关注的重点是有效地拒绝不合格的样品瓶，而不是确定检测到的颗粒类型并理解其存在的原因。
The situation faced
面临的情况
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.
根据Berdovich的说法，客户的这种心态正在发生变化。 我的大多数客户都希望了解他们的产品，而不仅是合规要求，例如颗粒形状 - 如果在配方中出现问题，这是非常宝贵的 - 但如果您使用激光散射法或光阻法，您就无法发现更多 他说。
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.
在另一个需要测量膜上形成的液体或凝胶中的颗粒的测试中，又一个问题出现了。 由于颗粒形状在几分钟内变形，因此需要快速完成分析。 手动微拷贝证明太慢了。 此外，颗粒在过滤并在载玻片之间挤压时变形。 我们碰到了一堵墙，并认为没有办法做到这一点， Berdovich说。
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. 
然后他在一本行业杂志上阅读了一种名为FlowCAM 的新仪器，这是一种颗粒成像分析系统，可自动检测样品中颗粒和微生物的存在，包括透明，半透明和亚可见颗粒。
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.
该仪器的软件不仅对颗粒数据进行典型的电子表格分析，同时又以图像形式呈现结果 - 而不仅仅是表格。 它结合了用于粒子颗粒表征的统计模式识别，这是对简单值过滤的重大改进。
Not relying on scatter
不依赖于分散
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的成像技术不是依赖光散射，而是捕获通过流动池前方的每个粒子的高分辨率数字图像。 在几秒钟内成像数千个颗粒，它根据其实际图像测量每个颗粒，根据粒子形状，大小和透明度产生数据。
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.
单击图像或触摸屏幕上的图像会显示测量数据，这些数据可能会以几种不同的方式表达，具体取决于数据需要如何显示以及所针对的颗粒类型。
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.
通过测量了30多种不同的特性，包括长度，宽度，直径，体积和纵横比，以及先进的形态类型，如圆形拟合，伸长率，周长和粗糙度。 每个图像都保存有相应的测量数据，以便在线或通过电子邮件进行查看，分析和共享。
You can get more information from the FlowCAM, says Berdovich.
Berdovich说，您可以从FlowCAM获得更多信息。
From the advent
自从出现
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.
现在，他和他的分析人员在仪器上运行分析已有三年多的经验，他说， 带有颗粒问题地去使用FlowCAM只是世界上最好的感觉，因为它可以将数据变成有用的信息，可以帮助客户解决问题。
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.
对于一种应用，FlowCAM可测量医疗设备上涂层的覆盖范围。 如果涂覆过程不充分，可能会释放可能影响患者的颗粒。 Berdovich说： 除了FlowCAM之外，这些类型的颗粒根本无法有效地表征。
Similarly, the FlowCAM may be used to optimize microencapsulation processes by looking at the shell layer around individual particles.
类似地，FlowCAM可用于通过观察单个颗粒周围的壳层来优化微囊化过程。
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.
自动区分一种粒子与另一种粒子的能力，例如来自卷曲纤维，圆形油滴或气泡的球状蛋白质聚集体，也证明是有用的。 例如，诸如硅油滴的异物可能与来自橡胶塞的样品接触。 对于光阻法仪器，这些不透明的油滴就像它们是蛋白质或任何其他颗粒一样被计数，因为可以不对它们进行区分。 这会人为地增加总颗粒数，并且可能触发破坏整批优质产品而不符合规范。
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.
虽然膜式显微镜分析可以证实这些液滴不是离散的，但由于它们穿过膜，因此不能充分表征它们的浓度。 这可能很麻烦，因为说明浓度来解决问题。
To the outcome
结果
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，FlowCAM对每个单独的油滴成像，并且液滴或任何其他类型颗粒的总数可以被表征为分离的群体，或甚至从总颗粒计数中去除。 再说一次，除了FlowCAM之外你不能做任何事情。 数据说明了很多， Berdovich补充道。
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.
该实验室为几个客户提供了FlowCAM数据，用于FDA的子任务。 Berdovich说： 当您向FDA提交数据时，您希望了解这些问题，而且您不希望对准确性产生任何疑问。 我对FlowCAM非常有信心，因为它帮助我们在解决问题时能够看到每一个颗粒，它也为我们打开了很多大门。
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以来，Berdovich已大幅增加其医疗设备和医药产品制造商的实验室服务客户群。 增长引发了设施扩建，增加了管理和工作人员。 通过FlowCAM和我们的知识库，我们可以接受其他实验室所不具备的应用程序， Berdovich说。