Ivacaftor/Lumacaftor

在过去的世纪中，通过连续制造实现的流程强化在钢铁、化工、食品和石化生产行业产生了革命性的影响，那么为什么生物制药行业花了这么长时间才接受连续处理呢？直到最近，第一个完全通过连续工艺生产的单克隆抗体才进入临床试验（BiosanaPharma 的奥马珠单抗生物类似药）。在此，我们回顾了抗体及相关产品的连续下游处理的最新进展，包括生物制药行业的现状、推动连续处理的变革性技术以及早期采用者所展示的连续生物处理的最新水平。 引言：生物制药生产的变革驱动因素——不断变化的行业格局 已有超过 80 种单克隆抗体治疗药物获得了监管批准，2018 年在欧盟或美国共有 12 种新的单克隆抗体获得批准。因此，该行业如今已可被视为成熟行业，与其他成熟行业一样，通过流程强化实现成本节约变得至关重要。降低成本的需求来自于针对同一适应症的多种单克隆抗体的竞争以及生物类似药的竞争。竞争导致需求难以预测。因此，降低成本和提高灵活性将成为未来生物生产设施的关键驱动因素。 生物类似药 单克隆抗体生物治疗药物的成功与一些备受瞩目的科学奖项密切相关。2018 年，诺贝尔化学奖共同授予了乔治·P·史密斯爵士和格雷戈里·P·温特爵士，以表彰他们在肽和抗体的噬菌体展示方面的贡献。阿达木单抗成为首个于 2002年获得市场批准的噬菌体展示衍生的单克隆抗体。阿达木单抗已发展成为销售额最高的治疗药物，全球年销售额接近 200 亿美元。然而，阿达木单抗以及许多其他生物治疗药物的专利近期已到期。表 4.1 展示了全球销售额最高的十大生物治疗药物及其专利到期日期。这些专利大多已到期或即将到期的事实，推动了生物类似药开发计划的开展，目前正在进行的此类计划超过 1000 个。由于针对每种单克隆抗体的多种生物类似药的开发，生物处理行业格局必然发生变化。这一领域的竞争已如此激烈，以至于 Momenta 基于原研公司与生物类似药公司之间的法律协议将于 2023 年到期时市场可能已饱和的判断，终止了其阿达木单抗生物类似药的开发。同样，Sandoz 在美国食品药品监督管理局要求提供更多数据后，基于假设利妥昔单抗的市场将在数据获批前就已饱和，停止了其利妥昔单抗生物类似药的申报。在饱和的市场格局中，成本将成为推动成功的关键因素，通过能够开拓新市场来实现，因此 BiosanaPharma 等生物类似药公司推动通过连续处理实现流程强化的创新也就不足为奇了。竞争还引入了对原研药和生物类似药的不可预测需求，使得灵活生产变得至关重要。 针对同一适应症的多种单克隆抗体 2018 年，诺贝尔生理学或医学奖授予了詹姆斯·P·艾利森和本庶佑，以表彰他们“发现了通过抑制免疫负性调节来治疗癌症”。自这一初步发现以来，许多免疫反应调节检查点已被发现，并开发了一系列针对这些检查点的抗体治疗药物：针对 CTLA-4（伊匹单抗），针对 PD-1（西米普利单抗、纳武利尤单抗、帕博利珠单抗），以及针对 PD-1 的配体（PD-L1；德瓦鲁单抗、阿维鲁单抗、阿特珠单抗）。众多治疗药物作用于相同或相似的通路，这种竞争最终将导致成本压力。目前，我们看到其中一些药物正在争夺新适应症的治疗。这导致需求难以预测，并推动了对灵活生产的需求。 强化的上游流程和下游瓶颈 对生物制剂需求的增加推动了上游流程的强化，以提高生产力并降低制造成本。随后，补料批培养生产工艺的滴度从 0.2 g/L 增加到 >3 g/L。此外，灌注工艺最初被用作满足大量需求或生产不稳定分子的工具，由于其生产力的提高而被重新审视。值得注意的是，高强度、低体积灌注（HILVOP）工艺比补料批培养生产具有更高的生产力。 然而，下游处理的进步未能跟上滴度的增加。因此，处理瓶颈已从上游转移到下游操作。即使是高容量色谱捕获介质也无法应对现代细胞培养工艺中常见的高滴度。这一缺陷导致人们对多柱色谱的兴趣增加，它可以有效地将柱尺寸与滴度解耦。可以根据流速选择柱子，并且随着滴度的增加，可以在工艺中增加更多的柱子。 小分子的成功和监管推动 小分子制药行业已经从转向连续流动技术中受益。在此，化学反应物的连续流被引入反应器以产生所需的产品。这种操作方式的优点包括快速混合、增强的传热、强化的传质以及由于高比表面积与体积比，能够进行放热反应。流动技术设备允许快速放大，避免了批量工艺放大所固有的许多问题。使用微反应器技术已广泛探索了受益于非常高或非常低温度（高于 200°C 和低于 -40°C）和更高压力的反应轮廓，这种技术已从各种制造商那里广泛商业化。其他优点包括最小化长期储存大量材料以及能够安全地进行涉及危险气体的反应。因此，连续技术使制药行业能够合成大量的活性药物成分和天然产物。 美国食品药品监督管理局在批准和审查市场上的新产品方面的支持，导致了连续方法在小分子行业中的广泛接受。自 2015 年以来，该机构已批准了四种通过连续工艺生产的产品——两种囊性纤维化药物（Vertex Pharmaceuticals 的 Orkambi 和 Symdeko）、一种乳腺癌药物（Eli Lilly and Company 的 Verzenio）和一种 HIV 药物（Janssen Pharmaceuticals 的 Prezista）。美国食品药品监督管理局对小分子药物连续制造的接受和鼓励为连续生物处理铺平了道路。美国食品药品监督管理局积极鼓励更新生物制造工艺，因为它有可能“提高产品的整体质量和对患者的可及性”。作为推动质量源于设计（QbD）方法的一部分，美国食品药品监督管理局组建了新兴技术团队，帮助连续制造的早期采用者“帮助解决实施挑战并指导使用这些现代方法生产的产品的应用审查过程”。 连续处理的利弊 鉴于生物制药行业的现状以及政府和社会对降低药品成本的压力，关注流程强化也就不足为奇了。尽管商品成本在销售价格中所占比例相对较小，但流程经济可能会有显著改善。已经有一系列关于连续处理的商品成本分析，依赖于多种方法。然而，这些结果强烈依赖于所使用的假设，由于尚未实施任何连续制造工艺且每家公司的制造场景都不同，直接的经济优势仍然具有推测性；这是生物制药运营集团的观点。 尽管连续处理的许多方面无法直接用金钱量化，但仍有一些特点可能有助于推动实施的有力理由。如果我们考虑产品在制造过程中的快速且均匀的流动，我们可以立即理解这项新技术有可能减少整体制造时间，并随后提高产品质量。在批量工艺中，所有材料都通过一个单元操作并收集在暂存罐中，然后才开始下一个操作。另一方面，连续方法依赖于最小化单元操作之间的暂存罐，并在材料准备好处理时立即开始下一个单元操作。因此，在连续生物处理中，所有单元操作并行运行，最大限度地利用设备和设施。对于每天可能只进行一两个单元操作的批量工艺，产品可能需要 >1 周时间才能从生物反应器进入配方。在连续处理中，我们看到了案例研究，其中从生物反应器到配方的产品处理时间 <20 小时。这种操作速度在处理不稳定的生物制剂时尤为重要。在这种情况下，连续制造可能是将这些产品推向市场的唯一方式。 增加设备利用率可以带来进一步的好处：尽管整体工艺时间缩短，但每个单独的单元操作可以持续更长时间。这引入了以较低流速运行和/或进行更多纯化循环的机会，从而实现更小的设备和更小的占地面积。 例如，在批量捕获色谱中，生物反应器通常进行 2-4 次色谱循环。然而，在连续色谱中，柱子可以以较短的停留时间运行，从而可以使用较小的柱子，这些柱子可以更频繁地循环，以更有效地利用树脂的使用寿命。较小的柱子可以预先装填，内径可达 80 厘米，进一步降低色谱操作的风险。 较小的设备还使得能够实施一次性（SU）流路，这在较大规模时可能会变得过于昂贵或技术上不可行。SU 带来了额外的好处：减少了清洁及其验证，以及更简单的设施，包括模块化的“舞厅”平面布置，这些设施更便宜、更快捷地建造，并且适合于不同产品之间的快速转换。这种类型的设施可以作为一个通用的处理套件，相同的设备可以在产品开发周期中使用，包括毒理学、临床试验以及全面生产。 通过灌注工艺能够通过不同的运行时间调整生产的药物量，这种灵活性得到了增强。因此，可以避免昂贵且耗时的放大，也许能够更快地获得药物。 另一个优势是增加循环次数，连续工艺在稳态下运行，已被证明比批量操作更可靠。稳态运行还带来了能够生成更多数据的能力，这些数据可以通过多变量数据分析等统计方法预测工艺何时偏离期望状态，从而在工艺失败之前进行调整。因此，连续处理可能通过动态控制推动质量提升。 然而，这些进步取决于克服重大的技术障碍。最终，进展不仅来自于将各个单元操作转移到连续模式，还来自于将单元操作耦合以开发工艺。下面我们将讨论一些变革性技术以及早期采用者如何拼凑他们自己的连续下游纯化平台。连续加工的关键使能技术 声波分离 在工艺开发方面的大量投资带来了更高强度、更高生产力的细胞培养。在许多情况下，这些生产力的提高也增加了生物反应器内的生物量。对于批次上游工艺，离心或一次性深层和传统过滤介质被用于减少药物产品中的细胞和细胞碎片。然而，在高细胞密度下，离心变得不够稳健，传统过滤介质可能会提前堵塞，需要增加过滤面积，这并不总能被现有设施容纳。对于灌注工艺，细胞保留通常通过中空纤维切向流过滤实现，无论是单向还是交替流动。然而，产品通过中空纤维的传输通常会随着时间的推移而减少，产品可能会被困在生物反应器中。因此，高细胞密度的挑战促使人们探索替代的一级澄清技术，包括声波分离。50多年来，非线性声学已在多个行业中得到应用；从医学超声到水下探测，从微流体到声悬浮。这些应用受到一组经过充分表征的物理波动方程的控制。从广义上讲，声学可以精确且智能地控制，以在连续、不结垢、可扩展的过程中凝聚并去除载体流中的颗粒。该技术的独特之处使其适合作为真正连续药物制造设施的一级澄清步骤。 声波分离器的核心处理部分是一个压电换能器和声反射器，它们跨越细胞培养液流经的流道（见图4.1）。在Pall的声波分离器中，在换能器和反射器之间建立了一个三维声驻波。在一次性谐振器内，驻波建立了一个高度调谐的压力场，其中高、低压力节点可预测地排列，颗粒根据其相对于细胞培养液的密度进行凝聚。将多个颗粒凝聚成较大的团簇，使重力能够轻松地将聚集的颗粒从悬浮液中拉出，收集并去除或重新加入到工艺中（具体取决于特定的流体学和工艺步骤的需求）。 在用于批次灌注工艺的澄清过程中，含细胞的生物反应器流体通过谐振器中的压力场。通过凝聚和去除流中的颗粒，流道将细胞从单克隆抗体（mAb）负载的渗透液中引导开，使它们能够被连续泵入浓缩的废流——渗透液作为澄清的液体传递到后续的加工步骤。在灌注工艺中，需要保留（而不是丢弃）细胞，生物反应器的进料流在声场下方切向循环，允许单克隆抗体作为渗透液不受阻碍地通过声学，而细胞则返回生物反应器进行进一步培养。在这两种应用中，专有的声学算法和精心设计的流道共同以连续、温和且高效的方式将单克隆抗体从产生单克隆抗体的细胞培养液中分离出来。 多柱色谱 多柱色谱是向连续加工转变的关键使能技术。人们一直致力于强化Protein A捕获色谱步骤。这在一定程度上是因为Protein A树脂的相对成本较高，它在下游加工成本中占了相当大的比例。Protein A树脂的一个共同特点是其对单克隆抗体的容量强烈依赖于加载步骤的停留时间。Protein A膜已被证明能够解除这种依赖关系。然而，由于膜的比表面积较低，其容量比树脂落后两倍甚至更多。为了最大化容量，Protein A树脂通常在4分钟或更长的停留时间下进行加载。长停留时间导致相对较低的生产率，这通常以每升树脂每小时纯化的产品克数来表示。在典型的批次制造场景中，Protein A柱的尺寸是根据处理生物反应器的能力以及两到四个色谱周期来确定的，这导致了大型柱和大量的资本支出。 为了解决批次Protein A步骤的不足，提出了一系列连续色谱解决方案。这些解决方案大多依赖于同时加载多个柱，从而带来更高的容量和生产率。由于可以使用次级和三级柱捕获未结合的产品，因此可以更快地进行加载（停留时间更短）。这为减少商业生物工艺中所需的树脂量以及通过更快的树脂循环改善制造经济性提供了可能性。 许多公司已经为生物制药市场开发了基于同时加载多个柱的连续色谱系统。这些系统主要根据可以操作的柱的数量来区分。例如，Chromacon Contichrom Cube有2个柱，GE PCC有3或4个柱，NovasepBio SC有6个柱，Semba Octave有8个柱，Pall BioSMB GMP有8个柱或Pall BioSMB PD有16个柱。所有这些系统都依赖于通过将主加载柱过载至产品突破点来提高效率。通过后续柱捕获突破第一柱的产品，避免了产品损失。柱过载可以被看作是将一个较大的柱分解成较小的部分，以便更积极地利用质量传递区。所有柱都经历相同的操作，产品质量通过重复的色谱周期保持一致。为了理解柱的数量和不同系统的操作的影响，将色谱周期视为两个阶段是有用的：加载和非加载阶段。后者包括色谱周期的所有非加载步骤，包括冲洗、洗脱、再生和再平衡。最简单的操作是使用三个柱，在任何时候，两个色谱柱串联加载，而第三个柱正在进行非加载步骤。所有柱都经历周期性的色谱操作。直接接收加载的柱最终会被产品饱和。此时，对该柱的加载可以停止，该柱被分配到非加载步骤。之前接收第一柱流过液的第二个加载柱现在直接接收加载，而再生的柱被放置在主捕获柱之后，以防止产品损失。在滴度适中时，例如1 g/L，加载步骤是速率限制步骤。在1分钟的停留时间下进行加载，可以实现>40 g/L的容量。非加载步骤也可以在1分钟的停留时间内完成，通常需要20到30个柱体积（CV），以冲洗、洗脱、清洁和再生一个Protein A柱。随着滴度的增加，加载时间减少。在10 g/L时，可能只需要4分钟的加载时间，容量为40 g/L，加载停留时间为1分钟。非加载步骤无法在这个时间框架内完成，成为速率限制步骤（见图4.2）。有多种可能的解决方案可以应对这种现象，包括减慢或停止加载，这会影响生产率。另一种选择是增加更多的柱来执行工艺的非加载步骤。这在高滴度下推动生产率。 由Chromatan开发的连续逆流切向色谱技术是多柱色谱的一种值得注意的替代方法。在这种方法中，树脂在系统中循环，减少了对固定床柱的依赖。其优势在于它促进了真正的连续洗脱，与提供离散洗脱液的柱方法不同。这代表了从当前批次柱工艺的进一步转变。这种方法的一个缺点集中在树脂的稳定性和寿命上，这必须经过验证。此外，与基于柱的色谱相比，洗脱浓度似乎较为适中，影响了下游单元操作的生产率。 其他连续色谱解决方案也已被探索。这些包括环状色谱、模拟移动床（SMB）及其变体多柱逆流溶剂梯度纯化（MCSGP）。这些方法在生物处理中引起了有限的兴趣。环状色谱依赖于在两个同心圆柱的环形空间内填充分离介质。柱缓慢旋转，而进料装置和分馏收集器保持静止。目前没有可用于操作连续环状色谱的商业系统。这可能是因为均匀填充分离基质和密封旋转部件的技术挑战。SMB通过定期切换柱入口来实现，形成分离基质和液体的模拟逆流。SMB特别适用于手性化合物的二元分离，这导致其在天然产物合成中间体中的常见应用。目前没有生物工艺使用SMB，但它可能对需要（SEC）尺寸排阻色谱的过程有用。 MCSGP是SMB的一个变体，其中部分纯化的分数被回收到加载中。这提供了高纯度和高收率的可能性，但重新加载材料可能导致低生产率，特别是当应用于SEC时。 低pH病毒灭活 低pH病毒灭活（VI）是确保产品安全的关键步骤，通常在Protein A捕获步骤之后直接进行。在当前的批次制造工艺中，VI大多手动进行：通过泵入酸和碱进行滴定，产品的pH通常通过取样和使用校准的台式pH计进行测试来验证。虽然GE、Sartorius和Pall有可用的批次系统，但该过程并不容易自动化。随着联合灌注上游工艺和连续捕获下游工艺的考虑，这使得VI单元操作成为焦点。正在探索几种不同的VI可能性。这些可以分为3类： 连续搅拌罐反应器 唯一商业可用的连续低pH病毒灭活解决方案是Pall的Cadence VI系统。Cadence VI依赖于一个两罐系统来操作半连续VI。罐的功能交替，一个罐接收来自上游工艺的新洗脱液。另一个罐用于进行低pH灭活步骤：当过程完成时，该罐被排空，然后可以接收新的洗脱液。与此同时，另一个罐中收集的洗脱液可以通过低pH步骤进行处理。这样，产品在同一个罐中被收集和处理。Cadence VI系统可以被认为是一种自动化的当前批次工艺版本，这可能为性能验证提供了一条相对简单的路径。然而，这种方法存在一些风险，这些风险被生物制药用户小组突出强调。许多风险在系统开发过程中被识别，并通过系统设计和测试得到了缓解或减少。 这些关键风险包括通过死腿（孤立的分支或一端封闭的管段）和悬挂滴液使灭活产品受到未灭活产品的污染。通过在混合器的低点引入液体、消除飞溅以及使用具有最小持液体积和循环回路的Artesyn阀门，这种风险得到了缓解。这些特点还减少了酸和碱的回混风险，消除了在保持步骤中pH漂移的机会。另一个关键风险是pH探头，它必须在较长时间内保持精确校准。在Pall，使用机械臂将pH探头在低pH和高pH缓冲液之间转移，以模拟VI进行测试。测试显示pH探头漂移很小，在48小时内为0.05个pH单位，使这些探头适用于连续VI。 循环流反应器 自动化当前批次VI工艺的挑战以及对更连续工艺的渴望促使几个小组追求循环（活塞）流反应器。在这里，所需的低pH保持时间由曲折的流道提供。为了最小化流的轴向分布以及最小化产品在活塞流装置中停留时间的分布，开发了一种交替卷绕方向的管，以引入迪安涡流并促进均匀性。尽管有一些专利活动，但目前没有商业可用的解决方案。生物制药用户论坛识别出一些技术挑战和风险。最大的挑战集中在滴定和pH测量上。Protein A的洗脱液在洗脱峰上不同位置的蛋白质电导率和pH值各不相同。这种变异性通过在滴定前汇集材料来解决，但这仍然存在确保材料足够均匀以在进入活塞流反应器之前达到灭活pH的挑战。随着pH探头存在滞后性，这种风险被放大：在恒定流动情况下，产品可能未达到灭活pH，并且未被pH探头检测到。 基于柱的策略 活塞流方法的轴向分散和随后的宽停留时间可能通过使用基于柱的方法来提供在低pH下所需的保持时间来解决。已经应用了多种介质，包括Protein A、阳离子交换、尺寸排阻和惰性珠子。Protein A和阳离子交换方法很有吸引力。使用这些方法可以将低pH VI与所需的色谱步骤结合起来，这可以被认为是在工艺强化方面的一个进一步发展。 然而，这种方法也有缺点。需要高电导率缓冲液以维持在低pH下mAb与ProteinA的结合。目前还不清楚这些缓冲液以及灭活后通常增加的浊度会对树脂的寿命产生什么影响。此外，声称Protein A步骤和低pH灭活都能清除病毒将是一个挑战。 使用SEC的缺点是介质的可压缩性。因此，SEC只能在低线速度下运行。因此，需要在工艺中引入一个相对较大的直径填充柱。对这种方法的一个改进可能是使用惰性珠子。它们通过增加柱的长度来扩大规模，这增加了更多的理论板并降低了分散。 替代低pH病毒灭活的方法 低pH可以被其他病毒灭活方法所替代。特别是溶剂/去污剂灭活可以使用相同的设备。溶剂/去污剂灭活的优点是它可以应用于对pH敏感的蛋白质。 另一种病毒灭活的替代方法是紫外线C辐照。紫外线C辐照与高温一起被应用于细胞培养材料，以减少进入工艺的病毒载量。紫外线C已经被Bayer开发成商业系统，并由Sartorius商业化。这些系统采用螺旋流道以确保一致的混合和紫外线C暴露。这种方法非常适合连续制造。无需添加任何东西，灭活可以在连续流动中进行。尽管具有这些优点，紫外线C系统似乎不再被市场推广。紫外线C在DNA中诱导嘧啶二聚体。这种损伤可以阻止DNA聚合酶，防止复制。然而，紫外线C辐照也可以通过光解或活性物种的生成来损伤蛋白质，导致蛋白质硫醇氧化。也许正是因为蛋白质损伤的可能性，紫外线C辐照在生物处理中的应用有限。 单程切向流过滤 单程切向流过滤（SPTFF） 传统的TFF在生物制药的配方中得到了广泛的应用。该过程通常是超滤（UF），通过脱水浓缩蛋白质，然后是透析（DF）以将产品放入正确的缓冲系统中，最后通过UF达到最终浓度。对于生物分子，配方浓度通常大于100 g/L，以最小化需要注入患者体内的产品体积。传统TFF的缺点是需要多次通过设备，这需要多次通过泵和TFF，可能会产生剪切力，从而损坏产品。此外，多次泵送需要使用相对较大的进料泵和大直径系统管道进行循环，这本质上是一个批次操作。 尽管自TFF引入以来的四十多年来，膜的性能和材料的制造已经取得了许多进展，但TFF的原理在这段时间内并没有发生实质性的变化。因此，SPTFF技术代表了自发明以来TFF实践的第一次重大变化，简化了纯化过程，并创造了传统TFF无法实现的新能力。因此，它将TFF技术提升到了一个新的制造能力、稳健性、复杂性水平，并实现了真正的连续操作。SPTFF通过在单次通过中实现高转化率，解决了传统TFF的缺点，从而提供了TFF的性能与直接流过滤的简单性。如图4.3所示，与传统TFF过程不同，SPTFF过程是连续的，没有循环回路，消除了对过程罐的需求，并能够在不需要积累批次的情况下，一旦可用就处理流。通常，TFF和SPTFF使用相同类型的模块，使用相同的盒式几何形状，包括相同的膜以及相同的进料和渗透筛。然而，SPTFF模块被配置为具有更长的流道，由稍低的流速和稍高的压力驱动，并且其流道的横截面沿流道变化。这种属性和特点的组合使得每次通过的转化率很高，与传统TFF相比具有同等或更高的生产率。 SPTFF过程执行与TFF过程相同的分离；今天可以用TFF过程完成的任何事情也可以用SPTFF完成。在SPTFF过程中，完整的分离在一个稳定状态下，在模块的流道中沿着流道在几分钟内完成。相比之下，在传统的TFF过程中，分离通常需要3到4小时的处理时间逐渐发生。这一特性可能导致对连续加工敏感的产品产量增加。 通过SPTFF进行在线浓缩已在生物工艺的多个位置得到应用，包括上游和下游工艺之间的界面，它可以被用来完全解耦它们，增加灌注工艺中捕获色谱的生产率，这些工艺的滴度适中，在色谱之前的缓冲液交换，或在最终配方之前的浓缩/缓冲液交换，然后进行无菌过滤。 用于在线透析（ILDF）的SPTFF SPTFF已被适应用于透析应用，以实现连续缓冲液交换。ILDF过程本质上是一个设备内的多个SPTFF浓缩步骤系列，每个浓缩步骤的流出液被DF缓冲液稀释，然后通过下一个阶段再次浓缩。这样的模块有一个DF分配器，这是一个流体装置，将DF缓冲液适当地分配到每个DF阶段。 与SPTFF浓缩模块相比，ILDF模块的流道通常是恒定横截面的，因为流体性质的变化不如SPTFF浓缩模块中的变化那么大。图4.2是一个ILDF过程的P&ID，显示了额外的DF流，需要第二个流量控制（FRC）来控制DF速率。在ILDF过程中，流出流可能与进料流具有相同的浓度，也可能没有。在下面的示例中，假设在ILDF过程中没有发生浓缩，因此FRC保持1的浓缩因子（见图4.4）。 正如在传统TFF中的透析一样，DF体积越大，去除因子越大，去除因子定义为不希望的溶质浓度的降低。通过一个ILDF设备的单次通过，可以进行三个对数或更多的缓冲液交换，该设备有六个相同的浓缩和稀释阶段。 尽管连续进行，但通过多次稀释和再浓缩（批次透析）步骤进行透析的一个缺点是，与传统TFF透析过程相比，缓冲液消耗可能更高。如果需要，可以通过将一些透析阶段的渗透液回收到早期阶段的DF进料流中来提高缓冲液使用的效率。这被称为逆流DF。 SPTFF浓缩和ILDF设备可以联合使用，以通过UF/DF/UF进行连续配方。这是能够完全连续操作的一个关键进步。 早期采用者的最新动态 连续加工的早期采用者采取了许多不同的强化路线。这些决策源于每家公司的商业案例，围绕当前制造需求和能力与由于药物管线而预测的未来需求之间的关系。在这里，我们旨在提供领先早期采用者的工作动态，尽管应该清楚的是，并非所有公司都分享他们强化计划的许多甚至任何细节，因为他们可能认为这是牺牲他们的竞争优势。鉴于这一限制，该列表并不全面。 Alvotech Alvotech专注于生物仿制药的开发和制造，其关键支柱之一是“投资于最高质量和差异化策略，以开发增值和患者友好的产品”。Alvotech的制造平台基于一个1000升的灌注生物反应器，运行时间可达45天，但基于15天的批次操作。细胞保留通过ATF实现，下游工艺包括使用Cadence BiosMB的连续捕获，随后是Cadence VI（Andrew Falconbridge，世界生物制药论坛，2019）。 Amgen Amgen的工艺强化之路是由更高强度的灌注工艺推动的。Amgen声称，一个2000升的灌注生物反应器，体积生产力为2.5 g/L-day，可以在15天内生产50公斤的药物物质。这相当于一个15000升的批次灌注生物反应器的产量。Amgen将灌注上游与下游工艺相结合，最小化步骤之间的保持罐，以减少占地面积并提高生产率。利用这些进步，Amgen在新加坡建立了一个120000平方英尺的制造设施。与传统工厂相比，Amgen的新加坡工厂以更低的价格建成，占地面积约为传统制药工厂的五分之一（约170000平方英尺，与Amgen在罗德岛的工厂相比为750000平方英尺）。一个包含制造、受控温度仓库、行政、质量控制和清洁公用设施的建筑在18个月内建成。工厂的宴会厅设计，采用一次性系统以缩短流道，省略了就地清洁系统，并提供了高度的灵活性。 连续生物处理使Amgen的大部分成功成为可能。上游进料直接进入两个色谱柱单元，中间有一个小的缓冲罐。第二柱的出流直接进入病毒过滤和UF/DF单元。整个过程的总时间仅为10到14小时。 然而，在Amgen使用一次性系统的挑战来自于复杂供应链的创建。为了评估聚合物薄膜的生物学影响，Amgen需要将原材料的变异性与工艺参数相关联。需要进行特征研究以了解用于生物处理的袋子的变异性。建立质量控制测试以确保一致的性能变得必要。一次性系统进一步需要持续监测和频繁验证性能。新加坡工厂的成功通过最近的新闻得到突出，即Amgen在罗德岛又破土动工了一个新一代生物制造工厂。 BiosanaPharma 正如已经讨论的那样，BiosanaPharma最近成为第一家将完全连续工艺生产的单克隆抗体推向临床的公司。BiosanaPharma将这一过程命名为3C，可能是因为它利用了一个连续的上游灌注生物反应器，结合了连续离心以控制细胞活性和一个连续的下游工艺。BiosanaPharma声称“3C技术平台是一个高生产率、灵活、占地面积小（50平方米）的制造平台，能够在50升生物反应器规模下每周生产1公斤药物物质抗体。通过多周期逆流操作使批次加工连续化。上游工艺基于高细胞密度连续灌注培养，交替使用生物反应器。下游工艺包括在BioSMBs中进行的Protein A和阳离子交换，结合流过式过滤。该工艺具有GMP状态，并设计为可运行长达2个月。所有步骤同时运行，通过智能软件（21 CFR第II部分）进行流程控制和数据采集。BiosanaPharma可能独一无二的是，他们与台湾的第三方Mycenax合作开发了他们的连续工艺，概念验证工艺在50升规模下进行——这也是计划用于最终制造工艺的规模。 勃林格殷格翰（BI）和辉瑞 BI和辉瑞合作实现了一个新一代制造平台。该平台的目标包括一个完全一次性使用的流道，通过连续操作实现工艺强化以及高度自动化；所有这些结合在一起，为多产品设施提供了高度的灵活性和敏捷性。该工艺的核心是新一代灌注平台HILVoP。这是一个非稳态过程，细胞密度可能达到每毫升2亿个细胞。BI/辉瑞声称该过程在类似时间内产生的产品比批次多10倍，比稳态灌注多5倍。这代表了在14天的运行期间，生物反应器的产量高达每升76克。产品收获持续10天，细胞通过中空纤维TFF保留。下游工艺似乎完全使用内部开发的系统运行。色谱使用iSKID进行，支持交替的Protein A捕获色谱。当一个柱正在加载时，另一个柱执行非加载步骤。这样可以实现连续加载。然而，为了简化操作，柱一次只加载一个，树脂的容量与批次操作相同。BI/辉瑞还开发了一种新的低pH VI解决方案，“盒中摇摆”（JIB）曲折流道连续系统。 总体而言，BI/辉瑞声称他们的新一代平台比批次操作的生产率提高了10倍，能够在14天-2000升生物反应器灌注活动中生产30到60公斤的产品。 Enzene 作为Alkem Laboratories的子公司，Enzene旨在通过在其位于印度浦那的世界级制造设施中开发颠覆性技术平台和先进分析技术，提供具有成本效益的生物仿制药。通过建立一个完全自动化的连续cGMP合规的单克隆抗体生产制造工厂，Enzene旨在在全球范围内颠覆生物技术产品的可负担性。为此，Enzene已从Novasep获得了一台BioSC Pilot仪器，并计划将其整合到其切向流过滤和病毒灭活流程中。 Enzene声称灌注相对于补料批培养方法的累积生产率提高了10倍。通过较小的工厂占地面积减少约5倍的资本支出（CAPEX），以及通过降低原材料、蒸汽、WFI和电力的消耗将运营支出（OPEX）减少一半，这是Enzene推动进入连续制造领域的驱动力。他们在实施连续制造方法的过程中遇到了一些障碍，例如需要找到工艺、自动化和PAT的正确组合。对于Enzene来说，生物反应器和色谱线的污染风险在上游和下游流程的相互作用中变得尤为突出。然而，通过最大限度地利用Protein A树脂所获得的节省已成为实施连续制造技术的一个主要因素。 Enzene的例子告诉我们，越来越多的公司不可避免地会选择在生物制品领域实施连续制造，因为竞争对手所获得的竞争优势将成为从开发和制造新的治疗药物和生物仿制药中获利的一个难以克服的障碍。 健赞 健赞实施灌注工艺，结合ATF技术进行细胞保留以及通过3C-PCC系统（GE Healthcare）进行Protein A捕获的描述之前已经有过。为了进一步发展这一制造平台，健赞最近开始建设一个生物制品研发开发设施。这个新的20000平方英尺的设施，以其位于马萨诸塞州弗雷明汉的纽约大道的位置而得名，31NYA试点工厂包括两个操作空间，其中包括一个一次性技术套件，配备波浪袋和高达500升尺寸的搅拌式SUBs。该设施的目标是评估并确认一次性生物反应器、细胞保留技术和周期性逆流色谱设计的选择。评估工艺的可制造性、展示“封闭系统”操作以及进行“按比例”研究以支持连续制造工艺开发也被提及为健赞旨在实施的“概念验证”努力的要素。所付出的努力和投资表明，健赞认真致力于实施连续下游制造，同时扩大其生物制品组合。 Just Biotherapeutics Just Biotherapeutics自称为“专注于将加速生物治疗药物开发并大幅降低其制造成本的技术的集成设计公司”。他们的目标是通过集成设计方法，在全球市场上提供对重要生物制品的获取。 Just Biotherapeutics在制造规模上展示了连续处理的概念验证（2019年佛罗里达州奥兰多ACS）。他们的连续工艺从一个500升灌注生物反应器的培养基浓缩物开始。通过使用中空纤维切向流装置保留细胞。澄清的进料被导向一个200升混合器，捕获步骤通过一个三柱Protein A工艺的Pall BioSMB Process 80进行。洗脱池在Pall病毒灭活系统Cadence VI中经过低pH处理并中和。从Protein A到病毒灭活的下游连续工艺进行了6天，但未来计划展示这一工艺更长时间。 这项工作展示了使用现成设备组装连续工艺的能力，并突出了能够以连续方式结合上游和下游工艺的协同作用。 默克 默克公司（新泽西州肯尼沃斯）采用连续处理的动机来自于其相对较低的安装生物反应器容量，将这一“劣势”转变为竞争优势。为此，默克继续成为集成连续生物处理的先驱之一。我们在2017年最后一次报道了默克的活动，重点关注其蛋白质精炼厂（PRO Lab）及其在六十天期间运行的联合上游和下游连续工艺。该工艺由Delta V控制系统完全自动化，包括一个10升灌注生物反应器，连接到一个连续的下游工艺，其中包括用于Protein A捕获的Cadence BioSMB PD和用于低pH病毒灭活的活塞流反应器。两个抛光色谱步骤，其中一个在Cadence BioSMB PD上进行，与病毒过滤和配方相结合。从那时起，默克继续在实验室开展活动，致力于质量源于设计和工艺分析技术，旨在实现实时产品放行。此外，默克还致力于Protein A捕获步骤的放大，将单柱工艺转移到多柱Cadence BioSMB PD，并最终在三周内放大到Cadence BioSMB Process。这一工艺比较表明，质量属性保持不变，但连续工艺的生产率提高了3倍以上（表4.2）。 诺华 诺华声称其接近满负荷制造能力。这提供了增加产能和创新制造平台的机会。全球制造目标包括降低成本、最大化净现值（NPV）、加快开发速度、最小化技术转移风险和实现灵活性。为此，诺华设想了一个重复制造设施，能够生产广泛的分子。诺华的目标是在开发、制造的所有阶段使用单一规模的设备，以消除放大的需要。 诺华的未来制造平台基于不锈钢，因其经过验证的稳健性。上游工艺基于高细胞密度的灌注细胞培养。这与使用AKTA PCC的连续捕获工艺相结合，随后是重复的批次VI。操作之间的缓冲罐被最小化到大约5分钟的保持时间，以平衡操作之间的流量。 有了这样的设施，诺华预测一个传统的批次制造设施，拥有6个11000升的生物反应器，每年生产4.5吨，可以被一个8个1000升的设施所取代。连续设施的建设时间仅为2年，比批次设施少3年，从而导致货物成本降低50%（Joel Schultz，世界生物制药论坛，2019）。 结论：临界点 我们看到生物制药领域的这些发展使行业逐渐接近那个传奇的临界点，在这一点上，连续处理的好处将超过风险。也许并不奇怪，这里描述的连续处理的早期采用者正在利用灌注来强化上游工艺，以在每个生物反应器体积中生产更多的产品。灌注工艺还使得“一刀切”的处理愿景成为可能，这可能消除了通过延长操作时间以生产更多材料的放大需求。然后，连续下游处理是灌注方法的逻辑延伸，消除了将产品汇集并作为离散批次进行纯化的需要。为了强化下游工艺，似乎每个单元操作都有选择。特别是，捕获色谱有多种不同的方法。持续的改进循环使我们相信，多柱方法将被采用以应对不断增加的产品滴度。 对于生物仿制药和已批准药物的更新工艺来说，上市时间可能远不如成本降低重要。也许并不奇怪，生物仿制药公司正在引领连续处理的道路。预计生物仿制药公司将克服技术和监管障碍，降低采用连续处理的门槛。到这一点时，尤其是那些达到当前产能极限的原研公司，将别无选择，只能迅速采用连续处理。识别微信二维码，添加生物制品圈小编，符合条件者即可加入生物制品微信群！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。

BOSTON--(BUSINESS WIRE)--Vertex Pharmaceuticals Incorporated (Nasdaq: VRTX) today reported consolidated financial results for the first quarter ended March 31, 2025, and raised the low end of its total revenue guidance range by $100 million, from $11.75 billion to $12 billion to a new range of $11.85 billion to $12 billion. “Vertex delivered a strong start to 2025 with notable execution across the business as we grow and diversify the revenue base, progress multiple launches and advance the R&D pipeline. We continued to expand our leadership in CF and build global momentum for CASGEVY, and we launched JOURNAVX in moderate-to-severe acute pain,” said Reshma Kewalramani, M.D., Chief Executive Officer and President of Vertex. “With multiple programs in pivotal development including povetacicept, which continues to make rapid progress in achieving its potential as a pipeline-in-a-product, and additional programs in early and mid-stage development, Vertex is poised to continue to deliver value for years to come.” First Quarter 2025 Results Total revenue increased 3% to $2.77 billion compared to the first quarter of 2024, primarily driven by the continued performance of TRIKAFTA®/KAFTRIO® and an early contribution from the U.S. launch of ALYFTREK. In the U.S., total revenue increased 9% to $1.66 billion due to continued strong patient demand and higher net realized pricing. Outside the U.S., total revenue decreased 5% to $1.11 billion as strong patient demand in both established and newer markets was offset by the expected revenue decline in Russia, where Vertex is experiencing violation of its intellectual property rights. Vertex has strong intellectual property protection for its medicines around the world and believes this violation in Russia is a limited and isolated matter. Combined GAAP and Non-GAAP R&D, Acquired IPR&D and SG&A expenses were $1.4 billion and $1.2 billion, respectively, compared to $1.2 billion and $1.0 billion, respectively, in the first quarter of 2024. The increases were primarily due to continued R&D investment in support of multiple mid- and late-stage clinical development programs and increased commercial investment to support the launch of JOURNAVX. Intangible asset impairment charge of $379.0 million associated with VX-264 (the “cells plus device” program) in patients with type 1 diabetes, which will not be advancing further in clinical development, was included in GAAP operating income in the first quarter of 2025. GAAP effective tax rate was 11.5% compared to 14.0% for the first quarter of 2024. Non-GAAP effective tax rate was 18.8% compared to 17.4% for the first quarter of 2024. The difference between the GAAP and non-GAAP effective tax rates was primarily due to tax benefits related to stock-based compensation. GAAP net income was $646 million compared to $1.1 billion for the first quarter of 2024, as a result of increased operating expenses and the intangible asset impairment charge. Non-GAAP net income was $1.1 billion compared to $1.2 billion in the first quarter of 2024, as a result of increased operating expenses. Cash, cash equivalents and total marketable securities as of March 31, 2025, were $11.4 billion, compared to $11.2 billion as of December 31, 2024. The increase was primarily due to cash flows from operating activities, partially offset by repurchases of Vertex’s common stock pursuant to its share repurchase program. Full Year 2025 Financial Guidance Vertex today raised the low end of its full-year 2025 revenue guidance range from $11.75 to $12 billion to $11.85 billion to $12 billion, which assumes continued growth in CF, including the launch of ALYFTREK; as well as continued uptake of CASGEVY in multiple regions; and early contributions from the launch of JOURNAVX. Vertex reiterated its guidance for both combined GAAP and Non-GAAP R&D, AIPR&D and SG&A expenses, which includes expectations for continued investment in multiple mid- and late-stage clinical development programs and commercial capabilities, and AIPR&D expenses of approximately $100 million. This guidance also includes an immaterial cost impact from tariffs in 2025 based on currently known tariff rates and regulations. Vertex’s financial guidance is summarized below: Current FY 2025 FY 2025 Total revenue $11.85 to $12.0 billion $11.75 to $12.0 billion Combined GAAP R&D, AIPR&D and SG&A expenses * Unchanged $5.55 to $5.7 billion Combined Non-GAAP R&D, AIPR&D and SG&A expenses * Unchanged $4.9 to $5.0 billion Non-GAAP effective tax rate Unchanged 20.5% to 21.5% *The difference between the combined GAAP R&D, AIPR&D and SG&A expenses and the combined non-GAAP R&D, AIPR&D and SG&A expenses guidance relates primarily to $650 million to $700 million of stock-based compensation expense. **Combined GAAP and Non-GAAP R&D, AIPR&D and SG&A expenses guidance includes approximately $100 million of AIPR&D expenses. Key Business Highlights Cystic Fibrosis (CF) Portfolio Marketed Products Vertex has worked for more than 20 years to discover and develop medicines to treat the underlying cause of CF. Vertex CFTR modulators can treat 95 percent of all people living with CF in markets with the highest prevalence and are approved for patients as young as one month old. ALYFTREK, the newest marketed CFTR modulator, is approved in the U.S. and the United Kingdom (U.K.) for the treatment of patients 6 years and older. Vertex anticipates the number of CF patients taking its medicines will continue to grow through new approvals and reimbursement, treatment of younger patients, increased survival, and expansion into additional geographies. Recent and anticipated progress includes: ALYFTREK, the once-daily, next-in-class combination CFTR modulator for the treatment of people with CF ages 6 years and older who have at least one F508del mutation or another responsive mutation in the CFTR gene, is now approved in the U.S. and the U.K. Vertex is working with the National Health Service (NHS) in the U.K. to secure coverage for eligible patients. In April, the European Medical Agency’s (EMA’s) Committee for Medicinal Products for Human Use (CHMP) adopted a positive opinion for ALYFTREK for the treatment of CF patients ages 6 years and older. European Commission approval is expected in the second half of 2025. Regulatory reviews are underway for ALYFTREK in Canada, Switzerland, Australia and New Zealand. Vertex secured European Commission approval for the label expansion of KAFTRIO in combination with ivacaftor for CF patients ages 2 years and older who have at least one non-class I mutation in the CFTR gene. With this approval, approximately 4,000 people in the European Union are newly eligible for a medicine that treats the underlying cause of their disease. Work to ensure access for all eligible patients is underway. CASGEVY for the treatment of sickle cell disease (SCD) and transfusion-dependent beta thalassemia (TDT) CASGEVY is a non-viral, ex vivo CRISPR/Cas9 gene-edited cell therapy for eligible patients with SCD or TDT that has been shown to reduce or eliminate vaso-occlusive crises (VOCs) for patients with SCD and transfusion requirements for patients with TDT. CASGEVY is approved in the U.S., Great Britain, the EU, the Kingdom of Saudi Arabia (KSA), the Kingdom of Bahrain (Bahrain), Canada, Switzerland and the United Arab Emirates (UAE) for the treatment of both SCD and TDT. Recent highlights include: In Europe, Vertex secured a formal reimbursement agreement with NHS England for SCD patients to access CASGEVY, following the agreement reached in August 2024 for eligible TDT patients to access CASGEVY. Vertex recently entered into a similar agreement in Wales for both eligible SCD and TDT patients. Following a positive assessment, Vertex also finalized national reimbursement in Austria. In the Middle East, following regulatory approval in the UAE, Vertex secured reimbursement in the majority of emirates. Since launch through May 1st, Vertex has activated more than 65 authorized treatment centers (ATCs) globally and approximately 90 patients have had their first cell collection. Vertex recently filed a manufacturing license submission with the FDA and expects to begin manufacturing CASGEVY in Portsmouth, NH, in the second half of 2025. This submission is part of the planned ramp of CASGEVY manufacturing capacity as demand for the therapy increases. JOURNAVX (suzetrigine) for the treatment of moderate-to-severe acute pain JOURNAVX is a first-in-class, oral, selective, non-opioid NaV1.8 pain signal inhibitor, approved in the U.S. for the treatment of moderate-to-severe acute pain. Vertex continues to advance a portfolio of selective NaV1.8 and NaV1.7 pain signal inhibitors for stand-alone use or in combination, with potential to provide effective pain relief without the limitations of opioids and other available medicines. On January 30, 2025, the FDA approved JOURNAVX for the treatment of adults with moderate-to-severe acute pain, and it is now available and stocked at pharmacies across the U.S., including major national and regional retail pharmacy chains. Since JOURNAVX became available in early March through April 18th, more than 20,000 prescriptions have been written and filled across the hospital and retail settings in different acute pain conditions, consistent with JOURNAVX’s broad label. As of May 1st, across commercial and government payers, approximately 94 million lives already have covered access to JOURNAVX, and approximately 42 million have unrestricted access (i.e., without the need for prior authorization or step edits). Vertex has reached a formal coverage agreement with a large national Pharmacy Benefit Manager (PBM) to make JOURNAVX available to their customers, representing approximately 22 million commercial lives. A total of ten state Medicaid plans are also providing unrestricted access to JOURNAVX and 20 more are currently evaluating their policies. More than 50 large healthcare systems have taken steps to initiate pharmacy and therapeutics (P&T) committee reviews of JOURNAVX, and some have already added it on formularies. Vertex recently initiated two Phase 4 studies in various moderate-to-severe acute pain conditions to provide additional data on the effectiveness and safety of JOURNAVX as part of real-world clinical practice, in both inpatient and outpatient settings. The Alternatives to Pain Act has been reintroduced in both the House and Senate of the new U.S. Congress, while nearly 35 U.S. states to date have passed or introduced new legislation in support of non-opioid options. Vertex also expects JOURNAVX to be added to the list of treatments eligible for an add-on payment under the NOPAIN Act, which became effective on January 1, 2025. Select Clinical-Stage R&D Pipeline Cystic Fibrosis Vertex is enrolling and dosing studies in younger age groups — a Phase 3 study of TRIKAFTA/KAFTRIO in 1- to 2-year-olds and a Phase 3 study of ALYFTREK in 2- to 5-year-olds — to expand the labels and enable earlier treatment of children with CF. Consistent with its commitment to serial innovation and bringing as many patients as possible to normal levels of CFTR function, Vertex continues to advance new oral small molecule combination therapies through preclinical and clinical development. Vertex expects to advance the once-daily, next-generation 3.0 VX-828 combination into a clinical trial in people with CF this year. Vertex has implemented a temporary pause to the multiple ascending dose (MAD) portion of the Phase 1/2 study of VX-522, a nebulized CFTR mRNA therapy, in order to assess a tolerability issue. Sickle Cell Disease and Transfusion-Dependent Beta Thalassemia Vertex has completed enrollment of children 5 to 11 years of age with SCD or TDT in two global Phase 3 studies of CASGEVY. Vertex continues to advance preclinical assets for gentler conditioning for CASGEVY, which could broaden the eligible patient population. Acute Pain Vertex expects to complete the Phase 2 study for an oral formulation of VX-993 this quarter. VX-993 is a next-generation selective NaV1.8 pain signal inhibitor, for the treatment of moderate-to-severe acute pain following bunionectomy surgery. Vertex expects to report results from the Phase 2 study in the second half of 2025. VX-993 has Fast Track designation for moderate-to-severe acute pain. Vertex has completed the Phase 1 study of an intravenous formulation of VX-993 in healthy volunteers. The FDA has granted the intravenous formulation of VX-993 Fast Track designation for moderate-to-severe acute pain. Peripheral Neuropathic Pain (PNP) Vertex continues to enroll and dose patients with diabetic peripheral neuropathy (DPN) in a Phase 3 pivotal trial of suzetrigine. The FDA has granted suzetrigine Fast Track designation in PNP and Breakthrough Therapy designation in DPN. Vertex continues to enroll and dose patients in a Phase 2 study for the oral formulation of VX-993 for the treatment of DPN. Type 1 Diabetes (T1D) Vertex is evaluating stem cell-derived, fully differentiated islet cell therapies for patients suffering from T1D, with the goal of developing a potential one-time functional cure for this disease. Vertex continues to enroll and dose patients in the Phase 3 portion of the Phase 1/2/3 study of zimislecel in patients with T1D with severe hypoglycemic events and impaired awareness of hypoglycemia in the U.S., Canada, U.K., and EU. Vertex expects to complete enrollment and dosing of the pivotal study in the second quarter of 2025 and submit marketing applications to global regulators in 2026. Zimislecel has been granted Regenerative Medicine Advanced Therapy (RMAT) and Fast Track designations from the U.S. Food and Drug Administration, Priority Medicines (PRIME) designation from the EMA, and has secured an Innovation Passport under the Innovative Licensing and Access Pathway (ILAP) from the UK Medicines and Healthcare products Regulatory Agency (MHRA). Vertex will present updated clinical data from the 12 participants who received the full dose of zimislecel as a single infusion with over one year of follow-up in the Phase 1/2 portion of the study in an oral presentation at the American Diabetes Association 85 th Scientific Sessions. Vertex will host an in-person investor event at the ADA conference on Friday, June 20, 2025 at 7:15 p.m. CT / 8:15 p.m. ET. The investor event will also be webcast live, and a link to the webcast can be accessed through Vertex’s website at www.vrtx.com in the “Investors” section. Vertex previously announced that it has discontinued development of the VX-264 (“cells plus device” program), as the Phase 1/2 study did not meet its efficacy endpoint. Vertex is pursuing research-stage programs to evaluate additional approaches that could provide transformative benefit to people with T1D and reduce or eliminate the need for standard immunosuppressive regimens. These approaches include alternative immunosuppressives, gene editing, and novel immunoprotection to encapsulate the islet cells. IgA Nephropathy (IgAN), Primary Membranous Nephropathy (pMN) and Other B Cell-Mediated Diseases Vertex is developing povetacicept, a dual antagonist of the BAFF and APRIL cytokines, which play key roles in the pathogenesis of multiple B cell-mediated diseases. Pove represents a potentially best-in-class approach to treat IgAN and pMN and has pipeline-in-a-product potential. The global Phase 3 RAINIER trial of povetacicept in patients with IgAN has completed enrollment of the interim analysis cohort. The interim analysis will be conducted once this cohort reaches 36 weeks of treatment, with the potential to file for Accelerated Approval in the U.S. in the first half of 2026, if results are supportive. Studies to support the launch of povetacicept for home administration are underway. Based on positive results of povetacicept in primary membranous nephropathy (pMN) in the RUBY-3 study, Vertex has reached agreement with the FDA to advance povetacicept into pivotal development for this disease. Vertex will initiate a single, adaptive Phase 2/3 trial of povetacicept vs. standard-of-care this year. In the Phase 2 portion, both 80 mg and 240 mg of povetacicept will be assessed and the selected dose will advance to Phase 3, where the primary endpoint of complete clinical remission at 72 weeks of treatment will be evaluated. Vertex plans to present updated data from the IgAN and pMN cohorts of the RUBY-3 study at upcoming medical congresses. Vertex expects to share data and next steps from other RUBY-3 renal diseases and the hematologic conditions in the RUBY-4 study later this year. APOL1-Mediated Kidney Disease (AMKD) Vertex has discovered and advanced multiple oral, small molecule inhibitors of APOL1 function, pioneering a new class of medicines that targets the underlying genetic driver of this kidney disease. Vertex expects to complete enrollment in the interim analysis cohort of the Phase 3 portion of the AMPLITUDE trial of inaxaplin in the second half of 2025. Vertex will conduct the pre-planned interim analysis once this cohort has been treated for 48 weeks, with potential to file for accelerated approval in the U.S. if the results are supportive. Vertex continues to enroll and dose patients in the AMPLIFIED Phase 2 study of inaxaplin in people with AMKD and diabetes or other co-morbidities. Myotonic Dystrophy Type 1 (DM1) Vertex is evaluating multiple approaches that target the underlying cause of DM1. Vertex’s lead approach, VX-670, is an oligonucleotide linked to a cyclic peptide, which holds the potential to address the underlying cause of DM1. Vertex continues to enroll and dose the MAD portion of the global Phase 1/2 clinical trial of VX-670 in people with DM1, which will assess both safety and efficacy. Autosomal Dominant Polycystic Kidney Disease (ADPKD) Vertex is developing small molecule correctors that restore function to the variant polycystin 1 (PC1) protein, with the goal of addressing the underlying cause of ADPKD. Vertex has completed the Phase 1 study of VX-407 in healthy volunteers. Vertex expects to advance VX-407 into a Phase 2 proof-of-concept study this year in patients with a subset of variants in the PKD1 gene, which encodes the PC1 protein, estimated to be up to ~30,000 (or ~10%) of the overall patient population. Non-GAAP Financial Measures In this press release, Vertex's financial results and financial guidance are provided in accordance with accounting principles generally accepted in the United States (GAAP) and using certain non-GAAP financial measures. In particular, non-GAAP financial results and guidance exclude from Vertex's pre-tax income (i) stock-based compensation expense, (ii) intangible asset amortization expense, (iii) gains or losses related to the fair value of the company's strategic investments, (iv) increases or decreases in the fair value of contingent consideration, (v) acquisition-related costs, (vi) an intangible asset impairment charge, and (vii) other adjustments. The company's non-GAAP financial results also exclude from its provision for income taxes the estimated tax impact related to its non-GAAP adjustments to pre-tax income described above and certain discrete items. These results should not be viewed as a substitute for the company’s GAAP results and are provided as a complement to results provided in accordance with GAAP. Management believes these non-GAAP financial measures help indicate underlying trends in the company's business, are important in comparing current results with prior period results and provide additional information regarding the company's financial position that the company believes is helpful to an understanding of its ongoing business. Management also uses these non-GAAP financial measures to establish budgets and operational goals that are communicated internally and externally, to manage the company's business and to evaluate its performance. The company’s calculation of non-GAAP financial measures likely differs from the calculations used by other companies. A reconciliation of the GAAP financial results to non-GAAP financial results is included in the attached financial information. The company provides guidance regarding combined R&D, AIPR&D and SG&A expenses and effective tax rate on a non-GAAP basis. Unless otherwise noted, the guidance regarding combined R&D, AIPR&D and SG&A expenses does not include estimates associated with any potential future business development transactions, including collaborations, asset acquisitions and/or licensing of third-party intellectual property rights. The company does not provide guidance regarding its GAAP effective tax rate because it is unable to forecast with reasonable certainty the impact of excess tax benefits related to stock-based compensation and the possibility of certain discrete items, which could be material. Vertex Pharmaceuticals Incorporated Consolidated Statements of Income (unaudited, in millions, except per share amounts) Three Months Ended March 31, 2025 2024 Revenues: Product revenues, net $ 2,760.2 $ 2,690.6 Other revenues 10.0 — Total revenues 2,770.2 2,690.6 Costs and expenses: Cost of sales 363.0 342.6 Research and development expenses 979.7 789.1 Acquired in-process research and development expenses 19.8 76.8 Selling, general and administrative expenses 396.4 342.7 Intangible asset impairment charge 379.0 — Change in fair value of contingent consideration 2.2 (0.1 ) Total costs and expenses 2,140.1 1,551.1 Income from operations 630.1 1,139.5 Interest income 120.9 181.2 Interest expense (3.0 ) (10.4 ) Other expense, net (17.6 ) (31.2 ) Income before provision for income taxes 730.4 1,279.1 Provision for income taxes 84.1 179.5 Net income $ 646.3 $ 1,099.6 Net income per common share: Basic $ 2.52 $ 4.26 Diluted $ 2.49 $ 4.21 Shares used in per share calculations: Basic 256.9 258.2 Diluted 259.5 261.1 Vertex Pharmaceuticals Incorporated Total Revenues (unaudited, in millions) Three Months Ended March 31, 2025 2024 TRIKAFTA/KAFTRIO $ 2,535.5 $ 2,483.6 ALYFTREK 53.9 — Other product revenues (1) 170.8 207.0 Product revenues, net 2,760.2 2,690.6 Other revenues 10.0 — Total revenues $ 2,770.2 $ 2,690.6 1: In the first quarter of 2025, "Other product revenues" included $14.2 million from CASGEVY, and an insignificant amount from JOURNAVX. In the first quarter of 2024, there were no revenues for these products. The remaining “Other product revenues” are related to KALYDECO, ORKAMBI, and SYMDEKO/SYMKEVI, our other CF products. Vertex Pharmaceuticals Incorporated Reconciliation of GAAP to Non-GAAP Financial Information (unaudited, in millions, except percentages) Three Months Ended March 31, 2025 2024 GAAP cost of sales $ 363.0 $ 342.6 Stock-based compensation expense (2.6 ) (1.8 ) Intangible asset amortization expense (5.0 ) (5.0 ) Non-GAAP cost of sales $ 355.4 $ 335.8 GAAP research and development expenses $ 979.7 $ 789.1 Stock-based compensation expense (100.1 ) (119.4 ) Intangible asset amortization expense (0.6 ) — Non-GAAP research and development expenses $ 879.0 $ 669.7 Acquired in-process research and development expenses $ 19.8 $ 76.8 GAAP selling, general and administrative expenses $ 396.4 $ 342.7 Stock-based compensation expense (63.4 ) (70.7 ) Non-GAAP selling, general and administrative expenses $ 333.0 $ 272.0 Combined non-GAAP R&D, AIPR&D and SG&A expenses $ 1,231.8 $ 1,018.5 GAAP other expense, net $ (17.6 ) $ (31.2 ) Decrease in fair value of strategic investments 15.0 27.0 Non-GAAP other expense, net $ (2.6 ) $ (4.2 ) GAAP provision for income taxes $ 84.1 $ 179.5 Tax adjustments (2) 160.1 81.6 Non-GAAP provision for income taxes $ 244.2 $ 261.1 GAAP effective tax rate 11.5 % 14.0 % Non-GAAP effective tax rate 18.8 % 17.4 % Vertex Pharmaceuticals Incorporated Reconciliation of GAAP to Non-GAAP Financial Information (continued) (unaudited, in millions, except per share amounts) Three Months Ended March 31, 2025 2024 GAAP operating income $ 630.1 $ 1,139.5 Stock-based compensation expense 166.1 191.9 Intangible asset impairment charge 379.0 — Intangible asset amortization expense 5.6 5.0 Increase (decrease) in fair value of contingent consideration 2.2 (0.1 ) Non-GAAP operating income $ 1,183.0 $ 1,336.3 GAAP net income $ 646.3 $ 1,099.6 Stock-based compensation expense 166.1 191.9 Intangible asset impairment charge 379.0 — Intangible asset amortization expense 5.6 5.0 Decrease in fair value of strategic investments 15.0 27.0 Increase (decrease) in fair value of contingent consideration 2.2 (0.1 ) Total non-GAAP adjustments to pre-tax income 567.9 223.8 Tax adjustments (2) (160.1 ) (81.6 ) Non-GAAP net income $ 1,054.1 $ 1,241.8 Net income per diluted common share: GAAP $ 2.49 $ 4.21 Non-GAAP $ 4.06 $ 4.76 Shares used in diluted per share calculations: GAAP and Non-GAAP 259.5 261.1 2: In the first quarter of 2025 and 2024, “Tax adjustments” included the estimated income taxes related to non-GAAP adjustments to the company's pre-tax income and excess tax benefits related to stock-based compensation. Vertex Pharmaceuticals Incorporated Condensed Consolidated Balance Sheets (unaudited, in millions) March 31, 2025 December 31, 2024 Assets Cash, cash equivalents and marketable securities $ 6,201.2 $ 6,115.9 Accounts receivable, net 1,805.1 1,609.4 Inventories 1,359.7 1,205.4 Prepaid expenses and other current assets 642.8 665.7 Total current assets 10,008.8 9,596.4 Property and equipment, net 1,295.9 1,227.8 Goodwill and other intangible assets, net 1,529.2 1,913.9 Deferred tax assets 2,544.3 2,331.1 Operating lease assets 1,338.5 1,356.8 Long-term marketable securities 5,156.5 5,107.9 Other long-term assets 1,007.3 999.3 Total assets $ 22,880.5 $ 22,533.2 Liabilities and Shareholders' Equity Accounts payable and accrued expenses $ 3,396.8 $ 3,201.6 Other current liabilities 386.4 363.0 Total current liabilities 3,783.2 3,564.6 Long-term operating lease liabilities 1,537.7 1,544.4 Long-term finance lease liabilities 111.4 112.8 Other long-term liabilities 951.9 901.8 Shareholders' equity 16,496.3 16,409.6 Total liabilities and shareholders' equity $ 22,880.5 $ 22,533.2 Common shares outstanding 257.0 256.9 About Vertex Vertex is a global biotechnology company that invests in scientific innovation to create transformative medicines for people with serious diseases. The company has approved medicines that treat the underlying causes of multiple chronic, life-shortening genetic diseases — cystic fibrosis, sickle cell disease and transfusion-dependent beta thalassemia — and continues to advance clinical and research programs in these diseases. Vertex also has a robust clinical pipeline of investigational therapies across a range of modalities in other serious diseases where it has deep insight into causal human biology, including acute and neuropathic pain, type 1 diabetes, APOL1-mediated kidney disease, IgA nephropathy, primary membranous nephropathy, autosomal dominant polycystic kidney disease, and myotonic dystrophy type 1. Vertex was founded in 1989 and has its global headquarters in Boston, with international headquarters in London. Additionally, the company has research and development sites and commercial offices in North America, Europe, Australia, Latin America and the Middle East. Vertex is consistently recognized as one of the industry's top places to work, including 14 consecutive years on Science magazine's Top Employers list and one of Fortune’s 100 Best Companies to Work For. For company updates and to learn more about Vertex's history of innovation, visit www.vrtx.com or follow us on LinkedIn, Facebook, Instagram, YouTube and Twitter/X. Special Note Regarding Forward-Looking Statements This press release contains forward-looking statements that are subject to risks, uncertainties and other factors. All statements other than statements of historical fact are statements that could be deemed forward-looking statements, including all statements regarding the intent, belief, or current expectation of Vertex and members of the Vertex senior management team. Forward-looking statements are not purely historical and may be accompanied by words such as “anticipates,” “may,” “forecasts,” “expects,” “intends,” “plans,” “potentially,” “believes,” “seeks,” “estimates,” and other words and terms of similar meaning. Such statements include, without limitation, Dr. Kewalramani's statements in this press release, the information provided regarding future financial performance and operations, the section captioned “Full Year 2025 Financial Guidance” and statements regarding (i) expectations for Vertex’s continued growth in CF, including by increasing the number of CF patients taking its medicines through new approvals and reimbursements, treatment of younger patients, increased survival, and expansion into additional geographies, (ii) expectations regarding securing coverage for ALYFTREK in the U.K., (iii) expectations regarding approval of ALYFTREK in the E.U. in the second half of 2025, (iv) beliefs regarding the anticipated benefits and commercial launch progress of CASGEVY, and with respect to increasing and initiating manufacturing activities for CASGEVY in the second half of 2025, (v) expectations regarding the potential benefits of JOURNAVX for the treatment of moderate-to-severe acute pain, including regarding the efficacy and safety of JOURNAVX, beliefs that JOURNAVX has potential to provide effective pain relief without the limitations of opioids and other available medicines, expectations with respect to the commercial launch progress and that commercial coverage of JOURNAVX will continue to expand throughout 2025, and expectations that JOURNAVX will be included on the list of treatments that quality for add-on payment under the NOPAIN Act, (vi) expectations to expand the labels for TRIKAFTA/KAFTRIO and ALYFTREK and enable earlier treatment of children with CF, (vii) expectations to advance the VX-828 combination into people with CF this year, (viii) expectations for the VX-522 clinical trial, (ix) expectations regarding Vertex’s SCD and TDT program, including with respect to gentler conditioning for CASGEVY broadening the eligible patient population, (x) plans with respect to the studies of the intravenous and oral formulation of VX-993 for the treatment of acute pain, including expectations to complete the Phase 2 study of the oral formulation of VX-993 in acute pain and expectations to report results from that study in the second half of 2025, (xi) expectations regarding the pivotal study evaluating zimislecel in T1D, including the expectations to complete enrollment and dosing during the fiscal quarter and submit marketing applications to global regulators in 2026, expectations to present updated data in upcoming medical conferences, and expectations and plans to pursue additional approaches to standard immunosuppression regimens, (xii) expectations with respect to povetacicept, including beliefs about its potential benefits and therapeutic scope, study designs, expectations regarding the Phase 3 RAINIER study, including plans to file for potential accelerated approval in the U.S. in the first half of 2026, beliefs with respect to the RUBY-3 basket study and plans to present updated data from this study at upcoming medical congresses, expectations and plans with respect to advancing povetacicept into pivotal development in pMN, beliefs with respect to the RUBY-4 basket study and expectations to share data and next steps later this year, (xiii) expectations regarding the AMPLITUDE trial in AMKD, including expectations to complete enrollment in the interim analysis cohort in the second half of 2025 and, assuming a positive interim analysis, apply for potential accelerated approval in the U.S., (xiv) expectations for the potential benefits and clinical status of VX-670 for the treatment in people with DM1, and (xv) expectations regarding the ADPKD program, including the potential benefits of VX-407 and expectations to advance VX-407 into a Phase 2 proof-of-concept study in 2025. While Vertex believes the forward-looking statements contained in this press release are accurate, these forward-looking statements represent the company's beliefs only as of the date of this press release and there are a number of risks and uncertainties that could cause actual events or results to differ materially from those expressed or implied by such forward-looking statements. Those risks and uncertainties include, among other things, that the company's expectations regarding its 2025 full year revenues, expenses and effective tax rates may be incorrect (including because one or more of the company's assumptions underlying its expectations may not be realized), that we may be unable to successfully commercialize ALYFTREK as a treatment for CF or JOURNAVX as a treatment for acute pain, that external factors may have different or more significant impacts on the company's business or operations than the company currently expects, that data from preclinical testing or clinical trials, especially if based on a limited number of patients, may not be indicative of final results or available on anticipated timelines, that patient enrollment in the company’s trials may be delayed, that the company may not realize the anticipated benefits from collaborations with third parties, that data from the company's development programs may not support registration or further development of its potential medicines in a timely manner, or at all, due to safety, efficacy or other reasons, and that anticipated commercial launches may be delayed, if they occur at all. Forward-looking statements in this press release should be evaluated together with the many uncertainties that affect Vertex’s business, particularly those risks listed under the heading “Risk Factors” and the other cautionary factors discussed in Vertex’s periodic reports filed with the SEC, including Vertex’s annual report on Form 10-K and its quarterly reports on Form 10-Q and current reports on Form 8-K, all of which are filed with the Securities and Exchange Commission (SEC) and available through the company's website at www.vrtx.com and on the SEC’s website at www.sec.gov. You should not place undue reliance on these statements, or the scientific data presented. Vertex disclaims any obligation to update the information contained in this press release as new information becomes available. Conference Call and Webcast The company will host a conference call and webcast at 4:30 p.m. ET. To access the call, please dial (833) 630-2124 (U.S.) or +1(412) 317-0651 (International) and reference the “Vertex Pharmaceuticals First Quarter 2025 Earnings Call.” The conference call will be webcast live and a link to the webcast can be accessed through Vertex's website at www.vrtx.com in the "Investors" section. To ensure a timely connection, it is recommended that participants register at least 15 minutes prior to the scheduled webcast. An archived webcast will be available on the company's website. (VRTX-E)